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aki is associated with short - term morbidity , a long - term risk of chronic kidney disease ( ckd ) and cardiovascular events and decreases survival [ 38 ] . hospitalized patients , particularly those with comorbidities and those undergoing complex procedures are at high risk for developing aki [ 7 , 9 ] . despite increasing attention in recent years [ 1 , 2 , 8 , 10 ] , little improvement in outcomes for aki has occurred and many authors have speculated that this is largely due to the inability to identify aki early , in a potentially easily reversible stage [ 1 , 2 ] . common functional markers such as serum creatinine or urine output are late indicators of aki . there are numerous potential strategies that could be deployed to mitigate aki if sufficient warning was provided . in this review , we will summarize the current evidence that supports the use of cell - cycle arrest biomarkers for risk assessment of aki , we will consider various responses to this warning and we will speculate on how this mechanism could be exploited to protect the kidney . serum creatinine ( scr ) , the most widely used marker of kidney function , is a component of the definition of aki , and yet as a single measurement is completely useless in differentiating aki from ckd . while scr is an adequate marker of glomerular filtration rate ( gfr ) , scr itself does not correlate with hospital survival whether measured at the time of presentation or the start of dialysis . what predicts short- and long - term outcomes is the change in renal function and herein lies the problem . changes in scr require several hours to days before they reach steady state following an injury to the kidney . thus the change in scr is an excellent tool for defining when a change in function has occurred but not particularly good for detecting that it is occurring ( or about to occur ) . worse yet , scr , like all functional markers , may be both insensitive to and non - specific for injury . uo will often decrease before scr increases making it a more time sensitive marker of gfr . while in the absence of obstruction if there is no uo , there can be no gfr , not all reductions in uo signal aki . sustained oliguria is invariably associated with aki but then , the timeliness of uo as an early indicator is lost . importantly , functional change is neither necessary nor sufficient to define aki as it is occurring . over time , a persistent renal functional change can be used to infer kidney injury in the same way that regional wall motion abnormalities or a decrement in ejection fraction can be used to infer myocardial injury . however conversely , the absence of functional change can not be used to exclude injury , especially in previously healthy individuals with normal renal reserve . as such , markers that can detect actual damage or warn of impending damage are required in the same way that functional measures alone ( e.g. left ventricular ejection fraction ) are insufficient to manage acute coronary syndrome . in 2013 , we reported the results of a prospective , observational , international investigation ( sapphire study ) of tissue inhibitor of metalloproteinases-2 ( timp-2 ) and insulin - like growth factor - binding protein 7 ( igfbp7 ) in a heterogeneous group of critically ill ( evidence of respiratory or cardiovascular failure ) patients . this report actually included two multi - center studies discovery and validation . in the discovery phase we enrolled 522 adults in three distinct cohorts including patients with sepsis , shock , major surgery and trauma and examined over 300 markers in urine and blood . in the validation study we enrolled 744 adults with critical illness and without evidence of aki at enrollment ; the final analysis cohort was a heterogeneous sample of 728 critically ill patients . the primary end point was moderate - severe aki [ kidney disease : improving global outcomes ( kdigo ) stage 23 ] within 12 h of sample collection which occurred in 14% of sapphire subjects . area under the receiver operating characteristic curve ( auc ) of 0.80 together ( 0.76 and 0.79 alone ) . [ timp-2][igfbp7 ] was significantly superior to all previously described markers of aki ( p < 0.002 ) including neutrophil gelatinase - associated lipocalin ( ngal ) and kidney injury molecule ( kim)-1 , none of which achieved an auc > 0.72 . however , this result might be due in part to varying kinetics of different early aki biomarkers . [ timp-2][igfbp7 ] significantly improved risk stratification when added to a 9-variable clinical model when analyzed using cox proportional hazards model , generalized estimating equation , integrated discrimination improvement or net reclassification improvement . finally , in sensitivity analyses [ timp-2][igfbp7 ] remained significant and superior to all other markers regardless of changes in reference creatinine method . other investigators have confirmed that [ timp-2 ] and [ igfbp7 ] are useful in the detection of aki . in a small study of patients undergoing cardiac surgery , wetz et al . found that levels of urinary [ timp-2][igfbp7 ] were higher in the patients who developed aki than those that did not . yamashita demonstrated that urinary [ timp-2 ] performed well to predict severe aki in critically ill patients with and without sepsis with a roc auc range of 0.810.84 . next , we developed and separately evaluated , two clinical cutoffs for a test using urinary [ timp-2][igfbp7 ] . in order to do so , we first had to consider the intended use of the test . there are no specific means to prevent aki , no equivalent to thrombolytic therapy and recommended actions are low - risk and generally applicable to most if not all critically ill patients ( see figure 1 ) . however , these actions are difficult to implement in all patients and some are time - consuming and potentially expensive . thus , the main purpose of the test would be to identify the majority of high - risk patients , and to ensure all such patients receive these recommended actions . as such , the test would need to have a primary cutoff that provides high sensitivity and high negative predictive value ( npv)the proportion of true negatives over all patients testing negative . although this would be the main use for the test , a cutoff with a high specificity and high positive predictive value ( ppv ) would have value as well . for example , in judging the risk - benefit to a patient for a decision to postpone some form of therapy that carries a risk of kidney damage ( e.g. cardiac surgery ) one might wish to have a test with a high ppv . shading of boxes indicates priority of action solid shading indicates actions that are equally appropriate at all stages whereas graded shading indicates increasing priority as intensity increases . aki , acute kidney injury ; icu , intensive care unit . source : www.kdigo.org . shading of boxes indicates priority of action solid shading indicates actions that are equally appropriate at all stages whereas graded shading indicates increasing priority as intensity increases . thus , we derived cutoffs for urinary [ timp-2][igfbp7 ] based on sensitivity and specificity ( as well as npv and ppv ) for prediction of aki using data from the sapphire study . we set the high sensitivity / high npv cutoff at 0.3 ( ng / ml)/1000 and the high specificity / high ppv cutoff at 2.0 ( ng / ml)/1000 . next , we verified these cutoffs in a new study ( opal ) enrolling 154 critically ill adults from six sites in the united states . in total , 27 subjects met the primary end point ( stage 23 aki within 12 h ) . the results of the opal study replicated those of sapphire ( figure 2 ) where the sensitivity at the 0.3 cutoff was 89% , and npv was 97% . for 2.0 , specificity was 95% and ppv was 49% . relative risk of aki for subjects testing in each strata is shown in figure 2 . compared with baseline risk , subjects in the middle strata ( > 0.32 ) were more than 4-fold and those in the highest strata ( > 2 ) were more than 10-fold more likely to manifest moderate to severe aki ( kdigo stage 23 ) in the next 12 h. in opal , 46% of patients tested 0.3 while the upper two strata included 39% and 16% of patients . we also examined these cutoffs for urinary [ timp-2][igfbp7 ] in sapphire with respect to subsequent development of major adverse kidney events at 30 days ( make30 ) , which included death , dialysis or persistence of renal dysfunction ( creatinine twice baseline ) . risk for make30 was lowest when [ timp-2][igfbp7 ] was 0.3 ( raw risk 18% ) and increased to 23% for > 0.32.0 and 40% for > 2.0 ( p < 0.001 ) . figure 2:top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . closed circles and triangles indicate [ timp-2][igfbp7 ] cutoffs of 0.3 and 2.0 , respectively . area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively . bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort . top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . closed area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively . bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort . finally we conducted a third study , topaz , and enrolled another heterogeneous cohort of 420 critically ill patients in order to prospectively validate the lower ( 0.3 ) biomarker cutoff value for risk assessment of aki . this was the first study to evaluate urinary [ timp-2][igfbp7 ] where the end point was determined by clinical adjudication by aki experts blinded to the results of the test . we found that the urinary [ timp-2][igfbp7 ] test significantly improved risk assessment by stratifying patients into distinct risk categories , with a 7-fold increase in risk for patients with a [ timp-2][igfbp7 ] test value > 0.3 compared with those 0.3 . when two cutoffs are used , relative risk for > 0.32.0 was 5 , and > 2.0 relative risk was 17 ( p < 0.002 ) . furthermore , using a multivariate model including clinical information , urinary [ timp-2][igfbp7 ] remained statistically significant and a strong predictor of aki . the clinical model alone exhibited an auc 0.70 , 95% ci 0.630.76 while the auc was 0.86 , 95% ci 0.800.90 for clinical variables plus [ timp-2][igfbp7 ] ) . thus , urinary [ timp-2][igfbp7 ] has now been shown to provide early risk stratification for imminent aki in over 1200 critically ill patients in three multi - center studies enrolling diverse groups of patients ( table 1 ) with the prevalence of major exposures for aki , such as sepsis , similar to other reports in the literature [ 19 , 20 ] . table 1.baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markerssapphireopaltopazall patients728153408male449 ( 62%)87 ( 57%)219 ( 54%)age , years64 ( 5373)65 ( 5477)65 ( 5476)race white573 ( 79%)119 ( 78%)339 ( 83% ) black87 ( 12%)13 ( 8%)56 ( 14% ) other / unknown68 ( 9%)21 ( 14%)13 ( 3%)history of ckd65 ( 9%)13 ( 8%)32 ( 8%)icu type medical225 ( 31%)53 ( 35%)180 ( 44% ) surgical179 ( 25%)13 ( 8%)70 ( 17% ) combined icu147 ( 20%)43 ( 28%)62 ( 15% ) cardiac surgery61 ( 8%)1 ( 1%)38 ( 9% ) neurologic39 ( 5%)2 ( 1%)14 ( 3% ) coronary care unit30 ( 4%)27 ( 18%)10 ( 2% ) trauma24 ( 3%)9 ( 6%)27 ( 7% ) other / unknown23 ( 3%)5 ( 3%)7 ( 2%)reason for icu admission respiratory310 ( 43%)81 ( 53%)206 ( 50% ) surgery247 ( 34%)23 ( 15%)128 ( 31% ) cardiovascular243 ( 33%)64 ( 42%)165 ( 40% ) sepsis136 ( 19%)29 ( 19%)97 ( 24% ) neurological70 ( 10%)15 ( 10%)52 ( 13% ) trauma55 ( 8%)12 ( 8%)44 ( 11% ) other126 ( 17%)57 ( 37%)120 ( 29%)enrollment scr , mg / dl0.9 ( 0.71.2)1.1 ( 0.81.6)0.9 ( 0.71.3)shown are n ( proportion ) or median ( interquartile range).results reproduced from table 1 of .results reproduced from table s1 of .results partially reproduced from table 1 of .subjects may have multiple reasons for icu admission . baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markers shown are n ( proportion ) or median ( interquartile range ) . taken together the results of these studies of urinary [ timp-2][igfbp7 ] should enable early triage and risk stratification in a wide range of critically ill patients from the emergency room , acute admission areas and intensive care . early identification of these at - risk patients should improve delivery of kdigo - recommended interventions and might also enable interventional studies for the treatment of aki in the not - so - distant future . importantly , both timp-2 and igfbp7 may increase in response to a wide variety of insults ( inflammation , oxidative stress , ultraviolet radiation , drugs and toxins ) [ 2123 ] . this may help explain why they correspond to risk for aki , a syndrome known for its multiple etiologies even in the same patient . however , these insults may not actually destroy cells and these molecules appear to be able to signal in autocrine and paracrine fashions [ 23 , 24 ] thus behaving more like an alarm spreading to adjacent cells ( figure 3 ) . in terms of timing , this signal could represent the earliest point of cellular stress . biomarkers that can detect cellular stress ( or conversely cell health ) may be more useful than markers of injury or cell death . figure 3:proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2 these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn , block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing . proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2 these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn , block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing . as urinary [ timp-2][igfbp7 ] can aid in the risk assessment for aki , it is important to recognize that , like all diagnostic tests , it does not take the place of clinical judgment . furthermore , while the test was designed to risk assess for stage 23 aki , the biomarker concentrations correspond to severity of aki across all three stages . however , the markers do not appear to persist in the urine for long after aki has occurred and thus they may be normal in patients who have already manifest aki by functional criteria ( e.g. scr ) . finally , the cutoffs for the test are based on overall behavior of the biomarkers in the majority of patients . while this performance appears to be very consistent across various exposures and susceptibilities for aki , specific groups of patients may require more fine - tuning . for example , meersch et al . examined sensitivity and specificity of [ timp-2][igfbp7 ] for aki ( stage 1 or greater ) in a high - severity group of patients undergoing cardiac surgery . these investigators found a sensitivity of 0.92 and specificity of 0.81 for a cutoff value of 0.5 ( auc 0.90 ) using the maximum urinary [ timp-2][igfbp7 ] concentration achieved in the first 24 h following surgery ( composite time point ) . taking the cardiac surgery example , we propose a set of actions based on pre - test assessment of risk and the urinary [ timp-2][igfbp7 ] test result ( figure 4 ) . low , moderate and high risk are based on a combination of ( i ) underlying clinical predisposition ( susceptibility ) which includes most of the same variables that determine sts predicted risk of mortality ; ( ii ) acute evidence of aki ( oliguria or increasing scr ) ; ( iii ) clinical suspicion of aki based on exposures and ( iv ) urinary [ timp-2][igfbp7 ] . when [ timp-2][igfbp7 ] is 0.3 ( ng / ml)/1000 , the high npv means that patients are low - risk unless clinical suspicion is high or unless they already have clinical evidence of aki . when [ timp-2][igfbp7 ] is > 0.3 but < 2.0 ( ng / ml)/1000 , risk is moderate but should be increased to high if any of the following exist : clinical evidence of aki , sts predicted risk of mortality 4% , clinical suspicion for aki high . finally , given the high ppv when urinary [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 , anyone regardless of clinical risk assessment should be considered to be at high risk . figure 4:proposed clinical application of risk assessment for patients immediately after cardiac surgery . sts , society of thoracic surgery ; i / o , input and output ; scr , serum creatinine ; cvp , central venous pressure ; nsaids , non - steroidal anti - inflammatory drug ; ace , angiotenson converting enzyme inhibitor ; scvo2 , central verous oxygen saturation ; h / o , history of ; lv fx , left ventricular function ; pa , pulmonary artery ; ci , cardiac index . sts , society of thoracic surgery ; i / o , input and output ; scr , serum creatinine ; cvp , central venous pressure ; nsaids , non - steroidal anti - inflammatory drug ; ace , angiotenson converting enzyme inhibitor ; scvo2 , central verous oxygen saturation ; h / o , history of ; lv fx , left ventricular function ; pa , pulmonary artery ; ci , cardiac index . each phase of the cell cycle has a specific function that is required for appropriate cell proliferation . begin the process of repair , they must enter and exit each phase of the cell cycle on schedule . if the cell exits a phase too soon , or stays in a phase too long , the normal repair and recovery process can become maladaptive . for instance , if epithelial cells remain arrested in g1 or g2 , it favors a hypertrophic and fibrotic phenotype [ 27 , 28 ] . cyclins and cyclin - dependent kinases , and inhibitors control each phase of the cell cycle . the cell uses cell - cycle arrest as a protective mechanism to avoid cell - division when potentially damaged . by initiating cell - cycle arrest , cells can thus avoid cell division during stress and injury , which is protective . however , if the cells do not re - initiate the cell - cycle and remain arrested at g1 or g2 ( or possibly other phases of cell cycle ) , a fibrotic phenotype can ensue . both timp2 and igfbp7 have been implicated in the g1 cell - cycle arrest phase noted to occur during the very early phases of cellular stress ( figure 3 ) [ 2123 ] . specifically , it has also been shown that renal tubular cells also go through this g1 cell - cycle arrest phase following stress due to a variety of insults . induction of cell - cycle arrest is not only associated with increased risk for aki but may also serve as a mechanistic link between aki and ckd . sustained cell - cycle arrest will result in a senescent cell phenotype and lead to fibrosis . interestingly , the various sub - types of timp proteins may play different roles in the kidney . have shown that timp-3 protects the cells from damage , whereas timp-2 appears to promote injury through matrix metalloproteinase activation . similarly , research studies in renal transplant allografts show that matrix metalloproteinase activity is important in mediating scarring in chronic allograft nephropathy . indeed there is already evidence that urinary [ timp-2][igfbp7 ] is strongly associated with a composite end point of death or need for dialysis . we found that [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 was associated with increased risk for mortality or receipt of rrt over the next 9 months ( hazard ratio [ hr ] , 2.11 ; 95% confidence interval [ 95% ci ] , 1.37 to 3.23 ; p,0.001 ) . interestingly , in a multivariate analysis adjusted for the clinical model , [ timp-2][igfbp7 ] > 0.3 ( ng / ml)/1000 were associated with death or rrt only in subjects who developed aki ( p = 0.002 ) . additionally , aregger et al . recently demonstrated that igfbp7 levels predicted mortality , renal recovery and severity / duration of aki in a small cohort of critically ill adults . as discussed above , cell - cycle arrest is nonetheless a protective mechanism to avoid the cell entering the cell - cycle when it is injured or even in an adverse environment . thus , temporary g1 cell - cycle arrest should reduce kidney damage . using an animal model of septic aki secondary to cecal ligation and puncture we hypothesized that a pharmacologically induced early cell - cycle arrest would be associated with less aki . we used cyclosporine a , a known inducer of cell - cycle arrest and previously shown to attenuate kidney damage in the setting of folic acid - induced aki , and found that a single dose given 18 h after cecal ligation and puncture and along with initial antibiotics was successful in reducing aki . thus manipulation of cell - cycle may represent a new therapeutic strategy in the prevention and treatment of aki . these results are also important because they have implications for how we understand the pathogenesis of aki . there is a growing appreciation for the concept of secondary injury to the kidney as danger signaling molecules ( damage and pathogen - associated molecular patterns ) are delivered to the renal tubule via both glomerular filtration and the blood stream . these molecules are detected by pattern recognition receptors on the tubular cell surface where they initiate a cascade leading to inflammation and/or apoptosis . in essence , the available data suggest that cell - cycle arrest signaling is a protective response , but when engaged by multiple cells such that increases in markers like timp-2 and igfbp7 can be detected in the urine , it is often followed by aki . furthermore , if cell - cycle arrest persists the result can become maladaptive and lead to a fibrosis phenotype . early protection of cells might be achievable by supporting the cell 's own self - preservation mechanisms including cell - cycle arrest . conversely , once the danger is past , it may be important to rapidly reverse this process so that the adverse consequences including cell senescence and fibrosis are avoided . thus , cell - cycle arrest activation and deactivation at critical clinical time points for a patient may prove to be targets of therapeutic intervention in the future . dark side where it heralds the onset of aki and persistence of this signaling may lead to maladaptive repair processes ultimately favoring fibrosis over regeneration . light side in that its detection can be used as an early warning prior to actual damage intriguingly though , we might also be able to manipulate cell - cycle in ways that can foster the innate protective aspects of this response while avoiding the maladaptive aspects turning the dark toward the light . cell - cycle arrest biomarkers : the light at the end of the acute kidney injury tunnel .

acute kidney injury ( aki ) is a common consequence of systemic illness or injury and it complicates several forms of major surgery . two major difficulties have hampered progress in aki research and clinical management . aki is difficult to detect early and its pathogenesis is still poorly understood . we recently reported results from multi - center studies where two urinary markers of cell - cycle arrest , tissue inhibitor of metalloproteinases-2 ( timp-2 ) and insulin - like growth factor - binding protein 7 ( igfbp7 ) were validated for development of aki well ahead of clinical manifestations azotemia and oliguria . cell - cycle arrest is known to be involved in the pathogenesis of aki and this dark side may also involve progression to chronic kidney disease . however , cell - cycle arrest has a light side as well , since this mechanism can protect cells from the disastrous consequences of entering cell division with damaged dna or insufficient bioenergetic resources during injury or stress . whether we can use the light side to help prevent aki remains to be seen , but there is already evidence that cell - cycle arrest biomarkers are indicators of both sides of this complex physiology .

INTRODUCTION WHY ARE BIOMARKERS NEEDED? DISCOVERY AND VALIDATION OF CELL-CYCLE ARREST MARKERS CLINICAL APPLICATION OF AKI BIOMARKERS TWO SIDES TO CELL-CYCLE ARREST CONCLUSIONS CONFLICT OF INTEREST STATEMENT

aki is associated with short - term morbidity , a long - term risk of chronic kidney disease ( ckd ) and cardiovascular events and decreases survival [ 38 ] . common functional markers such as serum creatinine or urine output are late indicators of aki . in this review , we will summarize the current evidence that supports the use of cell - cycle arrest biomarkers for risk assessment of aki , we will consider various responses to this warning and we will speculate on how this mechanism could be exploited to protect the kidney . serum creatinine ( scr ) , the most widely used marker of kidney function , is a component of the definition of aki , and yet as a single measurement is completely useless in differentiating aki from ckd . changes in scr require several hours to days before they reach steady state following an injury to the kidney . uo will often decrease before scr increases making it a more time sensitive marker of gfr . while in the absence of obstruction if there is no uo , there can be no gfr , not all reductions in uo signal aki . over time , a persistent renal functional change can be used to infer kidney injury in the same way that regional wall motion abnormalities or a decrement in ejection fraction can be used to infer myocardial injury . as such , markers that can detect actual damage or warn of impending damage are required in the same way that functional measures alone ( e.g. in 2013 , we reported the results of a prospective , observational , international investigation ( sapphire study ) of tissue inhibitor of metalloproteinases-2 ( timp-2 ) and insulin - like growth factor - binding protein 7 ( igfbp7 ) in a heterogeneous group of critically ill ( evidence of respiratory or cardiovascular failure ) patients . this report actually included two multi - center studies discovery and validation . in the discovery phase we enrolled 522 adults in three distinct cohorts including patients with sepsis , shock , major surgery and trauma and examined over 300 markers in urine and blood . in the validation study we enrolled 744 adults with critical illness and without evidence of aki at enrollment ; the final analysis cohort was a heterogeneous sample of 728 critically ill patients . the primary end point was moderate - severe aki [ kidney disease : improving global outcomes ( kdigo ) stage 23 ] within 12 h of sample collection which occurred in 14% of sapphire subjects . [ timp-2][igfbp7 ] was significantly superior to all previously described markers of aki ( p < 0.002 ) including neutrophil gelatinase - associated lipocalin ( ngal ) and kidney injury molecule ( kim)-1 , none of which achieved an auc > 0.72 . however , this result might be due in part to varying kinetics of different early aki biomarkers . other investigators have confirmed that [ timp-2 ] and [ igfbp7 ] are useful in the detection of aki . in a small study of patients undergoing cardiac surgery , wetz et al . found that levels of urinary [ timp-2][igfbp7 ] were higher in the patients who developed aki than those that did not . yamashita demonstrated that urinary [ timp-2 ] performed well to predict severe aki in critically ill patients with and without sepsis with a roc auc range of 0.810.84 . there are no specific means to prevent aki , no equivalent to thrombolytic therapy and recommended actions are low - risk and generally applicable to most if not all critically ill patients ( see figure 1 ) . however , these actions are difficult to implement in all patients and some are time - consuming and potentially expensive . although this would be the main use for the test , a cutoff with a high specificity and high positive predictive value ( ppv ) would have value as well . aki , acute kidney injury ; icu , intensive care unit . thus , we derived cutoffs for urinary [ timp-2][igfbp7 ] based on sensitivity and specificity ( as well as npv and ppv ) for prediction of aki using data from the sapphire study . next , we verified these cutoffs in a new study ( opal ) enrolling 154 critically ill adults from six sites in the united states . in total , 27 subjects met the primary end point ( stage 23 aki within 12 h ) . relative risk of aki for subjects testing in each strata is shown in figure 2 . compared with baseline risk , subjects in the middle strata ( > 0.32 ) were more than 4-fold and those in the highest strata ( > 2 ) were more than 10-fold more likely to manifest moderate to severe aki ( kdigo stage 23 ) in the next 12 h. in opal , 46% of patients tested 0.3 while the upper two strata included 39% and 16% of patients . we also examined these cutoffs for urinary [ timp-2][igfbp7 ] in sapphire with respect to subsequent development of major adverse kidney events at 30 days ( make30 ) , which included death , dialysis or persistence of renal dysfunction ( creatinine twice baseline ) . risk for make30 was lowest when [ timp-2][igfbp7 ] was 0.3 ( raw risk 18% ) and increased to 23% for > 0.32.0 and 40% for > 2.0 ( p < 0.001 ) . figure 2:top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively . bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort . top : [ timp-2][igfbp7 ] roc curves for the opal ( solid ) , sapphire ( short dash ) and topaz ( long dash ) cohorts . closed area under the roc curve ( 95% ci ) = 0.79 ( 0.690.88 ) , 0.80 ( 0.740.84 ) and 0.82 ( 0.760.88 ) for opal , sapphire and topaz , respectively . bottom : relative risk of aki stage 2 or 3 within 12 h in the opal ( light gray ) , sapphire ( medium gray ) and topaz ( dark gray ) cohort . finally we conducted a third study , topaz , and enrolled another heterogeneous cohort of 420 critically ill patients in order to prospectively validate the lower ( 0.3 ) biomarker cutoff value for risk assessment of aki . this was the first study to evaluate urinary [ timp-2][igfbp7 ] where the end point was determined by clinical adjudication by aki experts blinded to the results of the test . when two cutoffs are used , relative risk for > 0.32.0 was 5 , and > 2.0 relative risk was 17 ( p < 0.002 ) . furthermore , using a multivariate model including clinical information , urinary [ timp-2][igfbp7 ] remained statistically significant and a strong predictor of aki . thus , urinary [ timp-2][igfbp7 ] has now been shown to provide early risk stratification for imminent aki in over 1200 critically ill patients in three multi - center studies enrolling diverse groups of patients ( table 1 ) with the prevalence of major exposures for aki , such as sepsis , similar to other reports in the literature [ 19 , 20 ] . table 1.baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markerssapphireopaltopazall patients728153408male449 ( 62%)87 ( 57%)219 ( 54%)age , years64 ( 5373)65 ( 5477)65 ( 5476)race white573 ( 79%)119 ( 78%)339 ( 83% ) black87 ( 12%)13 ( 8%)56 ( 14% ) other / unknown68 ( 9%)21 ( 14%)13 ( 3%)history of ckd65 ( 9%)13 ( 8%)32 ( 8%)icu type medical225 ( 31%)53 ( 35%)180 ( 44% ) surgical179 ( 25%)13 ( 8%)70 ( 17% ) combined icu147 ( 20%)43 ( 28%)62 ( 15% ) cardiac surgery61 ( 8%)1 ( 1%)38 ( 9% ) neurologic39 ( 5%)2 ( 1%)14 ( 3% ) coronary care unit30 ( 4%)27 ( 18%)10 ( 2% ) trauma24 ( 3%)9 ( 6%)27 ( 7% ) other / unknown23 ( 3%)5 ( 3%)7 ( 2%)reason for icu admission respiratory310 ( 43%)81 ( 53%)206 ( 50% ) surgery247 ( 34%)23 ( 15%)128 ( 31% ) cardiovascular243 ( 33%)64 ( 42%)165 ( 40% ) sepsis136 ( 19%)29 ( 19%)97 ( 24% ) neurological70 ( 10%)15 ( 10%)52 ( 13% ) trauma55 ( 8%)12 ( 8%)44 ( 11% ) other126 ( 17%)57 ( 37%)120 ( 29%)enrollment scr , mg / dl0.9 ( 0.71.2)1.1 ( 0.81.6)0.9 ( 0.71.3)shown are n ( proportion ) or median ( interquartile range).results reproduced from table 1 of .results reproduced from table s1 of .results partially reproduced from table 1 of .subjects may have multiple reasons for icu admission . baseline characteristics of subjects included in three large multi - center trials evaluating cell - cycle arrest markers shown are n ( proportion ) or median ( interquartile range ) . taken together the results of these studies of urinary [ timp-2][igfbp7 ] should enable early triage and risk stratification in a wide range of critically ill patients from the emergency room , acute admission areas and intensive care . early identification of these at - risk patients should improve delivery of kdigo - recommended interventions and might also enable interventional studies for the treatment of aki in the not - so - distant future . this may help explain why they correspond to risk for aki , a syndrome known for its multiple etiologies even in the same patient . however , these insults may not actually destroy cells and these molecules appear to be able to signal in autocrine and paracrine fashions [ 23 , 24 ] thus behaving more like an alarm spreading to adjacent cells ( figure 3 ) . in terms of timing , this signal could represent the earliest point of cellular stress . biomarkers that can detect cellular stress ( or conversely cell health ) may be more useful than markers of injury or cell death . figure 3:proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2 these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn , block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing . proposed mechanistic involvement of the novel biomarkers in aki : initial tubular cells sustain injury by various insults . in response to dna and possibly other forms of damage igfbp7 and timp-2 these effects are conducted in an autocrine and paracrine manner via igfbp7 and timp-2 receptors . the p proteins in turn , block the effect of the cyclin - dependent protein kinase complexes ( cycld - cdk4 and cycle - cdk2 ) on the cell - cycle promotion , thereby resulting in g1 cell - cycle arrest for short periods of time presumably to avoid cells with possible damage from dividing . as urinary [ timp-2][igfbp7 ] can aid in the risk assessment for aki , it is important to recognize that , like all diagnostic tests , it does not take the place of clinical judgment . furthermore , while the test was designed to risk assess for stage 23 aki , the biomarker concentrations correspond to severity of aki across all three stages . however , the markers do not appear to persist in the urine for long after aki has occurred and thus they may be normal in patients who have already manifest aki by functional criteria ( e.g. finally , the cutoffs for the test are based on overall behavior of the biomarkers in the majority of patients . while this performance appears to be very consistent across various exposures and susceptibilities for aki , specific groups of patients may require more fine - tuning . examined sensitivity and specificity of [ timp-2][igfbp7 ] for aki ( stage 1 or greater ) in a high - severity group of patients undergoing cardiac surgery . these investigators found a sensitivity of 0.92 and specificity of 0.81 for a cutoff value of 0.5 ( auc 0.90 ) using the maximum urinary [ timp-2][igfbp7 ] concentration achieved in the first 24 h following surgery ( composite time point ) . taking the cardiac surgery example , we propose a set of actions based on pre - test assessment of risk and the urinary [ timp-2][igfbp7 ] test result ( figure 4 ) . low , moderate and high risk are based on a combination of ( i ) underlying clinical predisposition ( susceptibility ) which includes most of the same variables that determine sts predicted risk of mortality ; ( ii ) acute evidence of aki ( oliguria or increasing scr ) ; ( iii ) clinical suspicion of aki based on exposures and ( iv ) urinary [ timp-2][igfbp7 ] . when [ timp-2][igfbp7 ] is 0.3 ( ng / ml)/1000 , the high npv means that patients are low - risk unless clinical suspicion is high or unless they already have clinical evidence of aki . when [ timp-2][igfbp7 ] is > 0.3 but < 2.0 ( ng / ml)/1000 , risk is moderate but should be increased to high if any of the following exist : clinical evidence of aki , sts predicted risk of mortality 4% , clinical suspicion for aki high . finally , given the high ppv when urinary [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 , anyone regardless of clinical risk assessment should be considered to be at high risk . each phase of the cell cycle has a specific function that is required for appropriate cell proliferation . begin the process of repair , they must enter and exit each phase of the cell cycle on schedule . the cell uses cell - cycle arrest as a protective mechanism to avoid cell - division when potentially damaged . by initiating cell - cycle arrest , cells can thus avoid cell division during stress and injury , which is protective . however , if the cells do not re - initiate the cell - cycle and remain arrested at g1 or g2 ( or possibly other phases of cell cycle ) , a fibrotic phenotype can ensue . both timp2 and igfbp7 have been implicated in the g1 cell - cycle arrest phase noted to occur during the very early phases of cellular stress ( figure 3 ) [ 2123 ] . specifically , it has also been shown that renal tubular cells also go through this g1 cell - cycle arrest phase following stress due to a variety of insults . induction of cell - cycle arrest is not only associated with increased risk for aki but may also serve as a mechanistic link between aki and ckd . sustained cell - cycle arrest will result in a senescent cell phenotype and lead to fibrosis . interestingly , the various sub - types of timp proteins may play different roles in the kidney . have shown that timp-3 protects the cells from damage , whereas timp-2 appears to promote injury through matrix metalloproteinase activation . indeed there is already evidence that urinary [ timp-2][igfbp7 ] is strongly associated with a composite end point of death or need for dialysis . we found that [ timp-2][igfbp7 ] > 2.0 ( ng / ml)/1000 was associated with increased risk for mortality or receipt of rrt over the next 9 months ( hazard ratio [ hr ] , 2.11 ; 95% confidence interval [ 95% ci ] , 1.37 to 3.23 ; p,0.001 ) . recently demonstrated that igfbp7 levels predicted mortality , renal recovery and severity / duration of aki in a small cohort of critically ill adults . as discussed above , cell - cycle arrest is nonetheless a protective mechanism to avoid the cell entering the cell - cycle when it is injured or even in an adverse environment . thus , temporary g1 cell - cycle arrest should reduce kidney damage . using an animal model of septic aki secondary to cecal ligation and puncture we hypothesized that a pharmacologically induced early cell - cycle arrest would be associated with less aki . we used cyclosporine a , a known inducer of cell - cycle arrest and previously shown to attenuate kidney damage in the setting of folic acid - induced aki , and found that a single dose given 18 h after cecal ligation and puncture and along with initial antibiotics was successful in reducing aki . thus manipulation of cell - cycle may represent a new therapeutic strategy in the prevention and treatment of aki . these results are also important because they have implications for how we understand the pathogenesis of aki . there is a growing appreciation for the concept of secondary injury to the kidney as danger signaling molecules ( damage and pathogen - associated molecular patterns ) are delivered to the renal tubule via both glomerular filtration and the blood stream . in essence , the available data suggest that cell - cycle arrest signaling is a protective response , but when engaged by multiple cells such that increases in markers like timp-2 and igfbp7 can be detected in the urine , it is often followed by aki . furthermore , if cell - cycle arrest persists the result can become maladaptive and lead to a fibrosis phenotype . early protection of cells might be achievable by supporting the cell 's own self - preservation mechanisms including cell - cycle arrest . thus , cell - cycle arrest activation and deactivation at critical clinical time points for a patient may prove to be targets of therapeutic intervention in the future . dark side where it heralds the onset of aki and persistence of this signaling may lead to maladaptive repair processes ultimately favoring fibrosis over regeneration . light side in that its detection can be used as an early warning prior to actual damage intriguingly though , we might also be able to manipulate cell - cycle in ways that can foster the innate protective aspects of this response while avoiding the maladaptive aspects turning the dark toward the light . cell - cycle arrest biomarkers : the light at the end of the acute kidney injury tunnel .

currently the most efficient anti - cancer chemotherapy agents are the cytotoxic drugs that target cancer cells , in a most vulnerable state , during mitosis . these include the microtubule - binding taxanes ( e.g. , paclitaxel ) that stabilize and vinca alkaloids ( e.g. , vinblastine ) that destabilize microtubule polymers . both promote abnormal spindle assembly , chromosome misalignment , consequent activation of the spindle assembly checkpoint ( sac ) , and induce prolonged mitotic arrest and mitotic cell death . however , cancer cells often adapt to these drugs and exit mitosis in a process called mitotic slippage . moreover , these drugs target not only mitotic cells but also affect the microtubule cytoskeleton functions of non - proliferating cells . they disrupt the interphase microtubule bundles of quiescent neuronal cells , along which molecular and vesicular transport occurs . neurotoxicity is therefore one of the most common dose - limiting side effects of microtubule - targeting drugs . hence , there is a need to develop new anti - mitotic drugs , which by targeting specific mitotic proteins and mechanisms would achieve a similar level of anti - cancer efficacy without the unwanted side effects on non - proliferating cells . such new targets included mitotic kinases ( e.g. , plk1 , aurora kinases ) and mitotic motor proteins ( e.g. , eg5 , cenpe ) required for spindle assembly , chromosome alignment , and segregation . their inhibition also induces sac - mediated mitotic arrest and leads to mitotic cell death . although these new anti - mitotic drugs are highly specific in vitro and had some efficacy in xenograft models , they were so far less convincing in clinical trials than the microtubule - targeting drugs . the reasons for these disappointing clinical results might be the low mitotic index ( i.e. , low number of cells undergoing division ) of certain human tumors and strong neutropenia , the main dose - limiting toxicity of the new anti - mitotic drugs . neutropenia refers to an abnormally low number of neutrophil granulocytes , the most abundant type of white blood cells . the reason why this occurs as a side effect is that during their development neutrophils frequently divide . anti - mitotic drugs are not cancer cell - specific ; they target every dividing cell , including neutrophils , and thus neutropenia is a consequence of their activity . future anti - cancer drugs should therefore possess a larger therapeutic window and exert a higher - level cancer cell - specificity . one strategy to achieve this goal might be to develop new drugs that are synthetic lethal with mechanisms of cancer cell - specific hallmarks . in my opinion such new drug targets are the molecular mechanisms of nuclear assembly and organization because some of these mechanisms might get compromised during tumorigenesis , making the nuclei of these cancer cells more vulnerable than their normal counterparts . below i will discuss the nuclear organization of normal cells and cancer cells , the molecular mechanisms that govern nuclear assembly , and i will hypothesize about the usefulness of targeting some of these mechanisms in future anti - cancer therapies . the nucleus contains the genome of the eukaryotic cell , whose precise organization is essential for normal cell function . the structure that defines the nucleus is the nuclear envelope ( ne ) , a sub - domain of the endoplasmic reticulum ( er ) ( fig . 1 ) . the ne is composed of outer nuclear membranes ( onm ) and inner nuclear membranes ( inm ) with different protein compositions , which are fused at the sites of nuclear pore complex ( npc ) insertion . underlying the inm is the nuclear lamina , which is mainly composed of intermediate lamin filaments . in vertebrates , lamin proteins are grouped into a - type lamins ( lamin a , 10 , and c ) encoded by the lmna gene and b - type lamins ( lamin b1 and b2 ) encoded by lmnb1 and lmnb2 genes . while b - type lamins are expressed in every somatic cell , a - type lamins are absent from highly pluripotent and rapidly dividing cells and are present only in differentiated cells . lamins are alternatively spliced , and the ratio between the expressions of individual lamin isoforms is characteristic of each cell type . these polymers then laterally assemble into filaments , which form a lattice of remarkably regular arrangement underneath the inm . during maturation most lamins are c - terminally farnesylated . while this short lipid chain is retained on b - type lamins and anchors the proteins in the membranes , the farnesylated c - terminal part of lamin a is enzymatically cleaved after its incorporation into lamin polymers . the stabilization and organization of lamin filaments underneath the inm also requires lamin - associated membrane proteins , such as lamin b receptor ( lbr ) , diverse lap2/emerin / man1 ( lem ) domain - containing proteins , and others . figure 1 . nuclear envelope ( yellow ) is penetrated by the nuclear pore complexes ( red ) . underneath the nuclear envelope is the nuclear lamina ( green ) , which provides the stiffness to the ne and serves as a tethering surface for chromosomes ( blue lines ) . ( b ) several cancer types are characterized by abnormal expression of lamins ( particularly a - type ) , altered chromatin organization , and multi - lobulated nuclear shape . other changes involve enlargement or fragmentation of the nucleolus ( brown , large circles ) and promyelocytic leukemia ( pml ) bodies ( orange , small circles ) . ( c ) possible nuclear appearance of cancer cell nuclei after the treatment with drugs that interfere with mechanisms of nuclear assembly and organization . the stoichiometry of a - type and b - type lamins correlates well with the mechanical stress the cells experience within a tissue . in soft tissues such as liver or brain , a - type lamins are relatively low expressed , while in stiff tissues such as heart or muscle a - type lamins increase ( up to 30-fold ) to withstand the mechanical stress and to limit the potential disruption of the chromatin . b - type lamins are constitutively expressed and correlate much less with nuclear stiffness . by tethering the chromatin to the ne , nuclear lamina also contributes to the non - random chromatin organization within the nucleus ( fig . 1 ) . developmentally coregulated genes often form clusters on chromosomes , which are associated and corepressed at the nuclear periphery in cells where they are not expressed . genes and chromosomal domains that become internalized during certain differentiation steps are either immediately activated or are unlocked and prepared for activation during further differentiation steps . this suggests that the nuclear lamina often provides a repressive environment for chromosomal domains at the nuclear periphery whose three - dimensional organization is specific for certain cell types and differentiation states . mutation or downregulation of lamins or lamin - associated proteins results in disorganized lamin filaments and deformed , multi - lobulated , and fragile nuclei , which are often observed in human diseases such as cancer . cancer is initiated by genetic processes such as genome instability , genome rearrangements , or specific gene mutations , amplifications , or deletions , followed by epigenetic modifications , which ultimately lead to altered gene expression and result in deregulated cell proliferation . in cancer cells , nuclear size and shape cancer - related morphological changes include ne invaginations , multi - lobulation , malleable and passively distorted nuclei , and altered appearance of heterochromatin , nucleoli , and nuclear bodies ( fig . 1 ) . although all these features do not occur simultaneously , they are often used individually in the clinical diagnosis of cancer , in the assessment of the degree of the malignancy , and for prognostic and predictive indications of the disease state . lamins , the most important architectural elements of the nucleus , are often aberrantly expressed or localized in cancer cells , and it is likely that this contributes to the multi - lobulated nuclear shape often observed in different cancer types ( fig . 1 ) . many poorly differentiated cancer types exhibit downregulation of a - type lamins and concomitant irregularities in their nuclear shape . for example , in small cell lung cancer ( sclc ) cells a - type lamins are either not or only weakly expressed , while in non - sclc cells they are normally expressed but frequently mislocalized in the cytoplasm . in colon cancers , gastric cancers , breast cancers , and diffuse large b - cell lymphomas , the expression of a - type lamins is also strongly reduced , and this feature correlates with increased disease recurrence and poor patient prognosis . consequently , the nuclei of these cancer types are often fragile and lobulated , and in breast cancer cells , they were shown to contain massive ne membrane invaginations ( fig . 1 ) . the nuclei of prostate cancer cells and of some other cancer types are not only lobulated but also contain structures called nuclear blebs , protrusions from the nuclear surface enriched in lamin a / c but deficient in lamin b. further examples with multi - lobulated nuclear shape are ovarian cancers , papillary thyroid cancers , leukemias , and different b - cell lymphomas . in conclusion , altered lamin expression or localization and disrupted stoichiometry between a- and b - type lamins can change the elastic properties of the ne , which renders it unable to withstand cytoskeleton- and chromosome - based forces and leads to misshapen nuclei . consistently , downregulation of lamin a / c in non - cancer primary breast epithelial cells results in nuclear alterations similar to those observed in breast cancer cell . moreover , mutations in lmna and other genes encoding for proteins of nuclear lamina results in heritable diseases called laminopathies , which are also characterized by fragile and multi - lobulated nuclear shape . therefore the nuclear lamina alterations might directly account for the cancer - related changes in the nuclear morphology . however , it is important to note that several other cancer types display normal nuclear shape and that there is no simple universal pattern of lamin expressions for all cancer types . it is unclear whether the abnormal nuclear organization observed in cancer cells is the cause or the consequence of transformation and tumor progression . lamins might modulate gene expression not only indirectly through influencing global chromatin organization but also by directly interacting with transcription factors that affect cellular proliferation , differentiation , and apoptosis . therefore , the absence of lamins from tumors derived from tissues where they are normally present led to the hypothesis that lamins might be directly involved in tumorigenesis . however , this tempting hypothesis is contradicted by several lines of evidence . there are more than 20 distinct laminopathies associated with approximately 400 different mutations in the human lmna gene . most of these mutations affect the assembly , dynamics , or function of lamin filaments and result in deformed , multi - lobulated , and fragile nuclei and abnormal heterochromatin structure , similar to the nuclear abnormalities observed in cancer cells . however , none of the mutations in lamin or in lamin - organizing proteins are known to be tumorigenic , and patients with laminopathies are not more susceptible to cancer development than healthy individuals with normal nuclear architecture . moreover , loss of lamin a from human fibroblast cells or ovary surface epithelial cells results in reduced mitosis and retarded cell growth , which at least in part might be explained by the active role of lamin a in nuclear localization of the cell cycle regulator retinoblastoma protein ( rb ) . finally , although higher order chromatin organization is commonly altered in cancer cells , this does not necessarily lead to tumorigenic transcriptional changes . in breast cancer for example , several genes have been identified that specifically change their nuclear position only in cancer cells . however , the absence of transcriptional changes associated with the movement of these genes suggests that these changes are not responsible for tumorigenesis . in conclusion , although precise nuclear organization is essential for normal cellular function , up to now altered nuclear architecture has not been shown to drive cancer development , and therefore , it is more likely to be a consequence of cell transformation and tumor progression . since the nuclear architecture of many cancer cells is abnormal , this hallmark might improve cancer cell - selectivity in therapies using mechanisms of mitotic nuclear assembly and nuclear organization as targets ( fig . 1 ) . the nuclear structure is disassembled and reassembled during every cell division to allow cytoplasmic spindle microtubules to segregate the duplicated sister chromatids . mitotic nuclear disassembly is controlled by cyclin - dependent kinase 1 ( cdk1 ) and other mitotic kinases that function downstream of its activation , such as protein kinase c ( pkc ) , aurora a , polo - like kinase 1 ( plk1 ) , nima - related kinases , and vaccinia - related kinase 1 ( vrk1 ) . they phosphorylate proteins of nuclear lamina and npc to disrupt their interphase molecular interactions . in support of their critical role , inactivation or inhibition of these mitotic kinases either blocks or delays various steps of nuclear disassembly . for example , vrk1 kinase is activated during mitotic entry upon degradation of its interphase inhibitor macroh2a1 . it relocalizes to the nuclear periphery and phosphorylate barrier - to - autointegration factor ( baf or banf1 ) to release it from dna and lem domain - containing inm proteins ( fig . 2 ) . inhibition of vrk1 thus prevents a late but essential step of nuclear disassembly . as a consequence of massive protein phosphorylation events , during ne breakdown soluble proteins become dispersed into the cytosol while membrane proteins become mobile and , together with the ne membranes , absorbed into the oscillating er network . mitotic regulation of baf function . during interphase baf binds as a dimer ( light blue ) to one lem domain - containing integral nuclear envelope protein ( pink ) and to two dna helices ( dark blue ) . during mitotic entry vrk1 ( red ) phosphorylates baf to disrupt its interactions and contributes to nuclear envelope breakdown . during mitotic exit lem4 ( brown ) inhibits vrk1 ( red ) and promotes pp2a ( green ) to dephosphorylate baf and to enable its function in post - mitotic nuclear reassembly . cdk1 promotes mitotic progression until the alignment of mitotic chromosomes on the metaphase plate and the correct bipolar attachment of all the kinteochores with spindle poles is achieved . subsequently , members of the protein phosphatase 1 ( pp1 ) and 2a ( pp2a ) family are activated . they counteract cdk1 and other mitotic kinases to allow the assembly of the interphase nucleus . chromatin decondensation is an important step of post - mitotic nuclear assembly . although its exact mechanism is still unclear , likely players are pp1 , which acts by dephosphorylating histone h3 , aaa - atpase p97 , which extracts the polyubiquitylated aurora b histone kinase from chromosomes , and the small ras - like gtpase ran , which acts in a still poorly understood manner . the first step of ne reformation is the attachment of er membranes to the chromatin surface . although direct lipid - chromatin interactions might play a role , this interaction is mainly mediated by trans - membrane and membrane - associated proteins . a large population of inm proteins possesses a highly basic nucleoplasmic domain that can directly bind to dna . the inm proteins function redundantly , thus their individual inactivation has only a minor effect on ne assembly and even simultaneous inactivation of several only delays but does not prevent the recruitment of membranes to the chromatin surface . these interactions are controlled spatially by the small gtpase ran and temporally by protein dephosphorylation . gtp - loaded ran is generated in the vicinity of chromosomes and mediates the release of the inhibitory importin receptors from their target proteins , providing spatial control of ne reformation . as a specific example , during mitotic exit rangtp releases importin- from the chromatin - binding domain of lbr , thereby allowing its binding to histones h3/h4 and hp1 . in the subsequent steps , chromatin - attached membranes start to spread from the peripheral margins of the separating chromatin to surround the entire chromatin mass and enclose it in a single nuclear compartment . there is constant membrane supply from the er , and manipulation of er structure was shown to influence ne assembly . the recruited membranes on the chromatin surface are organized by baf , which as a dimer can bind to one lem domain of an inm protein and to two dna helices ( fig . 2 ) . consistently , inactivation of baf results in deformed , multi - lobulated nuclei , with ne membranes trapped between individual chromosomes . the localization and function of baf is regulated by phosphorylation , which is temporally controlled by lem4 ( fig . 2 ) . during mitotic exit lem4 binds to vrk1 , baf s mitotic kinase , and inhibits its further activity on baf . concomitantly , lem4 also binds to and activates a complex of pp2a ( i.e. , pp2a - b55 ) to dephosphorylate baf and to allow its re - association with chromatin and inm protein . this particular pp2a complex has been further implicated in other mitotic functions and is the only protein phosphatase essential for mitotic exit . recently pp4c , which is known to form complexes with different pp2a subunits , was also suggested to influence the phosphorylation state of baf . nevertheless , during mitotic exit baf rapidly and strongly accumulates on the central surface of the chromatin core regions , which surround the anaphase chromatin mass , on one side facing the spindle microtubules and on the other facing the midzone microtubules . one of the features of the core region is that it rapidly shrinks with mitotic progression prior to the spread of the ne membranes from the peripheral regions toward the central region . the exact function of baf during ne membrane organization is still unclear ; however , it may synchronize different membrane and chromatin events during ne assembly to enable the incorporation of the entire chromatin mass into a single nuclear compartment . concomitantly with ne reformation , npcs are also assembled post - mitotically via mechanisms that are different from those used during interphase npc assembly . npc sub - complexes form pre - pores on the chromatin surface , which are then incorporated in the ne during membrane spreading on the chromatin surface . in the final step of ne assembly , remaining holes in the ne membranes may be fused by snare proteins . however , since these holes can also be occupied by npcs , the membrane fusion machinery is less important during ne assembly than was initially anticipated . upon the formation of a closed ne , nucleocytoplasmic transport is reactivated . the lamin proteins are actively imported into the nuclei where they are polymerized and organized underneath the inm to provide shape , elasticity , and stiffness to the nuclei . in genetic studies using different experimental model organisms , synthetic lethality is based on the fact that inactivation of one gene makes the cell vulnerable for the inactivation of the other gene , while neither of these two genes is essential on its own . in anti - cancer therapies similar synthetic lethality might be achieved between cancer cells and drugs , in case the cancer cell - specific molecular lesions sensitize the cancer cells for drugs inhibiting particular protein functions . the value of such an approach is that normal cells without such lesions will be unaffected . as discussed earlier , the nuclear architecture of different cancer cells is compromised and unable to withstand cytoskeleton- and chromosome - based forces and often so malleable that they can be crushed during biopsies ( e.g. , sclc cells ) . this suggests that future drugs hitting mechanisms of nuclear assembly and organization to further weakening the ne structure might result in a synergistic effect and specific killing of such cancer cells ( fig . 1 ) . in support of this hypothesis , for example , b - type lamins were shown to be dispensable in mouse cells expressing a - type lamins but essential in those where lamin a was suppressed . drug targets that are potentially synthetic lethal with abnormal nuclear organization of some cancer types might be identified either via phenotypic and synthetic lethality screens or by literature mining . based on the latter one , in my opinion , baf represents one of the most promising targets for specifically killing cancer cells with altered nuclear appearance . i base this assumption on facts that baf is often highly expressed in some cancer types ( e.g. , ovarian cancer , endometrial cancer , breast cancer , colorectal cancer , lung cancer , prostate cancer , glioma , melanoma , and lymphoma ) , and it is an important player of post - mitotic nuclear assembly whose downregulation or mutation in humans delays ne formation and induces strong nuclear irregularities . due to its small size and relatively flat protein surface without suitable binding pockets for small molecular weight inhibitory compounds , new anti - mitotics might be designed against its mitotic regulators ( fig . 2 ) . first of all , the dephosphorylation of baf might be targeted because this is essential for its correct localization and function during mitotic exit . therefore , drugs interfering with either the inhibitory interaction between lem4 and vrk1 , the mitotic kinase of baf , or with the activating interaction between lem4 and pp2a , the mitotic phosphatase of baf , might be developed ( fig . consequently , they would enhance the phosphorylation of baf , hereby weakening its interactions with lem domain containing inm proteins and dna . a second strategy might rely on interfering with the nuclear structure during mitotic entry by inhibiting baf s kinases vrk1 and vrk2 . consistently , depletion of vrk1 from breast cancer cells results in retarded tumor growth and reduced incidence of metastasis in a murine orthotopic xenograft model . furthermore , the first inhibitor of baf phosphorylation with an in vitro anti - cancer activity was recently isolated from a species of tree used in traditional medicine . another approach might aim to target the inhibitory interaction between lbr and importin- because perturbation of this interaction also results in ne assembly failure , abnormal chromatin decondensation , and daughter cell death . finally , b - type lamins are also promising targets because they are highly expressed in several cancer types , and they might become essential for survival of cancer cells with reduced expression of a - type lamins . since lamins are also considered to be low - druggable , they might be targeted indirectly by inhibiting their regulators . such examples include akap149 , which via promoting pp1 mediates the assembly of b - type lamins into nuclear lamina , and farnesyltransferase enzymes that add a 15-carbon farnesyl group to the c - terminal of most of the lamins to keep the b - type lamins anchored to the membranes . currently , only a few drugs exist that directly target mechanisms of nuclear assembly and organization , but interestingly , they can induce cancer cell death . for example , the farnesyltransferase inhibitor r115777 inhibits the growth of b - cell lymphoma and breast and ovarian cancer cells in vitro and reduces the tumor growth in xenograft model systems . since lamins are not the only substrates of farnesyltransferases , it is possible that the main anti - tumor activity of this inhibitor is achieved via other substrates , such as the small gtpase ras proteins . betulinic acid inhibits the expression of lamin b1 in pancreatic cancer cells and induces dose - dependent anti - cancer activities in both in vitro cultures and xenograft model systems . covalent ( nms-859 ) and allosteric ( nms-873 ) inhibitors of p97 , an aaa - atpase known to extract the polyubiquitylated aurora b from chromatin during post - mitotic nuclear reassembly , have antiproliferative effect in vitro on few cancer types . however , it is possible that the anti - cancer activity is linked to other roles of p97 , such as the er - associated protein degradation . obtusilactone b is a new inhibitor of baf phosphorylation by vrk1 , and it also inhibits the proliferation of few cancer cells in vitro . in line with this , drugs that directly or indirectly activate pp2a ( e.g. , ceramide , fty7220 , dithiolethione , etc . ) , the mitotic phosphatase of baf , also have anti - cancer activities in different malignancies like prostate cancers , breast cancers , lung cancers , or leukemia . finally , microtubules have an influence on the nuclear shape , and they are directly involved in ne breakdown and reformation . therefore , microtubule - binding drugs , at least in part , could interfere with the abnormal nuclear structure of some cancer types . one possible side effect of targeting the mechanisms of nuclear assembly and organization in future anti - cancer therapies might be the artificial induction of laminopathy - like symptoms , such as muscular dystrophies or lipodystrophies . this is suggested by the fact that laminopathies are caused by mutations in different lamina proteins such as lamin a , baf , lbr , or emerin . however , laminopathies are developmental disorders , and therefore , it is possible that they might require more time to appear than the duration of the anti - cancer therapy itself . second , similarly to other anti - mitotic therapies , reduced levels of platelets and blood cells , such as thrombocytopenia or neutropenia , might be also induced . interestingly , however , neutrophils have multi - lobulated and malleable nuclear structures , probably required for the extrusion of their chromatin fibers to trap and kill bacteria at the sites of infection . it is likely that reduced expression of baf and its binding partners emerin , lap2 , lamin a / c , and lamin b2 might account for such nuclear appearance . therefore , targeting proteins that are highly expressed in cancer cells but repressed in neutrophils could lead to at least the terminally differentiated neutrophils being resistant to these new drugs . anti - cancer drugs , in addition to surgery , have been proven to be beneficial for patients with particular cancer types ; however , their effectiveness is often limited by dose - limiting toxicities . therefore , there is a need to develop new drugs that can achieve efficient and cancer cell - specific effects without undesirable side effects . in my opinion , particular molecular mechanisms of post - mitotic nuclear assembly and nuclear organization represent attractive new targets for such next - generation anti - cancer therapies . this strategy relies on putative synthetic lethality between the altered nuclear structure of some cancer types and drugs targeting , directly or indirectly , baf , b - type lamins , vrk1 , vrk2 , and other similar proteins that are important for proper assembly and organization of the nuclei and are clearly expressed or overexpressed in these cancer types .

current anti - cancer therapies have a great deal of undesirable side effects ; therefore , there is a need to develop efficient and cancer cell - specific new drugs without strong dose - limiting side effects . in my opinion , mechanisms of nuclear assembly and organization represent a novel platform for drug targets , which might fulfill these criteria . the nuclear stiffness and organization of some cancer types are often compromised , making them more vulnerable for further targeting the mechanisms of nuclear integrity than their normal counterparts . here i will discuss the nuclear organization of normal cells and cancer cells , the molecular mechanisms that govern nuclear assembly with emphasis on those that , in my view , might be considered as targets for future anti - cancer therapies .

Pitfalls of Current Anti-Mitotic Drugs Nuclear Organization of Normal Cells Nuclear Organization is Disrupted in Cancer Cells Mitotic Nuclear Dynamics Synthetic Lethality with Abnormal Nuclear Architecture of Cancer Cells Concluding Remarks

currently the most efficient anti - cancer chemotherapy agents are the cytotoxic drugs that target cancer cells , in a most vulnerable state , during mitosis . however , cancer cells often adapt to these drugs and exit mitosis in a process called mitotic slippage . they disrupt the interphase microtubule bundles of quiescent neuronal cells , along which molecular and vesicular transport occurs . neurotoxicity is therefore one of the most common dose - limiting side effects of microtubule - targeting drugs . hence , there is a need to develop new anti - mitotic drugs , which by targeting specific mitotic proteins and mechanisms would achieve a similar level of anti - cancer efficacy without the unwanted side effects on non - proliferating cells . although these new anti - mitotic drugs are highly specific in vitro and had some efficacy in xenograft models , they were so far less convincing in clinical trials than the microtubule - targeting drugs . the reasons for these disappointing clinical results might be the low mitotic index ( i.e. , low number of cells undergoing division ) of certain human tumors and strong neutropenia , the main dose - limiting toxicity of the new anti - mitotic drugs . neutropenia refers to an abnormally low number of neutrophil granulocytes , the most abundant type of white blood cells . anti - mitotic drugs are not cancer cell - specific ; they target every dividing cell , including neutrophils , and thus neutropenia is a consequence of their activity . future anti - cancer drugs should therefore possess a larger therapeutic window and exert a higher - level cancer cell - specificity . one strategy to achieve this goal might be to develop new drugs that are synthetic lethal with mechanisms of cancer cell - specific hallmarks . in my opinion such new drug targets are the molecular mechanisms of nuclear assembly and organization because some of these mechanisms might get compromised during tumorigenesis , making the nuclei of these cancer cells more vulnerable than their normal counterparts . below i will discuss the nuclear organization of normal cells and cancer cells , the molecular mechanisms that govern nuclear assembly , and i will hypothesize about the usefulness of targeting some of these mechanisms in future anti - cancer therapies . the nucleus contains the genome of the eukaryotic cell , whose precise organization is essential for normal cell function . the structure that defines the nucleus is the nuclear envelope ( ne ) , a sub - domain of the endoplasmic reticulum ( er ) ( fig . the ne is composed of outer nuclear membranes ( onm ) and inner nuclear membranes ( inm ) with different protein compositions , which are fused at the sites of nuclear pore complex ( npc ) insertion . underlying the inm is the nuclear lamina , which is mainly composed of intermediate lamin filaments . while b - type lamins are expressed in every somatic cell , a - type lamins are absent from highly pluripotent and rapidly dividing cells and are present only in differentiated cells . these polymers then laterally assemble into filaments , which form a lattice of remarkably regular arrangement underneath the inm . while this short lipid chain is retained on b - type lamins and anchors the proteins in the membranes , the farnesylated c - terminal part of lamin a is enzymatically cleaved after its incorporation into lamin polymers . the stabilization and organization of lamin filaments underneath the inm also requires lamin - associated membrane proteins , such as lamin b receptor ( lbr ) , diverse lap2/emerin / man1 ( lem ) domain - containing proteins , and others . nuclear envelope ( yellow ) is penetrated by the nuclear pore complexes ( red ) . underneath the nuclear envelope is the nuclear lamina ( green ) , which provides the stiffness to the ne and serves as a tethering surface for chromosomes ( blue lines ) . ( b ) several cancer types are characterized by abnormal expression of lamins ( particularly a - type ) , altered chromatin organization , and multi - lobulated nuclear shape . other changes involve enlargement or fragmentation of the nucleolus ( brown , large circles ) and promyelocytic leukemia ( pml ) bodies ( orange , small circles ) . ( c ) possible nuclear appearance of cancer cell nuclei after the treatment with drugs that interfere with mechanisms of nuclear assembly and organization . b - type lamins are constitutively expressed and correlate much less with nuclear stiffness . developmentally coregulated genes often form clusters on chromosomes , which are associated and corepressed at the nuclear periphery in cells where they are not expressed . this suggests that the nuclear lamina often provides a repressive environment for chromosomal domains at the nuclear periphery whose three - dimensional organization is specific for certain cell types and differentiation states . mutation or downregulation of lamins or lamin - associated proteins results in disorganized lamin filaments and deformed , multi - lobulated , and fragile nuclei , which are often observed in human diseases such as cancer . cancer is initiated by genetic processes such as genome instability , genome rearrangements , or specific gene mutations , amplifications , or deletions , followed by epigenetic modifications , which ultimately lead to altered gene expression and result in deregulated cell proliferation . in cancer cells , nuclear size and shape cancer - related morphological changes include ne invaginations , multi - lobulation , malleable and passively distorted nuclei , and altered appearance of heterochromatin , nucleoli , and nuclear bodies ( fig . although all these features do not occur simultaneously , they are often used individually in the clinical diagnosis of cancer , in the assessment of the degree of the malignancy , and for prognostic and predictive indications of the disease state . lamins , the most important architectural elements of the nucleus , are often aberrantly expressed or localized in cancer cells , and it is likely that this contributes to the multi - lobulated nuclear shape often observed in different cancer types ( fig . many poorly differentiated cancer types exhibit downregulation of a - type lamins and concomitant irregularities in their nuclear shape . for example , in small cell lung cancer ( sclc ) cells a - type lamins are either not or only weakly expressed , while in non - sclc cells they are normally expressed but frequently mislocalized in the cytoplasm . in colon cancers , gastric cancers , breast cancers , and diffuse large b - cell lymphomas , the expression of a - type lamins is also strongly reduced , and this feature correlates with increased disease recurrence and poor patient prognosis . consequently , the nuclei of these cancer types are often fragile and lobulated , and in breast cancer cells , they were shown to contain massive ne membrane invaginations ( fig . the nuclei of prostate cancer cells and of some other cancer types are not only lobulated but also contain structures called nuclear blebs , protrusions from the nuclear surface enriched in lamin a / c but deficient in lamin b. further examples with multi - lobulated nuclear shape are ovarian cancers , papillary thyroid cancers , leukemias , and different b - cell lymphomas . in conclusion , altered lamin expression or localization and disrupted stoichiometry between a- and b - type lamins can change the elastic properties of the ne , which renders it unable to withstand cytoskeleton- and chromosome - based forces and leads to misshapen nuclei . consistently , downregulation of lamin a / c in non - cancer primary breast epithelial cells results in nuclear alterations similar to those observed in breast cancer cell . moreover , mutations in lmna and other genes encoding for proteins of nuclear lamina results in heritable diseases called laminopathies , which are also characterized by fragile and multi - lobulated nuclear shape . therefore the nuclear lamina alterations might directly account for the cancer - related changes in the nuclear morphology . however , it is important to note that several other cancer types display normal nuclear shape and that there is no simple universal pattern of lamin expressions for all cancer types . it is unclear whether the abnormal nuclear organization observed in cancer cells is the cause or the consequence of transformation and tumor progression . therefore , the absence of lamins from tumors derived from tissues where they are normally present led to the hypothesis that lamins might be directly involved in tumorigenesis . most of these mutations affect the assembly , dynamics , or function of lamin filaments and result in deformed , multi - lobulated , and fragile nuclei and abnormal heterochromatin structure , similar to the nuclear abnormalities observed in cancer cells . moreover , loss of lamin a from human fibroblast cells or ovary surface epithelial cells results in reduced mitosis and retarded cell growth , which at least in part might be explained by the active role of lamin a in nuclear localization of the cell cycle regulator retinoblastoma protein ( rb ) . finally , although higher order chromatin organization is commonly altered in cancer cells , this does not necessarily lead to tumorigenic transcriptional changes . in breast cancer for example , several genes have been identified that specifically change their nuclear position only in cancer cells . however , the absence of transcriptional changes associated with the movement of these genes suggests that these changes are not responsible for tumorigenesis . in conclusion , although precise nuclear organization is essential for normal cellular function , up to now altered nuclear architecture has not been shown to drive cancer development , and therefore , it is more likely to be a consequence of cell transformation and tumor progression . since the nuclear architecture of many cancer cells is abnormal , this hallmark might improve cancer cell - selectivity in therapies using mechanisms of mitotic nuclear assembly and nuclear organization as targets ( fig . the nuclear structure is disassembled and reassembled during every cell division to allow cytoplasmic spindle microtubules to segregate the duplicated sister chromatids . they phosphorylate proteins of nuclear lamina and npc to disrupt their interphase molecular interactions . in support of their critical role , inactivation or inhibition of these mitotic kinases either blocks or delays various steps of nuclear disassembly . it relocalizes to the nuclear periphery and phosphorylate barrier - to - autointegration factor ( baf or banf1 ) to release it from dna and lem domain - containing inm proteins ( fig . inhibition of vrk1 thus prevents a late but essential step of nuclear disassembly . during mitotic entry vrk1 ( red ) phosphorylates baf to disrupt its interactions and contributes to nuclear envelope breakdown . during mitotic exit lem4 ( brown ) inhibits vrk1 ( red ) and promotes pp2a ( green ) to dephosphorylate baf and to enable its function in post - mitotic nuclear reassembly . cdk1 promotes mitotic progression until the alignment of mitotic chromosomes on the metaphase plate and the correct bipolar attachment of all the kinteochores with spindle poles is achieved . chromatin decondensation is an important step of post - mitotic nuclear assembly . although its exact mechanism is still unclear , likely players are pp1 , which acts by dephosphorylating histone h3 , aaa - atpase p97 , which extracts the polyubiquitylated aurora b histone kinase from chromosomes , and the small ras - like gtpase ran , which acts in a still poorly understood manner . the inm proteins function redundantly , thus their individual inactivation has only a minor effect on ne assembly and even simultaneous inactivation of several only delays but does not prevent the recruitment of membranes to the chromatin surface . there is constant membrane supply from the er , and manipulation of er structure was shown to influence ne assembly . the recruited membranes on the chromatin surface are organized by baf , which as a dimer can bind to one lem domain of an inm protein and to two dna helices ( fig . the localization and function of baf is regulated by phosphorylation , which is temporally controlled by lem4 ( fig . recently pp4c , which is known to form complexes with different pp2a subunits , was also suggested to influence the phosphorylation state of baf . nevertheless , during mitotic exit baf rapidly and strongly accumulates on the central surface of the chromatin core regions , which surround the anaphase chromatin mass , on one side facing the spindle microtubules and on the other facing the midzone microtubules . concomitantly with ne reformation , npcs are also assembled post - mitotically via mechanisms that are different from those used during interphase npc assembly . npc sub - complexes form pre - pores on the chromatin surface , which are then incorporated in the ne during membrane spreading on the chromatin surface . however , since these holes can also be occupied by npcs , the membrane fusion machinery is less important during ne assembly than was initially anticipated . in genetic studies using different experimental model organisms , synthetic lethality is based on the fact that inactivation of one gene makes the cell vulnerable for the inactivation of the other gene , while neither of these two genes is essential on its own . in anti - cancer therapies similar synthetic lethality might be achieved between cancer cells and drugs , in case the cancer cell - specific molecular lesions sensitize the cancer cells for drugs inhibiting particular protein functions . the value of such an approach is that normal cells without such lesions will be unaffected . as discussed earlier , the nuclear architecture of different cancer cells is compromised and unable to withstand cytoskeleton- and chromosome - based forces and often so malleable that they can be crushed during biopsies ( e.g. , sclc cells ) . this suggests that future drugs hitting mechanisms of nuclear assembly and organization to further weakening the ne structure might result in a synergistic effect and specific killing of such cancer cells ( fig . drug targets that are potentially synthetic lethal with abnormal nuclear organization of some cancer types might be identified either via phenotypic and synthetic lethality screens or by literature mining . based on the latter one , in my opinion , baf represents one of the most promising targets for specifically killing cancer cells with altered nuclear appearance . i base this assumption on facts that baf is often highly expressed in some cancer types ( e.g. , ovarian cancer , endometrial cancer , breast cancer , colorectal cancer , lung cancer , prostate cancer , glioma , melanoma , and lymphoma ) , and it is an important player of post - mitotic nuclear assembly whose downregulation or mutation in humans delays ne formation and induces strong nuclear irregularities . due to its small size and relatively flat protein surface without suitable binding pockets for small molecular weight inhibitory compounds , new anti - mitotics might be designed against its mitotic regulators ( fig . first of all , the dephosphorylation of baf might be targeted because this is essential for its correct localization and function during mitotic exit . therefore , drugs interfering with either the inhibitory interaction between lem4 and vrk1 , the mitotic kinase of baf , or with the activating interaction between lem4 and pp2a , the mitotic phosphatase of baf , might be developed ( fig . a second strategy might rely on interfering with the nuclear structure during mitotic entry by inhibiting baf s kinases vrk1 and vrk2 . consistently , depletion of vrk1 from breast cancer cells results in retarded tumor growth and reduced incidence of metastasis in a murine orthotopic xenograft model . furthermore , the first inhibitor of baf phosphorylation with an in vitro anti - cancer activity was recently isolated from a species of tree used in traditional medicine . finally , b - type lamins are also promising targets because they are highly expressed in several cancer types , and they might become essential for survival of cancer cells with reduced expression of a - type lamins . since lamins are also considered to be low - druggable , they might be targeted indirectly by inhibiting their regulators . such examples include akap149 , which via promoting pp1 mediates the assembly of b - type lamins into nuclear lamina , and farnesyltransferase enzymes that add a 15-carbon farnesyl group to the c - terminal of most of the lamins to keep the b - type lamins anchored to the membranes . currently , only a few drugs exist that directly target mechanisms of nuclear assembly and organization , but interestingly , they can induce cancer cell death . for example , the farnesyltransferase inhibitor r115777 inhibits the growth of b - cell lymphoma and breast and ovarian cancer cells in vitro and reduces the tumor growth in xenograft model systems . since lamins are not the only substrates of farnesyltransferases , it is possible that the main anti - tumor activity of this inhibitor is achieved via other substrates , such as the small gtpase ras proteins . betulinic acid inhibits the expression of lamin b1 in pancreatic cancer cells and induces dose - dependent anti - cancer activities in both in vitro cultures and xenograft model systems . covalent ( nms-859 ) and allosteric ( nms-873 ) inhibitors of p97 , an aaa - atpase known to extract the polyubiquitylated aurora b from chromatin during post - mitotic nuclear reassembly , have antiproliferative effect in vitro on few cancer types . however , it is possible that the anti - cancer activity is linked to other roles of p97 , such as the er - associated protein degradation . obtusilactone b is a new inhibitor of baf phosphorylation by vrk1 , and it also inhibits the proliferation of few cancer cells in vitro . , ceramide , fty7220 , dithiolethione , etc . ) , the mitotic phosphatase of baf , also have anti - cancer activities in different malignancies like prostate cancers , breast cancers , lung cancers , or leukemia . finally , microtubules have an influence on the nuclear shape , and they are directly involved in ne breakdown and reformation . therefore , microtubule - binding drugs , at least in part , could interfere with the abnormal nuclear structure of some cancer types . one possible side effect of targeting the mechanisms of nuclear assembly and organization in future anti - cancer therapies might be the artificial induction of laminopathy - like symptoms , such as muscular dystrophies or lipodystrophies . however , laminopathies are developmental disorders , and therefore , it is possible that they might require more time to appear than the duration of the anti - cancer therapy itself . second , similarly to other anti - mitotic therapies , reduced levels of platelets and blood cells , such as thrombocytopenia or neutropenia , might be also induced . therefore , targeting proteins that are highly expressed in cancer cells but repressed in neutrophils could lead to at least the terminally differentiated neutrophils being resistant to these new drugs . anti - cancer drugs , in addition to surgery , have been proven to be beneficial for patients with particular cancer types ; however , their effectiveness is often limited by dose - limiting toxicities . therefore , there is a need to develop new drugs that can achieve efficient and cancer cell - specific effects without undesirable side effects . in my opinion , particular molecular mechanisms of post - mitotic nuclear assembly and nuclear organization represent attractive new targets for such next - generation anti - cancer therapies . this strategy relies on putative synthetic lethality between the altered nuclear structure of some cancer types and drugs targeting , directly or indirectly , baf , b - type lamins , vrk1 , vrk2 , and other similar proteins that are important for proper assembly and organization of the nuclei and are clearly expressed or overexpressed in these cancer types .

fatigue is commonly reported by patients with multiple sclerosis and has a profound impact upon their daily activities and quality of life . evaluation of fatigue also brings about difficulties because of its lack of the objective biomarkers [ 1 , 2 ] . according to some theories , the background of fatigue is associated with disturbed bioelectrical neuronal activity due to demyelination and axonal loss [ 3 , 4 ] . studies with the use of motor evoked potentials and electroencephalography event - related desynchronization have indeed shown decreased neuronal excitability and frequency - dependent conduction block in fatigued ms patients [ 58 ] . visual and auditory evoked potentials are regarded as useful tools for recognizing and monitoring damage to central nervous system ascending pathways in the course of ms . however , these methods have not been used so far in studies on ms fatigue , investigating its origin and methods of its evaluation . the aim of our study was to assess visual and brainstem auditory evoked potentials ( baep ) in ms patients with regard to the presence and severity of fatigue , considering also the impact of other disease - related variables . the study comprised 86 patients with ms ( 24 men and 62 women , aged 1960 years , mean 39.55 ) who were under the care of the outpatient ms clinic , department of neurology , medical university of wroclaw . none of the patients had concomitant diseases known to cause fatigue or to affect parameters of visual and auditory evoked potentials in their history . 24 subjects had undergone treatment with interferon beta or glatiramer acetate for 13 years , but treatment had ceased at least 6 months prior to their inclusion in this study ( in 15 patients because of their transition into secondary progressive phase of ms ; nine subjects resigned from the treatment due to its side effects or for personal reasons ) . none of the patients were being treated with chronic immune - suppression . a washout period of at least 4 weeks was maintained between inclusion in the study and tapering treatment with corticosteroids due to the most recent ms relapse . the control group consisted of 40 healthy volunteers , who were matched for age and gender to the ms patients ( 12 men , 28 women , aged 2360 years , mean 38.8 ) . all the subjects gave their informed consent to participate in the study and the project was approved by the bioethical committee at the medical university of wroclaw . the patients underwent a neurological examination and their disability was assessed using the expanded disability status scale ( edss ) , with visual and brainstem functional systems ( fs ) scores separated for further analysis . on the basis of medical records , the duration of the disease was defined and the index of disability progression [ multiple sclerosis severity scale ( msss ) ] calculated ; the history of optic neuritis or loss of hearing was also determined . the level of fatigue in ms patients was evaluated using self - assessment questionnaires based on the fatigue severity scale ( fss ) and the modified fatigue impact scale ( mfis ) , with the results of fss re - evaluated using the rasch analysis applied by mills et al . the patients were divided into three subgroups : without fatigue ( subgroup i , fss / fss-5 < 3.5 ) , with moderate ( subgroup ii , fss / fss-5 = 3.55.5 ) or severe fatigue ( subgroup iii , fss / fss-5 > 5.5 ) . visual evoked potentials ( vep ) were performed by using a black and white checker board pattern on a screen , with a checker size of 36 cm and the frequency of pattern reversing being 1.9 hz . the subjects were sitting in the distance of 1 m from the screen , with the angle of vision 29. the stimuli were presented uni - ocularly . an active recording electrode was attached to the scalp on the midline at the occipital region ( oz according to the 1020 system ) , the reference electrode was placed on the midline frontal point ( fz ) and the ground electrode on the forearm . ag / agcl surface electrodes were used and their impedance was maintained below 5 k ohm . the responses were analyzed with a nicolet 1000 viking quest , with a 130 hz bandpass filter and a sweep time of 500 ms . a hundred responses were averaged in each run and two runs were performed for each eye . for each subject , the latency and amplitude ( peak to peak ) of the p100 component were determined for each eye , as well as relative p100 latency ( interocular latency difference ) . baep were performed with the use of stimuli presented to each ear separately via earphones . auditory stimuli were clicks of a duration of 0.1 ms , frequency 20.3 hz and an intensity 65 db higher than the hearing threshold initially established for each subject . a recording electrode was attached to the earlobe on the side of stimulation , with the reference electrode placed on the vertex ( fz ) and the ground electrode on the forearm . ag / agcl surface electrodes were used and their impedance was maintained below 5 k ohm . the responses were analyzed with a nicolet 1000 viking quest , with a 1503,000 hz bandpass filter and a sweep time of 10 ms . two hundred responses were averaged in each run and two runs were performed for each ear . latencies and amplitudes ( peak to peak ) were determined for components i , iii and v , as well as interpeak latencies i iii , iii v , i v and the proportion of amplitudes i / v . mean and median values with standard deviations were calculated for all the analyzed variables . the ep parameters obtained from the whole group of ms patients and subgroups i , ii and iii were compared with those from the controls , and the results were also compared between subgroups i , ii and iii with the use of post hoc test ( scheffe test ) and then analysis of variance ( anova ) , alternatively using the kruskal wallis test , when the variances in groups were not homogeneous ( the homogeneity of variance was determined by the bartelett s test ) or if the number of cases was too small . relation between continuous fatigue measures and continuous ep parameters was assessed using correlation analysis and pearson correlation coefficients were calculated . relation between continuous fatigue measures and categorized parameters ( visual fs , brainstem fs ) was assessed using correlation analysis and spearman correlation coefficients were calculated . multiple regression analysis was used to check the impact of age and ms - related variables upon correlations between fatigue measures and ep results . p < 0.05 was regarded as statistically significant and p < 0.07 sufficient to observe trends . the level of fatigue in ms patients was evaluated using self - assessment questionnaires based on the fatigue severity scale ( fss ) and the modified fatigue impact scale ( mfis ) , with the results of fss re - evaluated using the rasch analysis applied by mills et al . the patients were divided into three subgroups : without fatigue ( subgroup i , fss / fss-5 < 3.5 ) , with moderate ( subgroup ii , fss / fss-5 = 3.55.5 ) or severe fatigue ( subgroup iii , fss / fss-5 > 5.5 ) . visual evoked potentials ( vep ) were performed by using a black and white checker board pattern on a screen , with a checker size of 36 cm and the frequency of pattern reversing being 1.9 hz . the subjects were sitting in the distance of 1 m from the screen , with the angle of vision 29. the stimuli were presented uni - ocularly . an active recording electrode was attached to the scalp on the midline at the occipital region ( oz according to the 1020 system ) , the reference electrode was placed on the midline frontal point ( fz ) and the ground electrode on the forearm . ag / agcl surface electrodes were used and their impedance was maintained below 5 k ohm . the responses were analyzed with a nicolet 1000 viking quest , with a 130 hz bandpass filter and a sweep time of 500 ms . a hundred responses were averaged in each run and two runs were performed for each eye . for each subject , the latency and amplitude ( peak to peak ) of the p100 component were determined for each eye , as well as relative p100 latency ( interocular latency difference ) . baep were performed with the use of stimuli presented to each ear separately via earphones . auditory stimuli were clicks of a duration of 0.1 ms , frequency 20.3 hz and an intensity 65 db higher than the hearing threshold initially established for each subject . a recording electrode was attached to the earlobe on the side of stimulation , with the reference electrode placed on the vertex ( fz ) and the ground electrode on the forearm . ag / agcl surface electrodes were used and their impedance was maintained below 5 k ohm . the responses were analyzed with a nicolet 1000 viking quest , with a 1503,000 hz bandpass filter and a sweep time of 10 ms . two hundred responses were averaged in each run and two runs were performed for each ear . latencies and amplitudes ( peak to peak ) were determined for components i , iii and v , as well as interpeak latencies i iii , iii the ep parameters obtained from the whole group of ms patients and subgroups i , ii and iii were compared with those from the controls , and the results were also compared between subgroups i , ii and iii with the use of post hoc test ( scheffe test ) and then analysis of variance ( anova ) , alternatively using the kruskal wallis test , when the variances in groups were not homogeneous ( the homogeneity of variance was determined by the bartelett s test ) or if the number of cases was too small . relation between continuous fatigue measures and continuous ep parameters was assessed using correlation analysis and pearson correlation coefficients were calculated . relation between continuous fatigue measures and categorized parameters ( visual fs , brainstem fs ) was assessed using correlation analysis and spearman correlation coefficients were calculated . multiple regression analysis was used to check the impact of age and ms - related variables upon correlations between fatigue measures and ep results . p < 0.05 was regarded as statistically significant and p < 0.07 sufficient to observe trends . on the basis of fss / fss-5 results , 29 patients ( 8 men , 21 women ) were allocated to subgroup i ( non - fatigued ) , 31 patients ( 7 men , 24 women ) to subgroup ii ( moderately fatigued ) and 26 ( 8 men , 18 women ) to subgroup iii ( severely fatigued ) . no significant differences in terms of age or gender were found either between these groups , or between each of them and the healthy controls . the results of mfis on the whole group of patients ranged from 4 to 64 ( mean 36.3 ) . there was a significant correlation between mfis results and the age of the ms patients ( r = 0.24 , p = 0.02 ) . patients the duration of the disease was 130 years ( mean 8.57 ) , edss 16.5 ( mean 3.03 ) and msss 1.18.8 ( mean 4.4 ) . visual fs scores ranged from 0 to 3 ( mean 0.8 ) , and so did brainstem fs scores ( mean 1.2 ) . 42 patients had a history of optic neuritis , while none experienced loss of hearing during ms relapse . patients with or without the history of optic neuritis did not differ significantly in the mean values of fss / fss-5 ( 4.63 vs 4.02 , p = 0.1 ) or mfis ( 39.1 vs 33.7 , p = 0.12 ) . fss / fss-5 and mfis correlated significantly with edss , visual and brainstem fs ( table 1).table 1correlations between fatigue measures ( fss / fss-5 , mfis ) and degree of disability ( edss , visual fs , brainstem fs ) ; r spearman s correlation coefficientedssvisual fsbrainstem fsfss / fss-5 r = 0.48 r = 0.3 r = 0.26 p = 0.00001 p = 0.006 p = 0.018mfis r = 0.46 r = 0.32 r = 0.24 p = 0.0001 p = 0.003 p = 0.025 correlations between fatigue measures ( fss / fss-5 , mfis ) and degree of disability ( edss , visual fs , brainstem fs ) ; r spearman s correlation coefficient the mean duration of the disease was longer in subgroup iii in comparison with subgroup i ( 11.56 vs 5.38 years , p = 0.003 ) . the mean edss score was higher in subgroups ii and iii in comparison with subgroup i ( 2.98 vs 4.08 vs 2.12 , p = 0.027 and 0.0000001 , respectively ) . the mean msss score was higher in subgroup iii in comparison with subgroups ii and i ( 5.32 vs 4.16 vs 3.86 , p = 0.047 and 0.014 , respectively ) . the mean latency of the p100 component of vep for both eyes was significantly longer in ms patients than in the controls , and so was the mean relative p100 latency ( interocular latency difference ) . the mean p100 amplitude was significantly lower for ms patients than in controls , but only for the left eye ( table 2).table 2parameters of the p100 component of visual evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatiguedcontrols ( n = 40)ms patients ( n = 86)ms subgroup i ( n = 29)ms subgroup ii ( n = 31)ms subgroup iii ( n = 26)l : p100 latency ( ms ) mean101.8117.3 p ( ms - contr ) = 0.00001 113.9 p ( i - contr ) = 0.00001 118.3 p ( ii - contr ) = 0.00001 119.8 p ( iii - contr ) = 0.00001 sd5.021.614.916.1 p ( i ii ) = 0.2831.9 p ( i iii ) = 0.38 p ( ii iii ) = 0.82r : p100 latency ( ms ) mean102.2119.9 p ( ms - contr ) = 0.00001 113.8 p ( i - contr ) = 0.00001 121.1 p ( ii - contr ) = 0.00001 125.1 p ( iii - contr ) = 0.00001 sd4.715.812.914.6 p ( i ii ) = 0.02 18.4 p ( i iii ) = 0.006 p ( ii iii ) = 0.2relative p100 latency : mean2.149.95 p ( ms - contr ) = 0.00001 6.49 p p ( ii - contr ) = 0.00001 11.16 p ( iii - contr ) = 0.00001 sd1.9611.136.6313.13 p ( i ii ) = 0.1511.96 p ( i iii ) = 0.038 p ( ii iii ) = 0.89l : p100 amplitude ( v ) mean11.49.39 p ( ms - contr ) = 0.027 9.72 p ( i - contr ) = 0.159.34 p ( ii - contr ) = 0.0579.06 p ( iii - contr ) = 0.052 median4.54.925.254.63 p ( i ii ) = 0.495.08 p ( i iii ) = 0.65 sd p ( ii iii ) = 0.83r : p100 amplitude ( v ) mean10.68.97 p ( ms - contr ) = 0.0758.96 p ( i - contr ) = 0.159.25 p ( ii - contr ) = 0.228.63 p ( iii - contr ) = 0.097 sd4.45.058.437.92 p ( i ii ) = 0.835.12 p ( i iii ) = 0.012 5.075.13 p ( ii iii ) = 0.65relative p100 amplitude : mean0.971.02 p ( ms - contr ) = 0.471.07 p ( i - contr ) = 0.551.02 p ( ii - contr ) = 0.460.98 p ( iii - contr ) = 0.68 sd0.220.40.560.28 p ( i ii ) = 0.530.38 p ( i iii ) = 0.50 p ( ii iii ) = 0.36bold values are statistically significant at ( p < 0.05 ) l left eye , r right eye ) parameters of the p100 component of visual evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatigued bold values are statistically significant at ( p < 0.05 ) l left eye , r right eye ) p100 latency for the right eye was significantly longer in subgroups ii and iii than in subgroup i , and relative p100 latency was significantly longer in subgroup iii than in subgroup i ( table 2 ) . the amplitude of p100 for the left eye was lower in subgroups ii and iii than in the controls , and for the right eye lower in subgroup no significant correlations were found between the summated values of vep latency and amplitude and fss / fss-5 or mfis . the relative latency of p100 tended to correlate positively with fss / fss-5 ( r = 0.26 , p = 0.07 ) . there was a significant correlation between summated vep amplitude and visual fs ( r = 0.36 , p = 0.0006 ) and a correlation on the edge of significance between summated vep latency and visual fs ( r = 0.21 , p = 0.05 ) . no correlations were found between vep parameters and other clinical ms - related variables ( duration of the disease , edss or msss ) . the mean latencies of i , iii and v components of baep on both sides did not differ significantly between ms patients and controls . the mean amplitude of the v component was significantly lower in ms patients than in the controls , but only on the right side ( table 3).table 3parameters of brainstem auditory evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatiguedcontrols ( n = 40)ms patients ( n = 86)ms subgroup i ( n = 29)ms subgroup ii ( n = 31)ms subgroup iii ( n = 26 ) l : latency i mean1.691.68 p ( ms - contr ) = 0.671.66 p ( i - contr ) = 0.281.69 p ( ii - contr ) = 0.981.70 p ( iii - contr ) = 0.97 sd0.130.120.10.13 p ( i ii ) = 0.300.12 p ( i iii ) = 0.28 p ( ii iii ) = 0.99latency iii mean3.833.87 p ( ms - contr)=0.243.87 p ( i - contr ) = 0.263.860.16 p ( ii - contr ) = 0.143.91 p ( iii - contr ) = 0.06 sd0.120.170.17 p ( i ii ) = 0.840.20 p ( i iii ) = 0.43 p ( ii iii ) = 0.30latency v mean5.75.77 p ( ms - contr)=0.535.86 p ( i - contr ) = 0.235.78 p ( ii - contr ) = 0.925.65 p ( iii - contr ) = 0.049 sd0.210.730.440.24 p ( i ii ) = 0.831.23 p ( i iii ) = 0.39 p ( ii iii ) = 0.30interlat . iii mean2.132.19 p ( ms - contr ) = 0.046 2.2 p ( i - contr ) = 0.0532.16 p ( ii - contr ) = 0.392.21 p ( iii - contr ) = 0.054 sd0.140.160.170.16 p ( i ii ) = 0.310.17 p ( i iii ) = 0.63 p ( ii iii ) = 0.11interlat . iii v mean1.871.96 p ( ms - contr ) = 0.0721.99 p ( i - contr ) = 0.331.93 p ( ii - contr ) = 0.241.97 p ( iii - contr ) = 0.16 sd0.170.300.380.23 p ( i ii ) = 0.420.27 p ( i iii ) = 0.65 p ( ii iii ) = 0.63interlat . i v mean4.04.15 p ( ms - contr ) = 0.013 4.2 p ( i - contr ) = 0.0574.09 p ( ii - contr ) = 0.0984.19 p ( iii - contr ) = 0.025 sd0.190.340.430.25 p ( i ii ) = 0.690.34 p ( i iii ) = 0.64 p ( ii iii ) = 0.36amp . i mean0.30.32 p ( ms - contr ) = 0.580.32 p ( i - contr ) = 0.560.30 p ( ii - contr ) = 0.930.33 p ( iii - contr ) = 0.43 sd0.10.140.120.14 p ( i ii ) = 0.690.15 p ( i iii ) = 0.81 p ( ii iii ) = 0.58amp . p ( ms - contr ) = 0.330.39 p ( i - contr ) = 0.190.44 p ( ii - contr ) = 0.920.37 p ( iii - contr ) = 0.43 sd0.160.240.170.18 p ( i ii ) = 0.280.34 p ( i iii ) = 0.85 p ( ii iii ) = 0.37 r : latency i mean1.71.72 p ( ms - contr ) = 0.851.73 p ( i - contr ) = 0.611.73 p ( ii - contr ) = 0.691.69 p ( iii - contr ) = 0.64 sd0.140.190.190.18 p ( i ii ) = 0.910.21 p ( i iii ) = 0.47 p ( ii iii ) = 0.51latency iii mean3.853.88 p ( ms - contr ) = 0.633.850.42 p ( i - contr ) = 0.833.89 p ( ii - contr ) = 0.443.89 p ( iii - contr ) = 0.46 sd0.160.330.28 p ( i ii ) = 0.650.29 p ( i iii ) = 0.47 p ( ii iii ) = 0.99latency v mean5.735.81 p ( ms - contr ) = 0.525.78 p ( i - contr ) = 0.805.91 p ( ii - contr ) = 0.03 5.71 p ( iii - contr ) = 0.11 sd0.230.810.230.48 p ( i ii ) = 0.321.28 p ( i iii ) = 0.79 p ( ii iii ) = 0.42interlat . i iii mean2.142.18 p ( ms - contr ) = 0.262.18 p ( i - contr ) = 0.312.16 p ( ii - contr ) = 0.612.20 p ( iii - contr ) = 0.25 sd0.120.190.160.21 p ( i ii ) = 0.760.20 p ( i iii ) = 0.78 p ( ii iii ) = 0.31interlat . v mean1.872.0 p ( ms - contr ) = 0.013 1.95 p ( i - contr ) = 0.212.02 p ( ii - contr ) = 0.005 2.05 p ( iii - contr ) = 0.04 sd0.180.30.280.25 p ( i ii ) = 0.260.37 p ( i iii ) = 0.29 p ( ii iii ) = 0.99interlat . v mean4.024.19 p ( ms - contr ) = 0.042 4.12 p ( i - contr ) = 0.524.18 p ( ii - contr ) = 0.0584.26 p ( iii - contr ) = 0.025 sd0.20.390.350.39 p ( i ii ) = 0.520.43 p ( i iii ) = 0.19 p ( ii iii ) = 0.56amp . i mean0.30.29 p ( ms - contr ) = 0.680.32 p ( i - contr ) = 0.630.28 p ( ii - contr ) = 0.350.27 p ( iii - contr ) = 0.34 sd0.10.180.230.13 p ( i ii ) = 0.370.15 p ( i iii ) = 0.38 p ( ii iii ) = 0.92amp . v mean0.460.37 p ( ms - contr ) = 0.025 0.420.24 p ( i - ontr ) = 0.520.37 p ( ii - contr ) = 0.0540.31 p ( iii - contr ) = 0.003 sd0.190.20.19 p ( i ii ) = 0.310.17 p ( i iii ) = 0.057 p ( ii iii ) = 0.28bold values are statistically significant at ( p < 0.05 ) l left ear , r right ear , interlat . interlatency , amp amplitude parameters of brainstem auditory evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatigued bold values are statistically significant at ( p < 0.05 ) l left ear , r right ear , interlat . interlatency , amp amplitude the latency of the v component of baep , and interlatency i v for the left ear as well as interlatencies iii v and i v for the right ear were significantly longer in subgroup iii than in the controls . in subgroup ii , the latency of the v component and interlatencies iii v , and i v for the right ear were significantly longer than in the controls ( table 3 ) . the amplitude of the v component for the right ear was significantly lower in subgroups ii and iii than in the controls ( table 3 ) . no correlations were found between the baep parameters ( one - sided or summated values ) and fatigue measures ( fss / fss-5 , mfis ) . among the baep parameters , interlatencies iii v and i v showed significant positive correlations with msss ( iii v : r = 0.28 , p = 0.009 ; r = 0.25 , p = 0.024 ; i v : r = 0.25 , p = 0.021 ; r = 0.2 , p = 0.05 ; for left and right side , respectively ) . summated latencies of baep components correlated significantly with brainstem fs ( i : r = 0.23 , p = 0.04 ; iii : r = 0.23 , p = 0.03 ; v : r = 0.36 , p = 0.0008 ) and so did summated amplitude of v component ( r = 0.23 , p = 0.03 ) . no other correlations were found between baep parameters and remaining clinical ms - related variables ( duration of the disease , edss or msss ) . the importance of evoked potentials ( ep ) in the diagnosis of ms has decreased in the last decade as magnetic resonance has become the main diagnostic tool supporting clinical assessment . however , ep abnormalities are still regarded as good electrophysiological markers of disease progression and their prognostic value in the early stages of ms gains increasing attention [ 1517 ] . non - invasiveness and the availability of ep also encourage their use in clinical practice . fatigue constitutes an important aspect of non - physical disability in ms patients , which is still lacking objective biomarkers , so analysis of ep parameters with regards to fatigue in ms seemed worth investigating . we deliberately chose vep and baep as they have not been used in this field so far ( in contrast to mep ) . analysis of vep showed significantly prolonged latency of the p100 component in the whole group of ms patients as well as in each of the three subgroups , when compared to the controls . such a finding is common in ms subjects and indicates slowed conduction in the optic tract due to demyelination . it is worth noting that only severely fatigued patients in comparison with non - fatigued ones showed a significantly increased interocular latency difference ( relative p100 latency ) . this parameter tended to correlate ( although not significantly ) with one of the fatigue measures ( fss / fss-5 ) , but apart from visual fs score did not show significant relationships with other disease - related variables ( duration of ms , edss or msss ) . although no correlation was found between relative p100 latency and the result of mfis ( which allows more detailed assessment of fatigue than fss ) , it might be interesting to refer vep results to physical and cognitive aspects of fatigue . significant differences in p100 amplitude were also asymmetrical but they were found not only between fatigued and non - fatigued subgroups but also between ms patients and controls . the amplitude of vep components is usually regarded as a more variable and thus less sensitive parameter than latency , so we believe p100 latency deserves more attention in further investigation . subgroups i , ii and iii did not differ significantly as regards age and gender structure , so the influence of demographic factors upon vep parameters can be neglected . in the only available study comprising ms patients ( i.e. , regan et al . ) , vep were used to evaluate the fatigability of the visual pathway . in those patients with ms and glaucoma ( but not in parkinsonic ones ) , the amplitude of p100 increased when additional stimuli were superimposed on the basic pattern of stimulation . our results seem more consistent with the report of sobieszczaska et al . , who assessed vep as a measure of fatigability in healthy persons , professionally operating computer terminals . after a few hours of their constant gazing at the computer screen , there was an increase in p100 latency , a decrease in amplitude , and moreover , a decrease in correlation coefficients for the vep parameters obtained from both hemispheres . overall , the abnormalities of vep parameters in our material can be attributed to the impact of ms in general , but the asymmetry of these abnormalities might have been more specific for fatigue . unlike the optic tract , the auditory pathway is much less frequently affected by demyelination in the course of ms . in the whole group of our ms patients in comparison with the controls , we only found significantly prolonged interlatencies between i , iii and v components of baep , which indicate subtle conduction disturbances within the brainstem . on analysis of the subgroups of patients with and without fatigue , these abnormalities appeared to occur only in those with moderate and severe fatigue . it has to be considered that these subgroups also presented with higher level of disability and rate of its progression . the interlatencies of baep components indeed showed significant correlations with msss but not with any of the fatigue measures . moreover , the fatigued patients also showed prolonged latency of the v component of baep ( while non - fatigued ones and the whole ms group did not ) . this parameter , in turn , did not correlate significantly with the majority of disease - related variables , apart from brainstem fs . it is worth noting that significant findings in baep parameters mostly concerned only one side . to our knowledge , there have been no reports on baep with regards to fatigue in ms patients . described abnormalities of baep ( the lack of component i and prolonged interlatencies ) in subjects with chronic fatigue syndrome , which occurred only at higher frequencies of auditory stimulation , so were apparently revealed at a greater burden to the auditory pathway . the relationship between fatigue and other symptoms and signs of neurological deficit remains a disputable matter [ 1 , 2 ] . in our study , fatigue measures showed significant correlations with edss ( general degree of disability , although mostly determined by ambulation skills ) as well as with visual and brainstem fs scores . fatigue is a complex phenomenon , not limited to incapability of physical effort due to motor deficit , but also possibly associated with dysfunction of other systems . thus , vep and baep , as sensitive and objective markers of visual and brainstem pathways ( their parameters correlated significantly with corresponding fs scores ) , might provide measures of other aspects of disability contributing to fatigue . the asymmetry of ep abnormalities in our study seemed more specifically associated with fatigue than with ms itself . asymmetrical damage to cns pathways interferes with the perception and integration of stimuli of particular modality . to compensate for these dysfunctions , some additional areas of the brain this corresponds with the concept of fatigue as a result of excessive load of cns due to dysfunction of specific areas , as is supported by neuroimaging studies involving ms patients with fatigue [ 3 , 22 , 23 ] . to the best of our knowledge , so far there has been no report investigating visual and auditory ep with regards to fatigue in a large and well - defined group of ms patients . abnormalities of ep in fatigued patients , independent from ms - related variables , may support the hypothesis of disturbed bioelectrical activity due to cns damage as the background of fatigue , which contradicts the idea of its purely subjective origin . ep parameters seem promising as possible electrophysiological markers of fatigue with the asymmetry of their abnormalities deserving special attention . a limitation of our study is the fact that the assessment of fatigue and ep parameters was performed only once , without re - testing to check for reliability of the results . considering the common fluctuations of ms symptoms and the variability of ep parameters , we have already planned further study including parallel monitoring of fatigue and ep in the course of the disease to evaluate their relationships in prospective observation . in conclusion , the parameters of vep and baep undergo significant , mostly asymmetrical changes in ms patients with moderate and severe fatigue . these findings seem to support the hypothesis of neuronal pathways dysfunction as the background of ms fatigue . the role of ep parameters as electrophysiological markers of fatigue seems promising and deserves further investigation .

the aim of the study was to evaluate visual and brainstem auditory evoked potentials ( vep , baep ) in multiple sclerosis ( ms ) patients with regards to fatigue and disease - related variables . the study comprised 86 ms patients and 40 controls . fatigue was assessed using the fatigue severity scale ( fss / fss-5 ) and the modified fatigue impact scale ( mfis ) . latencies and amplitudes of the p100 component of vep and the i v components of baep were analyzed . the results of ep were compared between non - fatigued , moderately and severely fatigued ms patients and controls . p100 latency was increased and amplitude decreased in moderately and severely fatigued ms subjects . the latency of the v component of baep and interlatencies i - iii - v were increased in severely fatigued patients . the amplitude of the v component was lowered in fatigued patients . vep and baep abnormalities were usually one - sided . interocular p100 latency difference tended to correlate with fss / fss-5 . the parameters of vep and baep correlated with functional system scores but not with ms duration , overall degree of disability or its progression over time . significant , usually asymmetrical vep and baep abnormalities were found in fatigued ms patients , with no relationships to disease - related variables . ep may be considered an electrophysiological marker of fatigue in ms patients .

Introduction Materials and methods Assessment of fatigue Evoked potentials Statistical analysis Results Discussion

studies with the use of motor evoked potentials and electroencephalography event - related desynchronization have indeed shown decreased neuronal excitability and frequency - dependent conduction block in fatigued ms patients [ 58 ] . the aim of our study was to assess visual and brainstem auditory evoked potentials ( baep ) in ms patients with regard to the presence and severity of fatigue , considering also the impact of other disease - related variables . the study comprised 86 patients with ms ( 24 men and 62 women , aged 1960 years , mean 39.55 ) who were under the care of the outpatient ms clinic , department of neurology , medical university of wroclaw . none of the patients had concomitant diseases known to cause fatigue or to affect parameters of visual and auditory evoked potentials in their history . the patients underwent a neurological examination and their disability was assessed using the expanded disability status scale ( edss ) , with visual and brainstem functional systems ( fs ) scores separated for further analysis . on the basis of medical records , the duration of the disease was defined and the index of disability progression [ multiple sclerosis severity scale ( msss ) ] calculated ; the history of optic neuritis or loss of hearing was also determined . the level of fatigue in ms patients was evaluated using self - assessment questionnaires based on the fatigue severity scale ( fss ) and the modified fatigue impact scale ( mfis ) , with the results of fss re - evaluated using the rasch analysis applied by mills et al . visual evoked potentials ( vep ) were performed by using a black and white checker board pattern on a screen , with a checker size of 36 cm and the frequency of pattern reversing being 1.9 hz . for each subject , the latency and amplitude ( peak to peak ) of the p100 component were determined for each eye , as well as relative p100 latency ( interocular latency difference ) . latencies and amplitudes ( peak to peak ) were determined for components i , iii and v , as well as interpeak latencies i iii , iii v , i v and the proportion of amplitudes i / v . the ep parameters obtained from the whole group of ms patients and subgroups i , ii and iii were compared with those from the controls , and the results were also compared between subgroups i , ii and iii with the use of post hoc test ( scheffe test ) and then analysis of variance ( anova ) , alternatively using the kruskal wallis test , when the variances in groups were not homogeneous ( the homogeneity of variance was determined by the bartelett s test ) or if the number of cases was too small . the level of fatigue in ms patients was evaluated using self - assessment questionnaires based on the fatigue severity scale ( fss ) and the modified fatigue impact scale ( mfis ) , with the results of fss re - evaluated using the rasch analysis applied by mills et al . visual evoked potentials ( vep ) were performed by using a black and white checker board pattern on a screen , with a checker size of 36 cm and the frequency of pattern reversing being 1.9 hz . for each subject , the latency and amplitude ( peak to peak ) of the p100 component were determined for each eye , as well as relative p100 latency ( interocular latency difference ) . latencies and amplitudes ( peak to peak ) were determined for components i , iii and v , as well as interpeak latencies i iii , iii the ep parameters obtained from the whole group of ms patients and subgroups i , ii and iii were compared with those from the controls , and the results were also compared between subgroups i , ii and iii with the use of post hoc test ( scheffe test ) and then analysis of variance ( anova ) , alternatively using the kruskal wallis test , when the variances in groups were not homogeneous ( the homogeneity of variance was determined by the bartelett s test ) or if the number of cases was too small . on the basis of fss / fss-5 results , 29 patients ( 8 men , 21 women ) were allocated to subgroup i ( non - fatigued ) , 31 patients ( 7 men , 24 women ) to subgroup ii ( moderately fatigued ) and 26 ( 8 men , 18 women ) to subgroup iii ( severely fatigued ) . fss / fss-5 and mfis correlated significantly with edss , visual and brainstem fs ( table 1).table 1correlations between fatigue measures ( fss / fss-5 , mfis ) and degree of disability ( edss , visual fs , brainstem fs ) ; r spearman s correlation coefficientedssvisual fsbrainstem fsfss / fss-5 r = 0.48 r = 0.3 r = 0.26 p = 0.00001 p = 0.006 p = 0.018mfis r = 0.46 r = 0.32 r = 0.24 p = 0.0001 p = 0.003 p = 0.025 correlations between fatigue measures ( fss / fss-5 , mfis ) and degree of disability ( edss , visual fs , brainstem fs ) ; r spearman s correlation coefficient the mean duration of the disease was longer in subgroup iii in comparison with subgroup i ( 11.56 vs 5.38 years , p = 0.003 ) . the mean latency of the p100 component of vep for both eyes was significantly longer in ms patients than in the controls , and so was the mean relative p100 latency ( interocular latency difference ) . the mean p100 amplitude was significantly lower for ms patients than in controls , but only for the left eye ( table 2).table 2parameters of the p100 component of visual evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatiguedcontrols ( n = 40)ms patients ( n = 86)ms subgroup i ( n = 29)ms subgroup ii ( n = 31)ms subgroup iii ( n = 26)l : p100 latency ( ms ) mean101.8117.3 p ( ms - contr ) = 0.00001 113.9 p ( i - contr ) = 0.00001 118.3 p ( ii - contr ) = 0.00001 119.8 p ( iii - contr ) = 0.00001 sd5.021.614.916.1 p ( i ii ) = 0.2831.9 p ( i iii ) = 0.38 p ( ii iii ) = 0.82r : p100 latency ( ms ) mean102.2119.9 p ( ms - contr ) = 0.00001 113.8 p ( i - contr ) = 0.00001 121.1 p ( ii - contr ) = 0.00001 125.1 p ( iii - contr ) = 0.00001 sd4.715.812.914.6 p ( i ii ) = 0.02 18.4 p ( i iii ) = 0.006 p ( ii iii ) = 0.2relative p100 latency : mean2.149.95 p ( ms - contr ) = 0.00001 6.49 p p ( ii - contr ) = 0.00001 11.16 p ( iii - contr ) = 0.00001 sd1.9611.136.6313.13 p ( i ii ) = 0.1511.96 p ( i iii ) = 0.038 p ( ii iii ) = 0.89l : p100 amplitude ( v ) mean11.49.39 p ( ms - contr ) = 0.027 9.72 p ( i - contr ) = 0.159.34 p ( ii - contr ) = 0.0579.06 p ( iii - contr ) = 0.052 median4.54.925.254.63 p ( i ii ) = 0.495.08 p ( i iii ) = 0.65 sd p ( ii iii ) = 0.83r : p100 amplitude ( v ) mean10.68.97 p ( ms - contr ) = 0.0758.96 p ( i - contr ) = 0.159.25 p ( ii - contr ) = 0.228.63 p ( iii - contr ) = 0.097 sd4.45.058.437.92 p ( i ii ) = 0.835.12 p ( i iii ) = 0.012 5.075.13 p ( ii iii ) = 0.65relative p100 amplitude : mean0.971.02 p ( ms - contr ) = 0.471.07 p ( i - contr ) = 0.551.02 p ( ii - contr ) = 0.460.98 p ( iii - contr ) = 0.68 sd0.220.40.560.28 p ( i ii ) = 0.530.38 p ( i iii ) = 0.50 p ( ii iii ) = 0.36bold values are statistically significant at ( p < 0.05 ) l left eye , r right eye ) parameters of the p100 component of visual evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatigued bold values are statistically significant at ( p < 0.05 ) l left eye , r right eye ) p100 latency for the right eye was significantly longer in subgroups ii and iii than in subgroup i , and relative p100 latency was significantly longer in subgroup iii than in subgroup i ( table 2 ) . the amplitude of p100 for the left eye was lower in subgroups ii and iii than in the controls , and for the right eye lower in subgroup no significant correlations were found between the summated values of vep latency and amplitude and fss / fss-5 or mfis . the relative latency of p100 tended to correlate positively with fss / fss-5 ( r = 0.26 , p = 0.07 ) . no correlations were found between vep parameters and other clinical ms - related variables ( duration of the disease , edss or msss ) . the mean latencies of i , iii and v components of baep on both sides did not differ significantly between ms patients and controls . the mean amplitude of the v component was significantly lower in ms patients than in the controls , but only on the right side ( table 3).table 3parameters of brainstem auditory evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatiguedcontrols ( n = 40)ms patients ( n = 86)ms subgroup i ( n = 29)ms subgroup ii ( n = 31)ms subgroup iii ( n = 26 ) l : latency i mean1.691.68 p ( ms - contr ) = 0.671.66 p ( i - contr ) = 0.281.69 p ( ii - contr ) = 0.981.70 p ( iii - contr ) = 0.97 sd0.130.120.10.13 p ( i ii ) = 0.300.12 p ( i iii ) = 0.28 p ( ii iii ) = 0.99latency iii mean3.833.87 p ( ms - contr)=0.243.87 p ( i - contr ) = 0.263.860.16 p ( ii - contr ) = 0.143.91 p ( iii - contr ) = 0.06 sd0.120.170.17 p ( i ii ) = 0.840.20 p ( i iii ) = 0.43 p ( ii iii ) = 0.30latency v mean5.75.77 p ( ms - contr)=0.535.86 p ( i - contr ) = 0.235.78 p ( ii - contr ) = 0.925.65 p ( iii - contr ) = 0.049 sd0.210.730.440.24 p ( i ii ) = 0.831.23 p ( i iii ) = 0.39 p ( ii iii ) = 0.30interlat . i v mean4.04.15 p ( ms - contr ) = 0.013 4.2 p ( i - contr ) = 0.0574.09 p ( ii - contr ) = 0.0984.19 p ( iii - contr ) = 0.025 sd0.190.340.430.25 p ( i ii ) = 0.690.34 p ( i iii ) = 0.64 p ( ii iii ) = 0.36amp . interlatency , amp amplitude parameters of brainstem auditory evoked potentials in the controls , whole ms group and subgroups of ms patients : non - fatigued ( i ) , moderately ( ii ) and severely ( iii ) fatigued bold values are statistically significant at ( p < 0.05 ) l left ear , r right ear , interlat . interlatency , amp amplitude the latency of the v component of baep , and interlatency i v for the left ear as well as interlatencies iii v and i v for the right ear were significantly longer in subgroup iii than in the controls . in subgroup ii , the latency of the v component and interlatencies iii v , and i v for the right ear were significantly longer than in the controls ( table 3 ) . the amplitude of the v component for the right ear was significantly lower in subgroups ii and iii than in the controls ( table 3 ) . no correlations were found between the baep parameters ( one - sided or summated values ) and fatigue measures ( fss / fss-5 , mfis ) . summated latencies of baep components correlated significantly with brainstem fs ( i : r = 0.23 , p = 0.04 ; iii : r = 0.23 , p = 0.03 ; v : r = 0.36 , p = 0.0008 ) and so did summated amplitude of v component ( r = 0.23 , p = 0.03 ) . the importance of evoked potentials ( ep ) in the diagnosis of ms has decreased in the last decade as magnetic resonance has become the main diagnostic tool supporting clinical assessment . fatigue constitutes an important aspect of non - physical disability in ms patients , which is still lacking objective biomarkers , so analysis of ep parameters with regards to fatigue in ms seemed worth investigating . analysis of vep showed significantly prolonged latency of the p100 component in the whole group of ms patients as well as in each of the three subgroups , when compared to the controls . it is worth noting that only severely fatigued patients in comparison with non - fatigued ones showed a significantly increased interocular latency difference ( relative p100 latency ) . this parameter tended to correlate ( although not significantly ) with one of the fatigue measures ( fss / fss-5 ) , but apart from visual fs score did not show significant relationships with other disease - related variables ( duration of ms , edss or msss ) . significant differences in p100 amplitude were also asymmetrical but they were found not only between fatigued and non - fatigued subgroups but also between ms patients and controls . in those patients with ms and glaucoma ( but not in parkinsonic ones ) , the amplitude of p100 increased when additional stimuli were superimposed on the basic pattern of stimulation . the interlatencies of baep components indeed showed significant correlations with msss but not with any of the fatigue measures . moreover , the fatigued patients also showed prolonged latency of the v component of baep ( while non - fatigued ones and the whole ms group did not ) . to our knowledge , there have been no reports on baep with regards to fatigue in ms patients . thus , vep and baep , as sensitive and objective markers of visual and brainstem pathways ( their parameters correlated significantly with corresponding fs scores ) , might provide measures of other aspects of disability contributing to fatigue . to the best of our knowledge , so far there has been no report investigating visual and auditory ep with regards to fatigue in a large and well - defined group of ms patients . abnormalities of ep in fatigued patients , independent from ms - related variables , may support the hypothesis of disturbed bioelectrical activity due to cns damage as the background of fatigue , which contradicts the idea of its purely subjective origin . considering the common fluctuations of ms symptoms and the variability of ep parameters , we have already planned further study including parallel monitoring of fatigue and ep in the course of the disease to evaluate their relationships in prospective observation . in conclusion , the parameters of vep and baep undergo significant , mostly asymmetrical changes in ms patients with moderate and severe fatigue .

this was a 26 + 26-week , randomized , controlled , open - label , multinational , parallel - design , treat - to - target , noninferiority trial comparing the efficacy and safety of ideg and iglar , administered sc od in basal - bolus therapy , with insulin aspart ( iasp ) at mealtimes . the main study compared ideg in a forced - flexible regimen ( ideg forced - flex ) with ideg and iglar administered at the same time daily ( 3 treatment arms ) . an extension compared safety and efficacy of ideg given in a free - flexible regimen ( ideg free - flex ) with iglar ( 2 treatment arms ) . the trial was conducted in accordance with the declaration of helsinki ( 10 ) and good clinical practice guidelines ( 11 ) . the protocol and consent form were approved by local independent ethics committees or institutional review boards before trial initiation . adults 18 years old or older with t1 dm on basal - bolus therapy , with hba1c 10.0% or less and body mass index 35.0 kg / m or less participated . basal insulin allowed at screening included iglar , insulin detemir , or nph insulin ( as 1 or 2 daily injections ) and 3 or more daily injections of bolus insulin ( iasp , insulin lispro , insulin glulisine , or human insulin ) ; see supplemental data for inclusion / exclusion criteria . eligible participants were randomized 1:1:1 , using a central interactive voice / web response system , to receive ideg ( 100 u / ml , 3 ml flexpen ; novo nordisk , bagsvaerd , denmark ) ( given in either the forced - flex regimen or at the same time daily ) or iglar ( lantus , 100 u / ml , 3 ml solostar ; sanofi , paris , france ) , in combination with mealtime iasp ( novorapid / novolog , 100 u / ml , 3 ml flexpen ; novo nordisk ) . trial - product masking was maintained for titration surveillance monitors and statistical and medical personnel until data were locked for analysis . ongoing masked - data safety surveillance was performed by an internal novo nordisk safety committee and an independent , external committee adjudicated cardiovascular events in accordance with predefined classifications . eligible participants were switched to od ideg or iglar with mealtime iasp at randomization ( week 0 ) . if previous basal insulin was dosed od , initial doses were transferred 1:1 for ideg and iglar . if previous basal insulin was dosed more than od , total daily basal dose was calculated and transferred 1:1 for ideg , with dose reduction considered by the investigator 's judgment . for transfer to iglar , a 20%30% dose reduction was recommended , based on current prescribing information ( 12 ) . participants switched pretrial bolus insulin to iasp 1:1 at the pretrial dose ( supplemental data ) . at the end of the main and extension periods , basal insulin was switched to nph insulin for 7 days to minimize interference with antibody detection at a follow - up visit performed 1 week later . the trial was approximately 56 weeks including one screening week , two 26-week treatment periods , and two 7- to 12-day follow - up wash - out periods ( supplemental figure 1 ) . the main period compared ideg forced - flex [ ideg administered on monday , wednesday , and friday mornings and on tuesday , thursday , saturday , and sunday evenings ; ie , at fixed intervals with a minimum of 8 and a maximum of 40 hours between injections ( supplemental data ) ] with ideg ( given od with the evening meal ) and with iglar ( given od at the same time daily ) . all participants randomized to ideg in the main study were offered participation in the extension with instructions to take ideg od at any time of day ( ideg free - flex ) , provided they maintained a minimum of 8 and a maximum of 40 hours between doses . patients randomized to iglar in the main period who entered the extension continued the same - time - daily iglar . basal insulin doses in all groups were to be self - adjusted on mondays , wednesdays , and fridays based on mean prebreakfast self - measured plasma glucose ( smpg ) values of the preceding 23 days and titrated to a prebreakfast smpg target of 4.05.0 mmol / l ( titration algorithm details in supplemental data ) . glucose measurements were performed with drawn capillary blood automatically calibrated to plasma - equivalent glucose values . safety variables included adverse events ( aes ) , hypoglycemic episodes , insulin dose , body weight , antibodies , and standard laboratory and clinical safety assessments . confirmed hypoglycemia was defined as blood glucose measurements of less than 3.1 mmol / l or severe episodes requiring assistance ( 13 ) . hypoglycemic episodes occurring between 0001 and 0559 hours ( inclusive ) were classified as nocturnal . laboratory analyses were conducted at central laboratories ( quintiles laboratories europe , west lothian , united kingdom , and quintiles laboratories limited , marietta , georgia ) . antibody analyses were performed at celerion switzerland ag ( fehraltorf , switzerland ) , using a validated ria method ( 14 , 15 ) . the primary objective was to confirm the noninferiority of ideg forced - flex to iglar in hba1c change from baseline to week 26 . noninferiority was confirmed if the upper limit of the 2-sided 95% confidence interval ( ci ) for the treatment difference for mean change in hba1c was 0.4% or less , as recommended by regulatory guidelines ( 16 ) . sample size was determined on the basis of the primary objective under the assumption of a 1-sided t test of size 2.5% , a zero mean treatment difference , and standard deviation of 1.1% for hba1c . a total of 486 participants had to be randomized for 85% or greater power in the evaluation of the per - protocol ( pp ) analysis set to demonstrate noninferiority at 0.4% , after adjustment for a 15% dropout rate . the extension investigated long - term safety and efficacy of ideg ( given during the extension in a free - flex dosing regimen ) vs iglar . statistical analyses of efficacy end points , hypoglycemia , and body weight included all randomized participants [ full analysis set ( fas ) ] , following the intention - to treat principle . all other safety end points were evaluated in participants exposed to treatment ( safety analysis set ) . comparisons between ideg forced - flex and iglar and between ideg forced - flex and ideg were made after 26 treatment weeks . baseline characteristics , demographics , aes , and hypoglycemic episodes were presented using descriptive statistics . treatment differences in hba1c , fpg , smpg , and body weight after 26 and 52 weeks were estimated by anova with treatment , insulin therapy at screening , sex , and region as fixed factors and age and relevant baseline value as covariates . the robustness of results for change in hba1c was explored by additional analyses including analysis on the pp set . estimated rate ratios ( errs ) of hypoglycemic episodes were made using a negative binomial regression model including the same fixed factors with age as covariate and using the log of exposure time as offset , using all reported treatment - emergent episodes in randomized subjects . a mixed - effect model was fitted to 9-point smpg profile data with treatment , time , interaction between treatment and time , insulin therapy at screening , sex , and region as fixed factors ; age as covariate ; and subject as random effect . analyses were repeated for hba1c , treatment - emergent hypoglycemic episodes , antibodies , and central laboratory parameters at 52 weeks using the extension trial set ( ets ; subjects who attended the first visit of the extension trial ) to assess stability of key results . data were reported using a 95% ci and p values for 2-sided testing at = .05 . this was a 26 + 26-week , randomized , controlled , open - label , multinational , parallel - design , treat - to - target , noninferiority trial comparing the efficacy and safety of ideg and iglar , administered sc od in basal - bolus therapy , with insulin aspart ( iasp ) at mealtimes . the main study compared ideg in a forced - flexible regimen ( ideg forced - flex ) with ideg and iglar administered at the same time daily ( 3 treatment arms ) . an extension compared safety and efficacy of ideg given in a free - flexible regimen ( ideg free - flex ) with iglar ( 2 treatment arms ) . the trial was conducted in accordance with the declaration of helsinki ( 10 ) and good clinical practice guidelines ( 11 ) . the protocol and consent form were approved by local independent ethics committees or institutional review boards before trial initiation . adults 18 years old or older with t1 dm on basal - bolus therapy , with hba1c 10.0% or less and body mass index 35.0 kg / m or less participated . basal insulin allowed at screening included iglar , insulin detemir , or nph insulin ( as 1 or 2 daily injections ) and 3 or more daily injections of bolus insulin ( iasp , insulin lispro , insulin glulisine , or human insulin ) ; see supplemental data for inclusion / exclusion criteria . eligible participants were randomized 1:1:1 , using a central interactive voice / web response system , to receive ideg ( 100 u / ml , 3 ml flexpen ; novo nordisk , bagsvaerd , denmark ) ( given in either the forced - flex regimen or at the same time daily ) or iglar ( lantus , 100 u / ml , 3 ml solostar ; sanofi , paris , france ) , in combination with mealtime iasp ( novorapid / novolog , 100 u / ml , 3 ml flexpen ; novo nordisk ) . trial - product masking was maintained for titration surveillance monitors and statistical and medical personnel until data were locked for analysis . ongoing masked - data safety surveillance was performed by an internal novo nordisk safety committee and an independent , external committee adjudicated cardiovascular events in accordance with predefined classifications . eligible participants were switched to od ideg or iglar with mealtime iasp at randomization ( week 0 ) . if previous basal insulin was dosed od , initial doses were transferred 1:1 for ideg and iglar . if previous basal insulin was dosed more than od , total daily basal dose was calculated and transferred 1:1 for ideg , with dose reduction considered by the investigator 's judgment . for transfer to iglar , a 20%30% dose reduction was recommended , based on current prescribing information ( 12 ) . participants switched pretrial bolus insulin to iasp 1:1 at the pretrial dose ( supplemental data ) . at the end of the main and extension periods , basal insulin was switched to nph insulin for 7 days to minimize interference with antibody detection at a follow - up visit performed 1 week later . the trial was approximately 56 weeks including one screening week , two 26-week treatment periods , and two 7- to 12-day follow - up wash - out periods ( supplemental figure 1 ) . the main period compared ideg forced - flex [ ideg administered on monday , wednesday , and friday mornings and on tuesday , thursday , saturday , and sunday evenings ; ie , at fixed intervals with a minimum of 8 and a maximum of 40 hours between injections ( supplemental data ) ] with ideg ( given od with the evening meal ) and with iglar ( given od at the same time daily ) . all participants randomized to ideg in the main study were offered participation in the extension with instructions to take ideg od at any time of day ( ideg free - flex ) , provided they maintained a minimum of 8 and a maximum of 40 hours between doses . patients randomized to iglar in the main period who entered the extension continued the same - time - daily iglar . basal insulin doses in all groups were to be self - adjusted on mondays , wednesdays , and fridays based on mean prebreakfast self - measured plasma glucose ( smpg ) values of the preceding 23 days and titrated to a prebreakfast smpg target of 4.05.0 mmol / l ( titration algorithm details in supplemental data ) . glucose measurements were performed with drawn capillary blood automatically calibrated to plasma - equivalent glucose values . safety variables included adverse events ( aes ) , hypoglycemic episodes , insulin dose , body weight , antibodies , and standard laboratory and clinical safety assessments . confirmed hypoglycemia was defined as blood glucose measurements of less than 3.1 mmol / l or severe episodes requiring assistance ( 13 ) . hypoglycemic episodes occurring between 0001 and 0559 hours ( inclusive ) were classified as nocturnal . laboratory analyses were conducted at central laboratories ( quintiles laboratories europe , west lothian , united kingdom , and quintiles laboratories limited , marietta , georgia ) . antibody analyses were performed at celerion switzerland ag ( fehraltorf , switzerland ) , using a validated ria method ( 14 , 15 ) . the primary objective was to confirm the noninferiority of ideg forced - flex to iglar in hba1c change from baseline to week 26 . noninferiority was confirmed if the upper limit of the 2-sided 95% confidence interval ( ci ) for the treatment difference for mean change in hba1c was 0.4% or less , as recommended by regulatory guidelines ( 16 ) . sample size was determined on the basis of the primary objective under the assumption of a 1-sided t test of size 2.5% , a zero mean treatment difference , and standard deviation of 1.1% for hba1c . a total of 486 participants had to be randomized for 85% or greater power in the evaluation of the per - protocol ( pp ) analysis set to demonstrate noninferiority at 0.4% , after adjustment for a 15% dropout rate . the extension investigated long - term safety and efficacy of ideg ( given during the extension in a free - flex dosing regimen ) vs iglar . statistical analyses of efficacy end points , hypoglycemia , and body weight included all randomized participants [ full analysis set ( fas ) ] , following the intention - to treat principle . all other safety end points were evaluated in participants exposed to treatment ( safety analysis set ) . comparisons between ideg forced - flex and iglar and between ideg forced - flex and ideg were made after 26 treatment weeks . baseline characteristics , demographics , aes , and hypoglycemic episodes were presented using descriptive statistics . treatment differences in hba1c , fpg , smpg , and body weight after 26 and 52 weeks were estimated by anova with treatment , insulin therapy at screening , sex , and region as fixed factors and age and relevant baseline value as covariates . the robustness of results for change in hba1c was explored by additional analyses including analysis on the pp set . estimated rate ratios ( errs ) of hypoglycemic episodes were made using a negative binomial regression model including the same fixed factors with age as covariate and using the log of exposure time as offset , using all reported treatment - emergent episodes in randomized subjects . a mixed - effect model was fitted to 9-point smpg profile data with treatment , time , interaction between treatment and time , insulin therapy at screening , sex , and region as fixed factors ; age as covariate ; and subject as random effect . analyses were repeated for hba1c , treatment - emergent hypoglycemic episodes , antibodies , and central laboratory parameters at 52 weeks using the extension trial set ( ets ; subjects who attended the first visit of the extension trial ) to assess stability of key results . data were reported using a 95% ci and p values for 2-sided testing at = .05 . of the 493 randomized participants , 490 ( 99.4% ) received trial drug , and most [ 84.1% ( 138 of 164 ) ideg forced - flex , 84.2% ( 139 of 165 ) ideg , and 92.7% ( 152 of 164 ) iglar ] completed the main trial . the percentage of participants withdrawn during the main trial from the ideg forced - flex ( 15.9% ) and ideg ( 15.8% ) groups was higher than from the iglar group ( 7.3% ) ; see supplemental figure 2 and supplemental table 1 for details . more withdrawal - related aes , including hypoglycemic episodes , occurred among ideg - treated subjects , although the numbers overall were low . slightly more ideg - treated subjects met withdrawal criteria as specified in the protocol or were withdrawn for reasons classified as other . of 277 ideg - treated main trial completers , 239 entered the extension into the ideg free - flex arm and 67.8% ( 223 of 329 ) of those randomized in the main trial completed the extension . of 152 iglar - treated main trial completers , 133 entered the extension and 74.4% ( 122 of 164 ) of those randomized in the main trial completed the extension . the pattern and the percentage of participants withdrawn during the extension were similar with ideg free - flex ( 4.9% ) and iglar ( 6.7% ) . baseline characteristics were representative of a t1 dm population with moderate glycemic control ( mean hba1c 7.7% , table 1 ) . the pretrial insulin regimen of most participants [ 70.6% ( 348 of 493 ) ] comprised once - daily basal injection with 3 or more bolus doses daily . iglar and iasp were used by 63.7% ( 314 of 493 ) and 50.9% ( 251 of 493 ) of participants , respectively , before entering the trial . the following proportions of subjects took basal insulin od and more than od , respectively , at screening : ideg forced - flex , 68.3% ( 112 of 164 ) and 31.7% ( 52 of 164 ) , ideg , 70.9% ( 117 of 165 ) and 29.1% ( 48 of 165 ) , and iglar , 72.6% ( 119 of 164 ) and 27.4% ( 45 of 164 ) . demographics and baseline characteristics : full analysis set abbreviations : bid , twice daily ; tid , 3 times daily . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . one subject was randomized to iglar , although her antidiabetic treatment regimen at screening was basal od plus premix insulin tid . one subject was randomized to ideg forced - flex , although his antidiabetic treatment regimen at screening was bid premix insulin ; he was not withdrawn because this deviation was discovered late in the trial . consistent with the treat - to - target methodology , the observed mean decrease in hba1c from baseline to week 26 was similar among ideg forced - flex ( 0.40% points ) , ideg ( 0.41% points ) , and iglar ( 0.58% points ; figure 1a ) groups . the primary objective of the trial was met because ideg forced - flex was shown to be noninferior to iglar in reducing hba1c [ estimated treatment difference ( etd ) ( idegforced - flex - iglar ) : 0.17% points ( 0.04 ; 0.30)95%ci ] . the robustness of the primary analysis was further supported by pp and additional sensitivity analyses ( supplemental table 2 ) . there was no difference in observed mean decrease in hba1c from baseline to week 26 between ideg forced - flex ( 0.40% points ) and ideg ( 0.41% points ) ; etd [ idegforced - flex - ideg ] : 0.01% points [ 0.13 ; 0.14]95%ci . glycemic efficacy : mean hba1c sem over time ( a ) and mean fpg sem over time ( b ) last observation carried forward is used for each postbaseline time point in a and b. subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . laboratory - measured fpg decreased from baseline to week 26 ( ideg forced - flex , 1.28 ; ideg , 2.54 ; iglar , 1.33 mmol / l ) , with the most pronounced decline occurring during the first 12 weeks ( figure 1b ) . no significant difference was seen with ideg forced - flex vs iglar ( supplemental table 2 ) , but a greater reduction was seen with ideg than with ideg forced - flex [ etd ( idegforced - flex - ideg ) : 0.95 mmol / l ( 0.15 ; 1.75)95%ci , p = .021 ] . after 26 weeks , observed 9-point smpg means appeared similar among groups . despite varying dosing times with ideg forced - flex , the only difference in smpg vs iglar was before lunch [ etd ( idegforced - flex - iglar ) : 0.85 mmol / l ( 0.12 ; 1.57)95%ci , p = .022 ] . the proportion of participants who attained prebreakfast smpg target less than 5.0 mmol / l at week 26 was 11.3% ( ideg forced - flex ) , 23.8% ( ideg ) , and 18.4% ( iglar ) . the median time to achieving the prebreakfast titration target for the first time was 7 weeks ( ideg forced - flex ) , 4 weeks ( ideg ) , and 6 weeks ( iglar ) . although mean hba1c values increased slightly from 26-week levels during the extension , they remained below baseline at week 52 ; 0.13% points from baseline to 7.6% ( ideg free - flex ) and 0.21% points from baseline to 7.5% ( iglar ) . ideg free - flex was not significantly different from iglar in lowering hba1c in the fas or ets at week 52 , with an upper 95% ci limit below a predefined noninferiority mark of 0.4% ( supplemental table 2 ) . mean fpg decreased from baseline to week 52 with ideg free - flex ( 1.73 mmol / l ) and iglar ( 0.61 mmol / l ) , with significantly greater reduction observed with ideg free- flex [ etd ( idegfree - flex - iglar ) : 1.07 mmol / l ( 1.82 ; 0.32)95%ci , p = .005 ] . mean 9-point smpg profiles decreased from baseline to week 52 in both groups . at week 52 , smpg profiles were similar at all time points except at 90 minutes after the main evening meal , when it was significantly lower with ideg free - flex [ etd ( idegfree - flex - iglar ) : 0.82 mmol / l ( 1.53 ; 0.12)95%ci , p = .022 ] . the proportion of participants who attained prebreakfast smpg < 5.0 mmol / l at week 52 was 17.9% ( ideg free - flex ) and 13.8% ( iglar ) . mean basal doses increased slightly during weeks 026 with ideg forced - flex and iglar . however , basal doses remained stable with ideg during weeks 026 and in the ideg free - flex and iglar groups from week 27 to week 52 . mean daily iasp doses increased slightly during the main period in the iglar group . at week 52 , mean daily basal , bolus , and total insulin doses were lower by 4% , 18% , and 11% , respectively , with ideg free - flex vs iglar . the greater mean total daily insulin dose with iglar vs ideg free - flex was due mainly to a decrease in mean daily bolus insulin dose with ideg free - flex ( 0.39 to 0.35 u / kg ) and an increase with iglar ( 0.40 to 0.42 u / kg ; table 2 ) . mean daily insulin dose ( sd ) : safety analysis set abbreviation : n / a , not applicable . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . the data for ideg forced - flex and ideg arms from the first 26 weeks were pooled into the ideg free - flex arm in this table . mean ratio is the unadjusted ratio between mean doses at last treatment visit , in which missing data are imputed using last observation carried forward . overall , confirmed and severe hypoglycemia rates were similar across groups at week 26 ( figure 2 and supplemental table 3 ) . the nocturnal confirmed hypoglycemia rate was significantly lower with ideg forced - flex than iglar [ by 40% ; err ( idegforced - flex / iglar ) : 0.60 ( 0.44 ; 0.82)95%ci , p = .001 ] and ideg [ by 37% ; err ( idegforced - flex / ideg ) : 0.63 ( 0.46 ; 0.86)95%ci , p = .003 ] . nocturnal confirmed hypoglycemia rates were generally lower with ideg forced - flex than with iglar and ideg , regardless of the day of the week ( supplemental table 4 ) . no significant difference in overall confirmed hypoglycemia was seen at week 52 between ideg free - flex and iglar in the fas or ets . although the number of events was small , the severe hypoglycemia rate was numerically lower in the fas and significantly lower by 53% [ err ( idegfree - flex / iglar ) : 0.47 ( 0.23 ; 0.94)95%ci , p = .033 ] in the ets with ideg free - flex . nocturnal confirmed hypoglycemia rates were significantly lower with ideg free - flex by 25% [ err ( idegfree - flex / iglar ) : 0.75 ( 0.58 ; 0.97)95%ci , p = .026 ] in the fas and by 27% [ err ( idegfree - flex/ iglar ) : 0.73 ( 0.54 ; 0.98)95%ci , p = .035 ] in the ets . hypoglycemia over time , weeks 052 : severe hypoglycemia ( a ) ; overall confirmed hypoglycemia ( b ) ; and nocturnal confirmed hypoglycemia ( c ) . percentage risk reductions refer to delta risks based on estimated rate ratios ( fas ) . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . rates for aes were 443 ( ideg forced - flex ) , 550 ( ideg ) , and 527 ( iglar ) per 100 patient - years of exposure ( pye ) from week 0 to week 26 and 447 ( ideg free - flex ) and 481 ( iglar ) per 100 pye from week 0 to week 52 . most aes were mild or moderate and considered unrelated to basal insulin , with no treatment - specific patterns observed ( supplemental tables 5 and 6 ) . injection - site reaction rates were low over 52 weeks , with 6 ( ideg free - flex ) and 4 ( iglar ) events per 100 pye ; no reactions were classified as serious . mean weight gain from baseline to week 26 and during the extension was modest , with no between - group differences at week 52 ( ideg free - flex , 1.3 kg ; iglar , 1.9 kg ; supplemental table 2 ) . serious aes ( saes ) were reported by 5.5% ( ideg forced - flex ) , 4.2% ( ideg ) , and 5.0% ( iglar ) of participants from week 0 to week 26 and by 7.6% ( ideg free - flex ) and 7.5% ( iglar ) of participants in total from week 0 to week 52 ( supplemental table 5 ) . of randomized subjects who did not enter the extension , 10.0% ( 5 of 50 ) ideg forced - flex , 2.5% ( 1 of 40 ) ideg , and 6.5% ( 2 of 31 ) iglar - treated subjects reported saes during the main period . of randomized subjects who entered the extension , 3.5% ( 4 of 114 ) ideg forced - flex , 4.8% ( 6 of 125 ) ideg , and 4.5% ( 6 of 133 ) iglar - treated subjects reported saes during the main period . saes were distributed similarly among groups ; few were considered related to the trial product and the most frequently reported saes were related to hypoglycemia ( supplemental table 7 ) . one death , suicide by assumed - intentional insulin overdose , occurred in an ideg - treated female participant 158 days after starting the trial drug . concentrations of ideg - specific antibodies and antibodies cross - reacting between ideg and human insulin were low during the main and extension periods ( supplemental table 8) , with no apparent association between the development of antibodies and hba1c or insulin dose ( data not shown ) . no clinically relevant treatment - related differences were noted in physical examination findings , vital signs , electrocardiograms , funduscopy , or laboratory measurements . intensive insulin therapy in clinical trials is associated with lower rates of diabetes complications versus nonintensive therapy in people with t1 dm and is presently considered the standard of care ( 13 ) . basal - bolus regimens with insulins designed to closely mimic a physiologic profile allow many patients to reach glycemic targets . however , issues related to variability of action ( leading to unexpected hypoglycemia ) , and the need to adapt one 's lifestyle to the action profile of a prescribed insulin , can prevent the achievement of good glycemic control in a safe manner . ideg 's duration of action exceeds 42 hours , providing full 24-hour basal insulin coverage and offering a consistent glucose - lowering effect , thus potentially allowing administration within a broader dosing window ( 7 , 8) . in the present study , the aim was to evaluate whether ideg dosed in a flexible regimen would be safe and effective in patients with t1 dm . the prebreakfast titration target 4.05.0 mmol / l was narrower than targets described in some other intensive t1 dm basal - bolus trials ( 17 ) ; however , given that the pharmacokinetic profile of ideg shows potential to lower hypoglycemic risk compared with current therapies ( 8 , 18 ) and other recent trials in patients with t2 dm and t1 dm have investigated comparably low titration targets ( 1923 ) , an ambitious target was considered appropriate and the ultimate decision regarding insulin dose was the investigator 's . this was a novel trial both for investigators and participants because it challenged deeply ingrained habits by asking people with long - standing t1 dm to inject basal insulin at varying daily time points while simultaneously aiming for the ambitious fpg targets considered attainable given ideg 's pharmacokinetic profile . over 26 weeks , the forced - flex regimen proved effective ( noninferior ) compared with iglar and ideg and , even more importantly , proved safe . flexibly dosed ideg demonstrated lower rates of nocturnal hypoglycemia than iglar , reaffirming findings of previous phase 2 and 3 trials ( 9 , 24 ) . however , this trial was unique in that it captured daily 4-point smpg , which may partially explain higher overall hypoglycemia rates observed in this study vs a previous trial that compared od ideg and iglar in patients with t1 dm ( 9 ) . analysis between ideg and iglar od at 26 weeks was not a prespecified end point within this trial , and thus , no statistical analysis was performed , although observed efficacy and safety data are presented for each of the 3 treatment arms through week 26 . this comparison has been evaluated in other studies in patients with t1 dm ( 9 ) and t2 dm ( 25 ) . full - year results confirmed findings from the 26-week main trial , with the extension allowing all ideg subjects the option to dose more flexibly than at same time daily . this is important because such an option would positively impact patients ' abilities to lead the type of modern , hectic lives typical of people with t1 dm , including travel , dining out , changing bedtimes , etc , without compromising glucose control or safety . limitations of this study include a smaller population size compared with previous ideg studies ; the relatively short time frame of the forced - flex regimen ; and different recommendations for transferring subjects on more - than - od basal insulin to ideg and iglar at the study 's start ( total basal dose was transferred 1:1 for ideg , as is standard for switching long acting insulins , but reduced 20%30% for iglar ) . more ideg- than iglar - treated participants withdrew from the trial during the main period ; the rates of withdrawal could be related to the demanding nature of the study ( particularly in the forced - flex arm ) , including frequent blood glucose measurement requirements and study visits . however , reasons for withdrawal among the 3 treatment arms were sufficiently varied that no clear explanation for the higher rate in the ideg arms could be identified . data were not collected on dosing times during the extension ; therefore , it is unknown how many ideg free - flex participants varied dosing times . this was an open - label trial , and investigators may have been more alert in their management and documentation of the ideg forced - flex arm than the ideg or iglar arms ; caution on the part of investigators and subjects may partially explain some differences ( such as in fpg and nocturnal confirmed hypoglycemia ) in study results between ideg forced - flex and ideg . additionally , the 2 insulins tested used different delivery devices and unavailability of placebo - filled devices precluded double - dummy masking ( although such a design may be considered unethical in patients with t1 dm due to the 8 or more daily injections required ) . the authors do not advocate stretching insulin - dose timing to these limits in clinical practice , but this study has important implications for patients with t1 dm and may herald a departure from restrictive dosing regimens . this trial demonstrates that ideg 's ultralong duration of action allows variation of daily administration times without compromising efficacy or safety in patients with t1 dm who require multiple daily injections . it offers patients and clinicians insight into using this new basal insulin to assist patients in reaching glycemic targets despite ever - changing daily lifestyles .

objective : this study investigated the efficacy and safety of insulin degludec ( ideg ) once daily ( od ) , varying injection timing day to day in subjects with type 1 diabetes.research design and methods : this 26-week , open - label , treat - to - target , noninferiority trial compared ideg forced flexible ( forced - flex ) od ( given in a fixed schedule with a minimum 8 and maximum 40 hours between doses ) with ideg or insulin glargine ( iglar ) given at the same time daily od . in the 26-week extension , all ideg subjects were transferred to a free - flexible ( free - flex ) regimen , which allowed any - time - of - day dosing , and compared with subjects continued on iglar.results:after 26 treatment weeks , mean glycosylated hemoglobin was reduced with ideg forced- flex ( 0.40% ) , ideg ( 0.41% ) , and iglar ( 0.58% ) . ideg forced - flex noninferiority was achieved . fasting plasma glucose reductions were similar with ideg forced - flex and iglar but greater with ideg ( 2.54 mmol / l ) than ideg forced - flex ( 1.28 mmol / l ) ( p = .021 ) . at week 52 , ideg free - flex subjects had similar glycosylated hemoglobin but greater fasting plasma glucose reductions than iglar subjects ( 1.07 mmol / l ) ( p = .005 ) . confirmed hypoglycemia rates ( plasma glucose < 3.1 mmol / l or severe hypoglycemia ) were similar at weeks 26 and 52 . nocturnal confirmed hypoglycemia was lower with ideg forced - flex vs ideg ( 37% ; p = .003 ) and iglar ( 40% ; p = .001 ) at week 26 and 25% lower with ideg free - flex vs iglar ( p = .026 ) at week 52.conclusions:ideg can be administered od at any time of day , with injection timing varied without compromising glycemic control or safety vs same - time - daily ideg or iglar . this may improve basal insulin adherence by allowing injection - time adjustment according to individual needs .

Research Design and Methods Study design and participants Randomization and masking Procedures Statistical analyses Results Discussion Supplementary Material

this was a 26 + 26-week , randomized , controlled , open - label , multinational , parallel - design , treat - to - target , noninferiority trial comparing the efficacy and safety of ideg and iglar , administered sc od in basal - bolus therapy , with insulin aspart ( iasp ) at mealtimes . the main study compared ideg in a forced - flexible regimen ( ideg forced - flex ) with ideg and iglar administered at the same time daily ( 3 treatment arms ) . an extension compared safety and efficacy of ideg given in a free - flexible regimen ( ideg free - flex ) with iglar ( 2 treatment arms ) . eligible participants were randomized 1:1:1 , using a central interactive voice / web response system , to receive ideg ( 100 u / ml , 3 ml flexpen ; novo nordisk , bagsvaerd , denmark ) ( given in either the forced - flex regimen or at the same time daily ) or iglar ( lantus , 100 u / ml , 3 ml solostar ; sanofi , paris , france ) , in combination with mealtime iasp ( novorapid / novolog , 100 u / ml , 3 ml flexpen ; novo nordisk ) . the main period compared ideg forced - flex [ ideg administered on monday , wednesday , and friday mornings and on tuesday , thursday , saturday , and sunday evenings ; ie , at fixed intervals with a minimum of 8 and a maximum of 40 hours between injections ( supplemental data ) ] with ideg ( given od with the evening meal ) and with iglar ( given od at the same time daily ) . all participants randomized to ideg in the main study were offered participation in the extension with instructions to take ideg od at any time of day ( ideg free - flex ) , provided they maintained a minimum of 8 and a maximum of 40 hours between doses . patients randomized to iglar in the main period who entered the extension continued the same - time - daily iglar . basal insulin doses in all groups were to be self - adjusted on mondays , wednesdays , and fridays based on mean prebreakfast self - measured plasma glucose ( smpg ) values of the preceding 23 days and titrated to a prebreakfast smpg target of 4.05.0 mmol / l ( titration algorithm details in supplemental data ) . confirmed hypoglycemia was defined as blood glucose measurements of less than 3.1 mmol / l or severe episodes requiring assistance ( 13 ) . the primary objective was to confirm the noninferiority of ideg forced - flex to iglar in hba1c change from baseline to week 26 . the extension investigated long - term safety and efficacy of ideg ( given during the extension in a free - flex dosing regimen ) vs iglar . comparisons between ideg forced - flex and iglar and between ideg forced - flex and ideg were made after 26 treatment weeks . this was a 26 + 26-week , randomized , controlled , open - label , multinational , parallel - design , treat - to - target , noninferiority trial comparing the efficacy and safety of ideg and iglar , administered sc od in basal - bolus therapy , with insulin aspart ( iasp ) at mealtimes . the main study compared ideg in a forced - flexible regimen ( ideg forced - flex ) with ideg and iglar administered at the same time daily ( 3 treatment arms ) . an extension compared safety and efficacy of ideg given in a free - flexible regimen ( ideg free - flex ) with iglar ( 2 treatment arms ) . eligible participants were randomized 1:1:1 , using a central interactive voice / web response system , to receive ideg ( 100 u / ml , 3 ml flexpen ; novo nordisk , bagsvaerd , denmark ) ( given in either the forced - flex regimen or at the same time daily ) or iglar ( lantus , 100 u / ml , 3 ml solostar ; sanofi , paris , france ) , in combination with mealtime iasp ( novorapid / novolog , 100 u / ml , 3 ml flexpen ; novo nordisk ) . the main period compared ideg forced - flex [ ideg administered on monday , wednesday , and friday mornings and on tuesday , thursday , saturday , and sunday evenings ; ie , at fixed intervals with a minimum of 8 and a maximum of 40 hours between injections ( supplemental data ) ] with ideg ( given od with the evening meal ) and with iglar ( given od at the same time daily ) . all participants randomized to ideg in the main study were offered participation in the extension with instructions to take ideg od at any time of day ( ideg free - flex ) , provided they maintained a minimum of 8 and a maximum of 40 hours between doses . patients randomized to iglar in the main period who entered the extension continued the same - time - daily iglar . basal insulin doses in all groups were to be self - adjusted on mondays , wednesdays , and fridays based on mean prebreakfast self - measured plasma glucose ( smpg ) values of the preceding 23 days and titrated to a prebreakfast smpg target of 4.05.0 mmol / l ( titration algorithm details in supplemental data ) . confirmed hypoglycemia was defined as blood glucose measurements of less than 3.1 mmol / l or severe episodes requiring assistance ( 13 ) . the primary objective was to confirm the noninferiority of ideg forced - flex to iglar in hba1c change from baseline to week 26 . the extension investigated long - term safety and efficacy of ideg ( given during the extension in a free - flex dosing regimen ) vs iglar . comparisons between ideg forced - flex and iglar and between ideg forced - flex and ideg were made after 26 treatment weeks . of the 493 randomized participants , 490 ( 99.4% ) received trial drug , and most [ 84.1% ( 138 of 164 ) ideg forced - flex , 84.2% ( 139 of 165 ) ideg , and 92.7% ( 152 of 164 ) iglar ] completed the main trial . the percentage of participants withdrawn during the main trial from the ideg forced - flex ( 15.9% ) and ideg ( 15.8% ) groups was higher than from the iglar group ( 7.3% ) ; see supplemental figure 2 and supplemental table 1 for details . of 277 ideg - treated main trial completers , 239 entered the extension into the ideg free - flex arm and 67.8% ( 223 of 329 ) of those randomized in the main trial completed the extension . the pattern and the percentage of participants withdrawn during the extension were similar with ideg free - flex ( 4.9% ) and iglar ( 6.7% ) . the following proportions of subjects took basal insulin od and more than od , respectively , at screening : ideg forced - flex , 68.3% ( 112 of 164 ) and 31.7% ( 52 of 164 ) , ideg , 70.9% ( 117 of 165 ) and 29.1% ( 48 of 165 ) , and iglar , 72.6% ( 119 of 164 ) and 27.4% ( 45 of 164 ) . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . one subject was randomized to ideg forced - flex , although his antidiabetic treatment regimen at screening was bid premix insulin ; he was not withdrawn because this deviation was discovered late in the trial . consistent with the treat - to - target methodology , the observed mean decrease in hba1c from baseline to week 26 was similar among ideg forced - flex ( 0.40% points ) , ideg ( 0.41% points ) , and iglar ( 0.58% points ; figure 1a ) groups . the primary objective of the trial was met because ideg forced - flex was shown to be noninferior to iglar in reducing hba1c [ estimated treatment difference ( etd ) ( idegforced - flex - iglar ) : 0.17% points ( 0.04 ; 0.30)95%ci ] . there was no difference in observed mean decrease in hba1c from baseline to week 26 between ideg forced - flex ( 0.40% points ) and ideg ( 0.41% points ) ; etd [ idegforced - flex - ideg ] : 0.01% points [ 0.13 ; 0.14]95%ci . glycemic efficacy : mean hba1c sem over time ( a ) and mean fpg sem over time ( b ) last observation carried forward is used for each postbaseline time point in a and b. subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . laboratory - measured fpg decreased from baseline to week 26 ( ideg forced - flex , 1.28 ; ideg , 2.54 ; iglar , 1.33 mmol / l ) , with the most pronounced decline occurring during the first 12 weeks ( figure 1b ) . no significant difference was seen with ideg forced - flex vs iglar ( supplemental table 2 ) , but a greater reduction was seen with ideg than with ideg forced - flex [ etd ( idegforced - flex - ideg ) : 0.95 mmol / l ( 0.15 ; 1.75)95%ci , p = .021 ] . despite varying dosing times with ideg forced - flex , the only difference in smpg vs iglar was before lunch [ etd ( idegforced - flex - iglar ) : 0.85 mmol / l ( 0.12 ; 1.57)95%ci , p = .022 ] . the proportion of participants who attained prebreakfast smpg target less than 5.0 mmol / l at week 26 was 11.3% ( ideg forced - flex ) , 23.8% ( ideg ) , and 18.4% ( iglar ) . the median time to achieving the prebreakfast titration target for the first time was 7 weeks ( ideg forced - flex ) , 4 weeks ( ideg ) , and 6 weeks ( iglar ) . although mean hba1c values increased slightly from 26-week levels during the extension , they remained below baseline at week 52 ; 0.13% points from baseline to 7.6% ( ideg free - flex ) and 0.21% points from baseline to 7.5% ( iglar ) . ideg free - flex was not significantly different from iglar in lowering hba1c in the fas or ets at week 52 , with an upper 95% ci limit below a predefined noninferiority mark of 0.4% ( supplemental table 2 ) . mean fpg decreased from baseline to week 52 with ideg free - flex ( 1.73 mmol / l ) and iglar ( 0.61 mmol / l ) , with significantly greater reduction observed with ideg free- flex [ etd ( idegfree - flex - iglar ) : 1.07 mmol / l ( 1.82 ; 0.32)95%ci , p = .005 ] . at week 52 , smpg profiles were similar at all time points except at 90 minutes after the main evening meal , when it was significantly lower with ideg free - flex [ etd ( idegfree - flex - iglar ) : 0.82 mmol / l ( 1.53 ; 0.12)95%ci , p = .022 ] . the proportion of participants who attained prebreakfast smpg < 5.0 mmol / l at week 52 was 17.9% ( ideg free - flex ) and 13.8% ( iglar ) . mean basal doses increased slightly during weeks 026 with ideg forced - flex and iglar . however , basal doses remained stable with ideg during weeks 026 and in the ideg free - flex and iglar groups from week 27 to week 52 . at week 52 , mean daily basal , bolus , and total insulin doses were lower by 4% , 18% , and 11% , respectively , with ideg free - flex vs iglar . the greater mean total daily insulin dose with iglar vs ideg free - flex was due mainly to a decrease in mean daily bolus insulin dose with ideg free - flex ( 0.39 to 0.35 u / kg ) and an increase with iglar ( 0.40 to 0.42 u / kg ; table 2 ) . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . the data for ideg forced - flex and ideg arms from the first 26 weeks were pooled into the ideg free - flex arm in this table . overall , confirmed and severe hypoglycemia rates were similar across groups at week 26 ( figure 2 and supplemental table 3 ) . the nocturnal confirmed hypoglycemia rate was significantly lower with ideg forced - flex than iglar [ by 40% ; err ( idegforced - flex / iglar ) : 0.60 ( 0.44 ; 0.82)95%ci , p = .001 ] and ideg [ by 37% ; err ( idegforced - flex / ideg ) : 0.63 ( 0.46 ; 0.86)95%ci , p = .003 ] . nocturnal confirmed hypoglycemia rates were generally lower with ideg forced - flex than with iglar and ideg , regardless of the day of the week ( supplemental table 4 ) . no significant difference in overall confirmed hypoglycemia was seen at week 52 between ideg free - flex and iglar in the fas or ets . although the number of events was small , the severe hypoglycemia rate was numerically lower in the fas and significantly lower by 53% [ err ( idegfree - flex / iglar ) : 0.47 ( 0.23 ; 0.94)95%ci , p = .033 ] in the ets with ideg free - flex . nocturnal confirmed hypoglycemia rates were significantly lower with ideg free - flex by 25% [ err ( idegfree - flex / iglar ) : 0.75 ( 0.58 ; 0.97)95%ci , p = .026 ] in the fas and by 27% [ err ( idegfree - flex/ iglar ) : 0.73 ( 0.54 ; 0.98)95%ci , p = .035 ] in the ets . subjects randomized to the ideg forced - flex and ideg treatment arms during the main trial period had the opportunity to continue in the ideg free - flex arm after 26 weeks of treatment . rates for aes were 443 ( ideg forced - flex ) , 550 ( ideg ) , and 527 ( iglar ) per 100 patient - years of exposure ( pye ) from week 0 to week 26 and 447 ( ideg free - flex ) and 481 ( iglar ) per 100 pye from week 0 to week 52 . injection - site reaction rates were low over 52 weeks , with 6 ( ideg free - flex ) and 4 ( iglar ) events per 100 pye ; no reactions were classified as serious . mean weight gain from baseline to week 26 and during the extension was modest , with no between - group differences at week 52 ( ideg free - flex , 1.3 kg ; iglar , 1.9 kg ; supplemental table 2 ) . serious aes ( saes ) were reported by 5.5% ( ideg forced - flex ) , 4.2% ( ideg ) , and 5.0% ( iglar ) of participants from week 0 to week 26 and by 7.6% ( ideg free - flex ) and 7.5% ( iglar ) of participants in total from week 0 to week 52 ( supplemental table 5 ) . of randomized subjects who did not enter the extension , 10.0% ( 5 of 50 ) ideg forced - flex , 2.5% ( 1 of 40 ) ideg , and 6.5% ( 2 of 31 ) iglar - treated subjects reported saes during the main period . of randomized subjects who entered the extension , 3.5% ( 4 of 114 ) ideg forced - flex , 4.8% ( 6 of 125 ) ideg , and 4.5% ( 6 of 133 ) iglar - treated subjects reported saes during the main period . however , issues related to variability of action ( leading to unexpected hypoglycemia ) , and the need to adapt one 's lifestyle to the action profile of a prescribed insulin , can prevent the achievement of good glycemic control in a safe manner . the prebreakfast titration target 4.05.0 mmol / l was narrower than targets described in some other intensive t1 dm basal - bolus trials ( 17 ) ; however , given that the pharmacokinetic profile of ideg shows potential to lower hypoglycemic risk compared with current therapies ( 8 , 18 ) and other recent trials in patients with t2 dm and t1 dm have investigated comparably low titration targets ( 1923 ) , an ambitious target was considered appropriate and the ultimate decision regarding insulin dose was the investigator 's . over 26 weeks , the forced - flex regimen proved effective ( noninferior ) compared with iglar and ideg and , even more importantly , proved safe . however , this trial was unique in that it captured daily 4-point smpg , which may partially explain higher overall hypoglycemia rates observed in this study vs a previous trial that compared od ideg and iglar in patients with t1 dm ( 9 ) . analysis between ideg and iglar od at 26 weeks was not a prespecified end point within this trial , and thus , no statistical analysis was performed , although observed efficacy and safety data are presented for each of the 3 treatment arms through week 26 . full - year results confirmed findings from the 26-week main trial , with the extension allowing all ideg subjects the option to dose more flexibly than at same time daily . limitations of this study include a smaller population size compared with previous ideg studies ; the relatively short time frame of the forced - flex regimen ; and different recommendations for transferring subjects on more - than - od basal insulin to ideg and iglar at the study 's start ( total basal dose was transferred 1:1 for ideg , as is standard for switching long acting insulins , but reduced 20%30% for iglar ) . more ideg- than iglar - treated participants withdrew from the trial during the main period ; the rates of withdrawal could be related to the demanding nature of the study ( particularly in the forced - flex arm ) , including frequent blood glucose measurement requirements and study visits . this was an open - label trial , and investigators may have been more alert in their management and documentation of the ideg forced - flex arm than the ideg or iglar arms ; caution on the part of investigators and subjects may partially explain some differences ( such as in fpg and nocturnal confirmed hypoglycemia ) in study results between ideg forced - flex and ideg .

we identified a set of 193 genes that have been associated with feather development through mutant phenotypes or spaciotemporally restricted expression patterns ( supplementary materials and methods and supplementary table s1 , supplementary material online ) . to investigate the evolutionary history of these genes and their potential regulatory elements , we constructed a 19-way whole - genome alignment referenced on the chicken genome ( hillier et al . 2004 ) containing four birds , two crocodilians , two turtles , a lizard , four mammals , a frog , and five actinopterygian ( ray - finned ) fish . regions of the genome showing evolutionary constraint were identified using a phylogenetic hidden markov model to detect regions of the alignment evolving more slowly than synonymous sites in coding regions . overall , 957,409 conserved elements totaling approximately 71 mb and spanning approximately 7.2% of the chicken genome were identified , a higher percentage than the 5% often reported for the human genome . this result is consistent with the small ( 1.2 gb ) size of the chicken genome relative to the human genome , making the total amount of sequence annotated as constrained about half of what is currently reported for human ( siepel et al . 2005 ; lindblad - toh et al . 2011 ) . to identify putative regulatory elements we removed any regions overlapping an exon annotated in chicken , or another species , resulting in 602,539 cnees covering 4.4% of the chicken genome . we identified the gene that each cnee is likely to regulate by assigning each cnee to the gene with the closest transcription start site , and found that 13,307 of the cnees were associated with the 193 feather - related genes in the data set . although regulatory elements can act over long genomic distances that include genes not regulated by the elements ( kleinjan and van heyningen 2005 ) , experimentally identified enhancers tend to be closest to genes with expression in the same tissues and at the same times in development ( visel et al . additionally , many regulatory regions undergo rapid evolution and turnover ( wray 2007 , 2013 ) , and these will be missed by our analysis . due to their different functions , we split the list of 193 feather - related genes and their associated cnees into a structural set of 67 keratin genes and a patterning set of 126 nonkeratin genes and analyzed these groups separately . the genic and regulatory components of the keratin and nonkeratin sets show very different patterns across the 500-my backbone of our tree , on the lineage leading from the common ancestor of vertebrates to the chicken ( figs . 1 and 2 , supplementary fig . the most ancient branch in our analysis , leading to the common ancestor of ray - finned fishes and other vertebrates , shows the strongest enrichment for the nonkeratin feather genes ( 1.7 times expected ) , with smaller numbers of nonkeratin feather genes arising on branches leading to tetrapods and less inclusive clades ( figs . no members of this nonkeratin feather gene set are reconstructed to have arisen after the ancestor of birds and turtles . although ancient genes are more likely to be studied during chick development , the nonkeratin genes in our study were even more ancient than we would expect taking into account this bias ( mann whitney u test ; p < 0.022 ; supplementary fig the inferred first appearance of nonkeratin protein - coding regions that are involved , for example , in placode patterning and feather ontogeny in birds is consistent with these genes being part of an ancient developmental toolkit ( figs . evolutionary dynamics of ( a ) nonkeratin feather development genes and associated cnees ( n = 126 genes ) and ( b ) keratin genes and associated cnees ( n = 67 genes ) . the black horizontal line indicates the null expectation of the number of new genes ( comparison to all genes in the genome ) or cnees ( a uniform distribution throughout the genome ) . points above this line indicate lineages on which a higher - than - expected number of genes or cnees have arisen . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . 2006 ) . in ( b ) , the larger peak comprised -keratins arising from expansions of gene clusters on chicken chromosomes 27 and 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . 2013 ) . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . rates of origination of these three genomic classes are indicated by the colors for each stem internode and track in the tree , with blue colors indicating low origination rates and red colors indicating high origination rates . key events at the level of coding regions ( genes ) and regulatory elements are indicated . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . evolutionary dynamics of ( a ) nonkeratin feather development genes and associated cnees ( n = 126 genes ) and ( b ) keratin genes and associated cnees ( n = 67 genes ) . the black horizontal line indicates the null expectation of the number of new genes ( comparison to all genes in the genome ) or cnees ( a uniform distribution throughout the genome ) . points above this line indicate lineages on which a higher - than - expected number of genes or cnees have arisen . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . 2006 ) . in ( b ) , the larger peak comprised -keratins arising from expansions of gene clusters on chicken chromosomes 27 and 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . rates of origination of these three genomic classes are indicated by the colors for each stem internode and track in the tree , with blue colors indicating low origination rates and red colors indicating high origination rates . key events at the level of coding regions ( genes ) and regulatory elements are indicated . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . surprisingly , the cnees associated with nonkeratin feather - related genes show the highest rate of origin not on the internode between the ancestral archosaur and birds , where they exhibit a 25% higher - than - expected rate of origination , but instead on the branch leading to amniotes , where they exhibit a rate of origination 60% higher than expected ( figs . 1 and 2 , supplementary fig . s1 , supplementary material online ) . the rate of origination for these cnees is greater than what would be expected from cnees uniformly distributed throughout the genome for six of the eight branches along the lineage leading to chicken , suggesting a large amount of regulatory innovation over an extended time period ( figs . thus , the nonkeratin genic component of feather development arose deep in vertebrates and the greatest signal of regulatory innovation was coincident with the burst of phenotypic change associated with the transition to land . although information on the integument of the ancestral amniote remains exceptionally limited ( alibardi et al . 2009 ; alibardi 2012 ) , the accumulation of cnees inferred to have occurred at this time indicates a key role for regulatory change during this transition and in the subsequent evolution of vertebrate integumentary diversity . consistent with this hypothesis , 32 genes in our feather gene set are here identified as shared with those involved in the development of mammalian hair ( lowe et al . 2011 ) ( hypergeometric distribution , p < 1e-80 ; supplementary table s3 , supplementary material online ) and present in the amniote ancestor . genes driving hair development have been previously shown to exhibit an increase in regulatory innovation on the branch leading to amniotes , followed by a peak on the branch leading to mammals and a decline more recently ( lowe et al . our analysis suggests that nonavian dinosaurs , as part of archosauria , possessed the entirety of the known nonkeratin protein - coding toolkit for making feathers . moreover , assuming a constant rate of genome - wide accumulation of cnees throughout vertebrates , we estimate that 86% of nonkeratin feather gene cnees were also present in the archosaur ancestor . the cnees present in this ancestor may have less to do with feather origins but instead could be linked to the earlier amniote transition to land , with later , bird - specific cnees having feather - specific functions . these results are also consistent with new data on integumentary innovation and diversity in archosauria : filamentous or bristle structures either originated once early in the clade or three or more times ( clarke 2013 ) in pterosaurs ( kellner et al . thus , the genic and regulatory complement identified in the ancestral archosaur was either a flexible toolkit co - opted in multiple origins of new structures including feathers , or indicates an ancient origin in that clade for filamentous integumentary structures , often called feather precursors , on some part of the body or stage in development more than 100 my before the origin of pinnate feathers in dinosaurs . our analysis detects the well - known burst of duplication in -keratin genes within archosauria ( greenwold and sawyer 2010 ; li et al . 2013 ) on the branch leading to birds ( figs . 1b and 2 ) . the larger peak for keratin innovation comprised 57 -keratins arising as an expansion of a gene cluster on chicken chromosome 27 and 5 -keratins from duplications on chromosome 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . however , this keratin burst constitutes the only , albeit substantial , signal of innovation at the protein level in pinnate feather origins . notably , there is little evidence for regulatory innovation in the vicinity of -keratin genes . we detected little additional cross - species constraint outside of the exonic regions in the keratin clusters than we would expect if cnees were randomly distributed in the genome . we only detected 15 cnees neighboring feather - related keratins on the branch leading to birds , suggesting that regulatory evolution near -keratins is not exceptional . although the signature of cnees is likely complicated by a history of duplication and gene conversion in this multigene family , either the regulatory landscape around -keratins does not appear noteworthy or their regulatory elements are under less severe constraint . these data are consistent with the idea that the keratin component of feathers arose primarily as a result of genic innovations . aside from -keratin evolution after bonferroni correction , only 3 of the 126 nonkeratin feather genes showed signatures of positive selection on the archosaurian branch leading to birds ( supplementary table s2 , supplementary material online ) . these results indicate that most nonkeratin genes related to feather development exhibit regulatory , not protein - coding , innovations in the avian stem lineage , including living birds and nonavian dinosaurs , consistent with the hypothesis that regulatory innovations underlie adaptations in skin patterning and feather morphology . genes with an anomalously large number of regulatory elements arising in birds after their divergence from extant crocodilians may contribute to the origin of avian phenotypes . a genome - wide survey of 1-mb genomic windows revealed 23 segments of the chicken genome possessing anomalously high numbers of cnees arising on the branch leading to birds ( fig . 3a ; corrected p < 0.01 ; supplementary table s4 , supplementary material online ) . although gene ontology analysis does not reveal significant enrichment for any functions for the set of genes near these innovation - rich segments , a number of these segments flank genes involved in body size , limb development , and integument ( fig . the region showing the greatest enrichment for bird - specific cnees in the entire chicken genome , over 500% more than expected ( p < 10 ) , is centered in a 400-kb gene desert with insulin - like growth factor binding protein ( igfbp ) 2 and 5 being the two closest genes ( fig . igfbp2 is expressed in the chick apical ectodermal ridge and at the tips of the growth plates in the wing bud , contains single nucleotide polymorphisms linked to phenotypic variation in the limbs of chickens ( mcqueeney and dealy 2001 ; li et al . 2006 ) , and lies in the signaling pathway of both body size and limb length in mammals and birds ( fisher et al . igfbp5 also plays important roles in limb development ( mcqueeney and dealy 2001 ) and the reduction of body size ( salih et al . 2014 ) is consistent with a role for igfbp5-associated regulatory elements in body size reduction . body size and limb length are known to vary extensively across dinosauria and have been proposed to play a key role in dinosaur evolutionary dynamics ( benson et al . 2014 ) , with miniaturization indicated by the fossil record to have preceded the origin of flight in paraves ( turner et al . 2007 ; 2014 ) , and changes in limb scaling within maniraptora and continuing into birds associated with the origin of flight ( xu et al . thus , analysis of patterns of regulatory innovation offers the potential to link genome evolution to key shifts in shape and form occurring in deep time . 3.identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . red regions indicate those areas enriched compared with the distribution of cnees on other branches ( gray line in [ b ] ) and green squares indicate the 23 significant peaks of enrichment for bird - specific cnees relative to a uniform distribution throughout the genome . we examined the closest upstream and closest downstream genes and for select peaks a flanking gene is indicated along with a proposed role in avian morphological evolution ( key at top ) ; regulatory innovation may also have played a role in earlier dinosaur - lineage evolutionary dynamics . ( b ) the densest region for bird - specific cnees in the chicken genome is in a gene desert on chromosome 7 with igfbp2 being the closest well - annotated refseq gene and igfbp5 being the closest gene prediction . cnee density on all branches other than the one leading to birds is indicated in gray . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . red regions indicate those areas enriched compared with the distribution of cnees on other branches ( gray line in [ b ] ) and green squares indicate the 23 significant peaks of enrichment for bird - specific cnees relative to a uniform distribution throughout the genome . we examined the closest upstream and closest downstream genes and for select peaks a flanking gene is indicated along with a proposed role in avian morphological evolution ( key at top ) ; regulatory innovation may also have played a role in earlier dinosaur - lineage evolutionary dynamics . ( b ) the densest region for bird - specific cnees in the chicken genome is in a gene desert on chromosome 7 with igfbp2 being the closest well - annotated refseq gene and igfbp5 being the closest gene prediction . cnee density on all branches other than the one leading to birds is indicated in gray . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . we identified a set of 193 genes that have been associated with feather development through mutant phenotypes or spaciotemporally restricted expression patterns ( supplementary materials and methods and supplementary table s1 , supplementary material online ) . to investigate the evolutionary history of these genes and their potential regulatory elements , we constructed a 19-way whole - genome alignment referenced on the chicken genome ( hillier et al . 2004 ) containing four birds , two crocodilians , two turtles , a lizard , four mammals , a frog , and five actinopterygian ( ray - finned ) fish . regions of the genome showing evolutionary constraint were identified using a phylogenetic hidden markov model to detect regions of the alignment evolving more slowly than synonymous sites in coding regions . overall , 957,409 conserved elements totaling approximately 71 mb and spanning approximately 7.2% of the chicken genome were identified , a higher percentage than the 5% often reported for the human genome . this result is consistent with the small ( 1.2 gb ) size of the chicken genome relative to the human genome , making the total amount of sequence annotated as constrained about half of what is currently reported for human ( siepel et al . 2005 ; lindblad - toh et al . 2011 ) . to identify putative regulatory elements we removed any regions overlapping an exon annotated in chicken , or another species , resulting in 602,539 cnees covering 4.4% of the chicken genome . we identified the gene that each cnee is likely to regulate by assigning each cnee to the gene with the closest transcription start site , and found that 13,307 of the cnees were associated with the 193 feather - related genes in the data set . although regulatory elements can act over long genomic distances that include genes not regulated by the elements ( kleinjan and van heyningen 2005 ) , experimentally identified enhancers tend to be closest to genes with expression in the same tissues and at the same times in development ( visel et al . additionally , many regulatory regions undergo rapid evolution and turnover ( wray 2007 , 2013 ) , and these will be missed by our analysis . due to their different functions , we split the list of 193 feather - related genes and their associated cnees into a structural set of 67 keratin genes and a patterning set of 126 nonkeratin genes and analyzed these groups separately . the genic and regulatory components of the keratin and nonkeratin sets show very different patterns across the 500-my backbone of our tree , on the lineage leading from the common ancestor of vertebrates to the chicken ( figs . 1 and 2 , supplementary fig . the most ancient branch in our analysis , leading to the common ancestor of ray - finned fishes and other vertebrates , shows the strongest enrichment for the nonkeratin feather genes ( 1.7 times expected ) , with smaller numbers of nonkeratin feather genes arising on branches leading to tetrapods and less inclusive clades ( figs . no members of this nonkeratin feather gene set are reconstructed to have arisen after the ancestor of birds and turtles . although ancient genes are more likely to be studied during chick development , the nonkeratin genes in our study were even more ancient than we would expect taking into account this bias ( mann whitney u test ; p < 0.022 ; supplementary fig the inferred first appearance of nonkeratin protein - coding regions that are involved , for example , in placode patterning and feather ontogeny in birds is consistent with these genes being part of an ancient developmental toolkit ( figs . 1 and 2 ) . evolutionary dynamics of ( a ) nonkeratin feather development genes and associated cnees ( n = 126 genes ) and ( b ) keratin genes and associated cnees ( n = 67 genes ) . the black horizontal line indicates the null expectation of the number of new genes ( comparison to all genes in the genome ) or cnees ( a uniform distribution throughout the genome ) . points above this line indicate lineages on which a higher - than - expected number of genes or cnees have arisen . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . 2006 ) . in ( b ) , the larger peak comprised -keratins arising from expansions of gene clusters on chicken chromosomes 27 and 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . 2013 ) . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . rates of origination of these three genomic classes are indicated by the colors for each stem internode and track in the tree , with blue colors indicating low origination rates and red colors indicating high origination rates . key events at the level of coding regions ( genes ) and regulatory elements are indicated . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . evolutionary dynamics of ( a ) nonkeratin feather development genes and associated cnees ( n = 126 genes ) and ( b ) keratin genes and associated cnees ( n = 67 genes ) . the black horizontal line indicates the null expectation of the number of new genes ( comparison to all genes in the genome ) or cnees ( a uniform distribution throughout the genome ) . points above this line indicate lineages on which a higher - than - expected number of genes or cnees have arisen . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . , the larger peak comprised -keratins arising from expansions of gene clusters on chicken chromosomes 27 and 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . 2013 ) . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . rates of origination of these three genomic classes are indicated by the colors for each stem internode and track in the tree , with blue colors indicating low origination rates and red colors indicating high origination rates . key events at the level of coding regions ( genes ) and regulatory elements are indicated . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . surprisingly , the cnees associated with nonkeratin feather - related genes show the highest rate of origin not on the internode between the ancestral archosaur and birds , where they exhibit a 25% higher - than - expected rate of origination , but instead on the branch leading to amniotes , where they exhibit a rate of origination 60% higher than expected ( figs . 1 and 2 , supplementary fig . s1 , supplementary material online ) . the rate of origination for these cnees is greater than what would be expected from cnees uniformly distributed throughout the genome for six of the eight branches along the lineage leading to chicken , suggesting a large amount of regulatory innovation over an extended time period ( figs . thus , the nonkeratin genic component of feather development arose deep in vertebrates and the greatest signal of regulatory innovation was coincident with the burst of phenotypic change associated with the transition to land . although information on the integument of the ancestral amniote remains exceptionally limited ( alibardi et al . 2009 ; alibardi 2012 ) , the accumulation of cnees inferred to have occurred at this time indicates a key role for regulatory change during this transition and in the subsequent evolution of vertebrate integumentary diversity . consistent with this hypothesis , 32 genes in our feather gene set are here identified as shared with those involved in the development of mammalian hair ( lowe et al . 2011 ) ( hypergeometric distribution , p < 1e-80 ; supplementary table s3 , supplementary material online ) and present in the amniote ancestor . genes driving hair development have been previously shown to exhibit an increase in regulatory innovation on the branch leading to amniotes , followed by a peak on the branch leading to mammals and a decline more recently ( lowe et al . our analysis suggests that nonavian dinosaurs , as part of archosauria , possessed the entirety of the known nonkeratin protein - coding toolkit for making feathers . moreover , assuming a constant rate of genome - wide accumulation of cnees throughout vertebrates , we estimate that 86% of nonkeratin feather gene cnees were also present in the archosaur ancestor . the cnees present in this ancestor may have less to do with feather origins but instead could be linked to the earlier amniote transition to land , with later , bird - specific cnees having feather - specific functions . these results are also consistent with new data on integumentary innovation and diversity in archosauria : filamentous or bristle structures either originated once early in the clade or three or more times ( clarke 2013 ) in pterosaurs ( kellner et al . thus , the genic and regulatory complement identified in the ancestral archosaur was either a flexible toolkit co - opted in multiple origins of new structures including feathers , or indicates an ancient origin in that clade for filamentous integumentary structures , often called feather precursors , on some part of the body or stage in development more than 100 my before the origin of pinnate feathers in dinosaurs . our analysis detects the well - known burst of duplication in -keratin genes within archosauria ( greenwold and sawyer 2010 ; li et al . 2013 ) on the branch leading to birds ( figs . 1b and 2 ) . the larger peak for keratin innovation comprised 57 -keratins arising as an expansion of a gene cluster on chicken chromosome 27 and 5 -keratins from duplications on chromosome 2 . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . both of these results are consistent with previous studies of -keratin evolution ( greenwold and sawyer 2010 ; li et al . 2013 ) . however , this keratin burst constitutes the only , albeit substantial , signal of innovation at the protein level in pinnate feather origins . notably , there is little evidence for regulatory innovation in the vicinity of -keratin genes . we detected little additional cross - species constraint outside of the exonic regions in the keratin clusters than we would expect if cnees were randomly distributed in the genome . we only detected 15 cnees neighboring feather - related keratins on the branch leading to birds , suggesting that regulatory evolution near -keratins is not exceptional . although the signature of cnees is likely complicated by a history of duplication and gene conversion in this multigene family , either the regulatory landscape around -keratins does not appear noteworthy or their regulatory elements are under less severe constraint . these data are consistent with the idea that the keratin component of feathers arose primarily as a result of genic innovations . aside from -keratin evolution after bonferroni correction , only 3 of the 126 nonkeratin feather genes showed signatures of positive selection on the archosaurian branch leading to birds ( supplementary table s2 , supplementary material online ) . these results indicate that most nonkeratin genes related to feather development exhibit regulatory , not protein - coding , innovations in the avian stem lineage , including living birds and nonavian dinosaurs , consistent with the hypothesis that regulatory innovations underlie adaptations in skin patterning and feather morphology . genes with an anomalously large number of regulatory elements arising in birds after their divergence from extant crocodilians may contribute to the origin of avian phenotypes . a genome - wide survey of 1-mb genomic windows revealed 23 segments of the chicken genome possessing anomalously high numbers of cnees arising on the branch leading to birds ( fig . 3a ; corrected p < 0.01 ; supplementary table s4 , supplementary material online ) . although gene ontology analysis does not reveal significant enrichment for any functions for the set of genes near these innovation - rich segments , a number of these segments flank genes involved in body size , limb development , and integument ( fig . the region showing the greatest enrichment for bird - specific cnees in the entire chicken genome , over 500% more than expected ( p < 10 ) , is centered in a 400-kb gene desert with insulin - like growth factor binding protein ( igfbp ) 2 and 5 being the two closest genes ( fig . igfbp2 is expressed in the chick apical ectodermal ridge and at the tips of the growth plates in the wing bud , contains single nucleotide polymorphisms linked to phenotypic variation in the limbs of chickens ( mcqueeney and dealy 2001 ; li et al . 2006 ) , and lies in the signaling pathway of both body size and limb length in mammals and birds ( fisher et al . igfbp5 also plays important roles in limb development ( mcqueeney and dealy 2001 ) and the reduction of body size ( salih et al . 2014 ) is consistent with a role for igfbp5-associated regulatory elements in body size reduction . body size and limb length are known to vary extensively across dinosauria and have been proposed to play a key role in dinosaur evolutionary dynamics ( benson et al . 2014 ) , with miniaturization indicated by the fossil record to have preceded the origin of flight in paraves ( turner et al . 2007 ; lee et al . 2014 ) , and changes in limb scaling within maniraptora and continuing into birds associated with the origin of flight ( xu et al . 2001 ) . thus , analysis of patterns of regulatory innovation offers the potential to link genome evolution to key shifts in shape and form occurring in deep time . 3.identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . red regions indicate those areas enriched compared with the distribution of cnees on other branches ( gray line in [ b ] ) and green squares indicate the 23 significant peaks of enrichment for bird - specific cnees relative to a uniform distribution throughout the genome . we examined the closest upstream and closest downstream genes and for select peaks a flanking gene is indicated along with a proposed role in avian morphological evolution ( key at top ) ; regulatory innovation may also have played a role in earlier dinosaur - lineage evolutionary dynamics . ( b ) the densest region for bird - specific cnees in the chicken genome is in a gene desert on chromosome 7 with igfbp2 being the closest well - annotated refseq gene and igfbp5 being the closest gene prediction . cnee density on all branches other than the one leading to birds is indicated in gray . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . red regions indicate those areas enriched compared with the distribution of cnees on other branches ( gray line in [ b ] ) and green squares indicate the 23 significant peaks of enrichment for bird - specific cnees relative to a uniform distribution throughout the genome . we examined the closest upstream and closest downstream genes and for select peaks a flanking gene is indicated along with a proposed role in avian morphological evolution ( key at top ) ; regulatory innovation may also have played a role in earlier dinosaur - lineage evolutionary dynamics . ( b ) the densest region for bird - specific cnees in the chicken genome is in a gene desert on chromosome 7 with igfbp2 being the closest well - annotated refseq gene and igfbp5 being the closest gene prediction . cnee density on all branches other than the one leading to birds is indicated in gray . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . supplementary materials and methods , tables s1s4 , and figures s1 and s2 are available at molecular biology and evolution online ( http://www.mbe.oxfordjournals.org/ ) .

the evolution of avian feathers has recently been illuminated by fossils and the identification of genes involved in feather patterning and morphogenesis . however , molecular studies have focused mainly on protein - coding genes . using comparative genomics and more than 600,000 conserved regulatory elements , we show that patterns of genome evolution in the vicinity of feather genes are consistent with a major role for regulatory innovation in the evolution of feathers . rates of innovation at feather regulatory elements exhibit an extended period of innovation with peaks in the ancestors of amniotes and archosaurs . we estimate that 86% of such regulatory elements and 100% of the nonkeratin feather gene set were present prior to the origin of dinosauria . on the branch leading to modern birds , we detect a strong signal of regulatory innovation near insulin - like growth factor binding protein ( igfbp ) 2 and igfbp5 , which have roles in body size reduction , and may represent a genomic signature for the miniaturization of dinosaurian body size preceding the origin of flight .

Results and Discussion CNEEs and Constraint in the Avian Genome An Ancient Genic Toolkit and Extended Regulatory Evolution Are Associated with Feather Origins Limited Role of Protein Evolution in Feather Origins Body Size Genes Exhibit Exceptional Regulatory Innovation in Dinosauria Supplementary Material

to investigate the evolutionary history of these genes and their potential regulatory elements , we constructed a 19-way whole - genome alignment referenced on the chicken genome ( hillier et al . 2004 ) containing four birds , two crocodilians , two turtles , a lizard , four mammals , a frog , and five actinopterygian ( ray - finned ) fish . this result is consistent with the small ( 1.2 gb ) size of the chicken genome relative to the human genome , making the total amount of sequence annotated as constrained about half of what is currently reported for human ( siepel et al . we identified the gene that each cnee is likely to regulate by assigning each cnee to the gene with the closest transcription start site , and found that 13,307 of the cnees were associated with the 193 feather - related genes in the data set . the genic and regulatory components of the keratin and nonkeratin sets show very different patterns across the 500-my backbone of our tree , on the lineage leading from the common ancestor of vertebrates to the chicken ( figs . the most ancient branch in our analysis , leading to the common ancestor of ray - finned fishes and other vertebrates , shows the strongest enrichment for the nonkeratin feather genes ( 1.7 times expected ) , with smaller numbers of nonkeratin feather genes arising on branches leading to tetrapods and less inclusive clades ( figs . no members of this nonkeratin feather gene set are reconstructed to have arisen after the ancestor of birds and turtles . although ancient genes are more likely to be studied during chick development , the nonkeratin genes in our study were even more ancient than we would expect taking into account this bias ( mann whitney u test ; p < 0.022 ; supplementary fig the inferred first appearance of nonkeratin protein - coding regions that are involved , for example , in placode patterning and feather ontogeny in birds is consistent with these genes being part of an ancient developmental toolkit ( figs . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . surprisingly , the cnees associated with nonkeratin feather - related genes show the highest rate of origin not on the internode between the ancestral archosaur and birds , where they exhibit a 25% higher - than - expected rate of origination , but instead on the branch leading to amniotes , where they exhibit a rate of origination 60% higher than expected ( figs . the rate of origination for these cnees is greater than what would be expected from cnees uniformly distributed throughout the genome for six of the eight branches along the lineage leading to chicken , suggesting a large amount of regulatory innovation over an extended time period ( figs . thus , the nonkeratin genic component of feather development arose deep in vertebrates and the greatest signal of regulatory innovation was coincident with the burst of phenotypic change associated with the transition to land . 2009 ; alibardi 2012 ) , the accumulation of cnees inferred to have occurred at this time indicates a key role for regulatory change during this transition and in the subsequent evolution of vertebrate integumentary diversity . consistent with this hypothesis , 32 genes in our feather gene set are here identified as shared with those involved in the development of mammalian hair ( lowe et al . genes driving hair development have been previously shown to exhibit an increase in regulatory innovation on the branch leading to amniotes , followed by a peak on the branch leading to mammals and a decline more recently ( lowe et al . our analysis suggests that nonavian dinosaurs , as part of archosauria , possessed the entirety of the known nonkeratin protein - coding toolkit for making feathers . moreover , assuming a constant rate of genome - wide accumulation of cnees throughout vertebrates , we estimate that 86% of nonkeratin feather gene cnees were also present in the archosaur ancestor . thus , the genic and regulatory complement identified in the ancestral archosaur was either a flexible toolkit co - opted in multiple origins of new structures including feathers , or indicates an ancient origin in that clade for filamentous integumentary structures , often called feather precursors , on some part of the body or stage in development more than 100 my before the origin of pinnate feathers in dinosaurs . 2013 ) on the branch leading to birds ( figs . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . however , this keratin burst constitutes the only , albeit substantial , signal of innovation at the protein level in pinnate feather origins . notably , there is little evidence for regulatory innovation in the vicinity of -keratin genes . we only detected 15 cnees neighboring feather - related keratins on the branch leading to birds , suggesting that regulatory evolution near -keratins is not exceptional . these data are consistent with the idea that the keratin component of feathers arose primarily as a result of genic innovations . aside from -keratin evolution after bonferroni correction , only 3 of the 126 nonkeratin feather genes showed signatures of positive selection on the archosaurian branch leading to birds ( supplementary table s2 , supplementary material online ) . these results indicate that most nonkeratin genes related to feather development exhibit regulatory , not protein - coding , innovations in the avian stem lineage , including living birds and nonavian dinosaurs , consistent with the hypothesis that regulatory innovations underlie adaptations in skin patterning and feather morphology . genes with an anomalously large number of regulatory elements arising in birds after their divergence from extant crocodilians may contribute to the origin of avian phenotypes . a genome - wide survey of 1-mb genomic windows revealed 23 segments of the chicken genome possessing anomalously high numbers of cnees arising on the branch leading to birds ( fig . although gene ontology analysis does not reveal significant enrichment for any functions for the set of genes near these innovation - rich segments , a number of these segments flank genes involved in body size , limb development , and integument ( fig . the region showing the greatest enrichment for bird - specific cnees in the entire chicken genome , over 500% more than expected ( p < 10 ) , is centered in a 400-kb gene desert with insulin - like growth factor binding protein ( igfbp ) 2 and 5 being the two closest genes ( fig . 2006 ) , and lies in the signaling pathway of both body size and limb length in mammals and birds ( fisher et al . igfbp5 also plays important roles in limb development ( mcqueeney and dealy 2001 ) and the reduction of body size ( salih et al . 2014 ) is consistent with a role for igfbp5-associated regulatory elements in body size reduction . 2014 ) , with miniaturization indicated by the fossil record to have preceded the origin of flight in paraves ( turner et al . 2007 ; 2014 ) , and changes in limb scaling within maniraptora and continuing into birds associated with the origin of flight ( xu et al . thus , analysis of patterns of regulatory innovation offers the potential to link genome evolution to key shifts in shape and form occurring in deep time . 3.identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . to investigate the evolutionary history of these genes and their potential regulatory elements , we constructed a 19-way whole - genome alignment referenced on the chicken genome ( hillier et al . this result is consistent with the small ( 1.2 gb ) size of the chicken genome relative to the human genome , making the total amount of sequence annotated as constrained about half of what is currently reported for human ( siepel et al . we identified the gene that each cnee is likely to regulate by assigning each cnee to the gene with the closest transcription start site , and found that 13,307 of the cnees were associated with the 193 feather - related genes in the data set . the genic and regulatory components of the keratin and nonkeratin sets show very different patterns across the 500-my backbone of our tree , on the lineage leading from the common ancestor of vertebrates to the chicken ( figs . the most ancient branch in our analysis , leading to the common ancestor of ray - finned fishes and other vertebrates , shows the strongest enrichment for the nonkeratin feather genes ( 1.7 times expected ) , with smaller numbers of nonkeratin feather genes arising on branches leading to tetrapods and less inclusive clades ( figs . no members of this nonkeratin feather gene set are reconstructed to have arisen after the ancestor of birds and turtles . although ancient genes are more likely to be studied during chick development , the nonkeratin genes in our study were even more ancient than we would expect taking into account this bias ( mann whitney u test ; p < 0.022 ; supplementary fig the inferred first appearance of nonkeratin protein - coding regions that are involved , for example , in placode patterning and feather ontogeny in birds is consistent with these genes being part of an ancient developmental toolkit ( figs . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . points on the x axis correspond to the ancestors depicted in figure 2 , with spacing proportional to divergence times as recorded in timetree.org ( hedges et al . the colored backbone of the tree shows three tracks : cnees , nonkeratin feather genes ( n = 126 ) , and keratin genes ( n = 67 ) . the colors of the silhouettes at right indicate the percent of the feather regulatory component present in the chicken genome inferred to have arisen in the ancestor of each indicated taxon . for example , the fish are inferred to possess about 28% of the cnees associated with feather genes in chicken , whereas 86% of the observed chicken cnees are inferred to have arisen by the ancestral archosaur , including nonavian dinosaurs . surprisingly , the cnees associated with nonkeratin feather - related genes show the highest rate of origin not on the internode between the ancestral archosaur and birds , where they exhibit a 25% higher - than - expected rate of origination , but instead on the branch leading to amniotes , where they exhibit a rate of origination 60% higher than expected ( figs . the rate of origination for these cnees is greater than what would be expected from cnees uniformly distributed throughout the genome for six of the eight branches along the lineage leading to chicken , suggesting a large amount of regulatory innovation over an extended time period ( figs . thus , the nonkeratin genic component of feather development arose deep in vertebrates and the greatest signal of regulatory innovation was coincident with the burst of phenotypic change associated with the transition to land . 2009 ; alibardi 2012 ) , the accumulation of cnees inferred to have occurred at this time indicates a key role for regulatory change during this transition and in the subsequent evolution of vertebrate integumentary diversity . consistent with this hypothesis , 32 genes in our feather gene set are here identified as shared with those involved in the development of mammalian hair ( lowe et al . genes driving hair development have been previously shown to exhibit an increase in regulatory innovation on the branch leading to amniotes , followed by a peak on the branch leading to mammals and a decline more recently ( lowe et al . our analysis suggests that nonavian dinosaurs , as part of archosauria , possessed the entirety of the known nonkeratin protein - coding toolkit for making feathers . moreover , assuming a constant rate of genome - wide accumulation of cnees throughout vertebrates , we estimate that 86% of nonkeratin feather gene cnees were also present in the archosaur ancestor . thus , the genic and regulatory complement identified in the ancestral archosaur was either a flexible toolkit co - opted in multiple origins of new structures including feathers , or indicates an ancient origin in that clade for filamentous integumentary structures , often called feather precursors , on some part of the body or stage in development more than 100 my before the origin of pinnate feathers in dinosaurs . 2013 ) on the branch leading to birds ( figs . the small peak in the turtle - bird ancestor is due to the expansion of a -keratin gene cluster on chromosome 25 . however , this keratin burst constitutes the only , albeit substantial , signal of innovation at the protein level in pinnate feather origins . notably , there is little evidence for regulatory innovation in the vicinity of -keratin genes . we only detected 15 cnees neighboring feather - related keratins on the branch leading to birds , suggesting that regulatory evolution near -keratins is not exceptional . these data are consistent with the idea that the keratin component of feathers arose primarily as a result of genic innovations . aside from -keratin evolution after bonferroni correction , only 3 of the 126 nonkeratin feather genes showed signatures of positive selection on the archosaurian branch leading to birds ( supplementary table s2 , supplementary material online ) . these results indicate that most nonkeratin genes related to feather development exhibit regulatory , not protein - coding , innovations in the avian stem lineage , including living birds and nonavian dinosaurs , consistent with the hypothesis that regulatory innovations underlie adaptations in skin patterning and feather morphology . genes with an anomalously large number of regulatory elements arising in birds after their divergence from extant crocodilians may contribute to the origin of avian phenotypes . a genome - wide survey of 1-mb genomic windows revealed 23 segments of the chicken genome possessing anomalously high numbers of cnees arising on the branch leading to birds ( fig . although gene ontology analysis does not reveal significant enrichment for any functions for the set of genes near these innovation - rich segments , a number of these segments flank genes involved in body size , limb development , and integument ( fig . the region showing the greatest enrichment for bird - specific cnees in the entire chicken genome , over 500% more than expected ( p < 10 ) , is centered in a 400-kb gene desert with insulin - like growth factor binding protein ( igfbp ) 2 and 5 being the two closest genes ( fig . 2006 ) , and lies in the signaling pathway of both body size and limb length in mammals and birds ( fisher et al . igfbp5 also plays important roles in limb development ( mcqueeney and dealy 2001 ) and the reduction of body size ( salih et al . 2014 ) is consistent with a role for igfbp5-associated regulatory elements in body size reduction . 2014 ) , with miniaturization indicated by the fossil record to have preceded the origin of flight in paraves ( turner et al . 2014 ) , and changes in limb scaling within maniraptora and continuing into birds associated with the origin of flight ( xu et al . thus , analysis of patterns of regulatory innovation offers the potential to link genome evolution to key shifts in shape and form occurring in deep time . 3.identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) . identification of regions of the avian genome with signatures of exceptional regulatory innovation on the archosaur lineage that includes birds and other dinosaurs . ( a ) a genome - wide plot of the density of cnees arising on the archosaurian branch leading to the avian ancestor . ( c ) ucsc genome browser shot of a cnee - rich region in the vicinity of igfbp2 and igfbp5 , which functions in limb development and body size regulation ( see main text , supplementary table s4 , supplementary material online ) , showing cnees found only in birds ( red boxes ) or arising on deeper branches in the vertebrate tree ( gray boxes ) .

acute lung injury ( ali ) and its more severe form , acute respiratory distress syndrome ( ards ) , can arise after many types of injury to the lung , including sepsis , mechanical and chemical injury and bacterial and viral infections 1 . in ali , the mortality rate is in the range 2030% , with about 55% of the cases progressing to ards within a few days . ards causes significant morbidity and approximately 40% mortality , resulting in 75 000 deaths / year in the usa alone 2 . in the past two decades , five emerging viruses have been known to cause significant ards - related mortality , including influenza h1n1 2009 and , in particular , the highly pathogenic avian influenza h5n1 and h7n9 viruses and the sars and mers coronaviruses . in this review human coronavirus ( cov ) infections have traditionally caused a low percentage of annual upper and lower respiratory infections 3 , including severe disease outcomes in the elderly , immunocompromised patients and infants . hcov - oc43 ( oc43 ) and hcov-229e ( 229e ) were the first documented human covs but , more recently , hcov - nl63 ( nl63 ) 4 and hcov - hku1 ( hku1 ) 5 were identified as a consequence of increased viral surveillance efforts in the early twenty - first century ( table 1 ) . these four viruses usually cause acute infection of the upper respiratory tract and less frequently are associated with lower respiratory tract 6,7 diseases as well . severe disease is both rare and typically associated with co - morbidities and/or immunosenescence . the past 15 years have also seen the emergence of two new human coronaviruses that cause significant disease and mortality . sars - cov was identified in 2003 and caused an acute , atypical pneumonia and diffuse alveolar damage ( dad ) in roughly 8000 patients 8,9 . those over 65 years of age often developed ards , resulting in mortality rates that exceeded 50% . overall , sars - cov infection caused nearly 800 fatalities , representing a nearly 10% mortality rate . more recently , in 2012 , a new human coronavirus , designated mers - cov , was identified . mers - cov continues to circulate in camels and humans , with over 857 official cases and 334 deaths , representing an approximately 35% case fatality rate to date in humans 10,11 . mers - cov - induced disease is particularly severe in aged patients and those with pre - existing co - morbidities . human coronaviruses , their receptors , emergence , disease and infection data sars - cov and mers - cov have clear zoonotic origins , although their exact paths from animal reservoir to human infection are not yet clear . viruses with high nucleotide identity to sars - cov were found in key amplifying hosts such as palm civets and raccoon dogs in guangdong province china during the 20022003 sars epidemic 12 . later studies identified highly conserved viruses circulating in horseshoe bats , including some strains that are able to bind to , and infect , human cells 1316 . the existence of novel bat sars - like coronaviruses that also use bat , civet and human angiotensin 1 converting enzyme 2 ( ace2 ) receptors for entry , such as sars - cov , strongly suggests an opportunity for further zoonotic disease outbreaks in human and animal populations . sars causes an atypical pneumonia characterized by cough , fever and infiltrates with a ground - glass appearance on x - ray 17,18 . early - stage disease was characterized by acute dad , with oedema , fibrin and hyaline membranes in the alveolar spaces , typical of ali 19 . other patients predominantly showed an acute fibrinous and organizing pneumonia pattern or a mixture of the two patterns 20,21 . longer - term disease courses typically progressed to organizing phase dad and eventual deposition of fibrous tissue . autopsy of fatal sars - cov cases also revealed denuded airways , haemorrhage and increased macrophage populations in the lung 22,23 . during the sars epidemic , researchers noted that late - term disease progression was unrelated to viraemia but was more likely to be associated with immunopathological damage 24 . mers - cov has caused sporadic infections , along with several local outbreaks throughout the middle east since its discovery in 2012 25,26 . although much remains unknown , closely related viruses have been isolated from camels 27 and highly homologous mers - like bat covs have been identified in african neoromicia capensis bats 28 . local surveillance efforts have detected high levels of antibodies that recognize mers - cov in dromedary camels 29 ; furthermore , sampling of archived camel serum samples has revealed mers antibodies from as early as 1992 30 . these data suggest that bat to camel to human transmission routes may have seeded the 2012 outbreak in human populations , perhaps associated with the expanding camel trade that has emerged between equatorial africa and saudi arabia over the past 20 years . animal models of human disease should recapitulate many of the pathological and immune outcomes seen in human infections . numerous models have been established to better enable our understanding of the mechanics of sars - cov infection and pathogenesis , although few recapitulate the human disease phenotypes ( table 2 ) . initial studies utilized late epidemic strains in non - human primates 3133 , where mild to severe disease was observed , depending on the study location and animal age . to date , the differences in disease severity noted in primates have not been reconciled , but may reflect differences in virus strains or infection conditions . although still under development , mers - cov replication and disease have been reported in both rhesus macaques and common marmosets 34,35 . sars - cov replication resulted in limited disease in young models of immunocompetent mice 3638 ; however , mild clinical disease was noted in 1 year - old mice 39 . a mouse - adapted sars ( ma - sars ) strain was also developed that provides a model for moderate to lethal disease , depending on infectious dose , animal age and genetic background of the host 4042 ( table 2 ) . the ma - sars model faithfully replicates the age - dependent susceptibility observed in human patients , as well as key features of human lung pathology , including virus tropism to airway epithelial cells and type ii pneumocytes , pneumonia , hyaline membrane formation , development of dad and denudation of airway epithelial cells 40,43 . a limitation may be the rapid clearance of virus titres that is seen in younger and , to a much lesser extent , in aged animals . development of the ma - sars model has allowed for in - depth studies of viral pathogenesis and the host immune response , taking advantage of immunological tools and reagents for the mouse as well as the existence of knockout mouse strains . use of these tools has greatly added to our understanding of sars - cov pathogenesis , far beyond what could be learned in in vitro experiments or observational studies of human cases . because of receptor incompatibilities , mers - cov does not replicate in mice unless the animals are first transduced with adenovirus vectors encoding the receptor for entry , human dipeptidyl peptidase-4 ( dpp4 ) 44 . non - human primate and mouse models of sars - cov and mers - cov infection ; less common models include hamster 145 , ferret 146 and cat in this review we focus solely on hcov interactions within the context of the respiratory system and infection of relevant cell types . more specifically , we review some cov host interactions that alter cell - intrinsic antiviral defence programmes and other host pathways that contribute to pathological findings of ards , with its associated exudative and organizing phase diffuse alveolar damage and pulmonary fibrosis . nf-b signalling is an important component of numerous cellular responses , including stress , cytokine signalling , response to bacterial or viral infection and apoptosis 45,46 . the sars - cov envelope ( e ) protein stimulates nf-b signalling 47 , leading to lung cytokine signalling and inflammatory cell recruitment . the sars - cov papain - like protease ( plp ) has also been shown to antagonize nf-b signalling 48 in vitro . chemical inhibitors of nf-b signalling reduce lung pathology and inflammation following ma - sars infection , demonstrating the importance of this pathway 47 in pathogenesis . while the sars - cov e protein is not required for viral replication , it is important for inhibition of the host cellular stress response , apoptosis and unfolded protein response 49,50 . the e protein , along with the sars - cov orf3a and orf8a proteins , has ion channel activity 50 and may contribute to vascular permeability and fluid accumulation in the lung following sars - cov infection . sars - cov lacking e has been shown to be an effective vaccine 51,52 and a mers - cov clone lacking e has been generated 53 , although replication requires expression of e in trans . sars - cov , and to a greater extent mers - cov , are highly sensitive to interferon treatment in cell culture . interestingly , sars - cov pathogenesis does not significantly change in various type i interferon ( ifn ) knockout mouse models , except for a slight increase in overall virus titres 5456 . despite this , stat1- and myd88-deficient mice are significantly more vulnerable to lethal outcomes following infection 56,57 . like many viruses , covs encode a suite of genes that antagonize cell - intrinsic innate immune defence programmes in the infected host cell ( reviewed in 58 ) . numerous in vitro studies have demonstrated the ifn antagonist activity of both sars - cov and mers - cov proteins 5961 , and a detailed review of sars - cov evasion of the innate immune response was recently published by totura and baric 62 . analysis of ifn - stimulated gene ( isg ) expression in calu-3 human airway epithelial cells highlighted the ability of sars - cov and mers - cov to avoid detection by the host 63 . as compared with influenza a viruses , isg transcripts and proteins are not induced until late after sars - cov and mers - cov infection , when peak titres have already occurred in culture ( 1824 h ) . late in infection , isgs showed nearly universally increased expression following sars - cov infection , except for ace2 and serping1 . however , a much larger subset of isgs had significantly decreased expression following mers - cov infection . like mers - cov , h5n1 vn1203 infection also resulted in significant down - regulation of subsets of isgs . no consistent pattern in up - regulation or down - regulation of gene expression correlated with transcription factor usage , suggesting that a novel mechanism may be responsible for expression of the isg subsets . cells infected with mers - cov and h5n1 avian influenza were shown to have specifically altered open and closed chromatin structures , potentially limiting the ability of transcription factors to access and bind certain isg promoter regions . the mechanism by which mers - cov induces this chromatin structural alteration is as yet unknown . in contrast , the ns1 protein of h5n1 was responsible for the chromatin changes in influenza - infected cells . although speculative , it seems likely that many rna viruses may encode strategies to epigenetically alter host chromatin structure , influencing host gene expression . this mechanism may be partially mediated by the production of double - membrane vesicles , which could sequester rna replication intermediates away from the host - sensing machinery 64,65 . mda5 and ifit1 are important host antiviral sensor or antiviral defence isgs that detect viral rnas . ifit1 recognizes unmethylated 2-o rna 66 and alters efficient translation / stability of uncapped viral mrnas 67 . sars - cov and other coronavirus rnas are protected from ifit recognition because they encode a 2-o - methyltransferase ( 2-omt ) activity in the viral replicase protein , nsp16 68,69 . sars - cov is much more sensitive to interferon treatment in the absence of functional nsp16 methyltransferase activity , and mutant viral titres drop rapidly in both infected epithelial cells and mice . deletion or knockdown of either mda5 or ifit1 restored mutant sars - cov viral loads demonstrated the essential role of these host proteins in detecting pathogen - associated molecular patterns . ablation of the 2-omt activity may provide a universal strategy to rationally design live attenuated mutants of contemporary and newly emerging cov . both in vivo and in vitro studies have addressed the role of specific proteins in the innate immune system , often isgs , in sars - cov pathogenesis . transcriptional analysis on autopsy tissue from sars - cov - infected patients revealed increased expression of stat1 along with other ifn - induced cytokines 70 . the sars - cov accessory protein orf6 was identified as an interferon antagonist important for viral replication in low multiplicity of infection ( moi ) in vitro infections 71,72 . orf6 was subsequently found to sequester karyopherin 2 , a nuclear import factor , and block the nuclear translocation of stat1 after sars - cov infection . interestingly , stat1 translocation to the nucleus is not blocked in mers - cov - infected cells , so it remains uncertain whether antagonists of nuclear import are encoded in the viral genome 73 . transcriptional profiling of sars - cov - infected macaques revealed robust ifn signalling , including stat1 translocation to the nucleus , in the lung but not in the cells that stained positive for viral antigen 74 . these data highlight the importance of in vivo studies versus high moi in vitro studies . significantly , they also highlight the need to examine , or at least consider , expressing and signalling differences in specific cell types instead of global transcriptomic studies in those in vivo experiments . stat1 knockout mice have been studied extensively in the context of viral infection , typically showing a heightened susceptibility to disease , due to the lack of a type i ifn response 75 . these knockouts were first tested for sars - cov susceptibility using the tor2 strain in a sublethal model ; animals deficient in stat1 were unable to clear virus from the lung and developed a more severe and longer - lasting pneumonia than the control mice 76 . frieman et al showed that stat1 knockout mice are highly susceptible to infection with ma - sars in a novel , ifn - independent mechanism 56 . ma - sars - infection causes massive inflammatory cell influx in the lungs of stat1 knockout mice , including large numbers of macrophages , neutrophils and eosinophils . stat1 knockout mice have gross pathological changes in their lungs , including massive haemorrhage as well as increased lung size and stiffness . as seen in some humans , these mice develop severe pulmonary fibrosis and succumb to disease at late time points after infection . stained lung sections revealed the presence of collagen protein in alveolar exudates in stat1 knockouts , indicating development of early - stage pulmonary fibrosis . subsequent studies demonstrated that stat1 knockout animals developed a th2-skewed immune response and had significant numbers of alternatively activated or m2 macrophages in their lungs 77 . stat6 is required for the development of alternatively activated macrophages , and stat1/stat6 double knockout mice do not develop the severe lung disease and pro - fibrotic lesions observed in the stat1 single knockout 78 , thus demonstrating that these macrophages are essential for development of the pulmonary fibrosis phenotype . further elegant experiments showed that it is the stat1 deficiency in monocyte / macrophage cells , not the infected epithelial cells , that drives alternatively activated macrophage production and induction of fibrotic lung disease following sars - cov infection . alternatively activated macrophages are typically induced by the th2 cytokines il-4 and il-13 ; they have an anti - inflammatory role and play an important role in wound - healing processes 151 . ace2 is expressed on well - differentiated airway epithelial cells 79 and its expression increases following type i interferon treatment 63 . both ace2 protein levels and rna expression are down - regulated after either in vitro or in vivo sars - cov infection 63,80 and nl63 also down - regulates ace2 expression following in vitro infection 81 . it has previously been reported that ace2 and angiotensin-2 protect mice from sepsis- and acid aspiration - induced ali 82 . additionally , histopathological lung disease worsens when spike - fc is inoculated into mice with ali 80 . the normal function of ace2 is to inactivate angiotensin-2 , a negative regulator of the renin this system controls blood pressure and is involved in the development of pulmonary hypertension and pulmonary fibrosis . the renin multiple genome - wide association studies have investigated an association between genetic variation in ace and susceptibility to ards , with mixed results 86 . the role of dpp4 in mers - cov infection is discussed in detail by haagmans et al in a separate review in this issue 152 . furthermore , it has been suggested that unregulated ifn responses contributed to development of immunopathology and severe disease following sars - cov infection 88 . the data discussed above support this hypothesis and suggest that early control of isg signalling may be a means of preventing or controlling the development of severe lung disease . expression of the isg serping1 is also decreased following sars - cov infection of epithelial cells ; it functions by inhibiting the complement system as well as several proteases in the coagulation pathway . the role of the coagulation , fibrinolysis and wound healing in ards development are discussed below . the alveoli of the lung are where gas exchange occurs , providing oxygen to blood flowing through capillaries in the alveolar membrane . the alveolar walls are composed of type i and type ii pneumocytes , along with alveolar macrophages 89 . type i pneumocytes cover 95% of the alveolar surface area and allow for gas exchange with blood in the capillaries of the lung . type ii pneumocytes are the progenitors of type i pneumocytes and are also responsible for generating pulmonary surfactant 90 , a mixture of lipids and surfactant proteins that is crucial in reducing surface tension in the lung . sars - cov infection causes desquamation of pneumocytes in humans and mice , contributing to alveolar dysfunction , oedema and haemorrhage . alveolar macrophages play an essential role in surveillance of the local environment and inhibit an excessive immune response , although this inhibition can also block an effective response to sars - cov infection 91 . the functions of these cell types are critical in maintaining balance between inflammation , coagulation and wound repair , especially following lung injuries such as viral infection 92 . in ards patients , uncontrolled inflammation , fluid accumulation and developing fibrosis severely compromise gas exchange and lead to respiratory failure . sars - cov and influenza infect type i and type ii pneumocytes in the lung 93,94 . ards patients exhibit decreased surfactant levels 95 and ma - sars infection results in decreased surfactant transcript and protein levels 43 . decreased surfactant , and the consequent increase in surface tension , reduces the ability of the lung to expand and contract during normal respiration respiratory dysfunction occurs when the alveolar membranes are obstructed , or when the ability of the lung to expand and contract , circulating oxygenated air , is compromised . lethal sars - cov infection in the mouse and human is characterized by a breakdown of alveolar membrane integrity , resulting in accumulation of fluid exudates in the alveolar spaces . virus infection also results in an overwhelming cytokine response , severe lung tissue damage and respiratory failure 9699 . the progression from initial disease to diffuse alveolar damage and the exudative and organizing stage of dad is often independent of high - titre viral replication 24 , indicating that this severe disease outcome is primarily driven by an immunopathological response , including inflammatory cell recruitment and viral damage to type ii pneumocytes . this conclusion is further supported by non - human primate and mouse models of sars - cov infection , where lethal disease is more often associated with severe pulmonary lesions , alveolar exudates and respiratory dysfunction than with high viral load 43,100 . ma - sars infection results in peak viral titres at 12 days post - infection , along with airway denudation and resulting debris , which can occlude the small airways . severe lung disease , including inflammatory cell infiltrates , haemorrhage , alveolar oedema and hyaline membrane formation typical of the exudative stage of dad ( figure 1 ) , occurs at days 47 post - infection , when virus loads in the lung are dropping rapidly and/or are below the limit of detection . many isgs stimulated by sars - cov infection are involved in wound - healing responses and thus may contribute to sars - induced ali and ards . ma - sars lung immunopathology . ( a ) mock - infected lung stained with haematoxylin and eosin . ( b ) large airway of a c57bl/6 j ( b6 ) mouse , 7 days post - infection , with 10 plaque - forming units ( pfu ) ma - sars , shows denudation of the epithelial cells . ( c , d ) immunohistochemical staining of the sars - cov n protein at 2 days post - infection shows staining consistent with infection of airway epithelial cells and type ii pneumocytes , respectively . ( e ) msb staining highlights fibrin in the parenchyma of the lung ( red staining ) in b6 mice , 7 days post - infection with 10 pfu ma - sars . ( f ) perivascular cuffing in a b6 mouse , 4 days post - infection with 10 pfu ma - sars . ( g ) hyaline membranes in the parenchyma of the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( h ) inflammation in the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( i ) haemorrhage in the lung of a serpine1 mouse , 7 days post - infection with 10 pfu ma - sars . model of an infected alveolus in the lung . type i and type ii pneumocytes make up the alveolar walls and resident alveolar macrophages and pulmonary surfactant exist in the airspace ( a ) . in the acute phase of sars - cov infection ( b ) , type i and type ii pneumocytes are infected and secrete inflammatory cytokines , while surfactant levels decrease . during the late stage / tissue damage portion of viral infection , viral titres decrease , while airway debris , pulmonary oedema and hyaline membrane formation all impede respiration ( c ) . while sars - cov evades detection by the host immune system and causes minimal changes in transcript and protein levels for the first 24 h of infection 43 , it ultimately induces a massive signalling response in infected lungs . pro - inflammatory cytokines and chemokines , including il-6 , tnf , il-1 and ccl2 57 , recruit inflammatory cells to the site of infection . neutrophils and cytotoxic t cells , along with these cytokines , can induce tissue damage , including vascular leakage , and stimulate pulmonary fibrosis 101 . pro - fibrotic genes , including tgf1 , ctgf and pdgfa and numerous collagen transcripts , have increased expression following ma - sars infection . fluid exudates , haemorrhage and fibrin are all observed in the alveolar spaces of sars patients , as well as in animal models of disease 17,43,102 , increasing in severity as a function of age . in response , the coagulation cascade is activated , including increased factor 10 ( fx ) , f2 , f3 ( tissue factor ) , f11 , f12 and f7 transcript levels . activation of the coagulation cascade results in f10 cleavage of prothrombin into thrombin and subsequent thrombin cleavage of fibrinogen into fibrin 103 . fibrin clots in the alveoli are a prominent feature of sars - cov infection in humans and mice . the goal of this coagulation response is likely to protect the host by sealing the alveoli , preventing alveolar flooding and haemorrhage , which limit oxygen exchange and endanger patient survival . collagen accumulation , fibrin and fibrin clots all contribute to a developing fibrotic lung state , while at the same time stimulating the infected host to up - regulate fibrinolytic pathways 92 . profibrinolytic genes include members of the urokinase pathway , such as urokinase ( plau ) , tpa and plasmin ( plg ) 104 . the urokinase signalling pathway leads to cleavage and activation of plasmin into plasminogen ; this protease then cleaves fibrin clots . serpine1 and serpine2 are negative regulators of urokinase pathway and inhibit urokinase and tissue plasminogen activator ( tpa ) activity . urokinase signalling is highly active in the absence of serpine1 , and this imbalance often results in haemorrhage in knockout mice . serpine1 is highly expressed in sars patients , non - human primates and small animal models 43,74,105 . ards studies , independent of coronavirus infection , have attributed this serpine1 expression to alveolar macrophages and type ii pneumocytes 106,107 . ma - sars infection in a serpine1 knockout mouse model results in lethal disease with extreme lung pathology 43 . conversely , ma - sars - infected mice with a genetic deficiency in tpa have increased exudates in the lung . the dysregulation of these coagulation / anti - coagulation cascades can result in worsening end - stage lung disease conditions , resulting in death . profibrotic and profibrinolytic signalling are part of the wound - healing response , along with other extracellular matrix ( ecm ) remodelling pathways 101 . sars - cov infection causes massive tissue remodelling through urokinase and coagulation pathways activity , as discussed above . other important wound - healing pathways and ecm proteins with altered signalling following sars - cov infection include matrix metalloproteinases , egfr and collagens 43 . successful recovery from ali requires a delicate balance of pro - inflammatory , profibrotic and profibrinolytic responses . by altering isg expression , including ace2 , stat1 and serpine1 , sars - cov infection of alveolar epithelial cells sets the stage for the development of severe lung disease , including ards . sars and mers patients with severe lung disease exhibited lung consolidation , decreased blood oxygen saturation and often required intubation and ventilation 99,108,109 . small animal models of severe lung disease typically lack physiological readouts of respiratory function that can be directly correlated back to human signs of disease . whole - body plethysmography captures respiratory data in unrestrained animals , allowing for longitudinal measurement of pulmonary function ; these data can also be directly related to some human respiratory metrics 110,111 . sars - cov infection causes increased penh , a calculated measure of airway resistance and increased ef50 ( mid - breath exhalation force ) , indicating that respiratory function is compromised and animals must do more work to breathe 69 . unpublished data ( gralinski and menachery ) indicate that it is the exhalation portion of each breath that is impacted by sars - cov infection , likely due to extensive debris clogging the conducting airways . further experiments have shown that stat1 knockout mice have increased penh levels early after infection , and that at late timepoints they have reduced lung capacity , corresponding with the profibrotic histopathological changes observed in the lung ( gralinski , unpublished data ) . ards is a devastating end - stage lung disease with no cure . despite numerous clinical trials , improved several highly pathogenic emerging virus infections cause ards with high frequency , underscoring the critical public health importance of understanding the virological components and molecular mechanisms that drive this devastating end - stage lung disease . furthermore , a portion of ards cases may progress to pulmonary fibrosis , another clinically devastating end - stage lung disease with few treatment options . consequently , understanding the development of ards following viral infection remains a high - priority research topic that is germane to global health and pandemic disease control . the twenty - first century has demonstrated that zoonotic events will continue to introduce coronaviruses and other viruses into the human population , and that these viruses have the potential to spread rapidly , cause significant disease in communities and disrupt the global economy . an emerging theme is the connectivity between virus infection , complement and coagulation cascade activation , pro - inflammatory and profibrotic cytokine responses and disease severity . more studies are needed to unravel the complex interactions between these pathways that can interact to promote or dysregulate wound recovery after life - threatening respiratory virus infection . in particular there is a need for including well - articulated animal models that faithfully recapitulate disease processes across species . only through a better understanding of the interplay between a dysregulated host immune response and ali and ards

respiratory viruses can cause a wide spectrum of pulmonary diseases , ranging from mild , upper respiratory tract infections to severe and life - threatening lower respiratory tract infections , including the development of acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) . viral clearance and subsequent recovery from infection require activation of an effective host immune response ; however , many immune effector cells may also cause injury to host tissues . severe acute respiratory syndrome ( sars ) coronavirus and middle east respiratory syndrome ( mers ) coronavirus cause severe infection of the lower respiratory tract , with 10% and 35% overall mortality rates , respectively ; however , > 50% mortality rates are seen in the aged and immunosuppressed populations . while these viruses are susceptible to interferon treatment in vitro , they both encode numerous genes that allow for successful evasion of the host immune system until after high virus titres have been achieved . in this review , we discuss the importance of the innate immune response and the development of lung pathology following human coronavirus infection .

Introduction Innate immune response Acute respiratory distress syndrome (ARDS), coagulation, fibrinolysis and respiratory function Concluding thoughts Author contributions

acute lung injury ( ali ) and its more severe form , acute respiratory distress syndrome ( ards ) , can arise after many types of injury to the lung , including sepsis , mechanical and chemical injury and bacterial and viral infections 1 . in ali , the mortality rate is in the range 2030% , with about 55% of the cases progressing to ards within a few days . ards causes significant morbidity and approximately 40% mortality , resulting in 75 000 deaths / year in the usa alone 2 . in the past two decades , five emerging viruses have been known to cause significant ards - related mortality , including influenza h1n1 2009 and , in particular , the highly pathogenic avian influenza h5n1 and h7n9 viruses and the sars and mers coronaviruses . in this review human coronavirus ( cov ) infections have traditionally caused a low percentage of annual upper and lower respiratory infections 3 , including severe disease outcomes in the elderly , immunocompromised patients and infants . hcov - oc43 ( oc43 ) and hcov-229e ( 229e ) were the first documented human covs but , more recently , hcov - nl63 ( nl63 ) 4 and hcov - hku1 ( hku1 ) 5 were identified as a consequence of increased viral surveillance efforts in the early twenty - first century ( table 1 ) . these four viruses usually cause acute infection of the upper respiratory tract and less frequently are associated with lower respiratory tract 6,7 diseases as well . those over 65 years of age often developed ards , resulting in mortality rates that exceeded 50% . more recently , in 2012 , a new human coronavirus , designated mers - cov , was identified . mers - cov continues to circulate in camels and humans , with over 857 official cases and 334 deaths , representing an approximately 35% case fatality rate to date in humans 10,11 . later studies identified highly conserved viruses circulating in horseshoe bats , including some strains that are able to bind to , and infect , human cells 1316 . early - stage disease was characterized by acute dad , with oedema , fibrin and hyaline membranes in the alveolar spaces , typical of ali 19 . other patients predominantly showed an acute fibrinous and organizing pneumonia pattern or a mixture of the two patterns 20,21 . mers - cov has caused sporadic infections , along with several local outbreaks throughout the middle east since its discovery in 2012 25,26 . animal models of human disease should recapitulate many of the pathological and immune outcomes seen in human infections . numerous models have been established to better enable our understanding of the mechanics of sars - cov infection and pathogenesis , although few recapitulate the human disease phenotypes ( table 2 ) . initial studies utilized late epidemic strains in non - human primates 3133 , where mild to severe disease was observed , depending on the study location and animal age . although still under development , mers - cov replication and disease have been reported in both rhesus macaques and common marmosets 34,35 . sars - cov replication resulted in limited disease in young models of immunocompetent mice 3638 ; however , mild clinical disease was noted in 1 year - old mice 39 . a mouse - adapted sars ( ma - sars ) strain was also developed that provides a model for moderate to lethal disease , depending on infectious dose , animal age and genetic background of the host 4042 ( table 2 ) . the ma - sars model faithfully replicates the age - dependent susceptibility observed in human patients , as well as key features of human lung pathology , including virus tropism to airway epithelial cells and type ii pneumocytes , pneumonia , hyaline membrane formation , development of dad and denudation of airway epithelial cells 40,43 . a limitation may be the rapid clearance of virus titres that is seen in younger and , to a much lesser extent , in aged animals . development of the ma - sars model has allowed for in - depth studies of viral pathogenesis and the host immune response , taking advantage of immunological tools and reagents for the mouse as well as the existence of knockout mouse strains . use of these tools has greatly added to our understanding of sars - cov pathogenesis , far beyond what could be learned in in vitro experiments or observational studies of human cases . non - human primate and mouse models of sars - cov and mers - cov infection ; less common models include hamster 145 , ferret 146 and cat in this review we focus solely on hcov interactions within the context of the respiratory system and infection of relevant cell types . more specifically , we review some cov host interactions that alter cell - intrinsic antiviral defence programmes and other host pathways that contribute to pathological findings of ards , with its associated exudative and organizing phase diffuse alveolar damage and pulmonary fibrosis . nf-b signalling is an important component of numerous cellular responses , including stress , cytokine signalling , response to bacterial or viral infection and apoptosis 45,46 . the sars - cov papain - like protease ( plp ) has also been shown to antagonize nf-b signalling 48 in vitro . chemical inhibitors of nf-b signalling reduce lung pathology and inflammation following ma - sars infection , demonstrating the importance of this pathway 47 in pathogenesis . while the sars - cov e protein is not required for viral replication , it is important for inhibition of the host cellular stress response , apoptosis and unfolded protein response 49,50 . the e protein , along with the sars - cov orf3a and orf8a proteins , has ion channel activity 50 and may contribute to vascular permeability and fluid accumulation in the lung following sars - cov infection . sars - cov lacking e has been shown to be an effective vaccine 51,52 and a mers - cov clone lacking e has been generated 53 , although replication requires expression of e in trans . sars - cov , and to a greater extent mers - cov , are highly sensitive to interferon treatment in cell culture . interestingly , sars - cov pathogenesis does not significantly change in various type i interferon ( ifn ) knockout mouse models , except for a slight increase in overall virus titres 5456 . like many viruses , covs encode a suite of genes that antagonize cell - intrinsic innate immune defence programmes in the infected host cell ( reviewed in 58 ) . numerous in vitro studies have demonstrated the ifn antagonist activity of both sars - cov and mers - cov proteins 5961 , and a detailed review of sars - cov evasion of the innate immune response was recently published by totura and baric 62 . analysis of ifn - stimulated gene ( isg ) expression in calu-3 human airway epithelial cells highlighted the ability of sars - cov and mers - cov to avoid detection by the host 63 . as compared with influenza a viruses , isg transcripts and proteins are not induced until late after sars - cov and mers - cov infection , when peak titres have already occurred in culture ( 1824 h ) . however , a much larger subset of isgs had significantly decreased expression following mers - cov infection . no consistent pattern in up - regulation or down - regulation of gene expression correlated with transcription factor usage , suggesting that a novel mechanism may be responsible for expression of the isg subsets . this mechanism may be partially mediated by the production of double - membrane vesicles , which could sequester rna replication intermediates away from the host - sensing machinery 64,65 . sars - cov and other coronavirus rnas are protected from ifit recognition because they encode a 2-o - methyltransferase ( 2-omt ) activity in the viral replicase protein , nsp16 68,69 . sars - cov is much more sensitive to interferon treatment in the absence of functional nsp16 methyltransferase activity , and mutant viral titres drop rapidly in both infected epithelial cells and mice . ablation of the 2-omt activity may provide a universal strategy to rationally design live attenuated mutants of contemporary and newly emerging cov . both in vivo and in vitro studies have addressed the role of specific proteins in the innate immune system , often isgs , in sars - cov pathogenesis . the sars - cov accessory protein orf6 was identified as an interferon antagonist important for viral replication in low multiplicity of infection ( moi ) in vitro infections 71,72 . interestingly , stat1 translocation to the nucleus is not blocked in mers - cov - infected cells , so it remains uncertain whether antagonists of nuclear import are encoded in the viral genome 73 . transcriptional profiling of sars - cov - infected macaques revealed robust ifn signalling , including stat1 translocation to the nucleus , in the lung but not in the cells that stained positive for viral antigen 74 . these data highlight the importance of in vivo studies versus high moi in vitro studies . significantly , they also highlight the need to examine , or at least consider , expressing and signalling differences in specific cell types instead of global transcriptomic studies in those in vivo experiments . stat1 knockout mice have been studied extensively in the context of viral infection , typically showing a heightened susceptibility to disease , due to the lack of a type i ifn response 75 . these knockouts were first tested for sars - cov susceptibility using the tor2 strain in a sublethal model ; animals deficient in stat1 were unable to clear virus from the lung and developed a more severe and longer - lasting pneumonia than the control mice 76 . frieman et al showed that stat1 knockout mice are highly susceptible to infection with ma - sars in a novel , ifn - independent mechanism 56 . ma - sars - infection causes massive inflammatory cell influx in the lungs of stat1 knockout mice , including large numbers of macrophages , neutrophils and eosinophils . as seen in some humans , these mice develop severe pulmonary fibrosis and succumb to disease at late time points after infection . stained lung sections revealed the presence of collagen protein in alveolar exudates in stat1 knockouts , indicating development of early - stage pulmonary fibrosis . subsequent studies demonstrated that stat1 knockout animals developed a th2-skewed immune response and had significant numbers of alternatively activated or m2 macrophages in their lungs 77 . stat6 is required for the development of alternatively activated macrophages , and stat1/stat6 double knockout mice do not develop the severe lung disease and pro - fibrotic lesions observed in the stat1 single knockout 78 , thus demonstrating that these macrophages are essential for development of the pulmonary fibrosis phenotype . ace2 is expressed on well - differentiated airway epithelial cells 79 and its expression increases following type i interferon treatment 63 . both ace2 protein levels and rna expression are down - regulated after either in vitro or in vivo sars - cov infection 63,80 and nl63 also down - regulates ace2 expression following in vitro infection 81 . the normal function of ace2 is to inactivate angiotensin-2 , a negative regulator of the renin this system controls blood pressure and is involved in the development of pulmonary hypertension and pulmonary fibrosis . the renin multiple genome - wide association studies have investigated an association between genetic variation in ace and susceptibility to ards , with mixed results 86 . the role of dpp4 in mers - cov infection is discussed in detail by haagmans et al in a separate review in this issue 152 . furthermore , it has been suggested that unregulated ifn responses contributed to development of immunopathology and severe disease following sars - cov infection 88 . the data discussed above support this hypothesis and suggest that early control of isg signalling may be a means of preventing or controlling the development of severe lung disease . expression of the isg serping1 is also decreased following sars - cov infection of epithelial cells ; it functions by inhibiting the complement system as well as several proteases in the coagulation pathway . the role of the coagulation , fibrinolysis and wound healing in ards development are discussed below . the alveoli of the lung are where gas exchange occurs , providing oxygen to blood flowing through capillaries in the alveolar membrane . type i pneumocytes cover 95% of the alveolar surface area and allow for gas exchange with blood in the capillaries of the lung . type ii pneumocytes are the progenitors of type i pneumocytes and are also responsible for generating pulmonary surfactant 90 , a mixture of lipids and surfactant proteins that is crucial in reducing surface tension in the lung . alveolar macrophages play an essential role in surveillance of the local environment and inhibit an excessive immune response , although this inhibition can also block an effective response to sars - cov infection 91 . sars - cov and influenza infect type i and type ii pneumocytes in the lung 93,94 . decreased surfactant , and the consequent increase in surface tension , reduces the ability of the lung to expand and contract during normal respiration respiratory dysfunction occurs when the alveolar membranes are obstructed , or when the ability of the lung to expand and contract , circulating oxygenated air , is compromised . lethal sars - cov infection in the mouse and human is characterized by a breakdown of alveolar membrane integrity , resulting in accumulation of fluid exudates in the alveolar spaces . the progression from initial disease to diffuse alveolar damage and the exudative and organizing stage of dad is often independent of high - titre viral replication 24 , indicating that this severe disease outcome is primarily driven by an immunopathological response , including inflammatory cell recruitment and viral damage to type ii pneumocytes . severe lung disease , including inflammatory cell infiltrates , haemorrhage , alveolar oedema and hyaline membrane formation typical of the exudative stage of dad ( figure 1 ) , occurs at days 47 post - infection , when virus loads in the lung are dropping rapidly and/or are below the limit of detection . ( b ) large airway of a c57bl/6 j ( b6 ) mouse , 7 days post - infection , with 10 plaque - forming units ( pfu ) ma - sars , shows denudation of the epithelial cells . ( c , d ) immunohistochemical staining of the sars - cov n protein at 2 days post - infection shows staining consistent with infection of airway epithelial cells and type ii pneumocytes , respectively . ( e ) msb staining highlights fibrin in the parenchyma of the lung ( red staining ) in b6 mice , 7 days post - infection with 10 pfu ma - sars . ( g ) hyaline membranes in the parenchyma of the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( h ) inflammation in the lung of a b6 mouse , 7 days post - infection with 10 pfu ma - sars . ( i ) haemorrhage in the lung of a serpine1 mouse , 7 days post - infection with 10 pfu ma - sars . model of an infected alveolus in the lung . type i and type ii pneumocytes make up the alveolar walls and resident alveolar macrophages and pulmonary surfactant exist in the airspace ( a ) . in the acute phase of sars - cov infection ( b ) , type i and type ii pneumocytes are infected and secrete inflammatory cytokines , while surfactant levels decrease . while sars - cov evades detection by the host immune system and causes minimal changes in transcript and protein levels for the first 24 h of infection 43 , it ultimately induces a massive signalling response in infected lungs . pro - inflammatory cytokines and chemokines , including il-6 , tnf , il-1 and ccl2 57 , recruit inflammatory cells to the site of infection . pro - fibrotic genes , including tgf1 , ctgf and pdgfa and numerous collagen transcripts , have increased expression following ma - sars infection . fluid exudates , haemorrhage and fibrin are all observed in the alveolar spaces of sars patients , as well as in animal models of disease 17,43,102 , increasing in severity as a function of age . in response , the coagulation cascade is activated , including increased factor 10 ( fx ) , f2 , f3 ( tissue factor ) , f11 , f12 and f7 transcript levels . activation of the coagulation cascade results in f10 cleavage of prothrombin into thrombin and subsequent thrombin cleavage of fibrinogen into fibrin 103 . fibrin clots in the alveoli are a prominent feature of sars - cov infection in humans and mice . the goal of this coagulation response is likely to protect the host by sealing the alveoli , preventing alveolar flooding and haemorrhage , which limit oxygen exchange and endanger patient survival . profibrinolytic genes include members of the urokinase pathway , such as urokinase ( plau ) , tpa and plasmin ( plg ) 104 . the urokinase signalling pathway leads to cleavage and activation of plasmin into plasminogen ; this protease then cleaves fibrin clots . urokinase signalling is highly active in the absence of serpine1 , and this imbalance often results in haemorrhage in knockout mice . ards studies , independent of coronavirus infection , have attributed this serpine1 expression to alveolar macrophages and type ii pneumocytes 106,107 . ma - sars infection in a serpine1 knockout mouse model results in lethal disease with extreme lung pathology 43 . conversely , ma - sars - infected mice with a genetic deficiency in tpa have increased exudates in the lung . profibrotic and profibrinolytic signalling are part of the wound - healing response , along with other extracellular matrix ( ecm ) remodelling pathways 101 . successful recovery from ali requires a delicate balance of pro - inflammatory , profibrotic and profibrinolytic responses . by altering isg expression , including ace2 , stat1 and serpine1 , sars - cov infection of alveolar epithelial cells sets the stage for the development of severe lung disease , including ards . whole - body plethysmography captures respiratory data in unrestrained animals , allowing for longitudinal measurement of pulmonary function ; these data can also be directly related to some human respiratory metrics 110,111 . further experiments have shown that stat1 knockout mice have increased penh levels early after infection , and that at late timepoints they have reduced lung capacity , corresponding with the profibrotic histopathological changes observed in the lung ( gralinski , unpublished data ) . despite numerous clinical trials , improved several highly pathogenic emerging virus infections cause ards with high frequency , underscoring the critical public health importance of understanding the virological components and molecular mechanisms that drive this devastating end - stage lung disease . consequently , understanding the development of ards following viral infection remains a high - priority research topic that is germane to global health and pandemic disease control . the twenty - first century has demonstrated that zoonotic events will continue to introduce coronaviruses and other viruses into the human population , and that these viruses have the potential to spread rapidly , cause significant disease in communities and disrupt the global economy . more studies are needed to unravel the complex interactions between these pathways that can interact to promote or dysregulate wound recovery after life - threatening respiratory virus infection . only through a better understanding of the interplay between a dysregulated host immune response and ali and ards

at the beginning of the 1950s , the nobel laureate rita levi - montalcini discovered and characterized the nerve growth factor ( ngf ) as a key player in target - mediated regulation of peripheral innervations [ 1 , 2 ] . the pioneer studies performed by her and victor hamburger first showed that the nervous system requires an adequate supply of trophic factors ( neurotrophins nts ) from the environment for survival and development . in addition , and most intriguingly , these studies first raised the concept of cell death playing a pivotal role in this specific context . in particular , target tissues might determine the size and morphology of the peripheral innervations by regulating cell death among neurons and by making the nts available only in limited amounts , finally resulting in the selection only of those neurons which have established the quantitative and qualitative numbers of connections ( with the target tissue ) . subsequent studies have demonstrated that , during nervous system development , ngf is released by the target tissue , taken up in responsive neurons by receptor - mediated endocytosis and transported retrogradely to the cell body where it exerts its trophic / differentiative effects through its low ( p75ntr ) and high affinity receptors ( trka ) [ 37 ] . in particular , while the p75ntr can cause apoptosis in a variety of systems , when coexpressed with trka , it can modify their ligand - binding activity , dose - responsiveness , and kinase activity , leading to increased survival , neurite outgrowth , and synaptic plasticity [ 812 ] . since these first investigations on target - controlled neuronal survival , ngf has been one of the most thoroughly studied nts , regulating the survival , development , and trophism of specific neuronal populations in the peripheral and central nervous system ( cns ) . while in the periphery this nt was first recognized for its action on the sympathetic ganglia , in the adult brain the highest levels of ngf are found in hippocampus , cortex , and olfactory regions , which represent targets for basal forebrain cholinergic neurons [ 14 , 15 ] . ngf acts as a trophic factor for these neurons since its administration in vivo increases the levels of choline acetyltransferase [ 16 , 17 ] while rescuing from death basal forebrain neurons following transection of the septo - hippocampal pathways . in addition to the nervous system , nts exert their effects on various other tissue compartments [ 1926 ] . the largest amount of ngf is produced in the salivary glands of adult male mice , which are the largest and best available source of this nt ( smaller concentrations of this nt can be found in snake venom , guinea pig prostate , the seminal fluid of guinea pigs and bulls , in the human skin , and in numerous tissues and body fluids ) . furthermore , a number of other cells outside the nervous system , including epithelial cells , fibroblasts , lymphocytes , and activated macrophages , synthesize ngf [ 2729 ] . since ngf discovery , no explanation was available for almost three decades on ngf physiological role in adult animals . most neurobiologists , for a very long time , explained its presence in adult animals because of its ancestral physiological role during ontogeny . ngf was originally described as a protein exclusively acting on sympathetic peripheral neurons and only much later it was found to be also active on central nervous system neurons and on a variety of immune cell types . overall , the presence of ngf in those limbic areas of the cns involved in mood and cognition and in the orchestration of neuroendocrine responses and circadian activities , as well as in cells of the immune and endocrine system , indicates a much wider role for this nt than previously hypothesized and suggests that it might function as intercellular messenger or even a humoral factor to help regulate endocrine responses to stress [ 19 , 3135 ] . stress may be defined as any change of the internal or external milieu perturbing the maintenance of homeostasis of an organism . in complex organisms , stress responses involve a coordinated set of intercellular signals eventually resulting in the removal of the organism from , or adaptation to , the stressful situation . coping strategies are therefore important components of the stress response allowing an animal to fright , fight or flight . pivotal to these allostatic / adaptive responses is the neuroendocrine system that employs neuropeptides and hormones as mediators . the hypothalamic - pituitary - adrenal ( hpa ) axis ( which releases the glucocorticoids gcs hormones ) and sympathetic adrenomedullary systems ( which releases catecholamines ) coordinate the stress response . although the cascade of responses to acute stressful events originates at the cns level , peripheral structures , such as the adrenal glands , act in interplay and feedback on the brain to maintain body homeostasis [ 37 , 38 ] . by contrast , chronic stress can promote and exacerbate pathophysiological conditions through these same systems , leading to allostatic overload . the adaptive plasticity of chronic stress involves many mediators , including gcs , excitatory amino acids , and nts , such as ngf and also brain - derived neurotrophic factor ( bdnf ) . these latter can participate in the hpa axis response to stressful stimuli at different anatomical levels [ 38 , 40 ] . in the wild , adult male mice exhibit high levels of intraspecific aggressive behavior towards conspecifics mainly related to territorial defense . in the laboratory setting , the agonistic behavior of male mice can be elicited after 46 weeks of social isolation and represents a psychosocial stressful condition that has been shown to markedly alter ngf and bdnf levels both in plasma as well as in selected brain areas , including the hypothalamus and hippocampus [ 41 , 42 ] . in the mouse , ngf is mainly synthesized and released from the submaxillary salivary glands [ 5 , 43 ] . neurobehavioral studies demonstrated that both male intraspecific fighting and lactating females nest - defense , raised against a potentially infanticidal conspecific male , induce a massive release of ngf from salivary glands into the bloodstream of adults . in particular , circulating levels of ngf are highly correlated with the number of fighting episodes and most intriguingly to the social status achieved in fighting mice . thus , subjects experiencing repeated defeat and submissions ( i.e. , a subordinate status ) were characterized by a two - time increase in the amount of circulating ngf compared to attacking mice that achieved a dominant status [ 44 , 45 ] . in this specific context it is worth to note that physical stressors , such as cold water , foot shocks , or restraint stress exposure , exert a minor effect on ngf release [ 43 , 46 ] . following fighting behavior , adrenal weight increases quite markedly and quickly in male mice suggesting that social / aggressive behavior might control circulating ngf release from salivary glands and that these might in turn control adrenal morphology as well as adrenal functional status [ 47 , 48 ] . this hypothesis has been supported by data showing that exogenous ngf administration ( i.p . delivery , for 10 consecutive days in order to mimic 10 subsequent daily fighting sessions ) results in a marked adrenal gland hypertrophy [ 43 , 47 , 48 ] . the higher ngf release and the hypertrophy of the adrenals , occurring in subordinates male mice , suggest a regulative loop involving ngf - mediated increase of glucocorticoids secretion ( upon adrenals ) acting to enhance a submissive profile ( figure 1 ) [ 46 , 47 , 49 ] . further experimental evidence supports the idea that ngf expression may be regulated by behavioral activation . in particular , intermale aggressive behavior induces a large increase in ngf mrna and protein in hypothalamus , especially in the paraventricular nucleus , with no measurable changes in cerebral cortex , hippocampus , and cerebellum . the increase in hypothalamic ngf following intermale aggressive behavior is not abolished by sialoadenectomy , suggesting that hypothalamic ngf is not of salivary origin . alleva and aloe hypothesized that the rather rapid increase in levels of brain ngf , which follows a psychosocial stressful event , could allow some renewal of brain plasticity at adulthood . indeed , ngf could contribute to structural changes , such as the formation of dendritic spines or collateral sprouting , ultimately altering the structure of neural connections in the mature brain [ 46 , 47 ] . another possibility is that hypothalamic ngf might affect levels of other peptides or hormones present in the hypothalamus [ 5155 ] . taken together , these studies suggest that hypothalamic ngf levels are responsive to ( and modified by ) stimuli of a psychological nature , most likely associated with anxiety and fear . as hypothalamus is involved in the maintenance of physiological homeostasis , it is likely that hypothalamic ngf affects , or cooperates with , hormones and/or neurotransmitters present in this brain area to ultimately integrate behavioral and neuroendocrine responses [ 31 , 46 ] . in this complex scenario , peripheral organs , such as adrenal glands , such an extremely peripheral location provides most of the body with a remote station , in strict hormonal connection with some functionally integrated brain zones , to easily and efficaciously control the entire organisms . in addition and more importantly , these glands are morphologically ( and functionally ) endowed with a very high degree of plasticity leading to profound endocrine and behavioral modifications , and both short - term changes in behavioral reactivity at the individual level and changes in mouse population structure ( e.g. , major behavioral changes in reactivity to social stimulation ) could be expected [ 43 , 5659 ] . thus , ngf appears to play a role in stress - mediated changes in behavioral responses , leading to imprinting - like phenomena at adulthood , in which such social stimuli suddenly become relevant and produced long - lasting behavioral alterations . in particular , it could be hypothesized that variations in the hypothalamic ngf , caused by psychosocial stressor , and the related alterations in emotionality , could be functional to the development of proper strategies to cope with the stressor itself and thus to survive . ngf could shift some still unknown brain zones backwards to an immature - like stage [ 46 , 49 ] . belonging to the neuroendocrine system , the anterior pituitary gland represents an important relay station between the periphery and the cns under the control of hypothalamic hormones and peripheral signals . the integration operated by this gland determines the extent of hormonal secretion in different physiological situation and ngf and bdnf appear to play a neurotrophic role on the pituitary and stimulate synthesis of neuropeptides . the first evidence of such a role for nts came from studies suggesting that , following intravenous injection , ngf increased the secretion of adrenocorticotropic hormone ( acth ) as well as the concentration of gcs , and that following a psychosocial stress , a massive release of ngf occurred in the peripheral circulation , associated with increased hypothalamic ngf mrna and protein levels [ 43 , 60 , 61 ] . ngf is synthesized in the granular convoluted tubules within the submandibular glands soon after puberty ( much more in male than in female subjects ) [ 5 , 13 ] . the functional significance of the large amounts of ngf observed in the mouse submandibular glands is not fully elucidated ; however it has been shown to be released into the bloodstream as a result of intraspecific aggressive behavior acting on nerve , chromaffin , mast cells , and lymphocytes [ 43 , 61 , 62 ] . the adrenal glands are intrinsically characterized by a high degree of plasticity from a morphological ( and functional ) point of view . their size varies according to short- and medium - term life stressful events , and such a morpho - functional change is mirrored by subtle and rapid central modification in the expression of the hippocampal glucocorticoids receptor ( gr ) system , in the excitability of hippocampal neurons , neurogenesis of the dentate gyrus , synaptogenesis in the ca1 region , and dendritic remodeling in the ca3 region , just to mention few . ngf appears as key player in the ontogeny of adrenal glands as its administration induces the transformation of chromaffin cells in sympathetic nerve cells in the adrenal medulla . by contrast , injections of ngf antiserum ( from prenatal day 17 to postnatal day 10 in rats ) inhibit adrenal development and differentiation through a massive destruction of chromaffin cell precursors overall showing the main role played by ngf in critical developmental stages [ 37 , 38 ] . thus adrenal glands can be viewed as representing the last pair of vertebral ganglia . during early ( in altricial rodents late - gestational ) developmental stages , thanks to a complex interaction of neurohormonal factors ( including gcs and ngf , as reported just below ) , they do not fully differentiate as adult - like ganglia but maintain several fetal - type characteristics . therefore , their enhanced morphofunctional plasticity is maintained through adulthood , rendering them a remote ( control ) neurohormonal station involved in a continuous dialog with the brain for the maintenance of body homeostasis [ 37 , 38 ] . a large body of evidence suggests a link between adrenal size modification and coping strategies to face stressful events which might underlie the association often found for a pathologic increase in corticosteroid hormones and vulnerability to psychiatric disorders . since the adrenal gland appears to be one of the biological targets of ngf , one way this growth factor could exert a physiologic role is through its action on this structure . ngf administered for 610 consecutive days results in a dose - dependent increase in adrenal weight and volume by enlarging both the adrenal medulla and the cortex [ 43 , 49 ] . at present , it is not clear whether adrenal cortex hypertrophy following ngf administration in adult mice is the result of a direct action of this nt on adrenal cortex cells or whether it is mediated through its effects on acth release . interestingly , it has been shown that conditions exist in which adrenal activity is uncoupled from acth , such as during starvation , and this suggests a direct effect of ngf on the adrenals under specific conditions . furthermore , in animal models characterized by metabolic disorders involving dysregulation of the hpa axis , such as type i diabetes , corticosterone hypersecretion appears to occur independently from a surge in acth levels [ 38 , 66 ] . ngf might also contribute as an alternative mechanism in inducing such rapid changes in adrenal sensitivity . changes in adrenal hormone secretion ( e.g. , increased corticosterone levels ) resulting from an increase in ngf circulating levels could exert important effects on aggressive behavior : gcs enhance aggressive behavior in a context - dependent fashion , promoting social challenge - induced aggression without increasing aggressiveness under routine conditions in rats [ 67 , 68 ] . indeed , at present , there is no evidence of direct ngf effects on neural substrates of aggressive behavior , and the high molecular weight of the ngf protein seems to exclude the possibility that it could cross the blood - brain barrier , although a possible breakage of the ngf protein into fragments acting on the cns can be postulated . from a clinical application point of view , it appears interesting to point out that successful transport of biologically active ngf across the blood - brain barrier has been achieved by covalently linking it to an antitransferrin receptor antibody or by ocular delivery [ 38 , 70 , 71 ] . likewise , aloe and alleva , working in the team of levi - montalcini , analyzed all the evidences collected up to that point on the physiological activities exerted by ngf on cns and non - cns neurons . in those years , starting with mast cells [ 72 , 73 ] , and much later memory b lymphocytes , evidences were provided for a role of ngf on psychoneuroimmune regulation [ 31 , 46 ] . most of the studies on ngf were originally performed by rita - levi montalcini on the sympathetic ganglia explanted from chick embryos [ 5 , 75 ] , leading to an initial fascinating but incomplete vision of ngf as a protein molecule exerting specific although limited effects on peripheral sympathetic neurons . further studies , carried out , rather independently by barde and thoenen , and mobley , characterized ngf effects on cholinergic neurons at the cns level , opening the way to several lines of research aimed at understanding ngf physiological roles in the brain [ 7 , 12 , 76 ] . ngf is essential for the development and maintenance of sensory neurons , and for the formation of central pain circuitry , exogenous administration of this neurotrophin to rodents resulting in the rapid onset of hyperalgesia . together with its receptors ( p75 and trka ) , it plays a critical trophic role on forebrain cholinergic neurons ( fcns ) that degenerate during brain aging and neurodegenerative disorders [ 77 , 78 ] . during development , both ngf and bdnf regulate naturally occurring cell death , synaptic connectivity , fiber guidance , and dendritic morphology . furthermore , they participate to brain plasticity , being involved in activity - dependent neuronal functions [ 34 , 7981 ] . during early postnatal development , especially during critical periods , large changes in the connectivity and organization of neural networks take place [ 82 , 83 ] . in this period , the developing cns is particularly sensitive to external stimuli , and ngf and bdnf play a key role in modulating brain plasticity to better cope with environmental stimuli . as an example , mice reared in a communal nest ( cn , which consists in a single nest where three mothers keep their pups together and share care - giving behavior from birth to weaning ) are characterized by increased ngf and bdnf levels in selected brain areas , including hippocampus and hypothalamus , and prolonged survival of newly generated cells in the hippocampus . these features possibly underly the higher propensity of cn mice to interact socially and their better social skills when compared to subjects reared in standard laboratory conditions [ 84 , 85 ] . the adult brain still shows significant plasticity : for example , repeated agonistic intermale fighting in aged mouse selectively affects neurotrophin production . in particular , the social stress of being subordinate is able to increase ngf levels in the subventricular zone ( svz ) and hippocampus ( a brain area involved in learning and memory processes ) leading to the hypothesis of a regulatory role of ngf in the emotional status caused by psychosocial stressors and the physiological needs of the organism to remember the events leading to an appropriate coping with the stressor itself [ 41 , 47 ] . moreover , aggressive behavior can enhance the number of ki67-positive cells in the svz of aged animals . this suggests that , in the aged mouse , fighting behavior may increase neurogenesis , most probably throughout proliferation and/or differentiation of brain stem cells , possibly contributing to reduce the neuronal damage and loss caused by prolonged gc exposure as a consequence of the social stress related to fighting [ 87 , 88 ] . in addition , within the brain , the major increase in ngf following fighting has been observed in the hypothalamus a brain area involved in the activation of certain behaviors and in physiological modifications inducing changes in bodily homeostasis , suggesting a role for ngf in coping and neuroendocrine mechanisms . a correlation between changes in ngf levels and anxiety - like behaviors has been observed in humans . in particular , ngf levels were found to be increased in the blood of young soldiers experiencing their first parachute jump , implying that the release of ngf in the bloodstream is triggered by the stress of the novel and highly arousing experience . the key role of ngf in anxiety conditions is also suggested by findings demonstrating that alcohol or heroin withdrawal in human is also associated to changes in blood ngf levels . chronicity of stress system activation leads to the syndromal state that in 1936 selye described as the general adaptation syndrome . when facing a stressful situation , all physiological changes , related to coping strategies , lead both to protective and damaging effects on the body . in particular , on a short run they are essential for adaptation , maintenance of homeostasis , and survival ( allostasis is maintaining stability through changes ) . yet , over longer time intervals , they impose a cost ( allostatic load ) that can accelerate disease processes or participate to pathological changes possibly contributing to the development of neurodegenerative disorders and/or to precipitate psychopathological conditions . ngf , both in the bloodstream and in certain brain areas , is differentially expressed following environmental stressors . a large number of evidence show that chronic stress can lead to anxiety- and depression - like behaviors and may influence the distribution of ngf , both in animal models and in humans [ 46 , 92 ] . reduced ngf and bdnf signaling in the adult brain may be involved in the pathophysiology of psychiatric disorders , such as depression [ 38 , 81 , 9396 ] , and a role for ngf has been proposed in the etiopathogenesis of schizophrenia . nts themselves do not directly affect mood but rather play an important functional role in the modulation of networks determining how plastic changes influences mood . in fact , it has been hypothesized that , by activating nts systems , successful antidepressant treatments and cotreatments promote activity - dependent neuronal plasticity , possibly inducing proliferative or survival effects on neural stem cells . because of the fundamental role played by these neurotrophic factors in shaping brain function , pathological alterations in their concentration or action early during development could exert long - term effects on synaptic plasticity , impairing the ability of the organism to cope with novel / stressful situations , leading to psychopathology . during development , the expression of ngf and bdnf has been localized to the hippocampus and prefrontal cortex , two regions which are well - studied sites of both developmental and adult synaptic plasticity and playing a key role in psychiatric disorders [ 15 , 99 ] . stress during prenatal and early postnatal life may result in altered brain development and lead to a persistent sensitization of limbic circuits to even mild stress at adulthood , forming the basis for a greater susceptibility for mood and anxiety disorders ( levine , 1967 no.49 ; maccari , 2003 no.117 ) . numerous studies performed in rodents have indicated that nts are sensitive to the stress of maternal separation and to changes in the rearing conditions and that environmental stimulation can have both short- and long - term effects on nts levels [ 84 , 100107 ] . it is possible to hypothesize that while milder manipulations could promote neural plasticity , chronic stressful conditions could sensitize limbic structures to stress , decreasing brain plasticity and leading to higher susceptibility to psychopathology [ 102 , 108 ] . as an example , in rodents , separating mother and infant for brief periods results in increased ngf expression in the hippocampus , cerebral cortex , and hypothalamus in a time - dependent manner [ 101 , 104 ] while longer periods of maternal separation ( 24 h ) also result in increased rate of cell death in the neocortex , white matter , and granule cells of the dentate gyrus in 12-day - old rats . following chronic communal nesting increased bdnf levels in association with reduced neurogenesis and increased depression - like behavior have been also found in cd-1 mice . epidemiological studies indicate that anxiety and mood disorders are characterized by sex differences in prevalence , presentation or therapeutic outcomes . a recent study suggests that epigenetic changes , presumably in the male germ cells , might alter the exposure to the hormones that mediate brain masculinization , providing insight into the heritability and pathophysiology of sex - biased neurodevelopmental disorders . in addition , a very interesting work from cirulli and collaborators provides evidence for a specific role of nts in such a gender - specific vulnerability . in particular , bdnf and ngf were identified as neuroendocrine markers underlying differential responses to maternal deprivation in males and females rhesus macaques . the selective changes in bdnf levels in females suggest a possible mechanism for the greater vulnerability to mood disorders of this gender as reported in humans . the importance of early life experiences in the etiology of psychiatric disorders has been now recognized ; however it has been often underemphasized . early life stressful events , such as childhood trauma and neglect , are associated with depression and anxiety disorders and sustained changes in the hpa axis [ 111113 ] . these associations strengthen the hypothesis that environmental factors during development could lead to long - term enduring changes in the set - point of the neuroendocrine system physiology and emotional behavior . changes in the levels of ngf in the cns appear also to play an important role in the context of neurodegenerative disorders such as alzheimer disease ( ad ) . the disruption of ngf gene in transgenic mice leads to a lethal phenotype ; thus only the study of phenotypic knockout of ngf at adulthood is possible . transgenic mice expressing a neutralizing anti - ngf recombinant antibody are characterized by an age - dependent neurodegenerative pathology including amyloid plaques , insoluble and hyperphosphorylated tau , and neurofibrillary tangles in cortical and hippocampal neurons . furthermore , they show an extensive neuronal loss throughout the cortex , cholinergic deficit in the basal forebrain in addition to behavioral deficits , overall being strikingly reminiscent of human ad . ngf precursor ( pro - ngf ) has been shown to be highly expressed in the brains of ad patients and to be neurotoxic when bound in a heterotrimer with the p75 and the receptor sortilin . interestingly , sortilin levels are increased in aged central and peripheral neurons , possibly rendering neurons more vulnerable to the age - related increases in pro - ngf . more recently , capsoni and coworkers postulated that neurodegeneration occurring in a transgenic mouse model of ad is provoked by an imbalance of prongf / ngf and , consequently , of trka / p75 signaling such that the inactivation of trka determines a strong cholinergic deficit and ad - like neurodegeneration . thus , overall , the ngf / trka signaling pathway has emerged as a promising therapeutic target for the ad pathology . in addition to a possible therapeutic role in the context of neurodegenerative disorders , chiaretti and coworkers investigated the role of ngf in regard to severe traumatic brain injury ( tbi ) in pediatric patients . tbi is the most common cause of death and acquired disability among children and young adults in developed countries and its clinical outcome depends both or the primary cerebral lesions and or the extent of secondary brain damage which involves neuroinflammatory mechanisms . it has been suggested that interleukin-6 might participate to the mechanisms of recovery from tbi through the modulation of ngf biosynthesis , elevated levels of this neurotrophin representing a reliable marker of good prognosis , at least in children . moreover , and more recently , the same authors proposed that ngf might represent an effective and safe adjunct therapy in patients ( children and adults ) with severe hypoxicischemic injury suggesting a neuroprotective mechanism exerted by ngf on the residual viable neurological pathways of these patients .

nerve growth factor ( ngf ) was initially studied for its role as a key player in the regulation of peripheral innervations . however , the successive finding of its release in the bloodstream of male mice following aggressive encounters and its presence in the central nervous system led to the hypothesis that variations in brain ngf levels , caused by psychosocial stressor , and the related alterations in emotionality , could be functional to the development of proper strategies to cope with the stressor itself and thus to survive . years later this vision is still relevant , and the body of evidence on the role of ngf has been strengthened and expanded from trophic factor playing a role in brain growth and differentiation to a much more complex messenger , involved in psychoneuroendocrine plasticity .

1. Introduction 2. Stress Renewed CNS and Adrenal Plasticity 3. The Role of NGF at the Peripheral Level 4. The Role of NGF at the CNS

at the beginning of the 1950s , the nobel laureate rita levi - montalcini discovered and characterized the nerve growth factor ( ngf ) as a key player in target - mediated regulation of peripheral innervations [ 1 , 2 ] . the pioneer studies performed by her and victor hamburger first showed that the nervous system requires an adequate supply of trophic factors ( neurotrophins nts ) from the environment for survival and development . in addition , and most intriguingly , these studies first raised the concept of cell death playing a pivotal role in this specific context . in particular , target tissues might determine the size and morphology of the peripheral innervations by regulating cell death among neurons and by making the nts available only in limited amounts , finally resulting in the selection only of those neurons which have established the quantitative and qualitative numbers of connections ( with the target tissue ) . subsequent studies have demonstrated that , during nervous system development , ngf is released by the target tissue , taken up in responsive neurons by receptor - mediated endocytosis and transported retrogradely to the cell body where it exerts its trophic / differentiative effects through its low ( p75ntr ) and high affinity receptors ( trka ) [ 37 ] . in particular , while the p75ntr can cause apoptosis in a variety of systems , when coexpressed with trka , it can modify their ligand - binding activity , dose - responsiveness , and kinase activity , leading to increased survival , neurite outgrowth , and synaptic plasticity [ 812 ] . since these first investigations on target - controlled neuronal survival , ngf has been one of the most thoroughly studied nts , regulating the survival , development , and trophism of specific neuronal populations in the peripheral and central nervous system ( cns ) . while in the periphery this nt was first recognized for its action on the sympathetic ganglia , in the adult brain the highest levels of ngf are found in hippocampus , cortex , and olfactory regions , which represent targets for basal forebrain cholinergic neurons [ 14 , 15 ] . ngf acts as a trophic factor for these neurons since its administration in vivo increases the levels of choline acetyltransferase [ 16 , 17 ] while rescuing from death basal forebrain neurons following transection of the septo - hippocampal pathways . in addition to the nervous system , nts exert their effects on various other tissue compartments [ 1926 ] . the largest amount of ngf is produced in the salivary glands of adult male mice , which are the largest and best available source of this nt ( smaller concentrations of this nt can be found in snake venom , guinea pig prostate , the seminal fluid of guinea pigs and bulls , in the human skin , and in numerous tissues and body fluids ) . furthermore , a number of other cells outside the nervous system , including epithelial cells , fibroblasts , lymphocytes , and activated macrophages , synthesize ngf [ 2729 ] . most neurobiologists , for a very long time , explained its presence in adult animals because of its ancestral physiological role during ontogeny . ngf was originally described as a protein exclusively acting on sympathetic peripheral neurons and only much later it was found to be also active on central nervous system neurons and on a variety of immune cell types . overall , the presence of ngf in those limbic areas of the cns involved in mood and cognition and in the orchestration of neuroendocrine responses and circadian activities , as well as in cells of the immune and endocrine system , indicates a much wider role for this nt than previously hypothesized and suggests that it might function as intercellular messenger or even a humoral factor to help regulate endocrine responses to stress [ 19 , 3135 ] . in complex organisms , stress responses involve a coordinated set of intercellular signals eventually resulting in the removal of the organism from , or adaptation to , the stressful situation . although the cascade of responses to acute stressful events originates at the cns level , peripheral structures , such as the adrenal glands , act in interplay and feedback on the brain to maintain body homeostasis [ 37 , 38 ] . the adaptive plasticity of chronic stress involves many mediators , including gcs , excitatory amino acids , and nts , such as ngf and also brain - derived neurotrophic factor ( bdnf ) . these latter can participate in the hpa axis response to stressful stimuli at different anatomical levels [ 38 , 40 ] . in the wild , adult male mice exhibit high levels of intraspecific aggressive behavior towards conspecifics mainly related to territorial defense . in the laboratory setting , the agonistic behavior of male mice can be elicited after 46 weeks of social isolation and represents a psychosocial stressful condition that has been shown to markedly alter ngf and bdnf levels both in plasma as well as in selected brain areas , including the hypothalamus and hippocampus [ 41 , 42 ] . in the mouse , ngf is mainly synthesized and released from the submaxillary salivary glands [ 5 , 43 ] . neurobehavioral studies demonstrated that both male intraspecific fighting and lactating females nest - defense , raised against a potentially infanticidal conspecific male , induce a massive release of ngf from salivary glands into the bloodstream of adults . in particular , circulating levels of ngf are highly correlated with the number of fighting episodes and most intriguingly to the social status achieved in fighting mice . , a subordinate status ) were characterized by a two - time increase in the amount of circulating ngf compared to attacking mice that achieved a dominant status [ 44 , 45 ] . following fighting behavior , adrenal weight increases quite markedly and quickly in male mice suggesting that social / aggressive behavior might control circulating ngf release from salivary glands and that these might in turn control adrenal morphology as well as adrenal functional status [ 47 , 48 ] . this hypothesis has been supported by data showing that exogenous ngf administration ( i.p . the higher ngf release and the hypertrophy of the adrenals , occurring in subordinates male mice , suggest a regulative loop involving ngf - mediated increase of glucocorticoids secretion ( upon adrenals ) acting to enhance a submissive profile ( figure 1 ) [ 46 , 47 , 49 ] . in particular , intermale aggressive behavior induces a large increase in ngf mrna and protein in hypothalamus , especially in the paraventricular nucleus , with no measurable changes in cerebral cortex , hippocampus , and cerebellum . alleva and aloe hypothesized that the rather rapid increase in levels of brain ngf , which follows a psychosocial stressful event , could allow some renewal of brain plasticity at adulthood . another possibility is that hypothalamic ngf might affect levels of other peptides or hormones present in the hypothalamus [ 5155 ] . as hypothalamus is involved in the maintenance of physiological homeostasis , it is likely that hypothalamic ngf affects , or cooperates with , hormones and/or neurotransmitters present in this brain area to ultimately integrate behavioral and neuroendocrine responses [ 31 , 46 ] . in this complex scenario , peripheral organs , such as adrenal glands , such an extremely peripheral location provides most of the body with a remote station , in strict hormonal connection with some functionally integrated brain zones , to easily and efficaciously control the entire organisms . thus , ngf appears to play a role in stress - mediated changes in behavioral responses , leading to imprinting - like phenomena at adulthood , in which such social stimuli suddenly become relevant and produced long - lasting behavioral alterations . in particular , it could be hypothesized that variations in the hypothalamic ngf , caused by psychosocial stressor , and the related alterations in emotionality , could be functional to the development of proper strategies to cope with the stressor itself and thus to survive . belonging to the neuroendocrine system , the anterior pituitary gland represents an important relay station between the periphery and the cns under the control of hypothalamic hormones and peripheral signals . the integration operated by this gland determines the extent of hormonal secretion in different physiological situation and ngf and bdnf appear to play a neurotrophic role on the pituitary and stimulate synthesis of neuropeptides . the first evidence of such a role for nts came from studies suggesting that , following intravenous injection , ngf increased the secretion of adrenocorticotropic hormone ( acth ) as well as the concentration of gcs , and that following a psychosocial stress , a massive release of ngf occurred in the peripheral circulation , associated with increased hypothalamic ngf mrna and protein levels [ 43 , 60 , 61 ] . ngf is synthesized in the granular convoluted tubules within the submandibular glands soon after puberty ( much more in male than in female subjects ) [ 5 , 13 ] . the functional significance of the large amounts of ngf observed in the mouse submandibular glands is not fully elucidated ; however it has been shown to be released into the bloodstream as a result of intraspecific aggressive behavior acting on nerve , chromaffin , mast cells , and lymphocytes [ 43 , 61 , 62 ] . their size varies according to short- and medium - term life stressful events , and such a morpho - functional change is mirrored by subtle and rapid central modification in the expression of the hippocampal glucocorticoids receptor ( gr ) system , in the excitability of hippocampal neurons , neurogenesis of the dentate gyrus , synaptogenesis in the ca1 region , and dendritic remodeling in the ca3 region , just to mention few . ngf appears as key player in the ontogeny of adrenal glands as its administration induces the transformation of chromaffin cells in sympathetic nerve cells in the adrenal medulla . by contrast , injections of ngf antiserum ( from prenatal day 17 to postnatal day 10 in rats ) inhibit adrenal development and differentiation through a massive destruction of chromaffin cell precursors overall showing the main role played by ngf in critical developmental stages [ 37 , 38 ] . therefore , their enhanced morphofunctional plasticity is maintained through adulthood , rendering them a remote ( control ) neurohormonal station involved in a continuous dialog with the brain for the maintenance of body homeostasis [ 37 , 38 ] . a large body of evidence suggests a link between adrenal size modification and coping strategies to face stressful events which might underlie the association often found for a pathologic increase in corticosteroid hormones and vulnerability to psychiatric disorders . since the adrenal gland appears to be one of the biological targets of ngf , one way this growth factor could exert a physiologic role is through its action on this structure . interestingly , it has been shown that conditions exist in which adrenal activity is uncoupled from acth , such as during starvation , and this suggests a direct effect of ngf on the adrenals under specific conditions . indeed , at present , there is no evidence of direct ngf effects on neural substrates of aggressive behavior , and the high molecular weight of the ngf protein seems to exclude the possibility that it could cross the blood - brain barrier , although a possible breakage of the ngf protein into fragments acting on the cns can be postulated . likewise , aloe and alleva , working in the team of levi - montalcini , analyzed all the evidences collected up to that point on the physiological activities exerted by ngf on cns and non - cns neurons . in those years , starting with mast cells [ 72 , 73 ] , and much later memory b lymphocytes , evidences were provided for a role of ngf on psychoneuroimmune regulation [ 31 , 46 ] . most of the studies on ngf were originally performed by rita - levi montalcini on the sympathetic ganglia explanted from chick embryos [ 5 , 75 ] , leading to an initial fascinating but incomplete vision of ngf as a protein molecule exerting specific although limited effects on peripheral sympathetic neurons . further studies , carried out , rather independently by barde and thoenen , and mobley , characterized ngf effects on cholinergic neurons at the cns level , opening the way to several lines of research aimed at understanding ngf physiological roles in the brain [ 7 , 12 , 76 ] . ngf is essential for the development and maintenance of sensory neurons , and for the formation of central pain circuitry , exogenous administration of this neurotrophin to rodents resulting in the rapid onset of hyperalgesia . during development , both ngf and bdnf regulate naturally occurring cell death , synaptic connectivity , fiber guidance , and dendritic morphology . in this period , the developing cns is particularly sensitive to external stimuli , and ngf and bdnf play a key role in modulating brain plasticity to better cope with environmental stimuli . as an example , mice reared in a communal nest ( cn , which consists in a single nest where three mothers keep their pups together and share care - giving behavior from birth to weaning ) are characterized by increased ngf and bdnf levels in selected brain areas , including hippocampus and hypothalamus , and prolonged survival of newly generated cells in the hippocampus . in particular , the social stress of being subordinate is able to increase ngf levels in the subventricular zone ( svz ) and hippocampus ( a brain area involved in learning and memory processes ) leading to the hypothesis of a regulatory role of ngf in the emotional status caused by psychosocial stressors and the physiological needs of the organism to remember the events leading to an appropriate coping with the stressor itself [ 41 , 47 ] . moreover , aggressive behavior can enhance the number of ki67-positive cells in the svz of aged animals . this suggests that , in the aged mouse , fighting behavior may increase neurogenesis , most probably throughout proliferation and/or differentiation of brain stem cells , possibly contributing to reduce the neuronal damage and loss caused by prolonged gc exposure as a consequence of the social stress related to fighting [ 87 , 88 ] . in addition , within the brain , the major increase in ngf following fighting has been observed in the hypothalamus a brain area involved in the activation of certain behaviors and in physiological modifications inducing changes in bodily homeostasis , suggesting a role for ngf in coping and neuroendocrine mechanisms . a correlation between changes in ngf levels and anxiety - like behaviors has been observed in humans . in particular , ngf levels were found to be increased in the blood of young soldiers experiencing their first parachute jump , implying that the release of ngf in the bloodstream is triggered by the stress of the novel and highly arousing experience . the key role of ngf in anxiety conditions is also suggested by findings demonstrating that alcohol or heroin withdrawal in human is also associated to changes in blood ngf levels . when facing a stressful situation , all physiological changes , related to coping strategies , lead both to protective and damaging effects on the body . yet , over longer time intervals , they impose a cost ( allostatic load ) that can accelerate disease processes or participate to pathological changes possibly contributing to the development of neurodegenerative disorders and/or to precipitate psychopathological conditions . ngf , both in the bloodstream and in certain brain areas , is differentially expressed following environmental stressors . a large number of evidence show that chronic stress can lead to anxiety- and depression - like behaviors and may influence the distribution of ngf , both in animal models and in humans [ 46 , 92 ] . reduced ngf and bdnf signaling in the adult brain may be involved in the pathophysiology of psychiatric disorders , such as depression [ 38 , 81 , 9396 ] , and a role for ngf has been proposed in the etiopathogenesis of schizophrenia . nts themselves do not directly affect mood but rather play an important functional role in the modulation of networks determining how plastic changes influences mood . in fact , it has been hypothesized that , by activating nts systems , successful antidepressant treatments and cotreatments promote activity - dependent neuronal plasticity , possibly inducing proliferative or survival effects on neural stem cells . because of the fundamental role played by these neurotrophic factors in shaping brain function , pathological alterations in their concentration or action early during development could exert long - term effects on synaptic plasticity , impairing the ability of the organism to cope with novel / stressful situations , leading to psychopathology . during development , the expression of ngf and bdnf has been localized to the hippocampus and prefrontal cortex , two regions which are well - studied sites of both developmental and adult synaptic plasticity and playing a key role in psychiatric disorders [ 15 , 99 ] . numerous studies performed in rodents have indicated that nts are sensitive to the stress of maternal separation and to changes in the rearing conditions and that environmental stimulation can have both short- and long - term effects on nts levels [ 84 , 100107 ] . as an example , in rodents , separating mother and infant for brief periods results in increased ngf expression in the hippocampus , cerebral cortex , and hypothalamus in a time - dependent manner [ 101 , 104 ] while longer periods of maternal separation ( 24 h ) also result in increased rate of cell death in the neocortex , white matter , and granule cells of the dentate gyrus in 12-day - old rats . a recent study suggests that epigenetic changes , presumably in the male germ cells , might alter the exposure to the hormones that mediate brain masculinization , providing insight into the heritability and pathophysiology of sex - biased neurodevelopmental disorders . the importance of early life experiences in the etiology of psychiatric disorders has been now recognized ; however it has been often underemphasized . these associations strengthen the hypothesis that environmental factors during development could lead to long - term enduring changes in the set - point of the neuroendocrine system physiology and emotional behavior . changes in the levels of ngf in the cns appear also to play an important role in the context of neurodegenerative disorders such as alzheimer disease ( ad ) . the disruption of ngf gene in transgenic mice leads to a lethal phenotype ; thus only the study of phenotypic knockout of ngf at adulthood is possible . transgenic mice expressing a neutralizing anti - ngf recombinant antibody are characterized by an age - dependent neurodegenerative pathology including amyloid plaques , insoluble and hyperphosphorylated tau , and neurofibrillary tangles in cortical and hippocampal neurons . ngf precursor ( pro - ngf ) has been shown to be highly expressed in the brains of ad patients and to be neurotoxic when bound in a heterotrimer with the p75 and the receptor sortilin . thus , overall , the ngf / trka signaling pathway has emerged as a promising therapeutic target for the ad pathology . in addition to a possible therapeutic role in the context of neurodegenerative disorders , chiaretti and coworkers investigated the role of ngf in regard to severe traumatic brain injury ( tbi ) in pediatric patients . tbi is the most common cause of death and acquired disability among children and young adults in developed countries and its clinical outcome depends both or the primary cerebral lesions and or the extent of secondary brain damage which involves neuroinflammatory mechanisms . it has been suggested that interleukin-6 might participate to the mechanisms of recovery from tbi through the modulation of ngf biosynthesis , elevated levels of this neurotrophin representing a reliable marker of good prognosis , at least in children . moreover , and more recently , the same authors proposed that ngf might represent an effective and safe adjunct therapy in patients ( children and adults ) with severe hypoxicischemic injury suggesting a neuroprotective mechanism exerted by ngf on the residual viable neurological pathways of these patients .

the reported prevalence in the general population is 1%-9% in autopsy series1,2,3,4 and 0.5%-2% in imaging studies.4,5 this wide range in reported prevalence may be attributable to variations in evaluation methods and demographics . the diagnostic accuracy for unruptured intracranial aneurysms has increased6 following the recent advent of non - invasive vascular imaging tools such as multi - channel computed tomography angiography and magnetic resonance angiography ( mra ) , and incidental asymptomatic aneurysms have come to represent a substantial clinical burden on account of their controversial natural history . at our institution , a head mra is performed for not only patients with ischemic stroke or a high number of cerebrovascular risk factors but also preoperative evaluation of patients with a planned major cardiac or transplantation surgery . head mra has also recently become a component of routine health check - ups in selected cases when concerns regarding cerebrovascular disease are present . on several occasions , we were able to identify various cerebrovascular lesions such as steno - occlusive lesions . therefore , the aim of this study was to describe the incidental intracranial saccular aneurysms detected on mra performed at our institution over a 5-year period . we report the prevalence , anatomical location , size , and clinical follow - up findings of intracranial saccular aneurysms , as well as the gender and age of patients with incidental intracranial saccular aneurysms . a search of the radiology report database identified 19,171 sets of mra examinations from 18,237 patients that were obtained between january 2001 and december 2005 . this period was selected so that at least 5 years of follow - up data could be available for all patients . mra was performed to detect various cerebrovascular diseases , to evaluate nonspecific cerebrovascular symptoms such as dizziness or dementia , as part of a standard health check - up , or as part of a preoperative evaluation in patients with planned major cardiac or transplantation surgery . patients with incidentally detected aneurysms were identified from the radiology reports ( figure 1 ) . patients with known intracranial aneurysms prior to the mra examination and those with clear dissecting aneurysms or fusiform aneurysms were excluded . patients with subarachnoid hemorrhage detected at the time of magnetic resonance ( mr ) imaging were also excluded . patient data including age , gender , symptoms , clinical treatment course , and follow - up results were obtained from medical records . mr images were acquired on one of six 1.5-t mr scanners ( 2 siemens magnetom avanto , erlangen , germany ; 1 ge signa cv / i , milwaukee , usa ; and 3 philips gyroscan intera , best , the netherlands ) . a ramped radiofrequency pulse and three - directional zero filling were used during data acquisition . mra data were acquired using the three - dimensional time - of - flight method focusing on the circle of willis with the following parameters : echo time , 7 ms ; repetition time , 25 ms ; flip angle , 20 ; one excitation ; field of view , 160 or 200 mm ; matrix size , 256512 ; voxel size , approximately 0.90.91.0 mm ; slab number , 4 . the first set of 12 maximum intensity projection images was generated by rotating around the sagittal axis at 15. for the second and third sets of maximum intensity projection images , sub - volumes that contained the right and left circulation , respectively , were selected and 12 maximum intensity projection images were generated for each by rotating around the sagittal axis at 15 to construct 24 images . in total , 36 images providing 12 stereoscopic images in three orthogonal directions were examined on the picture archiving and communication system for each patient . if more images were required due to the delicate delineation of the arterial structures , random image projections of volume rendering were created by reconstructing the source images using an advantage workstation ( ge , milwaukee , wi , usa ) . " aneurysm " was used as a wild - card search word to identify the initial candidate cases of incidental aneurysms from among the 19,171 sets of mra examinations contained in the radiology report database . two experienced neuroradiologists ( c - wr and hwp ) used mra images and medical reports to confirm the presence of an aneurysm and to quantify the size and location of the aneurysm . if there was a discrepancy between the original report and the reader 's interpretation , or if the presence of an aneurysm was equivocal , another neuroradiologist ( dhl ) reviewed the records in order to reach a consensus . false - positive cases , in which the radiology report appeared in the initial search results but no aneurysm was found upon re - examination of the mra images and medical records , were recorded and the cause of the false - positive was noted . aneurysm size was defined as the largest diagonal measurement seen on the maximum intensity projection images and classified as one of four categories modified from a previous study:7 < 2 mm , 2 - 7 mm , 7 - 12 mm , and 13 - 24 mm . aneurysm location was described according to the secondary artery arising from the parent artery and classified as : paraclinoid internal carotid artery ( ica ) , distal ica posterior wall , ica bifurcation , anterior communicating artery , anterior cerebral artery , middle cerebral artery ( mca ) trunk , mca bifurcation , distal vertebral artery , basilar trunk , basilar top , posterior cerebral artery , superior cerebellar artery , or posterior inferior cerebellar artery . paraclinoid ica aneurysms included aneurysms originating near the anterior genu of the cavernous ica or near the ophthalmic artery , and distal ica posterior wall aneurysms included ica aneurysms originating at the posterior communicating artery or the anterior choroidal artery . multiplicity was checked and aneurysm numbers were recorded . in patients with multiple aneurysms , one index aneurysm with the largest size was selected for prevalence calculations . patient age was categorized as 20 - 29 years , 30 - 39 years , 40 - 59 years , 60 - 79 years , 70 - 79 years , or 80 years . paired t - tests were performed to evaluate statistical differences in prevalence according to overall gender . linear regression analyses were performed to test the relation between prevalence and age in men and women . a search of the radiology report database identified 19,171 sets of mra examinations from 18,237 patients that were obtained between january 2001 and december 2005 . this period was selected so that at least 5 years of follow - up data could be available for all patients . mra was performed to detect various cerebrovascular diseases , to evaluate nonspecific cerebrovascular symptoms such as dizziness or dementia , as part of a standard health check - up , or as part of a preoperative evaluation in patients with planned major cardiac or transplantation surgery . patients with incidentally detected aneurysms were identified from the radiology reports ( figure 1 ) . patients with known intracranial aneurysms prior to the mra examination and those with clear dissecting aneurysms or fusiform aneurysms were excluded . patients with subarachnoid hemorrhage detected at the time of magnetic resonance ( mr ) imaging were also excluded . patient data including age , gender , symptoms , clinical treatment course , and follow - up results were obtained from medical records . mr images were acquired on one of six 1.5-t mr scanners ( 2 siemens magnetom avanto , erlangen , germany ; 1 ge signa cv / i , milwaukee , usa ; and 3 philips gyroscan intera , best , the netherlands ) . a ramped radiofrequency pulse and three - directional zero filling were used during data acquisition . mra data were acquired using the three - dimensional time - of - flight method focusing on the circle of willis with the following parameters : echo time , 7 ms ; repetition time , 25 ms ; flip angle , 20 ; one excitation ; field of view , 160 or 200 mm ; matrix size , 256512 ; voxel size , approximately 0.90.91.0 mm ; slab number , 4 . the first set of 12 maximum intensity projection images was generated by rotating around the sagittal axis at 15. for the second and third sets of maximum intensity projection images , sub - volumes that contained the right and left circulation , respectively , were selected and 12 maximum intensity projection images were generated for each by rotating around the sagittal axis at 15 to construct 24 images . in total , 36 images providing 12 stereoscopic images in three orthogonal directions were examined on the picture archiving and communication system for each patient . if more images were required due to the delicate delineation of the arterial structures , random image projections of volume rendering were created by reconstructing the source images using an advantage workstation ( ge , milwaukee , wi , usa ) . " aneurysm " was used as a wild - card search word to identify the initial candidate cases of incidental aneurysms from among the 19,171 sets of mra examinations contained in the radiology report database . two experienced neuroradiologists ( c - wr and hwp ) used mra images and medical reports to confirm the presence of an aneurysm and to quantify the size and location of the aneurysm . if there was a discrepancy between the original report and the reader 's interpretation , or if the presence of an aneurysm was equivocal , another neuroradiologist ( dhl ) reviewed the records in order to reach a consensus . false - positive cases , in which the radiology report appeared in the initial search results but no aneurysm was found upon re - examination of the mra images and medical records , were recorded and the cause of the false - positive was noted . aneurysm size was defined as the largest diagonal measurement seen on the maximum intensity projection images and classified as one of four categories modified from a previous study:7 < 2 mm , 2 - 7 mm , 7 - 12 mm , and 13 - 24 mm . aneurysm location was described according to the secondary artery arising from the parent artery and classified as : paraclinoid internal carotid artery ( ica ) , distal ica posterior wall , ica bifurcation , anterior communicating artery , anterior cerebral artery , middle cerebral artery ( mca ) trunk , mca bifurcation , distal vertebral artery , basilar trunk , basilar top , posterior cerebral artery , superior cerebellar artery , or posterior inferior cerebellar artery . paraclinoid ica aneurysms included aneurysms originating near the anterior genu of the cavernous ica or near the ophthalmic artery , and distal ica posterior wall aneurysms included ica aneurysms originating at the posterior communicating artery or the anterior choroidal artery . multiplicity was checked and aneurysm numbers were recorded . in patients with multiple aneurysms , one index aneurysm with the largest size was selected for prevalence calculations . patient age was categorized as 20 - 29 years , 30 - 39 years , 40 - 59 years , 60 - 79 years , 70 - 79 years , or 80 years . paired t - tests were performed to evaluate statistical differences in prevalence according to overall gender . linear regression analyses were performed to test the relation between prevalence and age in men and women . of the 18,237 patients studied , 330 ( 115 men and 215 women ) had incidental aneurysms , resulting in a prevalence of 1.8% ( 95% confidence interval , 1.6%-2.0% ) . the median age of patients with incidental aneurysms was 63 years and the age range was 22 - 82 years . multiple aneurysms were found in 30 patients ( 9.1% ) , including 26 patients with 2 aneurysms each , 3 patients with 3 aneurysms each , and 1 patient with 4 aneurysms . of these patients with incidental aneurysms , those assessed as part of a health check - up ( n=165 ) had more frequent aneurysms than patients with a previous stroke history ( n=136 ) or cardiac risk ( n=29 ) . the prevalence of incidental aneurysms was significantly higher in women ( 215/8,112 ) than in men ( 115/10,125 ; p=0.02 ; table 1 ; figure 2 ) and nonsignificantly higher in men aged 70 - 79 years ( 28/1,444 ) than in men aged > 80 years ( 2/292 ; p= 0.30 ; table 1 ; figure 2 ) . among women , the prevalence of aneurysms was highest in women older than 80 years in a linear regression analysis ( p<0.01 ) . prevalence increased with age in women ( p<0.01 ) , but not in men ( p=0.30 ) . aneurysm size ranged from 1.5 mm to 13 mm , and the median size was 4.0 mm . most aneurysms ( 299/344 ; 86.9% ) were in the 2 - 7 mm category . the most common aneurysm location was the bifurcation of the mca ( 131/366 , 35.8% ) and the next most common location was the anterior communicating artery ( 72/366 , 19.7% ; table 2 ) . other frequent sites were the paraclinoid ica ( 51/366 , 13.9% ) , distal ica posterior wall ( 35/366 , 9.6% ) , basilar top ( 22/366 , 6.0% ) , and mca trunk ( 16/366 , 4.5% ; table 2 ) . the bifurcation of the mca was the most common location in both men and women ( table 2 ) . there were 2 paraclinoid aneurysms in patients aged 20 - 29 years , and 2 mca bifurcation aneurysms and 1 anterior communicating artery aneurysm in patients aged 30 - 39 years . in patients aged 40 - 49 years , the paraclinoid ica was the most common site ( n=10 ) followed by the anterior communicating artery ( n=7 ) , and bifurcation of the mca ( n = 6 ) . however , in patients aged 50 - 59 years , the mca bifurcation was the most common site ( n=23 ) followed by the anterior communicating artery ( n=13 ) and paraclinoid ica ( n=10 ) . a similar tendency was observed in patients aged 60 - 69 years ( mca bifurcation , 49 ; anterior communicating artery , 22 ; paraclinoid ica , 15 ) and those aged 70 - 79 years ( mca bifurcation , 34 ; anterior communicating artery , 21 ; paraclinoid ica , 8) . among patients aged 80 years , there were 3 mca bifurcation aneurysms , 3 distal ica posterior wall aneurysms , and 3 basilar top aneurysms . ninety false - positive cases , in which the radiology report contained the term " aneurysm " in the initial search results but no aneurysm was found on re - interpretation of the mra images , were recorded . this high rate of false positives was probably due to cases that had ' rule out aneurysm ' noted in the radiological report being included in the initial search results . in some of the cases , it was difficult to determine whether the lesion was an aneurysm even after a third neuroradiologist was consulted , mainly because of image resolution or image artifacts . the most common sites for false positives were the anterior communicating artery ( n=21 ) followed by the mca bifurcation ( n=20 ) and the posterior communicating artery ( n=14 ) . the causes of false positives were junctional dilatation ( n=35 ) , vascular ectasia ( n=24 ) , and broad - based focal bulging ( n=14 ) , which were all difficult to differentiate from mra artifacts and vascular variations such as fenestration ( n=17 ) . the duration of clinical follow - up ranged from 94 days to 2,641 days , and the median follow - up duration was 1,256 days , which was shorter than we intended . this was due to our inability to strictly follow up these patients . during the follow - up period , 43 of the 330 patients ( 13.0% ) were treated by clipping ( n=32 ) or coiling ( n=11 ) . the treatment decision was made at various time points after the initial diagnosis of the aneurysm . follow - up imaging was performed in 72 of the 287 untreated patients ( 25.1% ; computed tomography angiography in 38 , mra in 29 , and digital subtraction angiography in 5 ) . follow - up imaging showed enlargement of the aneurysm in 6 of the 72 patients ( 8.3% ) . two of these were mca bifurcation aneurysms , 2 were anterior communicating artery aneurysms , 1 was a distal ica posterior wall aneurysm , and 1 was a basilar top aneurysm . the initial mra showed a 9-mm aneurysm at the anterior cerebral artery a2 - 3 junction . subsequent computed tomography angiography showed a broad - neck aneurysm with a height of 4 mm . the patient was reluctant to undergo surgery and was followed with imaging at regular intervals . eight months after the initial diagnosis , the patient presented with a subarachnoid hemorrhage due to aneurysm rupture . the initial mra was obtained to evaluate a brainstem infarction and showed a 3.5-mm left mca bifurcation aneurysm . twenty - seven months after her initial diagnosis , while on antiplatelet therapy , the patient presented with a subarachnoid hemorrhage due to aneurysm rupture . of the 18,237 patients studied , 330 ( 115 men and 215 women ) had incidental aneurysms , resulting in a prevalence of 1.8% ( 95% confidence interval , 1.6%-2.0% ) . the median age of patients with incidental aneurysms was 63 years and the age range was 22 - 82 years . multiple aneurysms were found in 30 patients ( 9.1% ) , including 26 patients with 2 aneurysms each , 3 patients with 3 aneurysms each , and 1 patient with 4 aneurysms . of these patients with incidental aneurysms , those assessed as part of a health check - up ( n=165 ) had more frequent aneurysms than patients with a previous stroke history ( n=136 ) or cardiac risk ( n=29 ) . the prevalence of incidental aneurysms was significantly higher in women ( 215/8,112 ) than in men ( 115/10,125 ; p=0.02 ; table 1 ; figure 2 ) and nonsignificantly higher in men aged 70 - 79 years ( 28/1,444 ) than in men aged > 80 years ( 2/292 ; p= 0.30 ; table 1 ; figure 2 ) . among women , the prevalence of aneurysms was highest in women older than 80 years in a linear regression analysis ( p<0.01 ) . prevalence increased with age in women ( p<0.01 ) , but not in men ( p=0.30 ) . aneurysm size ranged from 1.5 mm to 13 mm , and the median size was 4.0 mm . most aneurysms ( 299/344 ; 86.9% ) were in the 2 - 7 mm category . the most common aneurysm location was the bifurcation of the mca ( 131/366 , 35.8% ) and the next most common location was the anterior communicating artery ( 72/366 , 19.7% ; table 2 ) . other frequent sites were the paraclinoid ica ( 51/366 , 13.9% ) , distal ica posterior wall ( 35/366 , 9.6% ) , basilar top ( 22/366 , 6.0% ) , and mca trunk ( 16/366 , 4.5% ; table 2 ) . the bifurcation of the mca was the most common location in both men and women ( table 2 ) . there were 2 paraclinoid aneurysms in patients aged 20 - 29 years , and 2 mca bifurcation aneurysms and 1 anterior communicating artery aneurysm in patients aged 30 - 39 years . in patients aged 40 - 49 years , the paraclinoid ica was the most common site ( n=10 ) followed by the anterior communicating artery ( n=7 ) , and bifurcation of the mca ( n = 6 ) . however , in patients aged 50 - 59 years , the mca bifurcation was the most common site ( n=23 ) followed by the anterior communicating artery ( n=13 ) and paraclinoid ica ( n=10 ) . a similar tendency was observed in patients aged 60 - 69 years ( mca bifurcation , 49 ; anterior communicating artery , 22 ; paraclinoid ica , 15 ) and those aged 70 - 79 years ( mca bifurcation , 34 ; anterior communicating artery , 21 ; paraclinoid ica , 8) . among patients aged 80 years , there were 3 mca bifurcation aneurysms , 3 distal ica posterior wall aneurysms , and 3 basilar top aneurysms . ninety false - positive cases , in which the radiology report contained the term " aneurysm " in the initial search results but no aneurysm was found on re - interpretation of the mra images , were recorded . this high rate of false positives was probably due to cases that had ' rule out aneurysm ' noted in the radiological report being included in the initial search results . in some of the cases , it was difficult to determine whether the lesion was an aneurysm even after a third neuroradiologist was consulted , mainly because of image resolution or image artifacts . the most common sites for false positives were the anterior communicating artery ( n=21 ) followed by the mca bifurcation ( n=20 ) and the posterior communicating artery ( n=14 ) . the causes of false positives were junctional dilatation ( n=35 ) , vascular ectasia ( n=24 ) , and broad - based focal bulging ( n=14 ) , which were all difficult to differentiate from mra artifacts and vascular variations such as fenestration ( n=17 ) . the duration of clinical follow - up ranged from 94 days to 2,641 days , and the median follow - up duration was 1,256 days , which was shorter than we intended . this was due to our inability to strictly follow up these patients . during the follow - up period , 43 of the 330 patients ( 13.0% ) were treated by clipping ( n=32 ) or coiling ( n=11 ) . the treatment decision was made at various time points after the initial diagnosis of the aneurysm . follow - up imaging was performed in 72 of the 287 untreated patients ( 25.1% ; computed tomography angiography in 38 , mra in 29 , and digital subtraction angiography in 5 ) . follow - up imaging showed enlargement of the aneurysm in 6 of the 72 patients ( 8.3% ) . two of these were mca bifurcation aneurysms , 2 were anterior communicating artery aneurysms , 1 was a distal ica posterior wall aneurysm , and 1 was a basilar top aneurysm . the initial mra showed a 9-mm aneurysm at the anterior cerebral artery a2 - 3 junction . subsequent computed tomography angiography showed a broad - neck aneurysm with a height of 4 mm . the patient was reluctant to undergo surgery and was followed with imaging at regular intervals . eight months after the initial diagnosis , the patient presented with a subarachnoid hemorrhage due to aneurysm rupture . the initial mra was obtained to evaluate a brainstem infarction and showed a 3.5-mm left mca bifurcation aneurysm . twenty - seven months after her initial diagnosis , while on antiplatelet therapy , the patient presented with a subarachnoid hemorrhage due to aneurysm rupture . with the advent of less invasive vascular imaging techniques , the incidental discovery of asymptomatic intracranial aneurysms is increasing.8 the clinical consequence and symptomatic conversion rate of these aneurysms is unknown and is a pressing clinical issue . there are many reports on the prevalence of unruptured saccular intracranial aneurysms.4,5,9,10 however , the prevalence varies significantly across reports , probably due to the diversity of study methods and/or ethnic differences among study populations . one angiography - based study in a large cohort of patients reported that the prevalence of asymptomatic intracranial aneurysms was 0.65%,9 and a recent meta - analysis reported a prevalence of 3.2%.4 a recent study evaluated the prevalence of unruptured intracranial aneurysms using 3-t mr imaging and reported a prevalence of 8.4%,6 which is substantially higher than previous reports.4,5 this can be explained by an increased rate of detection of aneurysms smaller than 3 mm due to the higher resolution of the 3-t mr system . we found a prevalence of 1.8% , which is somewhat lower than previous reports.4,10,11 there are several possible explanations for this difference . first , our study was not population - based and , although the patients were ethnically homogeneous , there may have been a bias in the study population . the lower rate might also be related to the low sensitivity of the 1.5-t mra , a possibility supported by the small number of cases with aneurysms measuring less than 2 mm . another important possible explanation is the existence of false - negative cases that were not included in the calculated prevalence . because we identified incidental aneurysms based only on cases returned during our initial search of the radiology report database , there may have been a significant number of false - negative cases that were not identified . in light of these limitations , we urge caution in interpreting our mra prevalence rate . however , our study represents the real - world experience of a single high - volume center with uniform non - invasive imaging that we believe can contribute to the overall view of prevalence , distribution of aneurysms in terms of sites , and age . our results confirmed a higher prevalence of intracranial aneurysms in women than in men.10 the prevalence of aneurysms in women increased with age , which is a well - known risk factor . one study hypothesized that this might be associated with the decrease in estrogen concentration and estrogen - receptor density that occurs in women during and after menopause.12 the prevalence of aneurysms in men older than 80 years was lower than that in men aged 70 - 79 years , although the difference was not statistically significant ( p=0.30 ) . aneurysm prevalence was highest in women older than 80 years , and was higher in this group than in women aged 70 - 79 years . this is probably attributable to the relatively small sample of men aged over 80 years , which is due to the increased number of deaths in this age group . a previous study demonstrated a gender difference in aneurysm location.13 however , there was no gender difference in aneurysm location in our study . although some studies report that the paraclinoid ica is the most common site for unruptured intracranial aneurysms,4,11,14 we found the most common site to be the mca bifurcation , and this is consistent with other previous studies.15,16,17,18 other frequent sites were the anterior communicating artery and the paraclinoid ica . before the age of 50 , the most frequent aneurysm location was the paraclinoid ica . however , the mca bifurcation became the main predilection site after the age of 50 . significant increases in the prevalence of mca bifurcation and anterior communicating artery aneurysms could be due to higher hemodynamic shear stress.19 most aneurysms were between 2 mm and 7 mm in size . aneurysms larger than 10 mm were rare , consistent with a previous report.9 aneurysm enlargement on follow - up imaging was present in 6 cases , and aneurysm rupture during follow - up occurred in 2 cases . if all patients had been left untreated , the incidence of symptomatic conversion may have been higher than 2.8% as the decision to treat an aneurysm may have been based on the clinical perception of a high - risk lesion . the causes of misclassification were junctional dilatation , vascular ectasia , broad - based focal bulging , and fenestration . this is consistent with a recent report on the false - positive causes of aneurysms on mra,20 and the causes of misclassification should be considered when analyzing mra findings , especially for anterior communicating artery aneurysms . on the distal ica many of the false positives in the current study could have been avoided with state - of - the - art mra techniques , as many of the instances of vascular ectasia and bulging contours were a consequence of poor image quality . limitations inherent to the study design limit the conclusions that can be drawn from our results . the prevalence of incidental aneurysms may have been underestimated because we were unable to include false - negative cases , and the use of 1.5-t mra data may have limited our ability to detect small aneurysms and thoroughly evaluate false - positive results due to limited resolution . in addition , the imaging follow - up data were limited because we could not strictly follow up these patients . we were therefore unable to obtain useful data on the natural history of incidental aneurysms . we conducted a retrospective review of 18,237 patients who underwent mra at our institution and found that the prevalence of unruptured intracranial aneurysms was 1.8% , which was significantly higher in women than in men . the prevalence increased with age in women , but not in men . before the age of 50 , however , the mca bifurcation became the main predilection site after the age of 50 . therefore , overall , the most common location for unruptured intracranial aneurysms was the bifurcation of the mca . there were a number of false - positive cases that appeared in our initial search , and the most common location for false - positive cases was the anterior communicating artery .

background and purposethe diagnostic accuracy for unruptured intracranial aneurysms has increased , and incidental asymptomatic aneurysms have come to represent a substantial clinical burden because of their controversial natural history . however , their prevalence may be attributable to variations in evaluation methods and demographics . we therefore describe the prevalence and magnetic resonance angiography ( mra ) findings of incidental intracranial saccular aneurysms over a 5-year period at a single large - volume center.methodsmra images from 18,237 patients obtained between january 2001 and december 2005 were retrieved from the radiology report database . patients diagnosed with incidental intracranial saccular aneurysms were identified and their mra data were reviewed . imaging and clinical follow - up data were evaluated.resultsduring the study period , 366 incidental intracranial saccular aneurysms were identified in 330 patients ( prevalence , 1.8% ; 95% confidence interval , 1.63%-2.01% ; 115 men and 215 women ; age range , 22 - 82 years ; median age , 63 years ) . the prevalence was higher in women ( 215/8,112 ) than in men ( 115/10,125 ; p=0.02 ) . the prevalence increased with age in women ( p<0.01 ) , but not in men ( p=0.30 ) . aneurysm size ranged from 1.5 mm to 13 mm , with a median size of 4 mm . the most common location was the bifurcation of the middle cerebral artery ( 131/366 ; 35.8%).conclusionsour real world experience indicated a slightly lower overall prevalence of incidental intracranial saccular aneurysms than previously reported . the prevalence increased with age in women but not in men .

Introduction Methods Patient selection Acquisition of MR imaging sequences Image interpretation Results Prevalence of incidental aneurysms Size and location of incidental aneurysms False-positive cases Follow-up results Discussion Conclusions

the reported prevalence in the general population is 1%-9% in autopsy series1,2,3,4 and 0.5%-2% in imaging studies.4,5 this wide range in reported prevalence may be attributable to variations in evaluation methods and demographics . the diagnostic accuracy for unruptured intracranial aneurysms has increased6 following the recent advent of non - invasive vascular imaging tools such as multi - channel computed tomography angiography and magnetic resonance angiography ( mra ) , and incidental asymptomatic aneurysms have come to represent a substantial clinical burden on account of their controversial natural history . therefore , the aim of this study was to describe the incidental intracranial saccular aneurysms detected on mra performed at our institution over a 5-year period . we report the prevalence , anatomical location , size , and clinical follow - up findings of intracranial saccular aneurysms , as well as the gender and age of patients with incidental intracranial saccular aneurysms . a search of the radiology report database identified 19,171 sets of mra examinations from 18,237 patients that were obtained between january 2001 and december 2005 . this period was selected so that at least 5 years of follow - up data could be available for all patients . patients with incidentally detected aneurysms were identified from the radiology reports ( figure 1 ) . patients with known intracranial aneurysms prior to the mra examination and those with clear dissecting aneurysms or fusiform aneurysms were excluded . patient data including age , gender , symptoms , clinical treatment course , and follow - up results were obtained from medical records . mra data were acquired using the three - dimensional time - of - flight method focusing on the circle of willis with the following parameters : echo time , 7 ms ; repetition time , 25 ms ; flip angle , 20 ; one excitation ; field of view , 160 or 200 mm ; matrix size , 256512 ; voxel size , approximately 0.90.91.0 mm ; slab number , 4 . aneurysm " was used as a wild - card search word to identify the initial candidate cases of incidental aneurysms from among the 19,171 sets of mra examinations contained in the radiology report database . false - positive cases , in which the radiology report appeared in the initial search results but no aneurysm was found upon re - examination of the mra images and medical records , were recorded and the cause of the false - positive was noted . aneurysm size was defined as the largest diagonal measurement seen on the maximum intensity projection images and classified as one of four categories modified from a previous study:7 < 2 mm , 2 - 7 mm , 7 - 12 mm , and 13 - 24 mm . aneurysm location was described according to the secondary artery arising from the parent artery and classified as : paraclinoid internal carotid artery ( ica ) , distal ica posterior wall , ica bifurcation , anterior communicating artery , anterior cerebral artery , middle cerebral artery ( mca ) trunk , mca bifurcation , distal vertebral artery , basilar trunk , basilar top , posterior cerebral artery , superior cerebellar artery , or posterior inferior cerebellar artery . paraclinoid ica aneurysms included aneurysms originating near the anterior genu of the cavernous ica or near the ophthalmic artery , and distal ica posterior wall aneurysms included ica aneurysms originating at the posterior communicating artery or the anterior choroidal artery . linear regression analyses were performed to test the relation between prevalence and age in men and women . a search of the radiology report database identified 19,171 sets of mra examinations from 18,237 patients that were obtained between january 2001 and december 2005 . this period was selected so that at least 5 years of follow - up data could be available for all patients . patients with incidentally detected aneurysms were identified from the radiology reports ( figure 1 ) . patients with known intracranial aneurysms prior to the mra examination and those with clear dissecting aneurysms or fusiform aneurysms were excluded . patient data including age , gender , symptoms , clinical treatment course , and follow - up results were obtained from medical records . mra data were acquired using the three - dimensional time - of - flight method focusing on the circle of willis with the following parameters : echo time , 7 ms ; repetition time , 25 ms ; flip angle , 20 ; one excitation ; field of view , 160 or 200 mm ; matrix size , 256512 ; voxel size , approximately 0.90.91.0 mm ; slab number , 4 . aneurysm " was used as a wild - card search word to identify the initial candidate cases of incidental aneurysms from among the 19,171 sets of mra examinations contained in the radiology report database . false - positive cases , in which the radiology report appeared in the initial search results but no aneurysm was found upon re - examination of the mra images and medical records , were recorded and the cause of the false - positive was noted . aneurysm size was defined as the largest diagonal measurement seen on the maximum intensity projection images and classified as one of four categories modified from a previous study:7 < 2 mm , 2 - 7 mm , 7 - 12 mm , and 13 - 24 mm . aneurysm location was described according to the secondary artery arising from the parent artery and classified as : paraclinoid internal carotid artery ( ica ) , distal ica posterior wall , ica bifurcation , anterior communicating artery , anterior cerebral artery , middle cerebral artery ( mca ) trunk , mca bifurcation , distal vertebral artery , basilar trunk , basilar top , posterior cerebral artery , superior cerebellar artery , or posterior inferior cerebellar artery . paraclinoid ica aneurysms included aneurysms originating near the anterior genu of the cavernous ica or near the ophthalmic artery , and distal ica posterior wall aneurysms included ica aneurysms originating at the posterior communicating artery or the anterior choroidal artery . linear regression analyses were performed to test the relation between prevalence and age in men and women . of the 18,237 patients studied , 330 ( 115 men and 215 women ) had incidental aneurysms , resulting in a prevalence of 1.8% ( 95% confidence interval , 1.6%-2.0% ) . the median age of patients with incidental aneurysms was 63 years and the age range was 22 - 82 years . multiple aneurysms were found in 30 patients ( 9.1% ) , including 26 patients with 2 aneurysms each , 3 patients with 3 aneurysms each , and 1 patient with 4 aneurysms . of these patients with incidental aneurysms , those assessed as part of a health check - up ( n=165 ) had more frequent aneurysms than patients with a previous stroke history ( n=136 ) or cardiac risk ( n=29 ) . the prevalence of incidental aneurysms was significantly higher in women ( 215/8,112 ) than in men ( 115/10,125 ; p=0.02 ; table 1 ; figure 2 ) and nonsignificantly higher in men aged 70 - 79 years ( 28/1,444 ) than in men aged > 80 years ( 2/292 ; p= 0.30 ; table 1 ; figure 2 ) . among women , the prevalence of aneurysms was highest in women older than 80 years in a linear regression analysis ( p<0.01 ) . prevalence increased with age in women ( p<0.01 ) , but not in men ( p=0.30 ) . aneurysm size ranged from 1.5 mm to 13 mm , and the median size was 4.0 mm . the most common aneurysm location was the bifurcation of the mca ( 131/366 , 35.8% ) and the next most common location was the anterior communicating artery ( 72/366 , 19.7% ; table 2 ) . the bifurcation of the mca was the most common location in both men and women ( table 2 ) . in patients aged 40 - 49 years , the paraclinoid ica was the most common site ( n=10 ) followed by the anterior communicating artery ( n=7 ) , and bifurcation of the mca ( n = 6 ) . however , in patients aged 50 - 59 years , the mca bifurcation was the most common site ( n=23 ) followed by the anterior communicating artery ( n=13 ) and paraclinoid ica ( n=10 ) . ninety false - positive cases , in which the radiology report contained the term " aneurysm " in the initial search results but no aneurysm was found on re - interpretation of the mra images , were recorded . in some of the cases , it was difficult to determine whether the lesion was an aneurysm even after a third neuroradiologist was consulted , mainly because of image resolution or image artifacts . the most common sites for false positives were the anterior communicating artery ( n=21 ) followed by the mca bifurcation ( n=20 ) and the posterior communicating artery ( n=14 ) . the duration of clinical follow - up ranged from 94 days to 2,641 days , and the median follow - up duration was 1,256 days , which was shorter than we intended . during the follow - up period , 43 of the 330 patients ( 13.0% ) were treated by clipping ( n=32 ) or coiling ( n=11 ) . follow - up imaging was performed in 72 of the 287 untreated patients ( 25.1% ; computed tomography angiography in 38 , mra in 29 , and digital subtraction angiography in 5 ) . follow - up imaging showed enlargement of the aneurysm in 6 of the 72 patients ( 8.3% ) . two of these were mca bifurcation aneurysms , 2 were anterior communicating artery aneurysms , 1 was a distal ica posterior wall aneurysm , and 1 was a basilar top aneurysm . subsequent computed tomography angiography showed a broad - neck aneurysm with a height of 4 mm . of the 18,237 patients studied , 330 ( 115 men and 215 women ) had incidental aneurysms , resulting in a prevalence of 1.8% ( 95% confidence interval , 1.6%-2.0% ) . the median age of patients with incidental aneurysms was 63 years and the age range was 22 - 82 years . multiple aneurysms were found in 30 patients ( 9.1% ) , including 26 patients with 2 aneurysms each , 3 patients with 3 aneurysms each , and 1 patient with 4 aneurysms . of these patients with incidental aneurysms , those assessed as part of a health check - up ( n=165 ) had more frequent aneurysms than patients with a previous stroke history ( n=136 ) or cardiac risk ( n=29 ) . the prevalence of incidental aneurysms was significantly higher in women ( 215/8,112 ) than in men ( 115/10,125 ; p=0.02 ; table 1 ; figure 2 ) and nonsignificantly higher in men aged 70 - 79 years ( 28/1,444 ) than in men aged > 80 years ( 2/292 ; p= 0.30 ; table 1 ; figure 2 ) . among women , the prevalence of aneurysms was highest in women older than 80 years in a linear regression analysis ( p<0.01 ) . prevalence increased with age in women ( p<0.01 ) , but not in men ( p=0.30 ) . aneurysm size ranged from 1.5 mm to 13 mm , and the median size was 4.0 mm . the most common aneurysm location was the bifurcation of the mca ( 131/366 , 35.8% ) and the next most common location was the anterior communicating artery ( 72/366 , 19.7% ; table 2 ) . other frequent sites were the paraclinoid ica ( 51/366 , 13.9% ) , distal ica posterior wall ( 35/366 , 9.6% ) , basilar top ( 22/366 , 6.0% ) , and mca trunk ( 16/366 , 4.5% ; table 2 ) . the bifurcation of the mca was the most common location in both men and women ( table 2 ) . in patients aged 40 - 49 years , the paraclinoid ica was the most common site ( n=10 ) followed by the anterior communicating artery ( n=7 ) , and bifurcation of the mca ( n = 6 ) . however , in patients aged 50 - 59 years , the mca bifurcation was the most common site ( n=23 ) followed by the anterior communicating artery ( n=13 ) and paraclinoid ica ( n=10 ) . ninety false - positive cases , in which the radiology report contained the term " aneurysm " in the initial search results but no aneurysm was found on re - interpretation of the mra images , were recorded . in some of the cases , it was difficult to determine whether the lesion was an aneurysm even after a third neuroradiologist was consulted , mainly because of image resolution or image artifacts . the most common sites for false positives were the anterior communicating artery ( n=21 ) followed by the mca bifurcation ( n=20 ) and the posterior communicating artery ( n=14 ) . the causes of false positives were junctional dilatation ( n=35 ) , vascular ectasia ( n=24 ) , and broad - based focal bulging ( n=14 ) , which were all difficult to differentiate from mra artifacts and vascular variations such as fenestration ( n=17 ) . the duration of clinical follow - up ranged from 94 days to 2,641 days , and the median follow - up duration was 1,256 days , which was shorter than we intended . during the follow - up period , 43 of the 330 patients ( 13.0% ) were treated by clipping ( n=32 ) or coiling ( n=11 ) . follow - up imaging was performed in 72 of the 287 untreated patients ( 25.1% ; computed tomography angiography in 38 , mra in 29 , and digital subtraction angiography in 5 ) . follow - up imaging showed enlargement of the aneurysm in 6 of the 72 patients ( 8.3% ) . two of these were mca bifurcation aneurysms , 2 were anterior communicating artery aneurysms , 1 was a distal ica posterior wall aneurysm , and 1 was a basilar top aneurysm . subsequent computed tomography angiography showed a broad - neck aneurysm with a height of 4 mm . there are many reports on the prevalence of unruptured saccular intracranial aneurysms.4,5,9,10 however , the prevalence varies significantly across reports , probably due to the diversity of study methods and/or ethnic differences among study populations . one angiography - based study in a large cohort of patients reported that the prevalence of asymptomatic intracranial aneurysms was 0.65%,9 and a recent meta - analysis reported a prevalence of 3.2%.4 a recent study evaluated the prevalence of unruptured intracranial aneurysms using 3-t mr imaging and reported a prevalence of 8.4%,6 which is substantially higher than previous reports.4,5 this can be explained by an increased rate of detection of aneurysms smaller than 3 mm due to the higher resolution of the 3-t mr system . we found a prevalence of 1.8% , which is somewhat lower than previous reports.4,10,11 there are several possible explanations for this difference . because we identified incidental aneurysms based only on cases returned during our initial search of the radiology report database , there may have been a significant number of false - negative cases that were not identified . however , our study represents the real - world experience of a single high - volume center with uniform non - invasive imaging that we believe can contribute to the overall view of prevalence , distribution of aneurysms in terms of sites , and age . our results confirmed a higher prevalence of intracranial aneurysms in women than in men.10 the prevalence of aneurysms in women increased with age , which is a well - known risk factor . one study hypothesized that this might be associated with the decrease in estrogen concentration and estrogen - receptor density that occurs in women during and after menopause.12 the prevalence of aneurysms in men older than 80 years was lower than that in men aged 70 - 79 years , although the difference was not statistically significant ( p=0.30 ) . aneurysm prevalence was highest in women older than 80 years , and was higher in this group than in women aged 70 - 79 years . although some studies report that the paraclinoid ica is the most common site for unruptured intracranial aneurysms,4,11,14 we found the most common site to be the mca bifurcation , and this is consistent with other previous studies.15,16,17,18 other frequent sites were the anterior communicating artery and the paraclinoid ica . before the age of 50 , the most frequent aneurysm location was the paraclinoid ica . significant increases in the prevalence of mca bifurcation and anterior communicating artery aneurysms could be due to higher hemodynamic shear stress.19 most aneurysms were between 2 mm and 7 mm in size . aneurysms larger than 10 mm were rare , consistent with a previous report.9 aneurysm enlargement on follow - up imaging was present in 6 cases , and aneurysm rupture during follow - up occurred in 2 cases . the prevalence of incidental aneurysms may have been underestimated because we were unable to include false - negative cases , and the use of 1.5-t mra data may have limited our ability to detect small aneurysms and thoroughly evaluate false - positive results due to limited resolution . in addition , the imaging follow - up data were limited because we could not strictly follow up these patients . we conducted a retrospective review of 18,237 patients who underwent mra at our institution and found that the prevalence of unruptured intracranial aneurysms was 1.8% , which was significantly higher in women than in men . the prevalence increased with age in women , but not in men . therefore , overall , the most common location for unruptured intracranial aneurysms was the bifurcation of the mca . there were a number of false - positive cases that appeared in our initial search , and the most common location for false - positive cases was the anterior communicating artery .

its properties like easy mould ability into any desired shape , easy availability of its constituent materials , cost effectiveness , and many other advantages make it popular construction material . however , concrete does have few deficiencies like brittleness , low tensile strength , and low ductility . the tensile strength of concrete is in the range of 710% of its compressive strength . durability is the basic requirement of any concrete structure as it should be able to withstand all stresses and remain functional throughout its designed life span . some of the reasons for failure of structures are poor design , use of poor quality materials , bad workmanship , deterioration of concrete due to ingress of harmful ingredients , and so forth . water is generally involved in any form of deterioration , and , in porous solids , permeability of the material to water usually determines the rate of deterioration . thus , of the many factors affecting the durability of concrete structures , permeability has been identified as one of the key factors . permeability of concrete is defined as the ease with which a fluid , liquid , or gas flows through it under pressure . the lower the permeability of concrete would be , the more durable the concrete would be . many factors like cement content , cement type , aggregate type , shape and size , admixtures in cement , super plasticizers , curing age , curing temperature , methods of curing , and so forth have been found to affect the permeability of concrete [ 2 , 3 ] . many investigators have carried out studies on the effect of the above - mentioned factors on the permeability of concrete . the effects of pozzolanic materials like silica fume , fly ash , and rice husk ash on permeability of concrete have also been investigated [ 47 ] . fibre reinforced concrete ( frc ) is a composite material comprising cement , aggregate , and randomly distributed discrete fibres . short , discrete , and randomly distributed fibres have been found to be very effective in overcoming some of the deficiencies of conventional concrete . a lot of research has been carried out on steel fibre reinforced concrete ( sfrc ) using mono steel fibres and it has been observed that the addition of fibres improves its compressive strength up to some extent and considerably increases the tensile strength , flexural strength , shear strength , and flexural toughness [ 816 ] . the degree of improvement in the above properties depends on many factors including size , type , shape , aspect ratio , and volume fraction of fibres used [ 1719 ] . in the last decade , attempts have been made to study the effect of addition of fibres on permeability of frc using mono fibres . researchers have used variety of mono fibres like steel , carbon , polypropylene , polyvinyl alcohol , nylon , and glass to study the permeability characteristics of cracked and uncracked concrete [ 2026 ] . mainly the fibres are divided into two types , that is , metallic and nonmetallic . steel and carbon fibres are termed as metallic fibres and fibres like polymeric , carbon , glass , and naturally occurring fibres are clubbed under the umbrella of nonmetallic fibres [ 2732 ] . further , depending upon the length of the fibre , these are classified as macro- and microfibres . hybridization of fibres means incorporating fibres of different materials or fibres of the same material having different lengths / aspect ratios [ 3338 ] . hybrid steel fibre reinforced concrete ( hysfrc ) is the most recent advancement in the field of frc . with the increase in the structural and mechanical properties of concrete , it is imperative to increase the durability of these materials . to open new application areas , hysfrc should be designed to perform adequately in terms of workability , strength , ductility , and , most importantly , the durability . utilizing the concept of hybridization , concrete with superior properties can be developed . the moisture which enters into the concrete can lead to corrosion of steel reinforcement and considerably reduces the life of the structures . as such , the durability of concrete depends largely upon the permeability of concrete which is defined as the ease with which it allows the fluids to pass through it . even though durability is a key factor affecting longevity of concrete structures , only limited studies were carried out to investigate the effect of addition of different steel fibres on the durability of concrete . a comprehensive review of literature , which was carried out but reported briefly in the preceding paragraphs , indicates that the effect of addition of hybrid steel fibres on the permeability and strength characteristic of hysfrc has not been investigated so far and the information on the subject is scanty . in this study , therefore , an attempt has been made to investigate the combined effect of addition of hybrid steel fibres on the water permeability and strength characteristics such as compressive strength and split tensile strength of hysfrc mixes made with different fibre volume fractions , each volume fraction containing different combinations of steel fibres of varying lengths . the proportion of the ingredients by weight constituting the reference concrete mix was 1 : 1.52 : 1.88 with a water - cement ratio of 0.46 by weight . pozzolanic portland cement , crushed stone coarse aggregates having maximum size of 12 mm , and locally available river sand were used . corrugated steel fibres , 12.5 mm , 25 mm , and 50 mm long , each with constant diameter of 0.6 mm , were used in different combinations by weight . three volume fractions of the fibres , that is , 0.5% , 1.0% , and 1.5% , were used , each volume fraction containing different mix proportions of steel fibres of different aspect ratios in mono , binary , and ternary combinations . table 1 presents 13 such fibre mix combinations corresponding to each fibre volume fraction of 0.5% , 1.0% , and 1.5% resulting in a total of 39 fibre concrete mixes . in addition , concrete containing no fibres was also cast for reference purpose . in all 40 concrete mixes the inverted slump cone test was used to measure the workability of concrete containing different combinations of steel fibres . this test has been specifically developed to measure workability of frc . the time taken to empty the cone for all the mixes used in this investigation was within limits prescribed for frc except for some mixes made with 1.5% volume fraction of fibres where it was rather difficult to maintain the workability within limits even with increased dose of superplasticizer and fibre balling each mix of concrete consisted of standard cube specimens of 100100100 mm size for compressive strength , split tensile strength , and water permeability tests which were conducted after 7 , 28 , 90 , and 120 days of curing in potable water . for all the 40 concrete mixes , a total number of 1440 cube specimens , 480 specimens each for compressive strength , split tensile strength , and water permeability , were cast . compressive strength tests were conducted in accordance with is : 5161956 on a 2000 kn compression testing machine . the bearing surfaces of the machine were cleaned and the test specimen was placed in the machine such that the load was applied to the faces other than the cast face of the specimen . the maximum compressive load on the specimen was recorded as the load at which the specimen failed to take further increase in the load . the average of three specimens was taken as the representative value of compressive strength for each mix . the compressive strength was calculated by dividing the maximum compressive load by the cross - sectional area of the cube specimen over which the load was applied . the split tensile strength was conducted on the compression testing machine by placing the specimen diagonally . the procedure adopted in this investigation for split tensile strength tests was the same as used in some previous investigations [ 41 , 42 ] . the split tensile strength was determined by using the following formula : ( 1)spt=0.544pa2 , where spt is split tensile strength in mpa , p is splitting load in n , and a is size of cube specimen in mm . water permeability of fibrous concrete was obtained by testing specimens in a water permeability tester . the water permeability was conducted as per the procedure laid out in is : 30851965 . the test cells of the permeability testing equipment have a cross section of 115 mm 115 mm . the annular space between the test mould and the cube was filled with a mix of resin and wax mixed in the ratio of 2 : 1 by volume . prior to testing , the specimens were surface - dried and painted with a mixture of resin and wax , on all the faces except the two faces through which unidirectional flow of water under requisite pressure was planned to take place . the annular space in the bottom portion of the test mould was tightly packed with jute pieces soaked in the molten mixture . this mixture in its smoking state was added to fill the remaining portion of the annular space and was compressed by a steel ruler to release any entrapped air . the seal was allowed to harden for 24 hours and then was checked by passing air from the bottom while covering the top surface with the layer of water . absence of any air bubble emerging out of the seal confirmed that the seal was perfect . the sealing ensured a unidirectional flow from top to bottom through the sample and not from sides . the test cell was designed to withstand a working pressure of 1.5 mpa and the specimens in the present study were tested at pressure ranging from 0.8 mpa to 1.0 mpa . the discharge measurements were taken at regular intervals till the attainment of steady state which was confirmed when the discharge passing through the specimen becomes constant . after attaining the steady state , readings were taken at regular intervals for 48 hours . the steady state was normally attained within a test period of 7 days to 15 days . the coefficient of permeability was calculated by using the following formula : ( 2)k = qlah , where k is coefficient of permeability , m / s , q is rate of discharge , cumecs , l is dimension of the specimen measured in the direction of flow , a is area of cross section of the specimen , and h is water head causing flow measured , m. in all , 39 mixes , 13 each for volume fraction of 0.5% , 1.0% , and 1.5% containing different combinations of steel fibres , were tested for compressive strength , split tensile strength , and water permeability at 7 , 28 , 90 , and 120 days of curing . results for different mixes containing different combinations of steel fibres at 28 days of curing are presented in the following sections as the trends are more or less similar at other curing ages . for mono steel fibre mixes , at a volume fraction of 0.5% , the compressive strength was found to increase by 5.28% , 24.06% , and 16.82% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . similarly , for a mix containing 1.0% volume fraction , the compressive strength was found to increase by 8.85% , 26.61% , and 20.32% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . for 1.5% volume fraction , increase in the compressive strength of the order of 13.69% , 29.54% , and 14.45% was observed with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete . figure 1 presents typical trends for compressive strength of mixes containing mono steel fibres at a fibre volume fraction of 1.0% . for mono steel fibre mixes , with the addition of 100% 12.5 mm long fibres , the compressive strength was increased by 5.28% , 8.85% , and 13.69% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , with the addition of 100% 25 mm long fibres , the compressive strength was increased by 24.06% , 26.61% , and 29.54% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . for 100% 50 mm long fibres , an increase in the compressive strength of the order of 16.82% , 20.32% , and 14.45% the drop in compressive strength at 1.5% volume fraction for a mix with 100% 50 mm long fibres may be attributed to the balling effect of the steel fibres during mixing . figure 2 presents the influence of fibre volume fraction on the compressive strength of mono steel fibre mixes made with 100% 25 mm long fibres . the optimum fibre length for compressive strength for all the volume fractions tested for sfrc mixes is 25 mm , whereas 12.5 mm long fibres performed poorly . this may be due to the fact that some minimum fibre length is required to resist the cracks and their propagation . for 50 mm long fibres , their number for particular volume fraction is less as compared to 25 mm fibres and thus may not be as effective in arresting the propagation of cracks , thus giving poor performance compared to 25 mm fibres . first , the results of hysfrc mixes containing binary combinations of steel fibres are discussed and the results of hysfrc mixes containing ternary combinations of steel fibres are presented later . it has been observed that , with the addition of 75% 12.5 mm + 25% 25 mm long steel fibres presented in figure 5 , increase in the compressive strength of the order of 8.82% , 11.06% , and 10.88% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the compressive strength was increased by 11.65% , 13.51% , and 10.55% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . figure 3 presents a typical trend for hysfrc mixes containing binary combinations of 25 mm and 50 mm long steel fibres for a fibre volume fraction of 0.5% . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm , and the increase in the compressive strength over plain concrete mix was 14.96% , 17.26% , and 15.47% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for binary steel fibre mixes of 100% 12.5 mm and 100% 50 mm long steel fibres , with the addition of 75% 12.5 mm + 25% 50 mm long steel fibres , increase in the compressive strength of the order of 15.73% , 12.67% , and 10.55% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 50 mm long steel fibres , the compressive strength was increased by 10.88% , 14.89% , and 12.41% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 50 mm , and the increase in the compressive strength over plain concrete mix was 14.22% , 17.51% , and 14.84% for 0.5% , 1.0% , and 1.5% fibre content , respectively . in the binary mix combinations of 100% 25 mm and 100% 50 mm long steel fibres , with the addition of 75% 25 mm + 25% 50 mm long steel fibres , increase in the compressive strength of the order of 21.59% , 21.72% , and 13.87% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 25 mm + 50% 50 mm long steel fibres , the compressive strength was increased by 18.79% , 23.88% , and 21.34% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 25 mm + 75% 50 mm , and the increase in the compressive strength over plain concrete mix was 12.82% , 22.18% , and 16.67% for 0.5% , 1.0% , and 1.5% fibre content , respectively . however , for the ternary mix combination containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres , results are presented in figure 4 ; the increase in the compressive strength over plain concrete was observed to be 19.35% , 23.37% , and 21.92% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , at 28 days of curing . for a particular fibre volume fraction , the highest compressive strength is obtained for a mix made with 100% 25 mm steel fibres . similarly , for a particular fibre volume fraction , the lowest value of the compressive strength is given by a mix made with 100% 12.5 mm long fibre followed by a mix made of 75% 12.5 mm + 25% 25 mm long fibres . as the amount of short fibres in a particular mix is increased or short fibres are partially or fully replaced by relatively long fibres , a decrease in the compressive strength is observed . further , at all the fibre mixes tested in this investigation , the lowest compressive strength is obtained for a mix made with 100% 12.5 mm long fibres at a fibre volume fraction of 0.5% , whereas the highest compressive strength is given by a mix made of 100% 25 mm long fibres at a fibre volume fraction of 1.5% . it may also be noted that the compressive strength increase is more sensitive to fibre length than volume fraction in case of binary combinations of fibre . however , in case of ternary combinations , the influence of fibre volume fraction is almost negligible . for mono steel fibre mixes , at a volume fraction of 0.5% , the split tensile strength was found to increase by 4.88% , 18.16% , and 26.29% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at curing of 28 days . similarly , for a mix containing 1.0% volume fraction of fibres , the split tensile strength was found to increase by 10.57% , 24.66% , and 37.40% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . for 1.5% volume fraction , increase in the split tensile strength of the order of 12.74% , 31.98% , and 14.09% was observed with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete . figure 5 presents typical trends for split tensile strength of mixes containing mono steel fibres at a fibre volume fraction of 0.5% . for mono steel fibre mixes , for mixes made with 100% 12.5 mm long fibres , the split tensile strength was increased by 4.88% , 10.57% , and 12.74% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for mixes containing 100% 25 mm long fibres , the split tensile strength was increased by 18.16% , 24.66% , and 31.98% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . for mixes with 100% 50 mm long fibres , an increase in the split tensile strength of the order of 26.29% , 37.40% , and 14.09% was observed for 0.5% , 1.0% , and 1.5% fibre content , respectively . it was observed that the difference in the split tensile strength for all the mixes at fibre contents of 1.0% and 1.5% was not significant except for 100% 50 mm long fibres , which may be attributed to the balling effect of the steel fibres during mixing at 1.5% volume fraction . figure 6 presents the influence of fibre volume fraction on the split tensile strength of mono steel fibre mixes made with 100% 50 mm long fibres . like compressive strength results , the split tensile strength results of hysfrc mixes containing binary combinations of steel fibres are discussed first and the results of hysfrc mixes containing ternary combinations of steel fibres are presented later . + 25% 25 mm long steel fibres , increase in the split tensile strength of the order of 7.05% , 13.82% , and 14.36% for 0.5% , 1.0% , and 1.5% fibre volume fraction , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the split tensile strength was increased by 10.84% , 17.07% , and 15.72% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm and the increase in the split tensile strength over plain concrete mix was 12.47% , 20.87% , and 19.24% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for binary steel fibre mixes of 100% 12.5 mm and 100% 50 mm long steel fibres , with the addition of 75% 12.5 mm + 25% 50 mm long steel fibres , increase in the split tensile strength of the order of 8.67% , 18.70% , and 21.68% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 50 mm long steel fibres , the split tensile strength was increased by 15.72% , 22.76% , and 17.34% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 50 mm , and the increase in the split tensile strength over plain concrete mix was 19.78% , 32.25% , and 22.76% for 0.5% , 1.0% , and 1.5% fibre content , respectively . in the binary mix combinations of 100% 25 mm and 100% 50 mm long steel fibres , with the addition of 75% 25 mm + 25% 50 mm long steel fibres , increase in the split tensile strength of the order of 14.09% , 27.37% , and 17.89% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix which contained 50% 25 mm + 50% 50 mm long steel fibres , the split tensile strength was increased by 30.08% , 33.60% , and 31.71% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 25 mm + 75% 50 mm , and the increase in the split tensile strength over plain concrete mix was 21.95% , 30.62% , and 27.10% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . figure 7 presents a typical trend for hysfrc mixes containing binary combinations of 25 mm and 50 mm long steel fibres for a fibre volume fraction of 0.5% . however , for hysfrc ternary mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres for which the results are presented in figure 8 , the increase in the split tensile strength over plain concrete was observed to be 35.77% , 46.07% , and 39.30% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , at 28 days of curing . for a particular fibre volume fraction , the highest split tensile strength is obtained for a mix made with 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres followed by a mix made with 50% 25 mm + 50% 50 mm long fibres . similarly , for a particular fibre volume fraction , the lowest value of the split tensile strength is given by a mix made with 100% 12.5 mm long fibre followed by a mix made of 75% 12.5 mm + 25% 25 mm long fibres . further , at all the fibre mixes tested in this investigation , the lowest split tensile strength is obtained for a mix made with 100% 12.5 mm long fibres at a fibre volume fraction of 0.5% , whereas the highest split tensile strength is given by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres at a fibre volume fraction of 1.0% . it may also be noted that the split tensile strength increase is more sensitive to fibre length than volume fraction in case of binary combinations of fibre . however , in case of ternary combinations , the influence of fibre volume fraction is almost negligible . for mono steel fibre reinforced concrete ( sfrc ) mixes , for volume fraction of 0.5% and at 28 days of curing , the coefficient of water permeability was found to decrease over plain concrete mix ( control mix ) by 68.42% , 45.45% , and 31.33% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively . similarly , for a mix containing 1.0% volume fraction of steel fibres and at 28 days of curing , the coefficient of water permeability was found to decrease over plain concrete mix by 79.21% , 65.23% , and 51.94% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively . for 1.5% volume fraction , decrease in the coefficient of water permeability of the order of 45.94% , 25% , and 2.61% at 28 days of curing was observed with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete . the increase in coefficient of water permeability at 1.5% volume fraction may be attributed to the balling effect of the steel fibres during mixing . figure 9 presents the variation of coefficient of water permeability with curing period for different mixes containing mono steel fibres for vf = 0.5% . the decrease in the water permeability of sfrc mixes may be attributed to the fact that a considerable reduction is reported in the occurrence of shrinkage cracks with the addition of fibres . the reduction in the shrinkage cracks helps in reducing the water permeability of concrete . also , the drying shrinkage cracks get reduced to the extent of 25% up to an age of 28 days with the addition of steel fibres . further , addition of impermeable steel fibres helps in breaking the continuity or interconnectivity of porous channels present in the concrete , thus resulting in lower water permeability . also the maximum decrease in the water permeability of sfrc mixes made with 12.5 mm long fibres as compared to relatively long fibres such as 25 mm and 50 mm may be attributed to the fact that , for particular fibre volume fraction , the short fibres are more in number and are more effective in breaking the paths through which water can travel in concrete . first , the results of hysfrc mixes containing binary combinations of steel fibres are discussed and the results of hysfrc mixes containing ternary combinations of steel fibres are presented later . it has been observed that , with the addition of 75% 12.5 mm + 25% 25 mm long steel fibres , decrease in the coefficient of water permeability of the order of 66.57% , 74.75% , and 42.46% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed after 28 days of curing . similarly , for hysfrc mix , which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the coefficient of water permeability was decreased by 59.79% , 73.08% , and 50.80% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix after 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm , and the decrease in the coefficient of water permeability over plain concrete mix was 55.46% , 67.44% , and 29.66% for 0.5% , 1.0% , and 1.5% fibre content , respectively . figure 10 presents typical trends for hysfrc mixes containing binary combinations of 12.5 mm and 25 mm long steel fibres for a fibre volume fraction of 1.0% . for binary mixes made with 12.5 mm and 50 mm long steel fibres and 25 mm and 50 mm long steel fibres , similar trends were observed and the detailed results are not presented here as the same are submitted to another journal . however , for the ternary mix combination containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres ( figure 11 ) , the decrease in the coefficient of water permeability over plain concrete was observed to be 65.99% , 75.74% , and 30.46% for 0.5% , 1.0% , and 1.5% volume fraction , respectively . for a particular fibre volume fraction , the lowest values of the coefficient of water permeability are obtained for a mix made with 100% 12.5 mm steel fibres followed by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres . similarly , for a particular fibre volume fraction , the highest value of the water permeability is given by a mix made with 100% 50 mm long fibre followed by a mix made of 25% 12.5 mm + 75% as the amount of short fibres in a particular mix is decreased or short fibres are partially or fully replaced by relatively long fibres , an increase in the water permeability is observed . further , at all the fibre mixes tested in this investigation , the lowest coefficient of water permeability is obtained for a mix made with 100% 12.5 mm long fibres at a fibre volume fraction of 1.0% , whereas the highest coefficient of water permeability is given by a mix made of 100% 50 mm long fibres at a fibre volume fraction of 1.5% . it may be noted that the mix made up of 100% 50 mm long fibres at fibre volume fraction of 1.5% has the water permeability even higher than plain concrete which may be as a result of fibre balling taking place during mixing . in general , as the amount of short fibres in a particular mix is decreased or short fibres are partially or fully replaced by relatively long fibres , an increase in the water permeability is observed . further , as already mentioned , for all the fibre mixes tested in this investigation , the lowest coefficient of water permeability is obtained for a mix made with 100% 12.5 mm long fibres at a fibre volume fraction of 1.0% , whereas the highest coefficient of water permeability is given by a mix made of 100% 50 mm long fibres at a fibre volume fraction of 1.5% . it can be seen from the results of water permeability , compressive strength , and split tensile strength tests obtained in this investigation that no single fibre combination can be adjudged as the best combination for all these properties ; this may be due to the reason that a large number of fibre combinations have been tested in this investigation . however , for most of the fibre combinations employed in this investigation , it can be concluded that materials achieve the best performance at a fibre volume fraction of 1.0% in all the tests conducted on the hardened concrete , that is , water permeability , compressive strength , and split tensile strength . it will be in the fitness of things to examine the results in more detail and to arrive at an acceptable fibre combination for all the tests conducted . it can be easily observed that the best performance in terms of split tensile strength is given by a mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres at a volume fraction of 1.0% . the best fibre combination for water permeability is 100% 12.5 mm long fibres , whereas the mix made with 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres gives the second best performance . similarly , the mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres gives the second best performance in terms of compressive strength also . hence , it can be concluded without much loss of accuracy that , for water permeability , compressive strength , and split tensile strength , a fibre combination of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres can be adjudged as the best combination . properties of hysfrc containing different combinations of steel fibres of different lengths and plain concrete in hardened state have been investigated . tests such as compressive strength , split tensile strength , and water permeability were conducted on hardened concrete after 7 , 28 , 90 , and 120 days of curing . a total number of 40 mixes were considered and approximately 1440 specimens were tested in this investigation . for convenience , the results of various tests at the curing age of 28 days are presented and discussed briefly in this paper as the trends are more or less similar at other curing ages . in case of water permeability , a maximum decrease in coefficient of water permeability of the order of 79.21% for a mix with 100% 12.5 mm long steel fibres , at a fibre volume fraction of 1.0% , was observed followed by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres . for compressive strength , maximum increase of the order of 29.54% over plain concrete was observed in case of mix containing 100% 25 mm long steel fibres at fibre volume fraction of 1.5% . similarly , 46.07% increase in split tensile strength of hysfrc was observed with respect to plain concrete with a fibre mix ratio of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres at a fibre volume fraction of 1.0% . a careful examination of the results indicates that a fibre combination of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres can be taken as the most appropriate combination for water permeability , compressive strength , and split tensile strength of hysfrc .

results of an investigation conducted to study the effect of fibre hybridization on the strength characteristics such as compressive strength , split tensile strength , and water permeability of steel fibre reinforced concrete ( sfrc ) are presented . steel fibres of different lengths , that is , 12.5 mm , 25 mm , and 50 mm , having constant diameter of 0.6 mm , were systematically combined in different mix proportions to obtain mono , binary , and ternary combinations at each of 0.5% , 1.0% , and 1.5% fibre volume fraction . a concrete mix containing no fibres was also cast for reference purpose . a total number of 1440 cube specimens of size 100100100 mm were tested , 480 each for compressive strength , split tensile strength , and water permeability at 7 , 28 , 90 , and 120 days of curing . it has been observed from the results of this investigation that a fibre combination of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres can be adjudged as the most appropriate combination to be employed in hybrid steel fibre reinforced concrete ( hysfrc ) for optimum performance in terms of compressive strength , split tensile strength and water permeability requirements taken together .

1. Introduction 2. Research Significance 3. Experimental Procedure 4. Results and Discussion 5. Conclusions

in this study , therefore , an attempt has been made to investigate the combined effect of addition of hybrid steel fibres on the water permeability and strength characteristics such as compressive strength and split tensile strength of hysfrc mixes made with different fibre volume fractions , each volume fraction containing different combinations of steel fibres of varying lengths . corrugated steel fibres , 12.5 mm , 25 mm , and 50 mm long , each with constant diameter of 0.6 mm , were used in different combinations by weight . three volume fractions of the fibres , that is , 0.5% , 1.0% , and 1.5% , were used , each volume fraction containing different mix proportions of steel fibres of different aspect ratios in mono , binary , and ternary combinations . the time taken to empty the cone for all the mixes used in this investigation was within limits prescribed for frc except for some mixes made with 1.5% volume fraction of fibres where it was rather difficult to maintain the workability within limits even with increased dose of superplasticizer and fibre balling each mix of concrete consisted of standard cube specimens of 100100100 mm size for compressive strength , split tensile strength , and water permeability tests which were conducted after 7 , 28 , 90 , and 120 days of curing in potable water . for all the 40 concrete mixes , a total number of 1440 cube specimens , 480 specimens each for compressive strength , split tensile strength , and water permeability , were cast . the coefficient of permeability was calculated by using the following formula : ( 2)k = qlah , where k is coefficient of permeability , m / s , q is rate of discharge , cumecs , l is dimension of the specimen measured in the direction of flow , a is area of cross section of the specimen , and h is water head causing flow measured , m. in all , 39 mixes , 13 each for volume fraction of 0.5% , 1.0% , and 1.5% containing different combinations of steel fibres , were tested for compressive strength , split tensile strength , and water permeability at 7 , 28 , 90 , and 120 days of curing . for mono steel fibre mixes , at a volume fraction of 0.5% , the compressive strength was found to increase by 5.28% , 24.06% , and 16.82% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . similarly , for a mix containing 1.0% volume fraction , the compressive strength was found to increase by 8.85% , 26.61% , and 20.32% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . for mono steel fibre mixes , with the addition of 100% 12.5 mm long fibres , the compressive strength was increased by 5.28% , 8.85% , and 13.69% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , with the addition of 100% 25 mm long fibres , the compressive strength was increased by 24.06% , 26.61% , and 29.54% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . it has been observed that , with the addition of 75% 12.5 mm + 25% 25 mm long steel fibres presented in figure 5 , increase in the compressive strength of the order of 8.82% , 11.06% , and 10.88% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the compressive strength was increased by 11.65% , 13.51% , and 10.55% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm , and the increase in the compressive strength over plain concrete mix was 14.96% , 17.26% , and 15.47% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for binary steel fibre mixes of 100% 12.5 mm and 100% 50 mm long steel fibres , with the addition of 75% 12.5 mm + 25% 50 mm long steel fibres , increase in the compressive strength of the order of 15.73% , 12.67% , and 10.55% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 50 mm long steel fibres , the compressive strength was increased by 10.88% , 14.89% , and 12.41% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 50 mm , and the increase in the compressive strength over plain concrete mix was 14.22% , 17.51% , and 14.84% for 0.5% , 1.0% , and 1.5% fibre content , respectively . in the binary mix combinations of 100% 25 mm and 100% 50 mm long steel fibres , with the addition of 75% 25 mm + 25% 50 mm long steel fibres , increase in the compressive strength of the order of 21.59% , 21.72% , and 13.87% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 25 mm + 50% 50 mm long steel fibres , the compressive strength was increased by 18.79% , 23.88% , and 21.34% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 25 mm + 75% 50 mm , and the increase in the compressive strength over plain concrete mix was 12.82% , 22.18% , and 16.67% for 0.5% , 1.0% , and 1.5% fibre content , respectively . however , for the ternary mix combination containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres , results are presented in figure 4 ; the increase in the compressive strength over plain concrete was observed to be 19.35% , 23.37% , and 21.92% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , at 28 days of curing . for mono steel fibre mixes , at a volume fraction of 0.5% , the split tensile strength was found to increase by 4.88% , 18.16% , and 26.29% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at curing of 28 days . similarly , for a mix containing 1.0% volume fraction of fibres , the split tensile strength was found to increase by 10.57% , 24.66% , and 37.40% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete mix at 28 days of curing . for mono steel fibre mixes , for mixes made with 100% 12.5 mm long fibres , the split tensile strength was increased by 4.88% , 10.57% , and 12.74% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for mixes containing 100% 25 mm long fibres , the split tensile strength was increased by 18.16% , 24.66% , and 31.98% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . + 25% 25 mm long steel fibres , increase in the split tensile strength of the order of 7.05% , 13.82% , and 14.36% for 0.5% , 1.0% , and 1.5% fibre volume fraction , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the split tensile strength was increased by 10.84% , 17.07% , and 15.72% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm and the increase in the split tensile strength over plain concrete mix was 12.47% , 20.87% , and 19.24% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . similarly , for binary steel fibre mixes of 100% 12.5 mm and 100% 50 mm long steel fibres , with the addition of 75% 12.5 mm + 25% 50 mm long steel fibres , increase in the split tensile strength of the order of 8.67% , 18.70% , and 21.68% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix , which contained 50% 12.5 mm + 50% 50 mm long steel fibres , the split tensile strength was increased by 15.72% , 22.76% , and 17.34% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 50 mm , and the increase in the split tensile strength over plain concrete mix was 19.78% , 32.25% , and 22.76% for 0.5% , 1.0% , and 1.5% fibre content , respectively . in the binary mix combinations of 100% 25 mm and 100% 50 mm long steel fibres , with the addition of 75% 25 mm + 25% 50 mm long steel fibres , increase in the split tensile strength of the order of 14.09% , 27.37% , and 17.89% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed at 28 days of curing . for hysfrc mix which contained 50% 25 mm + 50% 50 mm long steel fibres , the split tensile strength was increased by 30.08% , 33.60% , and 31.71% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix at 28 days of curing . similar trends were observed for a mix containing 25% 25 mm + 75% 50 mm , and the increase in the split tensile strength over plain concrete mix was 21.95% , 30.62% , and 27.10% for 0.5% , 1.0% , and 1.5% fibre content , respectively , at 28 days of curing . however , for hysfrc ternary mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres for which the results are presented in figure 8 , the increase in the split tensile strength over plain concrete was observed to be 35.77% , 46.07% , and 39.30% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , at 28 days of curing . for a particular fibre volume fraction , the highest split tensile strength is obtained for a mix made with 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres followed by a mix made with 50% 25 mm + 50% 50 mm long fibres . further , at all the fibre mixes tested in this investigation , the lowest split tensile strength is obtained for a mix made with 100% 12.5 mm long fibres at a fibre volume fraction of 0.5% , whereas the highest split tensile strength is given by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres at a fibre volume fraction of 1.0% . for mono steel fibre reinforced concrete ( sfrc ) mixes , for volume fraction of 0.5% and at 28 days of curing , the coefficient of water permeability was found to decrease over plain concrete mix ( control mix ) by 68.42% , 45.45% , and 31.33% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively . similarly , for a mix containing 1.0% volume fraction of steel fibres and at 28 days of curing , the coefficient of water permeability was found to decrease over plain concrete mix by 79.21% , 65.23% , and 51.94% with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively . for 1.5% volume fraction , decrease in the coefficient of water permeability of the order of 45.94% , 25% , and 2.61% at 28 days of curing was observed with the addition of 100% 12.5 mm , 100% 25 mm , and 100% 50 mm long fibres , respectively , over plain concrete . it has been observed that , with the addition of 75% 12.5 mm + 25% 25 mm long steel fibres , decrease in the coefficient of water permeability of the order of 66.57% , 74.75% , and 42.46% for 0.5% , 1.0% , and 1.5% fibre content , respectively , over plain concrete mix was observed after 28 days of curing . similarly , for hysfrc mix , which contained 50% 12.5 mm + 50% 25 mm long steel fibres , the coefficient of water permeability was decreased by 59.79% , 73.08% , and 50.80% for 0.5% , 1.0% , and 1.5% volume fraction , respectively , over plain concrete mix after 28 days of curing . similar trends were observed for a mix containing 25% 12.5 mm + 75% 25 mm , and the decrease in the coefficient of water permeability over plain concrete mix was 55.46% , 67.44% , and 29.66% for 0.5% , 1.0% , and 1.5% fibre content , respectively . however , for the ternary mix combination containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres ( figure 11 ) , the decrease in the coefficient of water permeability over plain concrete was observed to be 65.99% , 75.74% , and 30.46% for 0.5% , 1.0% , and 1.5% volume fraction , respectively . for a particular fibre volume fraction , the lowest values of the coefficient of water permeability are obtained for a mix made with 100% 12.5 mm steel fibres followed by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres . it can be seen from the results of water permeability , compressive strength , and split tensile strength tests obtained in this investigation that no single fibre combination can be adjudged as the best combination for all these properties ; this may be due to the reason that a large number of fibre combinations have been tested in this investigation . however , for most of the fibre combinations employed in this investigation , it can be concluded that materials achieve the best performance at a fibre volume fraction of 1.0% in all the tests conducted on the hardened concrete , that is , water permeability , compressive strength , and split tensile strength . it can be easily observed that the best performance in terms of split tensile strength is given by a mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres at a volume fraction of 1.0% . similarly , the mix containing 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres gives the second best performance in terms of compressive strength also . hence , it can be concluded without much loss of accuracy that , for water permeability , compressive strength , and split tensile strength , a fibre combination of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres can be adjudged as the best combination . tests such as compressive strength , split tensile strength , and water permeability were conducted on hardened concrete after 7 , 28 , 90 , and 120 days of curing . in case of water permeability , a maximum decrease in coefficient of water permeability of the order of 79.21% for a mix with 100% 12.5 mm long steel fibres , at a fibre volume fraction of 1.0% , was observed followed by a mix made of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres . similarly , 46.07% increase in split tensile strength of hysfrc was observed with respect to plain concrete with a fibre mix ratio of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long fibres at a fibre volume fraction of 1.0% . a careful examination of the results indicates that a fibre combination of 33% 12.5 mm + 33% 25 mm + 33% 50 mm long steel fibres can be taken as the most appropriate combination for water permeability , compressive strength , and split tensile strength of hysfrc .

the observation at the lhc of a higgs particle with a mass of 125 gev [ 1 , 2 ] has important implications for supersymmetric ( susy ) and , in particular , for the minimal supersymmetric standard model ( mssm ) . in this extension , the higgs sector consists of two scalar doublet fields hu and hd that lead , after electroweak symmetry breaking , to five higgs states , two cp - even h and h , a cp - odd a and two charged h bosons [ 36 ] . at tree level , the masses of these particles and their mixings are described by only two parameters usually chosen to be the ratio of the vacuum expectations values of the two doublet fields tan=vd / vu and the mass ma of the pseudoscalar higgs boson . however , as is well known , the radiative corrections play a very important role as their dominant component grows like the fourth power of the top quark mass , logarithmically with the supersymmetry breaking scale ms and quadratically with the stop mixing parameter at ; see e.g. refs . , it gives support to the mssm in which the lightest higgs boson is predicted to have a mass below 130 gev when the radiative corrections are included [ 611 ] . on the other hand , the fact that the measured value mh125 gev is close to this upper mass limit implies that the susy - breaking scale ms might be rather high . this is backed up by the presently strong limits on supersymmetric particle masses from direct searches that indicate that the susy partners of the strongly interacting particles , the squarks and gluinos , are heavier than 1 tev . hence , the mssm that we currently have , and that we call hmssm ( habemus mssm ? ) in the subsequent discussion , appears to have mh125 gev and ms1 tev . [ 1315 ] that when the information mh=125 gev is taken into account , the mssm higgs sector with solely the dominant radiative correction to the higgs boson masses included , can be again described with only the two free parameters tan and ma as it was the case at tree level . in other words , the dominant radiative corrections that involve the susy parameters are fixed by the value of mh . in this paper , we show that to a good approximation , this remains true even when the full set of radiative corrections to the higgs masses at the two - loop level is included . this is demonstrated in particular by performing a full scan on the mssm parameters that have an impact on the higgs sector such as for instance tan and the stop and sbottom mass and mixing parameters . the subleading radiative corrections are shown to have little impact on the mass and mixing of the heavier higgs bosons when these susy parameters are varied in a reasonable range . nevertheless , there are also possibly large direct susy radiative corrections that modify the higgs boson couplings and which might alter this simple picture . among such corrections are , for instance , the stop contribution [ 1619 ] to the dominant higgs production mechanism at the lhc , the gluon fusion process ggh , and to the important decay into two photons h , and the additional one - loop vertex corrections to the h couplings to b - quarks that grow with tan . in the most general case , besides mh , seven couplings need to be considered to fully describe the properties of the observed h boson : those to gluons , photons , massive gauge bosons , t , b , c - quarks and leptons . however , we show that given the accuracy that is foreseen at the lhc , a good approximation is to consider the three effective couplings to t , b quarks and to v = w / z bosons , ct , cb and cv , as it was suggested in ref . . [ 2224 ] for the inclusion of the current theoretical and experimental uncertainties , we perform a fit of these three couplings using the latest lhc data on the production and decay rates of the lighter h boson and the limits from the negative search of the heavier h , a and h mssm states . almost one year after the higgs discovery at the lhc , these two aspects will be discussed in the next two sections . a brief discussion and a conclusion are given in sect . 4 and a short appendix collects a set of formulas used in this analysis . in the cp - conserving mssm,1 the tree - level cp - even h and h masses depend on ma , tan and the z boson mass . however , many parameters of the mssm such as the susy scale , taken to be the geometric average of the stop masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{s}= \sqrt{m_{\tilde{t}_{1 } } m_{\tilde{t}_{2 } } } $ \end{document } , the stop / sbottom trilinear couplings at / b or the higgsino mass enter mh and mh through radiative corrections . in the basis ( hd , hu ) , the cp - even higgs mass matrix can be written as 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } m_{s}^2 = & m_{z}^2 \left ( \begin{array}{c@{\quad } c } c^2_\beta & -s_\beta c_\beta\\ -s_\beta c_\beta & s^2_\beta\\ \end{array } \right ) + m_{a}^2 \left ( \begin{array}{c@{\quad } c } s^2_\beta & -s_\beta c_\beta\\ -s_\beta c_\beta & c^2_\beta\\ \end{array } \right ) \\ & { } + \left ( \begin{array}{c@{\quad } c } \vardelta \mathcal { m}_{11}^2 & \vardelta \mathcal { m}_{12}^2 \\ \vardelta \mathcal { m}_{12}^2 & \vardelta \mathcal { m}_{22}^2 \\ \end{array } \right ) \end{aligned}$$ \end{document } where we use the short - hand notation ssin etc . and have introduced the radiative corrections by a 22 general matrix \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}_{ij}^{2}$\end{document}. one can then easily derive the neutral cp - even higgs boson masses and the mixing angle that diagonalizes the h , h states,2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h= \cos\alpha h_{d}^{0 } + \sin\alpha h_{u}^{0}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h= -\sin\alpha h_{d}^{0 } + \cos\alpha h_{u}^{0}$\end{document } : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & m_{h / h}^2=\frac{1}{2 } \bigl ( m_{a}^2+m_{z}^2 + \vardelta \mathcal { m}_{11}^2 + \vardelta \mathcal { m}_{22}^2 \mp \sqrt { m_{a}^4+m_{z}^4 - 2 m_{a}^2 m_{z}^2 c_{4\beta } + c } \bigr ) \end{aligned}$$ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & \tan\alpha= \frac{2\vardelta \mathcal { m}_{12}^2 - ( m_{a}^2 + m_{z}^2 ) s_{\beta } } { \vardelta \mathcal { m}_{11}^2 - \vardelta \mathcal { m}_{22}^2 + ( m_{z}^2-m_{a}^2 ) c_{2\beta } + \sqrt{m_{a}^4 + m_{z}^4 - 2 m_{a}^2 m_{z}^2 c_{4\beta } + c } } \end{aligned}$$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c= 4 \vardelta \mathcal { m}_{12}^4 + \bigl ( \vardelta \mathcal { m}_{11}^2 - \vardelta \mathcal { m}_{22}^2 \bigr)^2 - 2 \bigl(m_{a}^2 - m_{z}^2\bigr ) \bigl ( \vardelta \mathcal { m}_{11}^2 - \vardelta m_{22}^2\bigr ) c_{2\beta } - 4 \bigl(m_{a}^2 + m_{z}^2\bigr ) \vardelta \mathcal { m}_{12}^2 s_{2\beta } \end{aligned}$$ \end{document } in previous analyses [ 1315 ] , we have assumed that in the 22 matrix for the radiative corrections , only the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } entry which involves the by far dominant stop top sector correction , is relevant , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22 } \gg\vardelta \mathcal { m}^{2}_{11 } , \vardelta \mathcal { m}^{2}_{12}$\end{document}. this occurs , for instance , in the so - called approximation [ 79 ] and its refinements [ 10 , 11 ] that are given in eqs . , one can simply trade \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } for the by now known mh using 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } \vardelta \mathcal { m}^{2}_{22}= \frac{m_{h}^2(m_{a}^2 + m_{z}^2 -m_{h}^2 ) - m_{a}^2 m_{z}^2 c^{2}_{2\beta } } { m_{z}^2 c^{2}_{\beta } + m_{a}^2 s^{2}_{\beta } -m_{h}^2 } \end{aligned}$$ \end{document } in this case , one can simply write mh and in terms of ma , tan and mh : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & \mathrm{hmssm } { : } \\ & \quad m_{h}^2 = \frac{(m_{a}^2+m_{z}^2-m_{h}^2)(m_{z}^2 c^{2}_{\beta}+m_{a}^2 s^{2}_{\beta } ) - m_{a}^2 m_{z}^2 c^{2}_{2\beta } } { m_{z}^2 c^{2}_{\beta}+m_{a}^2 s^{2}_{\beta } - m_{h}^2 } \\ & \quad \alpha= -\arctan \biggl(\frac { ( m_{z}^2+m_{a}^2 ) c_{\beta } s_{\beta}}{m_{z}^2 c^{2}_{\beta}+m_{a}^2 s^{2}_{\beta } - m_{h}^2 } \biggr ) \end{aligned}$$ \end{document } in this section , we will check the validity of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}= \vardelta \mathcal { m}^{2}_{12}=0$\end{document } approximation . to do so , we first consider the radiative corrections when the subleading contributions proportional to ,at or ab are included in the form of eq . ( a.4 ) of the appendix , which is expected to be a good approximation [ 6 , 25 ] , and in which one has \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } \neq{\vardelta \mathcal { m}^{2}_{12 } \neq0}$\end{document}. as a first step we only consider the stop - top sector corrections which enter the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{ij}$\end{document } terms and confront in fig . 1 , the values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } for three different scenarios with ma=300 gev ( i.e. before the onset of the decoupling regime mamz ) : ms=3 tev and tan=2.5 , ms=1.5 tev and tan=5 , ms=1 tev and tan=30 . the parameter at is adjusted in order to accommodate a light higgs boson with a mass mh=1263 gev , including an expected theoretical and experimental uncertainty of 3 gev [ 2628 ] . one observes that for reasonable values , one obtains naturally \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11},\vardelta \mathcal { m}^{2}_{12 } \ll\vardelta \mathcal { m}^{2}_{22}$\end{document}. fig . 1the entries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } ( solid ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } ( dashed ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } ( dotted dashed lines ) of the radiative corrections matrix as functions of with a fixed m a=300 gev for three different ( m s , tan ) sets and a t such that it accommodates the mass range m h=123129 gev the entries \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } ( solid ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } ( dashed ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } ( dotted dashed lines ) of the radiative corrections matrix as functions of with a fixed m a=300 gev for three different ( m s , tan ) sets and a t such that it accommodates the mass range m h=123129 gev we have verified that the situation is not very different if the corrections in the sbottom sector are also included : assuming ab = at , we also obtain the hierarchy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11},\vardelta \mathcal { m}^{2}_{12 } \ll\vardelta \mathcal { m}^{2}_{22}$\end{document } for 3 tev even for tan=30 where contributions tan become important . taking into account only the dominant top 2 displays the mass of the heavy cp - even higgs state ( left ) and the mixing angle ( right ) as a function of when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } are set to zero ( dashed lines ) and when they are included ( solid lines ) . we have assumed the same ( ms , tan ) sets as above and for each value of , we calculate approximate and exact mh and values assuming mh=1263 gev . even for large values of the parameter ( but 3 tev ) , the relative variation for mh never exceeds the 0.5 % level while the variation of the angle is bounded by 0.015 . hence , in this scenario for the radiative corrections , the approximation of determining the parameters mh and from tan,ma and the value of mh is extremely good . we have again verified that it stays the case when the corrections in the sbottom sector , with ab = at , are included . 2the mass of the heavier cp - even h boson ( left ) and the mixing angle ( right ) as a function of with ( solid lines ) and without ( dashed ) the off - diagonal components for m a=300 gev and three ( m s , tan ) sets . a t is such that m h=123129 gev and a b=0 the mass of the heavier cp - even h boson ( left ) and the mixing angle ( right ) as a function of with ( solid lines ) and without ( dashed ) the off - diagonal components for m a=300 gev and three ( m s , tan ) sets . a t is such that m h=123129 gev and a b=0 we should note that for higher ma values , ma300 gev , the approximation is even better as we are closer to the decoupling limit in which one has mh = ma and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha= \frac{\pi}{2}-\beta$\end{document}. lower values , ma300 gev , are disfavored by the observed h rates [ 14 , 15 ] as seen later . in order to check more thoroughly the impact of the subleading corrections \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } , we perform a scan of the mssm parameter space using the program suspect [ 29 , 30 ] in which the full two - loop radiative corrections to the higgs sector are implemented . for a chosen ( tan , ma ) input set , the soft - susy parameters that play an important role in the higgs sector are varied in the following ranges : ||3 tev , |at , ab|3ms , 1 tevm33 tev and 0.5 tevms3 tev ( 3 tev is the scale up to which programs such as suspect are expected to be reliable ) . we assume the usual relation between the weak scale gaugino masses 6m1=3m2=m3 and set au , ad , a=0 ( these last parameters have little impact ) . we have computed the mssm higgs sector parameters all across the parameter space selecting the points which satisfy the constraint 123mh129 gev . for each of the points , we have compared the higgs parameters to those obtained in the simplified mssm approximation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } = \vardelta \mathcal { m}^{2}_{12 } = 0$\end{document } , with the lightest higgs boson mass as input . we also required mh to lie in the range 123129 gev , but allowed it to be different from the one obtained in the exact case \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } , \vardelta \mathcal { m}^{2}_{12 } \neq0$\end{document}. for the mass mh and the angle , we display in fig . 3 the difference between the values obtained when the two possibilities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } = \vardelta \mathcal { m}^{2}_{12 } = 0$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } , \vardelta \mathcal { m}^{2}_{12 } \neq 0$\end{document } are considered . this is shown in the plane [ ms , xt ] with xt = atcot when all other parameters are scanned as above . again , we have fixed the pseudoscalar higgs mass to ma=300 gev and used the two representative values tan=5 and 30 . 3the variation of the mass m h ( left ) and the mixing angle ( right ) , are shown as separate vertical colored scales , in the plane [ m s , x t ] when the full two loop corrections are included with and without the subleading matrix elements \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document}. we take m a=300 gev , tan=5 ( top ) and 30 ( bottom ) and the other parameters are varied as stated in the text the variation of the mass m h ( left ) and the mixing angle ( right ) , are shown as separate vertical colored scales , in the plane [ m s , x t ] when the full two loop corrections are included with and without the subleading matrix elements \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document}. we take m a=300 gev , tan=5 ( top ) and 30 ( bottom ) and the other parameters are varied as stated in the text in all cases , the difference between the two mh values is very small ( in fact , much smaller than the total decay width h ) , less than a few percent , while for the difference does not exceed 0.025 for low values of tan but at high tan values , one can reach the level of 0.05 in some rare situations ( large values of , which enhance the tan contributions ) . nevertheless , at high enough tan , we are far in the decoupling regime already for ma200 gev and such a difference does not significantly affect the couplings of the h and h bosons which , phenomenologically , are the main ingredients . hence , even when including the full set of radiative corrections up to two loops , it is a good approximation to use eqs . ( 5 ) to derive the parameters mh and in terms of the inputs tan,ma and the measured value of mh . in the case of the charged higgs boson mass , the radiative corrections are much smaller for large enough ma and one has , at the few percent level ( which is again smaller than the total h decay width ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{h^{\pm } } \simeq\sqrt { m_{a}^{2 } + m_{w}^{2}}$\end{document } except in very rare situations3 . a second important issue is the mssm higgs couplings . in principle and as discussed earlier , knowing two parameters such as the pair of inputs [ tan,ma ] and fixing the value of mh to its measured value , the couplings of the higgs bosons , in particular h , to fermions and gauge bosons can be derived , including the generally dominant radiative corrections that enter in the mssm higgs masses . indeed , in terms of the angles and , one has for the reduced couplings ( i.e. normalized to their sm values ) of the lighter h state to third generation t , b fermions and gauge bosons v = w / z , 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c_v^0 = \sin(\beta- \alpha ) , \qquad c_t^0 = \frac{\cos \alpha}{\sin\beta } , \qquad c_b^0 = - \frac{\sin \alpha}{\cos\beta } \end{aligned}$$ \end{document } however , outside the regime in which the pseudoscalar a boson and some supersymmetric particles are very heavy , there are also direct radiative corrections to the higgs couplings not contained in the mass matrix of eq . first , in the case of b - quarks , additional one - loop vertex corrections modify the tree - level \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h b \bar{b}$\end{document } coupling : they grow as mbtan and are thus very large at high tan. the dominant component comes from the susy - qcd corrections with sbottom gluino loops that can be approximated by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta _ { b } \simeq 2\alpha_{s}/(3\pi ) \times\mu m_{\tilde{g } } \tan\beta /{\rm max } ( m_{\tilde{g}}^{2 } , m_{\tilde{b}_{1}}^{2},m_{\tilde{b}_{2}}^{2 } ) $ \end{document } . outside the decoupling regime , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$hb\bar{b}$\end{document } coupling receives the possibly large correction 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & c_b \approx c_b^0 \times\bigl[1- \vardelta _ b/(1+\vardelta _ b ) \times(1 + \cot \alpha\cot\beta ) \bigr ] \\ & \quad \mathrm{with}\ \tan\alpha\stackrel{m_a \gg m_z}{\to}-1/\tan\beta \end{aligned}$$ \end{document } which would significantly alter the partial width of the decay \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h \to b\bar{b}$\end{document } that is , in principle , by far the dominant one and , hence , affect the branching fractions of all other decay modes . in addition , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ht\bar{t}$\end{document } coupling is derived indirectly from the ggh production cross section and the h decay branching ratio , two processes that are generated via triangular loops . in the mssm , these loops involve not only the top quark ( and the w boson in the decay h ) but also contributions from supersymmetric particles , if they are not too heavy . in the case of the ggh process , only the contributions of stops is generally important . including the latter and working in the limit \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{h } \ll m_{t } , m_{\tilde { t}_{1 } } , m_{\tilde { t}_{2 } } $ \end{document } , the hgg amplitude can be ( very well ) approximated by the expression [ 1618 ] 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c_t \approx & c_t^0 \times \biggl [ 1 + \frac{m_t^2 } { 4 m_{\tilde{t}_1}^2 m_{\tilde{t}_2}^2 } \bigl ( m_{\tilde{t}_1}^2 + m_{\tilde{t}_2}^2 \\ & { } - ( a_t -\mu\cot\alpha ) ( a_t+\mu\tan\alpha ) \bigr ) \biggr ] \end{aligned}$$ \end{document } which shows that indeed , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde{t}$\end{document } contributions can be very large for sufficiently light stops and in the presence of large stop mixing . in the h decay rate , because the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t , \tilde{t}$\end{document } electric charges are the same , the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ht\bar{t}$\end{document } coupling is shifted by the same amount as above . if one ignores the usually small \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde{b}$\end{document } contributions in the ggh production and h decay processes ( in the latter case , it is suppressed by powers of the b electric charge \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{b}^{2}/e_{t}^{2 } = \frac{1}{4}$\end{document } in addition ) as well as the contributions of other susy particles such as charginos and staus in the h decay rate,4 the leading corrections to the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ht\bar{t}$\end{document } vertex can be simply accounted for by using the effective coupling given in eq . note that in the case of associated production of the h boson with top quarks , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$gg / q\bar{q } \to h t\bar{t}$\end{document } , it is the parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{t}^{0}$\end{document } which should be considered for the direct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ht\bar{t}$\end{document } coupling . however , for the time being ( and presumably for a long time ) , the constraints on the h properties from this process are very weak as the cross section has very large uncertainties . one also should note that the couplings of the h boson to leptons and charm quarks do not receive the direct corrections of , respectively , eqs . ( 7 ) and ( 8) and one should still have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{c}=c_{t}^{0}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{\tau}= c_{b}^{0}$\end{document}. however , using ct , b or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{t , b}^{0}$\end{document } in this case has almost no impact in practice as these couplings appear only in the branching ratios for the decays \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h \to c\bar{c}$\end{document } and which are small , below 5 % , and the direct corrections can not be very large ( these are radiative corrections after all ) . one can thus , in a first approximation , ignore them and assume that cc = ct and c=cb . note that br(\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h\to c\bar{c}$\end{document } ) can not be measured at the lhc while the h rate is presently measured only at the level of 40 % or so . another caveat is that possible invisible decays ( which at present are probed directly only for rates that are at the 50 % to 100 % level ) , can also affect the properties of the observed h particle . however , a large invisible rate implies that the neutralinos that are considered as the lightest susy particles , are relatively light and couple significantly to the h boson , a situation that is rather unlikely ( if the lsp is very light , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$2m_{\chi_{1}^{0 } } \lesssim m_{h}$\end{document } , it should be mostly bino - like and , hence , has very suppressed couplings to the higgs bosons that prefer to couple to mixtures of higgsinos and gauginos ; see for instance ref . ) . in the case of large direct corrections , the higgs couplings can not be described only by the parameters and as in eq . one should consider at least three independent h couplings , namely cc = ct , c=cb and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{v}=c_{v}^{0}$\end{document } as advocated in ref . . this is equivalent to excluding the h data from the global fit which , in practice , has no significant impact as the experimental error on the signal strength in this channel is presently large . note that a future determination of the theoretically clean ratio of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b\bar{b}$\end{document } and signals in pphv gives a direct access to the b correction outside the decoupling regime [ 2224 ] . to study the h state at the lhc , we thus define the following effective lagrangian : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } \mathcal { l}_h = & c_v g_{hww } h w_{\mu}^+ w^{- \mu } + c_v g_{hzz } h z_{\mu}^0 z^{0 \mu } \\ & { } - c_t y_t h \bar{t}_l t_r - c_t y_c h \bar{c}_l c_r - c_b y_b h \bar{b}_l b_r \\ & { } - c_b y_\tau h \bar{\tau}_l \tau_r + \mathrm{h.c . } \end{aligned}$$ \end{document } where yt , c , b,=mt , c , b,/v are the sm yukawa coupling constants in the mass eigenbasis ( l / r indicates the fermion chirality and we consider only the heavy fermions that have substantial couplings to the higgs boson ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g_{hww } = 2m^{2}_{w}/v$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g_{hzz } = m^{2}_{z}/v$\end{document } are the electroweak gauge boson couplings and v is the higgs vacuum expectation value . we present the results for the fits of the higgs signal strengths in the various channels 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } \mu_x \simeq & \sigma ( pp \to h ) \times{\rm br}(h \to xx)/ \sigma ( pp \to h)_{\rm sm } \\ & { } \times{\rm br}(h \to xx)_{\rm sm } \end{aligned}$$ \end{document } closely following the procedure of refs . all the higgs production / decay channels are considered and the data used are the latest ones using the full 25 fb statistics for the ,zz , ww channels as well as the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h\to b\bar{b}$\end{document } and modes for cms , but only 17 fb data for the atlas fermionic channels . we have performed the appropriate three - parameter fit in the three - dimensional space5 [ ct , cb , cv ] , assuming cc = ct and c=cb as discussed above and of course the custodial symmetry relation cv = cw = cz which holds in supersymmetric models . the results of this fit are presented in fig . 4 for ct , cb , cv0 , as motivated by the supersymmetric structure of the higgs couplings ( there is also an exact reflection symmetry under , cc or equivalently + , leaving the squared amplitudes of the higgs rates unaffected ) . [ 2224 ] , we have treated the theoretical uncertainty as a bias and not as if it were associated to a statistical distribution and have performed the fit for values of the signal strength \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mu_{i } \vert _ { \rm exp } [ 1 \pm\vardelta \mu _ { i}/\mu_{i } \vert_{\rm th } ] $ \end{document } with the theoretical uncertainty \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mu_{i}/ \mu_{i } \vert_{\rm th}$\end{document } conservatively assumed to be 20 % for both the gluon and the vector boson fusion mechanisms ( because of contamination ) and 5 % for h production in association with v = w / z [ 44 , 45 ] . 4best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green , left ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray , right ) for the higgs signal strengths in the three - dimensional space [ c t , c b , c v ] . the three overlapped regions are associated to central and two extreme choices of the theoretical prediction for the higgs rates best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green , left ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray , right ) for the higgs signal strengths in the three - dimensional space [ c t , c b , c v ] . the three overlapped regions are associated to central and two extreme choices of the theoretical prediction for the higgs rates the best - fit value for the couplings , when the atlas and cms data are combined , is ct=0.89,cb=1.01 and cv=1.02 with =64.8 ( =66.7 in the sm ) . in turn , in scenarios where the direct corrections in eqs . ( 7)(8 ) are not quantitatively significant ( i.e. considering either not too large values of tan or high stop / sbottom masses ) , one can use the mssm relations of eq . for instance , using ct = cos/sin and cv = sin( ) , one can derive the following relation : cbsin/cos=(1cvct)/(cvct ) . this allows to perform the two - parameter fit in the plane [ cv , ct ] . similarly , one can study the planes [ cv , cb ] and [ ct , cb ] . 5 . as in the mssm one has [/2,0 ] and tan[1,50 ] , one obtains the following variation ranges : cv , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_{t } \in[0,\sqrt{2}]$\end{document } and cb>0 . 5best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray ) for the higgs signal strengths in the planes [ c t , c v ] ( left ) , [ c b , c v ] ( center ) and [ c t , c b ] ( right ) . the theoretical uncertainty on the higgs signal strengths is taken into account as a bias . the best - fit contours at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( dashed ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( dotted ) from the fit of signal strength ratios the sm points are indicated in red and the best - fit points in blue ( color figure online ) best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray ) for the higgs signal strengths in the planes [ c t , c v ] ( left ) , [ c b , c v ] ( center ) and [ c t , c b ] ( right ) . the theoretical uncertainty on the higgs signal strengths is taken into account as a bias . the best - fit contours at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( dashed ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( dotted ) from the fit of signal strength ratios the sm points are indicated in red and the best - fit points in blue ( color figure online ) we also show on these figures the potential constraints obtained from fitting ratios of the higgs signal strengths ( essentially the two ratios r=/zz and r=/ww ) that are not or much less affected by the qcd uncertainties at the production level [ 2224 ] . in this two - dimensional case , the best - fit points are located at ( ct=0.88 , cv=1.0 ) , ( cb=0.97 , cv=1.0 ) and ( ct=0.88 , cb=0.97 ) . note that although for the best - fit point one has cb1 , actually cb1 in most of the 1 region . ( 6 ) , one can also realize a two - parameter fit in the [ tan, ] plane6 and with the expressions of eq . ( 5 ) for the mixing angle and fixing mh to the measured value mh125 gev , one can even perform a fit in the plane [ tan,ma ] . 6 where the 68 % cl , 95 % cl and 99 % cl contours from the signal strengths only are displayed when , again , the theoretical uncertainty is considered as a bias . we also display the best - fit contours for the signal strength ratios at the 68 % cl and 95 % cl . the best - fit point for the signal strengths when the theoretical uncertainty is set to zero , is obtained for the values tan=1 and ma=557 gev . one should note , however , that the value is relatively flat all over the 1 region shown in fig . hence , larger values of tan and lower values of ma could also be accommodated reasonably well by the fit . in any case , the best - fit point when taken literally , implies for the other parameters ( using the information mh=125 gev to derive the radiative corrections ) : mh=580 gev , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{h^{\pm}}= 563$\end{document } gev and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha=-0.837~{\rm rad}$\end{document } which leads to cos()0.05 . such a point with tan1 implies an extremely large value of the susy scale , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{s } = \mathcal { o}(100)$\end{document } tev , for mh125 gev . 6 left : best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$95\ \%\ { \rm cl}$\end{document } ( yellow ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray ) for the higgs signal strengths in the plane [ tan,m a ] ; the best - fit point is shown in blue and the theoretical uncertainty is taken into account as a bias as in the previous figures . the best - fit contours at 1 ( dashed ) and 2 ( dotted ) for the signal strength ratios are also shown . right : we superimpose on these constraints the excluded regions ( in red , and as a shadow when superimposed on the best - fit regions ) from the direct searches of the heavier higgs bosons at the lhc following the analysis of ref . ( color figure online ) left : best - fit regions at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( green ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$95\ \%\ { \rm cl}$\end{document } ( yellow ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( light gray ) for the higgs signal strengths in the plane [ tan,m a ] ; the best - fit point is shown in blue and the theoretical uncertainty is taken into account as a bias as in the previous figures . the best - fit contours at 1 ( dashed ) and 2 ( dotted ) for the signal strength ratios are also shown . right : we superimpose on these constraints the excluded regions ( in red , and as a shadow when superimposed on the best - fit regions ) from the direct searches of the heavier higgs bosons at the lhc following the analysis of ref . ( color figure online ) it is interesting to superimpose on these indirect limits in the [ tan,ma ] plane , the direct constraints on the heavy h / a / h boson searches performed by the atlas and cms collaborations as shown in the right - hand side of fig . 6 . as discussed in ref . ( see also ref . ) , besides the limits from the a / h and to a lesser extent tbhb searches which exclude high tan values and which can be extended to very low tan as well , there are also limits from adapting to the mssm the high mass sm higgs searches in the channels7hww and zz as well as the searches for heavy resonances decaying into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t\bar{t}$\end{document } final states that exclude low values of tan and ma . for values we have discussed the hmssm , i.e. the mssm that we seem to have after the discovery of the higgs boson at the lhc that we identify with the lighter h state . the mass mh125 gev and the non - observation of susy particles , seems to indicate that the soft - susy breaking scale might be large , ms1 tev . we have shown , using both approximate analytical formulas and a scan of the mssm parameters , that the mssm higgs sector can be described to a good approximation by only the two parameters tan and ma if the information mh=125 gev is used . one could then ignore the radiative corrections to the higgs masses and their complicated dependence on the mssm parameters and use a simple formula to derive the other parameters of the higgs sector , , mh and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{h^{\pm}}$\end{document}. this will considerably simplify phenomenological analyses in the mssm which up to now rely either on large scans of the parameter space ( as e.g. refs . [ 26 , 27 ] ) or resort to benchmark scenarios in which most of the mssm parameters are fixed ( as is the case of ref . for instance ) . in a second step , we have shown that to describe accurately the h properties when the direct radiative corrections are also important , the three couplings ct , cb and cv are needed besides the h mass . we have performed a fit of these couplings using the latest lhc data and taking into account properly the theoretical uncertainties . the fit turns out to very slightly favor the low tan region with a not too high cp - odd higgs mass . first , the heavier higgs particles could be searched for in the next lhc run in the channels \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a \to t \bar{t } , hz$\end{document } and and in the modes hww , zz , hh for which the rates can be substantial for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tan\beta = \mathcal { o}(1)$\end{document}. this is shown in fig . 7 where the cross sections times decay branching ratios for a and h are displayed as a function of tan for the choice ma=557 gev for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{s}=14$\end{document } tev . furthermore , the correct relic abundance of the lsp neutralino can be easily obtained through \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\chi_{1}^{0 } \chi_{1}^{0 } \to a \to t\bar{t}$\end{document } annihilation by allowing the parameters and m1 to be comparable and have an lsp mass close to the a - pole , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{\chi_{1}^{0 } } \approx\frac{1}{2 } m_{a}$\end{document}. this low tan region will be discussed in more detail in a separate publication . 7the cross section times branching fractions for the a ( left ) and h ( right ) mssm higgs bosons at the lhc with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{s}=14$\end{document } tev as a function of tan for the best - fit mass m a=557 gev and with m h=125 gev . for the production , we have taken into account only the gluon and bottom quark fusion processes and followed the analysis given in ref . the cross section times branching fractions for the a ( left ) and h ( right ) mssm higgs bosons at the lhc with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt{s}=14$\end{document } tev as a function of tan for the best - fit mass m a=557 gev and with m h=125 gev . for the production , we have taken into account only the gluon and bottom quark fusion processes and followed the analysis given in ref . the radiative corrections to the cp - even higgs boson mass matrix can be written as a.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } \mathcal { m}^2 = \left [ \begin{array}{c@{\quad } c } \mathcal { m}_{11}^2 + \vardelta \mathcal { m}_{11}^2 & \mathcal { m}_{12}^2 + \vardelta \mathcal { m}_{12}^2 \\ \mathcal { m}_{12}^2 + \vardelta \mathcal { m}_{12}^2 & \mathcal { m}_{22}^2 + \vardelta \mathcal { m}_{22}^2 \end{array } \right ] \end{aligned}$$ \end{document } the leading one - loop radiative corrections \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}_{ij}^{2}$\end{document } to the mass matrix are controlled by the top yukawa coupling t = mt / vsin which appears with the fourth power . one can obtain a very simple analytical expression if only this contribution is taken into account [ 79 ] : a.2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \vardelta \mathcal { m}_{11}^2 \sim \vardelta \mathcal { m}_{12}^2 \sim0 \\ & \vardelta \mathcal { m}_{22}^2 \sim \epsilon\\ & \hphantom{\vardelta \mathcal { m}_{22}^2 } = \frac{3 \bar { m}_t^4}{2\pi^2 v^2\sin^ 2\beta } \biggl [ \log\frac{m_s^2}{\bar{m}_t^2 } + \frac{x_t^2}{m_s^2 } \biggl ( 1 - \frac{x_t^2}{12m_s^2 } \biggr ) \biggr]\end{aligned } $ $ \end{document } where ms is the geometric average of the stop masses \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{s } = \sqrt { m_{\tilde{t}_{1}}m_{\tilde{t}_{2 } } } $ \end{document } , xt is the stop mixing parameter given by xt = at/tan and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar{m}_{t}$\end{document } is the running \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm \overline{ms}}$\end{document } top quark mass to account for the leading two - loop qcd corrections in a renormalization - group improvement . a better approximation , with some more renormalization - group improved two - loop qcd and electroweak corrections included is given by [ 10 , 11 ] a.3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } \vardelta \mathcal { m}_{22}^2 = & \frac{3}{2\pi^2 } \frac{m_t^4}{v^2 \sin ^2\beta } \biggl [ \frac{1}{2 } \tilde{x}_t + \ell_s \\ & { } + \frac{1}{16\pi^2 } \biggl(\frac{3}{2 } \frac{m_t^2}{v^2}-32\pi\alpha_s \biggr ) \bigl(\tilde{x}_t \ell_s+ \ell_s^2 \bigr ) \biggr ] \end{aligned}$$ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\ell_{s}= \log(m_{s}^{2}/m_{t}^{2})$\end{document } and using xt = xt / ms=(atcot)/ms one has \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde{x}_{t } = 2 x_{t}^{2 } ( 1 - x_{t}^{2}/12)$\end{document } with at the trilinear higgs - stop coupling and the higgsino mass parameter . other soft susy - breaking parameters , in particular and ab ( and in general the corrections controlled by the bottom yukawa coupling b = mb / vcos which at large value of the product tan , provide a non - negligible correction to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathcal { m}_{ij}^{2}$\end{document } ) can also have an impact on the loop corrections . including these subleading contributions at one - loop , plus the leading logarithmic contributions at two loops , the radiative corrections to the cp - even mass matrix elements can still be written in a compact form a.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \vardelta \mathcal { m}_{11}^2 & = - \frac{v^2 \sin^2\beta}{32 \pi^2 } \bar { \mu}^2 \bigl [ x_t^2 \lambda_t^4 ( 1 + c_{11 } \ell_s)\\ & \quad { } + a_b^2 \lambda_b^4 ( 1 + c_{12 } \ell_s ) \bigr ] \\ \vardelta \mathcal { m}_{12}^2 & = - \frac{v^2 \sin^2\beta}{32 \pi^2 } \bar { \mu } \bigl [ x_t \lambda_t^4 ( 6- x_t a_t ) ( 1 + c_{31 } \ell_s)\\ & \quad { } - \bar{\mu}^2 a_b \lambda_b^4 ( 1 + c_{32 } \ell_s ) \bigr ] \\ \vardelta \mathcal { m}_{22}^2 & = \frac{v^2 \sin^2\beta}{32 \pi^2 } \bigl [ 6 \lambda_t^4 \ell_s ( 2 + c_{21 } \ell_s)\\ & \quad { } + x_t a_t \lambda_t^4 ( 12 - x_t a_t ) ( 1 + c_{21 } \ell_s)\\ & \quad { } - \bar{\mu}^4 \lambda_b^4 ( 1 + c_{22 } \ell_s ) \bigr ] \end{aligned}$$\end{document } where the additional abbreviations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar{\mu}=\mu / m_{s}$\end{document } and at , b = at , b / ms have been used . the factors cij take into account the leading two - loop corrections due to the top and bottom yukawa couplings and to the strong coupling constant \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g_{s}=\sqrt{4\pi \alpha_{s}}$\end{document } ; they read a.5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c_{ij}= \frac{1}{32\pi^2 } \bigl(t_{ij}\lambda_{t}^2 + b_{ij}\lambda_{b}^2 -32 g_s^2 \bigr ) \end{aligned}$$ \end{document } with the various coefficients given by ( t11,t12,t21,t22,t31,t32)=(12,4,6,10,9,7 ) and ( b11,b12,b21,b22,b31,b32)=(4,12,2,18,1,15 ) . the expressions eq . however , one needs to include the full set of corrections to have precise predictions for the higgs boson masses and couplings as discussed at the end of sect . 2 .

we analyze the minimal supersymmetric extension of the standard model that we have after the discovery of the higgs boson at the lhc , the hmssm ( habemus mssm ? ) , i.e. a model in which the lighter h boson has a mass of approximately 125 gev which , together with the non - observation of superparticles at the lhc , indicates that the susy - breaking scale ms is rather high , ms1 tev . we first demonstrate that the value mh125 gev fixes the dominant radiative corrections that enter the mssm higgs boson masses , leading to a higgs sector that can be described , to a good approximation , by only two free parameters . in a second step , we consider the direct supersymmetric radiative corrections and show that , to a good approximation , the phenomenology of the lighter higgs state can be described by its mass and three couplings : those to massive gauge bosons and to top and bottom quarks . we perform a fit of these couplings using the latest lhc data on the production and decay rates of the light h boson and combine it with the limits from the negative search of the heavier h , a and h states , taking into account the current uncertainties .

Introduction Post Higgs discovery parametrization ofradiativecorrections Determination of the Conclusion Appendix:Approximating the radiative corrections

the observation at the lhc of a higgs particle with a mass of 125 gev [ 1 , 2 ] has important implications for supersymmetric ( susy ) and , in particular , for the minimal supersymmetric standard model ( mssm ) . in this extension , the higgs sector consists of two scalar doublet fields hu and hd that lead , after electroweak symmetry breaking , to five higgs states , two cp - even h and h , a cp - odd a and two charged h bosons [ 36 ] . at tree level , the masses of these particles and their mixings are described by only two parameters usually chosen to be the ratio of the vacuum expectations values of the two doublet fields tan=vd / vu and the mass ma of the pseudoscalar higgs boson . on the other hand , the fact that the measured value mh125 gev is close to this upper mass limit implies that the susy - breaking scale ms might be rather high . hence , the mssm that we currently have , and that we call hmssm ( habemus mssm ? ) [ 1315 ] that when the information mh=125 gev is taken into account , the mssm higgs sector with solely the dominant radiative correction to the higgs boson masses included , can be again described with only the two free parameters tan and ma as it was the case at tree level . in other words , the dominant radiative corrections that involve the susy parameters are fixed by the value of mh . in this paper , we show that to a good approximation , this remains true even when the full set of radiative corrections to the higgs masses at the two - loop level is included . among such corrections are , for instance , the stop contribution [ 1619 ] to the dominant higgs production mechanism at the lhc , the gluon fusion process ggh , and to the important decay into two photons h , and the additional one - loop vertex corrections to the h couplings to b - quarks that grow with tan . however , we show that given the accuracy that is foreseen at the lhc , a good approximation is to consider the three effective couplings to t , b quarks and to v = w / z bosons , ct , cb and cv , as it was suggested in ref . [ 2224 ] for the inclusion of the current theoretical and experimental uncertainties , we perform a fit of these three couplings using the latest lhc data on the production and decay rates of the lighter h boson and the limits from the negative search of the heavier h , a and h mssm states . one can then easily derive the neutral cp - even higgs boson masses and the mixing angle that diagonalizes the h , h states,2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h= \cos\alpha h_{d}^{0 } + \sin\alpha h_{u}^{0}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h= -\sin\alpha h_{d}^{0 } + \cos\alpha h_{u}^{0}$\end{document } : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & m_{h / h}^2=\frac{1}{2 } \bigl ( m_{a}^2+m_{z}^2 + \vardelta \mathcal { m}_{11}^2 + \vardelta \mathcal { m}_{22}^2 \mp \sqrt { m_{a}^4+m_{z}^4 - 2 m_{a}^2 m_{z}^2 c_{4\beta } + c } \bigr ) \end{aligned}$$ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } & \tan\alpha= \frac{2\vardelta \mathcal { m}_{12}^2 - ( m_{a}^2 + m_{z}^2 ) s_{\beta } } { \vardelta \mathcal { m}_{11}^2 - \vardelta \mathcal { m}_{22}^2 + ( m_{z}^2-m_{a}^2 ) c_{2\beta } + \sqrt{m_{a}^4 + m_{z}^4 - 2 m_{a}^2 m_{z}^2 c_{4\beta } + c } } \end{aligned}$$ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c= 4 \vardelta \mathcal { m}_{12}^4 + \bigl ( \vardelta \mathcal { m}_{11}^2 - \vardelta \mathcal { m}_{22}^2 \bigr)^2 - 2 \bigl(m_{a}^2 - m_{z}^2\bigr ) \bigl ( \vardelta \mathcal { m}_{11}^2 - \vardelta m_{22}^2\bigr ) c_{2\beta } - 4 \bigl(m_{a}^2 + m_{z}^2\bigr ) \vardelta \mathcal { m}_{12}^2 s_{2\beta } \end{aligned}$$ \end{document } in previous analyses [ 1315 ] , we have assumed that in the 22 matrix for the radiative corrections , only the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22}$\end{document } entry which involves the by far dominant stop top sector correction , is relevant , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{22 } \gg\vardelta \mathcal { m}^{2}_{11 } , \vardelta \mathcal { m}^{2}_{12}$\end{document}. in order to check more thoroughly the impact of the subleading corrections \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{12}$\end{document } , we perform a scan of the mssm parameter space using the program suspect [ 29 , 30 ] in which the full two - loop radiative corrections to the higgs sector are implemented . for each of the points , we have compared the higgs parameters to those obtained in the simplified mssm approximation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vardelta \mathcal { m}^{2}_{11 } = \vardelta \mathcal { m}^{2}_{12 } = 0$\end{document } , with the lightest higgs boson mass as input . in principle and as discussed earlier , knowing two parameters such as the pair of inputs [ tan,ma ] and fixing the value of mh to its measured value , the couplings of the higgs bosons , in particular h , to fermions and gauge bosons can be derived , including the generally dominant radiative corrections that enter in the mssm higgs masses . normalized to their sm values ) of the lighter h state to third generation t , b fermions and gauge bosons v = w / z , 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ \begin{aligned } c_v^0 = \sin(\beta- \alpha ) , \qquad c_t^0 = \frac{\cos \alpha}{\sin\beta } , \qquad c_b^0 = - \frac{\sin \alpha}{\cos\beta } \end{aligned}$$ \end{document } however , outside the regime in which the pseudoscalar a boson and some supersymmetric particles are very heavy , there are also direct radiative corrections to the higgs couplings not contained in the mass matrix of eq . the best - fit contours at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$68\ \%\ { \rm cl}$\end{document } ( dashed ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$99\ \%\ { \rm cl}$\end{document } ( dotted ) from the fit of signal strength ratios the sm points are indicated in red and the best - fit points in blue ( color figure online ) we also show on these figures the potential constraints obtained from fitting ratios of the higgs signal strengths ( essentially the two ratios r=/zz and r=/ww ) that are not or much less affected by the qcd uncertainties at the production level [ 2224 ] . right : we superimpose on these constraints the excluded regions ( in red , and as a shadow when superimposed on the best - fit regions ) from the direct searches of the heavier higgs bosons at the lhc following the analysis of ref . right : we superimpose on these constraints the excluded regions ( in red , and as a shadow when superimposed on the best - fit regions ) from the direct searches of the heavier higgs bosons at the lhc following the analysis of ref . , besides the limits from the a / h and to a lesser extent tbhb searches which exclude high tan values and which can be extended to very low tan as well , there are also limits from adapting to the mssm the high mass sm higgs searches in the channels7hww and zz as well as the searches for heavy resonances decaying into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t\bar{t}$\end{document } final states that exclude low values of tan and ma . the mssm that we seem to have after the discovery of the higgs boson at the lhc that we identify with the lighter h state . the mass mh125 gev and the non - observation of susy particles , seems to indicate that the soft - susy breaking scale might be large , ms1 tev . we have shown , using both approximate analytical formulas and a scan of the mssm parameters , that the mssm higgs sector can be described to a good approximation by only the two parameters tan and ma if the information mh=125 gev is used . in a second step , we have shown that to describe accurately the h properties when the direct radiative corrections are also important , the three couplings ct , cb and cv are needed besides the h mass . we have performed a fit of these couplings using the latest lhc data and taking into account properly the theoretical uncertainties .

malaria , a major tropical disease for which no long - term sustainable treatment is available , continues to affect large parts of the world . according to the latest world malaria report , 3.2 billion people in 96 countries are at risk of being infected . in 2015 alone , 214 million cases were reported globally resulting in an estimated 438,000 deaths mainly consisting of african children and pregnant women ( who , 2015 ) . this life - threatening disease is caused by plasmodium species with plasmodium falciparum ( p. falciparum ) being the most deadly . currently , first - line therapy includes artemisinin - based combination therapies ( act ) ( delves et al . , 2012 , wells et al . , 2015 ) ; however , in recent years parasite resistance against artemisinin and its derivatives has emerged and spread along the cambodia - thailand border ( ashley et al . , 2014 , tun et al . , 2015 ) . although recent efforts in antimalarial drug discovery have focused on new targets , nonclassical chemical scaffolds , and vaccines ( biamonte et al . , 2013 , barnett and guy , 2014 , wells et al . , 2015 ) , extensive studies on classical antimalarial chemotypes or drug repurposing are needed due to the high cost and time required in the drug discovery and development process ( andrews et al . , 2014 , biamonte , 2014 ) . one classic antimalarial chemotype present in potent antimalarial drugs , including quinine ( 1 ) , mefloquine ( 2 ) , lumefantrine ( 3 ) , and halofantrine ( 4 ) , is the arylamino alcohol ( - amino or -amino alcohol moiety ) . the structural requirements for antiplasmodial activity include the presence of an aromatic and amino alcohol portion linked by a carbon chain of two or three atoms in length ( bhattacharjee and karle , 1996 ) ( fig . 1 ) . based on this antiplasmodial pharmacophore , antimalarial amino alcohols continue to attract the interest of various research groups due to their high biological activity and admet values . representative studies of the - amino alcohol moiety include the works of guy et al . who performed the optimization of propafenone analogues ( lowes et al . , 2011 , lowes et al . , 2012 ) as a product of high throughput screening ( weisman et al . , 2006 ) , and smith and chibale et al . who developed totarol ( clarkson et al . , 2003 , 2012 ) and chalcone ( hans et al . , 2010 ) , respectively , as natural product - like hybrid derivatives . in addition , mefloquine and 4-aminoquinoline derivatives are valid synthetic approaches explored by milner et al . ( 2012 ) , respectively , among other studies ( robin et al . , 2007 , dhooghe et al . , 2011 ) . however , there are only a few examples of the -amino alcohol moiety reported in the last thirteen years ( dhooghe et al . , 2009 , perez - silanes et al . , 2009 , mendoza et al . , 2011 , ( 2009 ) reported a wide range of antiplasmodial activity against the chloroquine sensitive p. falciparum strain d10 ; however , submicromolar values were not reached ( 6 m ic50 175 m ) . thus , further studies on the unexplored -moiety as a source of new antimalarial drugs are needed . our research on -amino alcohols explored the structure - activity relationship ( sar ) of 1-aryl-3-susbtituted propanol derivatives ( apd ) with promising results ( perez - silanes et al . , 2009 , mendoza et al . , 2011 , quiliano and aldana , 2013 ) . initial sar studies showed that all of the aryl - ketone derivatives were inactive against 3d7 , nf54 , and fcr-3 strains of p. falciparum ( perez - silanes et al . in contrast , apd were active against 3d7 ( 0.19 ic50 0.38 m ) ( perez - silanes et al . , 2009 ) , nf54 ( 1.3 ic50 8 m ) ( mendoza et al . , 2011 ) , and fcr-3 ( 0.5 ic50 10 m ) ( mendoza et al . , 2011 ) strains of p. falciparum . however , low parasitemia reduction ( 65% ) was observed in the in vivo plasmodium berghei ( p. berghei ) mouse model . interestingly , linker reduction in one carbon atom between the alcohol and amine portion ( -to - amino alcohol ) in apd reduced the activity by half . previous , in silico studies proposed plasmepsin ii ( pm2 ) as a putative target for apd ( mendoza et al . , 2011 ) , but has yet to be confirmed experimentally . nonetheless , more studies with apd are necessary to be validated as potential antimalarial hits ( mmv , 2008 ) . thus , exploration and development of apd as antimalarials requires ( 1 ) expanding sar studies , ( 2 ) generating additional analogues with high potency against both chloroquine sensitive and multidrug resistant strains of p. falciparum , ( 3 ) improving parasitemia reduction in the p. berghei mouse model , ( 4 ) establishing a safe toxicological profile , and ( 5 ) exploring biological targets in p. falciparum . in this study , we expanded the chemical scope in the antimalarial framework by preparing new apd for testing their antiplasmodial activity in vitro against p. falciparum ( sensitive and resistant strains ) and in vivo against p. berghei . since target validation is a crucial step in the drug discovery process , apd compounds were evaluated for their ability to inhibit both the pm2 enzyme and the hemozoin formation pathway . the methods used for synthesizing the final compounds ( 1426 ) are presented in scheme 1 , scheme 2 . the synthetic method has been published previously ( perez - silanes et al . , 2009 , mendoza et al . , 2011 ) . the starting arylamines 2-nitro-4-trifluoromethyl phenyl piperazine , 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine and 4-trifluoromethyl phenyl piperazine were commercially available . non - commercially available arylamines were synthesized using the corresponding boc - amine ( i ) and 2-fluoro-4-nitrobenzotrifluoride by an ar - sn reaction via meisenheimer complex formation and subsequent removal of the boc - group with hcl and acetic acid ( scheme 1 ) . all methyl ketone precursors ( v ) were commercially available . the ketone intermediates ( -amino ketones ) ( 113 ) were prepared by condensation of the corresponding methyl ketone ( v ) with the different aryl amines ( iv ) via mannich reaction ( scheme 2 ) . the hydroxyl derivatives ( -amino alcohols ) ( 1426 ) were obtained by reduction of the corresponding carbonyl group with nabh4 in methanol ( scheme 2 ) . chemical reagents and solvents were purchased from commercial companies and were used without further purification . all of the synthesized final compounds were chemically characterized by thin layer chromatography ( tlc ) , melting point ( mp ) and proton and carbon nuclear magnetic resonance ( h nmr and c nmr ) spectra as well as by elemental microanalysis . h nmr and c nmr spectra were recorded on a bruker 400 ultrashield ( rheinstetten , germany ) operating at 400 and 100 mhz , respectively , using tetramethylsilane ( tms ) as the internal standard and chloroform ( cdcl3 ) or dimethyl sulfoxide - d6 ( dmso - d6 ) as solvents . the chemical shifts are reported in ppm ( ) and the coupling constant ( j ) values are given in hertz ( hz ) . elemental microanalyses were obtained on an elemental analyzer ( leco chn-900 , michigan , usa ) from vacuum - dried samples . the analytical results for c , h , and n were within 0.4 of the theoretical values . alugram sil g / uv254 ( layer : 0.2 mm ) ( macherey - nagel , germany ) was used for tlc and silica gel 60 ( 0.0400.063 mm and 0.0630.200 nm ) was used for column chromatography ( merck ) . some final derivatives were purified by automated flash chromatography with a binary gradient of dichloromethane ( dcm ) ( synthesis grade sds - carlo erba reactifs , france ) and methanol ( meoh ) ( panreac qumica s.a . ) and uv variable dual - wavelength detection . usa ) with dcm / meoh as solvents and a normal phase of 12 g flash column ( redisep rf columns by teledyne isco , inc . hplc experiments were developed on an ultimate 3000 chromatograph ( dionex ) with chromeleon v.6.8 software . the measurements were performed using an rp 18 column ( lichrospher 100 rp 18 e.c . 5 m ; 10 0.46 cm ; teknokroma ) as the stationary phase at a flow rate of 1 ml / min and with meoh / water ( 80:20 ) as the mobile phase . the retention times ( tr ) are expressed in minutes and the reference wavelength is set at 254 nm . compounds ii and iii were synthesized as previously described ( perez - silanes et al . , 2009 , mendoza et al . , 2011 ) . the corresponding substituted aryl methyl ketone ( v ) ( 1.0 equiv ) and the aryl amine ( iv ) ( 1.0 equiv ) was dissolved in 1,3-dioxolane ( 1.4% ) and concentrated hcl ( 1 ml ) was added until ph 12 was reached . the resulting mixture was heated at reflux for 12 h. naoh ( 2 m ) was then added ( 50 ml ) and the product was extracted with dcm ( 3 50 ml ) . the organic phase was dried with anhydrous na2so4 , filtered , and evaporated to dryness under reduced pressure . the residue was purified by column chromatography on silica gel using dcm / meoh 95:5 ( v / v ) as eluent or automated flash chromatography eluting with dcm / meoh 99:1 ( v / v ) . in other cases , the hydrochloride salt was prepared by adding a hydrogen chloride ethereal solution to the stirred compounds . sodium borohydride ( 3.0 equiv ) was added dropwise to a pre - cooled suspension ( 0 c ) of the corresponding ketone ( v ) ( 1.0 equiv ) in meoh over a period of 3060 min . the solvent was removed under reduced pressure and the residue was dissolved in dcm ( 40 ml ) and then washed with water ( 3 30 ml ) . the organic phase was dried with anhydrous na2so4 and filtered . after evaporating the solvent to dryness under reduced pressure , the compound was purified by column chromatography ( sp : silica gel ) eluting with dcm / meoh 99:1 ( v / v ) or automated flash chromatography eluting with dcm / meoh 99:1 ( v / v ) . selected compounds were converted to hydrochloride salts by adding a hydrogen chloride ethereal solution to the stirred compounds . h nmr ( dmso - d6 , 400 mhz ) ppm : 8.46 , 8.49 ( dd , 1h , j = 9.0 hz , j = 2.5 hz ) ; 8.42 ( d , 1h , j = 2.6 hz ) ; 8.04 ( q , 1h , j = 8.8 hz , j = 5.1 hz ) ; 7.82,7.83 ( dd , 1h , j = 10.3 hz , j = 2.4 hz ) ; 7.77 ( s , 1h ) ; 7.65 ( d , 1h , j = 9.0 hz ) ; 7.27 ( dt , 1h , j = 8.9 hz , j = 2.4 hz ) ; 5.82 ( br s , 1h ) ; 5.05 ( br s , 1h ) ; 3.64 ( d , 2h ) ; 3.423.50 ( m , 6h ) ; 3.17 ( br s , 2h ) ; 2.23,2.30 ( dd , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 162.07 ; 159.65 ; 156.10 ; 151.77 ; 143.58 ; 140.66 ( d , j = 4.6 hz ) ; 139.18 ( d , j = 8.4 hz ) ; 136.75 ; 129.60 ; 126.26 ; 125.43 ( d , j = 9.0 hz ) ; 125.13 ; 124.56 ( d , j = 6.8 hz ) ; 113.78 ( d , j = 25.4 hz ) ; 109.15 ( d , j = 23.6 hz ) ; 66.45 ; 54.19 ; 51.92 ( 2c ) ; 50.05 ( 2c ) ; 31.81 ppm . calcd for c22h21n3f4o3s : c , 54.65% ; h , 4.38% ; n , 8.64% ; found : c , 54.83% ; h , 4.44% ; n , 8.99% . h nmr ( 400 mhz , dmso - d6 ) : 8.20 ( s , 1h ) , 8.02 ( dd , 1h , j = 8.9 hz , 5.1 hz ) , 7.90 ( d , 1h , j = 8.4 hz ) , 7.77 ( d , 1h , j = 10.2 hz ) , 7.72 ( s , 1h ) , 7.49 ( d , 1h , j = 8.5 hz ) , 7.26 ( dt , 1h , j = 9.1 hz , 8.8 hz , 2.2 hz ) , 5.00 ( br s , 1h ) , 3.70 ( br s , 2h ) , 3.24 ( br s , 4h ) , 2.92 ( bs , 4h ) , 2.06 ( br s , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 161.98 ; 159.61 ; 147.87 ; 140.78 ; 139.33 ( d , j = 9.0 hz ) ; 136.73 ; 131.20 ( d , j = 3.1 hz ) ; 126.02 ; 125.68 ; 125.41 ( d , j = 9.1 hz ) ; 124.60 ( q , j = 4.1 hz ) ; 122.98 ; 122.74 ; 113.71 ( d , j = 25.4 hz ) ; 109.06 ( d , j = 23.2 hz ) ; 74.12 ; 66.99 ; 54.88 ( 2c ) ; 52.35 ( 2c ) ; 48.23 ppm . calcd for c22h21n3f4o3s.hcl : c , 50.81% ; h , 4.23% ; n , 8.08% ; found : c , 51.20% ; h , 3.85% ; n , 8.11% . h nmr ( 400 mhz , cdcl3 ) : 7.80 ( br s , 1h ) , 7.507.54 ( m , 4h ) , 7.107.15 ( m , 1h ) , 6.96 ( d , 2h , j = 8.4 hz ) , 5.32 ( dd , 1h , j = 4.1 hz , 6.7 hz ) , 3.45 ( br s , 4h ) , 2.932.94 ( m , 3h ) , 2.82 ( br s , 3h ) , 2.21 ( bs , 2h ) ppm . c nmr ( cdcl3 , 100 mhz ) : 162.20 ; 159.80 ; 153.44 ; 139.83 ( d , j = 4.4 hz ) ; 138.71 ( d , j = 9.1 hz ) ; 136.76 ( d , j = 1.4 hz ) ; 126.88 ( 2c , q , j = 3.8 hz ) ; 126.44 ; 124.64 ; 124.41(d , j = 9.4 hz ) ; 115.19 ( 2c ) ; 113.51 ( d , j = 25.2 hz ) ; 108.29 ( d , j = 23.4 hz ) ; 71.97 ; 57.57 ; 53.40 ( 2c ) ; 48.51 ( 2c ) ; 31.87 ppm . calcd for c22h22n2f4os : c , 60.26% ; h , 5.06% ; n , 6.39% ; found : c , 60.30% ; h , 4.95% ; n , 6.24% . h nmr ( 400 mhz , cdcl3 ) : 8.54 ( s , 1h ) , 8.28 ( dd , 1h , j = 9.2 hz , 2.7 hz ) , 7.80 ( dd , 1h , j = 8.8 hz , 5.0 hz ) , 7.51 ( dd , 1h , j = 9.8 hz , 1.5 hz ) , 7.50 ( s , 1h ) , 7.22 ( d , 1h , j = 9.2 hz ) , 7.12 ( dd , 1h , j = 8.8 hz , 2.4 hz ) , 5.62 ( br s , 2h ) , 5.30 ( t , 1h , j = 5.7 hz ) , 3.54 ( br s , 2h ) , 2.982.99 ( m , 2h ) , 2.872.93 ( m , 4h ) , 2.08 ( br s , 4h ) ppm . c nmr ( cdcl3 , 100 mhz ) : 162.18 ; 159.78 ; 157.28 ; 151.76 ; 140.69 ; 139.90 ( d , j = 4.7 hz ) ; 138.68 ( d , j = 8.9 hz ) ; 136.76 ( d , j = 1.3 hz ) ; 127.92 ; 125.78 ( q , j = 5.9 hz ) ; 124.62 ; 124.40 ( d , j = 9.4 hz ) ; 121.74 ; 113.49 ( d , j = 25.0 hz ) ; 108.26 ( d , j = 23.4 hz ) ; 72.07 ; 57.69 ; 56.35 ; 55.68 ; 55.37 ; 54.64 ; 32.60 ; 28.04 ppm . calcd for c23h23n3f4o3s : c , 55.53% ; h , 4.66% ; n , 8.45% ; found : c , 55.15% ; h , 4.64% ; n , 8.26% . h nmr ( 400 mhz , cdcl3 ) : 8.53 ( br s , 1h ) , 8.33 ( dd , 1h , j = 8.5 hz , 1.8 hz ) , 7.79 ( dd . 1h , j = 8.2 hz , 3.1 hz ) , 7.52 ( s , 1h ) , 7.50 ( dd , 1h , j = 8.5 hz , 4.9 hz ) , 7.29 ( d , 1h , j = 8.6 hz ) , 7.12 ( t , 1h , j = 7.7 hz ) , 5.30 ( d , 1h , j = 7.7 hz ) , 3.42 ( d , 2h , j = 9.0 hz ) , 3.13 ( br s , 2h ) , 2.95 ( t , 2h , j = 9.7 hz ) , 2.75 ( br s , 1h ) , 2.11 ( br s , 3h ) , 1.951.97 ( m , 1h ) , 1.561.71 ( m , 2h ) ppm . c nmr ( cdcl3 , 100 mhz ) : 162.16 ; 159.76 ; 157.67 ( d , j = 1.4 hz ) ; 151.76 ; 142.37 ; 140.10 ( d , j = 4.4 hz ) ; 138.67 ( d , j = 9.5 hz ) ; 136.78 ( d , j = 1.2 hz ) ; 128.18 ; 125.11 ( q , j = 5.7 hz ) ; 124.65 ; 124.38 ( d , j = 9.4 hz ) ; 122.71 ; 113.43 ( d , j = 25.0 hz ) ; 108.25 ( d , j = 23.4 hz ) ; 72.13 ; 54.52 ; 52.34 ( 2c ) ; 45.72 ; 35.80 ; 32.94 ( 2c ) ppm . calcd for c23h23n3f4o3s : c , 55.53% ; h , 4.66% ; n , 8.45% ; found : c , 55.55% ; h , 4.62% ; n , 8.44% . h nmr ( 400 mhz , cdcl3 ) : 7.91 ( s , 1h ) , 7.87 ( dd , 1h , j = 7.8 hz , 2.3 hz ) , 7.85 ( d , 2h , j = 8.3 hz ) , 7.51 ( dd , 1h , j = 7.4 hz , 1.6 hz ) , 7.457.50 ( m , 2h ) , 7.37 ( dd , 2h , j = 8.7 hz , 5.4 hz ) , 7.04 ( t , 2h , j = 8.7 hz ) , 6.02 ( s , 1h ) , 5.17 ( br s , 1h ) , 3.31 ( d , 2h , j = 16.7 hz ) , 2.722.96 ( m , 4h ) , 2.64 ( s , 2h ) , 1.992.12 ( m , 2h ) ppm . c nmr ( cdcl3 , 100 mhz ) : 163.81 ; 161.35 ; 142.52 ; 136.88 ( d , j = 2.9 hz ) ; 134.67 ; 133.82 ; 133.13 ; 128.36 ( d , j = 4.4 hz ) ; 128.06 ; 126.86 ( 2c , d , j = 7.9 hz ) ; 126.40 ; 125.95 ; 124.48 ( 2c , d , j = 12.8 hz ) ; 121.08 ; 115.60 ( 2c , d , j = 21.3 hz ) ; 75.80 ; 56.89 ; 53.55 ; 50.42 ; 34.29 ; 28.27 ppm . calcd for c24h24nfo.h2o : c , 77.74% ; h , 6.74% ; n , 3.77% ; found : c , 77.87% ; h , 6.82% ; n , 3.40% . yield : 40% , mp 116 - 117 c . h nmr ( 400 mhz , dmso - d6 ) : 7.68 ( d , 2h , j = 8.1 hz ) , 7.57 ( d , 2h , j = 7.9 hz ) , 7.47 ( dd , 2h , j = 8.4 hz , 5.6 hz ) , 7.15 ( t , 2h , j = 8.8 hz ) , 6.12 ( s , 1h ) , 5.70 ( br s , 1h ) , 4.754.80 ( m , 1h ) , 3.08 ( s , 2h ) , 2.62 ( t , 2h , j = 5.5 hz ) , 2.452.50 ( m , 4h ) , 1.81 ( dd , 2h , j = 13.7 hz , 7.3 hz ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 160.98 ; 151.87 ; 137.40 ( d , j = 3.0 hz ) ; 133.82 ; 128.30 ; 127.99 ; 127.30 ( 2c ) ; 127.22 ; 126.64 ; 125.73 ( 2c , d , j = 4.0 hz ) ; 122.82 ; 115.89 ( 2c , d , j = 21.2 hz ) ; 71.49 ; 55.32 ; 53.60 ; 50.74 ; 36.96 ; 28.36 ppm . calcd for c21h21nf4o : c , 66.49% ; h , 5.54% ; n , 3.69% ; found : c , 66.11% ; h , 5.67% ; n , 3.81% . h nmr ( 400 mhz , dmso - d6 ) : 11.12 ( br s , 1h ) , 8.29 ( d , 1h , j = 8.8 hz ) , 8.10 ( d , 1h , j = 6.6 hz ) , 7.67 ( br s , 3h ) , 7.56 ( d , 2h , j = 8.4 hz ) , 7.36 ( dd , 1h , j = 8.2 hz , 9.7 hz ) , 7.13 ( d , 2h , j = 8.1 hz ) , 5.90 ( br s , 1h ) , 5.41 ( d , 1h , j = 7.5 hz ) , 3.97 ( br s , 2h ) , 3.203.30 ( m , 6h ) , 3.55 ( br s , 2h ) , 2.21 ( d , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 159.34 ; 152.87 ; 137.93 ; 129.53 ; 127.72 ; 127.21 ( 2c ) ; 126.81 ; 125.01 ; 124.62 ; 124.34 ; 123.92 ( d , j = 9.1 hz ) ; 123.67 ; 121.37 ( d , j = 6.0 hz ) ; 115.76 ( 2c ) ; 109.94 ; 67.62 ; 54.46 ; 51.64 ( 2c ) ; 45.23 ( 2c ) ; 32.94 ppm . n , 5.98% ; found : c , 61.35% ; h , 5.02% ; n , 5.96% . yield : 90% , mp 93 - 95 c . h nmr ( 400 mhz cdcl3 ) : 8.53 ( d , 1h , j = 2.7 hz ) , 8.33 ( dd , 1h , j = 9.0 hz , 2.7 hz ) , 8.16 ( dd , 1h , j = 7.2 hz , 3.3 hz ) , 8.04 ( d , 1h , j = 7.8 hz ) , 7.677.72 ( m , 1h ) , 7.537.61 ( m , 2h ) , 7.277.30 ( m , 1h ) , 7.17 ( dd , 1h , j = 10.2 hz , 8.1 hz ) , 5.71 ( dd , 1h , j = 8.2 hz , 2.3 hz ) , 3.42 ( d , 2h ) , 3.083.15 ( m , 1h ) , 3.003.03 ( m , 1h ) , 2.98 ( t , 2h , j = 11.5 hz ) , 2.752.84 ( m , 1h ) , 2.12 ( d , 1h , j = 12.7 hz ) , 2.152.23 ( m , 2h ) , 1.862.01 ( m , 1h ) , 1.681.83 ( m , 2h ) ppm . c nmr ( cdcl3 , 100 mhz ) : 159.75 ; 157.66 ; 157.26 ; 142.39 ; 136.42 ( d , j = 3.7 hz ) ; 131.74 ( d , j = 4.2 hz ) ; 128.18 ; 127.16 ; 126.06 ; 125.12 ( d , j = 5.4 hz ) ; 124.66 ; 124.22 ; 123.33 ( d , j = 2.8 hz ) ; 123.24 ( d , j = 8.5 hz ) ; 122.72 ; 121.76 ( d , j = 5.8 hz ) ; 109.25 ( d , j = 19.5 hz ) ; 72.58 ; 54.56 ; 52.32 ( 2c ) ; 45.74 ; 36.95 ; 32.88 ( 2c ) ppm . calcd for c25h25n3f4o3 : c , 61.10% ; h , 5.09% ; n , 8.55% ; found : c , 60.77% ; h , 5.43% ; n , 8.30% . dmso - d6 ) : 8.20 ( dd , 1h , j = 7.0 hz , 2.5 hz ) , 8.07 ( d , 1h , j = 8.2 hz ) , 7.70 ( dd , 1h , j = 8.0 hz , 5.6 hz ) , 7.547.61 ( m , 2h ) , 7.357.40 ( m , 2h ) , 7.18 ( dd , 1h , j = 10.2 hz , 8.1 hz ) , 7.007.08 ( m , 2h ) , 6.006.05 ( m , 1h ) , 5.74 ( br s , 1h ) , 3.47 ( d , 1h , j = 11.3 hz ) , 3.41 ( d , 1h , j = 11.2 hz ) , 2.843.12 ( m , 4h ) , 2.73 ( br s , 2h ) , 2.142.20 ( m , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 160.99 ; 159.06 ; 139.11 ( d , j = 4.2 hz ) ; 137.42 ( d , j = 3.2 hz ) ; 133.87 ; 131.99 ( d , j = 4.3 hz ) ; 127.79 ; 127.31 ( 2c , d , j = 8.0 hz ) ; 127.02 ( d , j = 1.6 hz ) ; 124.55 ( d , j = 2.6 hz ) ; 123.77 ( d , j = 1.0 hz ) ; 123.65 ( d , j = 8.8 hz ) ; 122.91 ( d , j = 1.4 hz ) ; 121.30 ( d , j = 5.8 hz ) ; 115.92 ( 2c , d , j = 21.2 hz ) ; 109.82 ( d , j = 19.2 hz ) ; 68.80 ; 55.72 ; 53.85 ; 50.82 ; 36.75 ; 28.43 ppm . calcd for c24h23nf2o : c , 75.99% ; h , 6.07% ; n , 3.69% ; found : c , 75.65% ; h , 6.45% ; n , 3.50% . yield : 20% , mp 163 - 165 c , h nmr ( 400 mhz , dmso - d6 ) : 11.20 ( br s , 1h ) , 8.47 ( d , 1h , j = 8.8 hz ) , 8.42 ( s , 1h ) , 7.64 ( d , 1h , j = 9.0 hz ) , 7.42 ( dd , 2h , j = 8.0 hz , 6.6 hz ) , 7.18 ( t , 2h , j = 8.1 hz ) , 5.65 ( s , 1h ) , 4.69 ( s , 1h ) , 3.60 ( br s , 2h ) , 3.353.44 ( m , 4h ) , 3.153.20 ( m , 4h ) , 2.09 ( bs , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 162.08 ; 159.50 ; 154.72 ; 150.40 ; 142.21 ; 140.75 ( d , j = 2.3 hz ) ; 128.22 ( 2c ) ; 127.09 ( d , j = 8.1 hz ) ; 123.74 ; 123.21 ( d , j = 5.2 hz ) ; 114.35 ( 2c , d , j = 21.0 hz ) ; 66.47 ; 54.20 ; 52.04 ( 2c ) ; 50.07 ( 2c ) ; 31.83 ppm . anal . calcd for c20h21n3f4o3 .hcl.h2o : c , 49.85% ; h , 5.02% ; n , 8.72% ; found : c , 49.50% ; h , 4.76% ; n , 8.63% . yield : 11% , mp 185 - 187 c , h nmr ( 400 mhz , dmso - d6 ) : 8.37 ( br s , 2h ) , 7.50 ( d , 1h , j = 8.0 hz ) , 7.36 ( br s , 2h ) , 7.13 ( t , 2h , j = 8.1 hz ) , 5.61 ( s , 1h ) ; 4.69 ( t , 1h , j = 9.0 hz ) , 3.203.30 ( m , 2h ) , 2.95 ( t , 2h , j = 12.1 hz ) , 2.632.64 ( m , 2h ) , 2.58 ( br s , 1h ) , 1.90 ( d , 2h , j = 11.0 hz ) , 1.70 ( br s , 2h ) , 1.38 ( br s , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 163.06 ; 160.66 ; 157.70 ; 143.19 ( d , j = 2.7 hz ) ; 141.73 ; 129.21 ; 128.34 ( 2c , d , j = 8.0 hz ) ; 124.98 ( q , j = 6.0 hz ) ; 123.88 ; 122.82 ; 115.49 ( 2c , d , j = 21.1 hz ) ; 71.59 ; 54.24 ; 52.08 ( 2c ) ; 44.00 ; 35.62 ; 32.34 ( 2c ) ppm . calcd for c21h23n3f4o3 .hcl : c , 52.51% ; h , 5.03% ; n , 8.80% ; found : c , 52.13% ; h , 4.91% ; n , 8.49% . h nmr ( 400 mhz , dmso - d6 ) : 7.46 ( dd , 2h , j = 7.7 hz , 5.5 hz ) , 7.37 ( dd , 2h , j = 7.9 hz , 6.0 hz ) , 7.14 ( dd , 4h , j = 12.6 hz , 8.0 hz ) , 6.12 ( br s , 1h ) , 5.49 ( br s , 1h ) , 4.66 ( t , 1h , j=6.3 hz ) , 3.06 ( br s , 2h ) , 2.572.62 ( m , 2h ) , 2.45 ( br s , 4h ) , 1.761.80 ( m , 2h ) ppm . c nmr ( dmso - d6 , 100 mhz ) : 160.98 ; 159.80 ; 141.53 ; 139.87 ( d , j = 3.0 hz ) ; 133.85 ; 128.40 ( 2c , d , j = 8.0 hz ) ; 127.78 ; 127.25 ( 2c , d , j = 8.0 hz ) ; 115.80 ( 2c , d , j = 21.2 hz ) ; 115.29 ( 2c , d , j = 21.0 hz ) ; 71.89 ; 55.87 ; 53.57 ; 50.62 ; 36.96 ; 28.34 ppm . anal . calcd for c20h21nf2o.h2o : c , 71.88% ; h , 6.43% ; n , 4.19% ; found : c , 72.06% ; h , 6.33% ; n , 4.03% . the separation of enantiomers involved two steps : analytical scale chiral chromatography and preparative scale chiral chromatography . for analytical chromatography , an isocratic method was developed on the acquity ultraperformance convergence chromatography system ( upc ) from waters . the fast screening strategy was performed testing organic modifiers ( meoh , isopropanol ( i - pr ) , and diethylamine ( dea ) , all hplc grade in the co2 mobile phase and 5 chiral columns ( chiralpak ia , od , ad , oj and ic , 100 4.6 mm i.d . the outlet pressure was 130 bar and the columns were maintained at 40 c . the best conditions were obtained using chiralpak ic column with 20% of i - pr + 0.1% dea as an additive in the co2 mobile phase . the retention times for each of the enantiomers were rt1 = 4.65 min and rt2 = 5.60 min . for the enantiomeric separation of compound 25 , supercritical preparative scale chromatography was performed on a purification prep 80 system ( waters ) . chiral separation was run on chiralpak ic column , 5 m ( 20 250 ) mm column ( chiral technologies , daicel group ) eluted with a mixture of co2 and 15% of i - pr + 0.1% dea . the automated back pressure valve was regulated to 130 bar and the oven temperature was 40 c . the injection volume was 1 ml of an upstream filtered concentrated solution of 5 all fractions containing enantiomers 1 and 2 were collected in the same bottles a and b , respectively . finally , each enantiomer and racemic mixture of compound 25 were evaluated against p. falciparum f32 tanzania strain ( chloroquine sensitive ) according to bouquet et al . the multidrug resistant fcr-3 strain of p. falciparum was cultured at 37 c in a pure gas mixture of 5% o2 , 5% co2 , and 90% n2 environment in rpmi 1640 medium supplemented with 25 mm hepes , 5% ( w / v ) nahco3 , 0.1 mg / ml gentamicin , and 10% a heat - inactivated human serum , as previously described ( trager and jensen , 1976 ) . compounds were dissolved in dmso and tested with final concentrations ranging between 0.1 and 200 m . was measured using the [ h]-hypoxanthine ( mp biomedicals , usa ) incorporation assay ( desjardins et al . , 1979 ) with some modifications . results were expressed as the concentration resulting in 50% inhibition ( ic50 ) which was calculated by linear interpolation ( huber and koella , 1993 ) as follow : log(ic50)=log(x1)+(50y1)(y2y1)[log(x2)log(x1)]where : x1 : concentration of the drug that gives a % inhibition of the parasitemia y1>50%x2 : concentration of the drug that gives a % inhibition of the parasitemia y2<50%% inhibition of the incorporation of labeled hypoxanthine = 100-(p / t*100)p : counts per minute for every concentrationt : negative control ( red blood cells without drug ) x1 : concentration of the drug that gives a % inhibition of the parasitemia y1>50% x2 : concentration of the drug that gives a % inhibition of the parasitemia y2<50% % inhibition of the incorporation of labeled hypoxanthine = 100-(p / t*100 ) p : counts per minute for every concentration t : negative control ( red blood cells without drug ) studies were conducted according to the french and colombian guidelines on laboratory animal use and care ( n 2001 - 464 and n 008430 , respectively ) . the classical 4-day suppressive test was carried out as follows ( peters , 1970 ) . swiss male mice weighing 20 2 g , were infected with 10 p. berghei anka parasitized cells ( day 0 ) . two hours after infection and at the same time during 4 consecutive days , batches of three to five mice were orally treated at a dose of 50 mg / kg / day ( drugs were dissolved in vehicle , water : dmso ) . a control group received the vehicle while a reference group was orally administered chloroquine diphosphate at 3 mg / kg / day . survival of the mice was checked daily and the percentage of parasitized erythrocytes was determined on day 4 by giemsa - stained thin blood smears made from peripheral blood . vero cells ( african green monkey kidney epithelial cells ) were seeded ( 5 10 cells / ml , 100l / well ) in a 96-well flat - bottom plate at 37 c and with 5% co2 in phenol red free rpmi 1640 ( sigma ) supplemented with 10% heat - inactivated fetal bovine serum . compounds were added at varying concentrations and the cells were cultured for 48 h. the effect was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) viability assay ( mosmann , 1983 ) . four hours after the addition of mtt , 100 l of lysis buffer ( 50% i - pr , 30% water , 20% ( w / v ) sds ) was added and the cells were incubated at room temperature for 15 min under agitation . finally , optical density was read at 590 nm with a 96-well scanner ( bio - rad ) . the gi50 determined by linear regression analysis was defined as the concentration of test sample resulting in 50% inhibition of cell proliferation compared to controls . virtual computational chemistry laboratory ( tetko et al . , 2005 ) ( http://www.vcclab.org/ ) and molinspiration online property calculation toolkit ( http://www.molinspiration.com/cgi-bin/properties ) were used to calculate topological polar surface area ( tpsa ) ( ertl et al . , 2000 ) , alogps2.1 , number of rotatable bonds and violations of lipinski 's rule of five ( lipinski et al . , 1997 ) . in addition , human intestinal absorption ( % abs ) was calculated using the following approach of zhao et al . ( 2002):%abs = 109 (0.345 tpsa ) the sos / umu test was used to determine the dna - damaging effect and was carried out according to the method of oda et al . the test strain salmonella typhimurium ta1535/psk1002 ( german collection for microorganisms and cell cultures ( dsmz ) was thawed from stock ( 80 c ; in tga medium containing 10% dmso as cryoprotective agent ) and 0.5 ml of bacteria was resuspended in 100 ml tga medium supplemented with ampicillin ( 50 g / ml ) . the test strain suspension was incubated overnight at 37 c with slight orbital shaking ( 155 rpm ) until an optical density was reached ( od600 between 0.5 and 1.5 ) . then , the overnight culture was diluted with fresh tga medium ( not supplemented with ampicillin ) and incubated for 2 h at 37 c , 155 rpm , in order to obtain a log - phase bacterial growth culture ( od600 between 0.15 and 0.4 ) . the test was performed in the absence and presence of an external metabolic activation system ( 10% of rat s9 mix , prepared from s9 sd rat liver aroclor kcl frozen , trinova , germany ) , in order to also determine the possible genotoxic effects of any metabolite . in each test performed negative and positive controls were included with dmso used as the negative control and 4-nitroquinoline - n - oxide ( 4-nqo ) ( sigma - aldrich , china ) and 2-aminoanthracene ( 2-aa ) ( sigma - aldrich , germany ) used as positive controls in the absence and presence of s9 mix , respectively . test procedure was as follows : first , each compound tested was dissolved in dmso at 40 mg / ml ( for a final assay concentration of 1 mg / ml ) and 11 serial dilutions were prepared in a 96-well plate ( plate a ; final volume in each well was 10 l ) . in case where cell survival was < 80% in the lowest concentration tested , new 11 serial dilutions at lower concentrations were prepared . the highest concentrations of dmso used for the positive controls were 100 g / ml for 4-nqo ( final concentration : 2.5 g / ml ) and 0.5 mg / ml for 2-aa ( final concentration : 0.0125 mg / ml ) . then , 70 l of water was added to each well and evaluated in order to detect any precipitation of the compounds . in two other 96-well plates ( plates b ; one with s9 and the other without s9 ) , 10 l s9 mix or 10 l pbs , respectively were added and afterwards , 25 l of each concentration of compound previously prepared . finally , 90 l of exponentially growing bacteria was added to each well and both plates were incubated for 4 h by shaking ( 500 rpm ) at 37 c . after the incubation period , the od600 was measured in order to evaluate toxicity on s. typhimurium ta1535/psk1002 . toxicity was calculated as follows : survival percentage = ( a600 nm for each concentration testedmedia a600 nm for negative control ) 100% afterwards , for the determination of -galactosidase activity , in two new 96-well plates ( plates c ) 150 l onpg solution ( 2-nitrophenyl--d - galactopyranoside , sigma - aldrich , switzerland ) ( 0.9 mg / ml in b - buffer prepared according to reifferscheid et al . ( 1991 ) was added to each well and 30 l of the content of each well of the plates b was transferred to plates c. both plates were incubated 30 min by shaking ( 500 rpm ) at 28 c avoiding direct light exposure . after the incubation period , the reaction was stopped by adding 120 l of na2co3 ( 1 m ) . absorbance at 420 nm was then measured immediately , and -galactosidase activity ( relative units ; ru ) was calculated as follows: galactosidase enzymatic units = a420 nm for each concentration testeda600 nm for each concentration testedand finally , the induction factor ( if ) was calculated as follows : if = galactosidase ru for each concentration testedaverage galactosidase ru for negative controlwhere : average galactosidase ru for negative control = average a420 nm for negative controlaverage a600 nm for negative controlin the same way , -galactosidase relative units were calculated for both positive controls and the test was only considered valid if the positive controls reached an induction factor 2 under the given test conditions . thus , a compound was considered genotoxic when in any of the conditions studied ( with or without metabolic activation ) the if of the umu operon was 2 at non - cytotoxic concentrations ( bacteria survival percentage 80% ) . a chloroquine sensitive ( d6 , obtained from walter reed army institute of research ) and multidrug resistant ( c235 , obtained from walter reed army institute of research ) strain of p. falciparum were continuously cultured following modifications to the original trager and jensen method ( 1976 ) . each strain was maintained at 5% hematocrit using human a erythrocytes and incubated at 37 c in a pure gas mixture of 5% o2 , 5% co2 , and 90% n2 . culture media included rpmi 1640 with l - glutamine and supplemented with 25 mm hepes , 11 mm glucose , 29 m hypoxanthine , 29 mm sodium bicarbonate , and 10% human a heat - inactivated human plasma . drug susceptibility was determined using the malaria sybr green - i fluorescence ( msf ) assay ( johnson et al . , 2007 ) . compounds were dissolved in dmso at a concentration of 10 mm and were subsequently transferred to assay plates ( nunc microwell 384-well optical bottom ) in duplicate using an echo 555 liquid handler . final compound concentrations ranged between 0.03 and 24.90 m ( 0.25% dmso ) to establish ic50 values . controls of 0.25% dmso , no dmso , and 110 m amodiaquine were used to assess the quality of each plate . the d6 and c235 strains were sorbitol synchronized to the ring stage , added to the plate at an initial 0.3% parasitemia level and 2% hematocrit , and incubated for 72 h. the parasites were then lysed with 10 mm edta , 100 mm tris - hcl ( ph 7.5 ) , 0.16% ( w / v ) saponin , and 1.6% ( v / v ) triton x-100 , mixed with sybr green-1 ( 1:10,000 dilution ) and incubated in the dark for 24 h. fluorescence was read using a synergy h4 hybrid plate reader ( biotek ) ( exc : 485 nm , em : 535 nm ) with concentration response curves generated using the sigmoidal dose response variable slope curve fit on graphpad prism v5.0 . the commercially available intermediate 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine ( am01 ) and the synthesized intermediate 4-(4-nitro-2-trifluoromethylphenyl)aminopiperidine ( am02 ) were tested against p. falciparum fcr-3 multidrug resistant strain using the methodology followed to evaluate final compounds ( 1426 ) . genotoxicity test sos / umu was performed on am01 and am02 using the protocol followed to evaluate final compounds ( 22 , 23 , 24 , and 25 ) . metasite software 3.0.4 ( molecular discovery ltd . ) was used to predict human liver metabolism ( site of metabolism ) and potential metabolites using the p450 liver model that involves the three major liver isoforms ( cyp3a4 , cyp2c9 , and cyp2d6 ) . although metasite does not have mouse cpy models , it is well known that these human isoforms have mouse homologs ( cui et al . , 2012 ) . to perform the predictions , metasite only used 3d structures of potential substrates as described by cruciani et al . structures were submitted to metasite as simplified molecular input line entry system notation ( smiles ) , the reactivity correction option and a minimal mass threshold of 50 da for predicted metabolites were used . the site of metabolism is shown as soft spots graphic with a report as a histogram bar chart showing the probability of metabolism for any of the atoms . pm2 ( 200 nm ) was preincubated for 5 min , 37 c in 0.1 m sodium formate , ph 4.4 . compounds dissolved in 100% dmso were added to the enzyme for a final concentration of 5 m ( final dmso concentration of 2% ) and incubated with enzyme for 5 min . the preincubation time allows for complete conversion of the zymogen to active mature form of the enzyme . the reaction was initiated by the addition of 20 m rs6 peptide ( k - p - i - e - f - nph - r - l ) . the substrate cleavage reaction was monitored by the decrease in the average absorbance from 284 to 324 nm on a cary 50 bio varian spectrophotometer equipped with an 18-cell multitransport system . an in vitro assay developed by sandlin et al . was used to test for -hematin , abiological hemozoin , inhibition activity ( sandlin et al . , 2011 ) . test compounds were screened between 0.44 and 110 m with a final dmso concentration of 0.25% in a clear 384-well flat bottom microtiter plate ( corning ) . positive controls of 80 m amodiaquine and negative controls of 0.25% dmso were used to evaluate the validity of the assay . following the addition of each compound , 20 l of water and nonidet p-40 detergent ( 30.5 m final concentration ) a 25 mm hemin chloride stock was prepared in dmso and was passed through a 0.22 m pvdf membrane filter . from this stock solution , a 228-m hematin suspension was prepared in 2 m acetate buffer ( ph 4.8 ) and added to the plate for a final hematin concentration of 100 m . after a 6 h shaking incubation at 37 c , pyridine was added to the plate ( 5% v / v final concentration ) and was shaken an additional hour . the absorbance of the pyridine - ferrochrome complex was measured at 405 nm using a synergy h4 hybrid plate reader ( biotek ) . dose response curves were generated from the maximum absorbance values using a nonlinear regression of sigmoidal dose response variable slope curve fit on graphpad prism v5.0 . the compound 's ability to affect the hemozoin formation pathway as a drug target was validated with the heme speciation assay ( combrinck et al . , 2015 ) . a d6 culture was sorbitol synchronized to the early ring stage and diluted to 5% parasitemia and 2% hematocrit . this culture was distributed in 2 ml aliquots to a 24-well flat bottom cell culture plate containing the test compound . a control sample of no drug was included alongside five varying concentrations , each in quadruplicate , based on the ic50 values previously determined by the msf assay . the plates were incubated at 37 c , 5% o2 , 5% co2 , and 90% n2 for 32 h to allow for the parasite to reach the trophozoite stage . the mature parasites were then isolated from the erythrocytes through selective lysis with saponin ( 0.05% final concentration ) and washed and resuspended in pbs . a 10 l aliquot was removed from each sample for cell counting with the countess ii fl automated cell counter . the trophozoite pellet in pbs was fractionated into the three heme species : p. falciparum hemoglobin , free intracellular heme , and hemozoin as described by combrinck et al . the maximum absorbance of the fe ( iii ) heme - pyridine complex that results from each of the fractionation steps was recorded at 405 nm in order to calculate the ratios of heme species in each sample . spectrophotometric titrations were conducted to further understand the interaction of each compound with the -oxo heme dimer in vitro . first , each compound was dissolved in 40% aqueous dmso and 0.02 m hepes ( ph 7.4 ) and serial diluted into a clear 96-well microtiter plate in triplicate . a hemin solution in the same solvent system was added to the plate to a final concentration of 5 m . the plate was incubated for 1 h in the dark prior to measuring the absorbance at 400 nm using a synergy h4 hybrid plate reader ( biotek ) . titrations were conducted without the heme addition to account for any absorbance from the compound itself at 400 nm and these values were subtracted from the final results . absorbance values were plotted using a nonlinear least squares fit with curvefit v1.00 to determine the equilibrium association constant . the ligandscout software ( wolber and langer , 2005 ) 4.09.2 ( inteligand gmbh ltd . ) models were obtained using the ligand - based pharmacophore design that is implemented in the espresso module . for the apd model , the most active compounds were used as a training set ( 22 , 23 , 24 , and 25 ; fcr-3 ic50 0.5 m ) , which included both possible enantiomers ( r and s configurations ) for each compound . for the classical arylamino alcohols ( caa ) , a consensus model was generated using the four molecules ( quinine , mefloquine , lumefantrine , and halofantrine ) and respective stereoisomers . all molecules used in the pharmacophore study were drawn using chemdraw ultra software 7.0 ( cambridgesoft ltd . ) with stereoisomerism manually checked . then , structures were submitted to ligandscout as smiles . for each molecule , a three - dimensional ( 3d ) multiconformational calculation was performed using the icon conformer generation tool in ligandscout . the best setting option was used to generate the conformations ( maximum number of conformations = 400 , timeout = 600 , rms threshold = 0.8 , energy window = 20 , maximum pool size = 4000 , and maximum fragment build time = 30 ) . to generate models , the merge feature pharmacophore option was used to take all the features into account and assemble them into one pharmacophore model ( consensus model ) . the scoring function used to rank the models was pharmacophore - fit and atom overlap . the model with the highest score was manually checked and considered as valid , since the model exhibited the molecules properly aligned . pharmacophore representation including hydrophobic features ( hpf ) , hydrogen bond donor features ( hbdf ) , hydrogen bond acceptor features ( hbaf ) , positive ionizable features ( pif ) , and aromatic ring features ( arf ) were projected on molecules . for comparison of the final pharmacophore models ( apd and caa ) , a consensus model was constructed for apd . the r and s models were aligned using the alignment module in ligandscout . finally , the new pharmacophore model for apd was aligned with the consensus pharmacophore model for caa where only common features in both models were calculated and extracted . chiral separation of compound 25 was made using ultra performance convergence chromatography with a chiralpack ic column ( supplementary data fig . 1 ) . there were no significant potency differences between the compound 25 enantiomers and racemic mixture against p. falciparum f32 tanzania , chloroquine sensitive strain ( f32 ic50 0.50 m ) . the biological data for the two enantiomers of the compound 25 is shown in the supplementary data table 1 . based on these results , we continued with synthesis and testing of the apd as racemates . the activity of thirteen newly synthesized hydroxyl analogues ( 1426 ) was determined against fcr-3 multidrug resistant strain ( resistant to chloroquine , cycloguanil , and pyrimethamine ) ( table 1 ) . chloroquine was used as a reference drug in all experiments for comparison ( fcr-3 ic50 = 0.13 m ) . compounds 17 , 18 , 21 , 22 , 23 , 24 , 25 , and 26 all resulted in submicromolar activity ( fcr-3 ic50 < 1 m ) with four of these below 0.5 m ( 22 , 23 , 24 and 25 ) . overall , incorporation of a single fluorine atom in the ar ( hydrophobic ) region provided potency across the different benzo[b]thiophene , naphthalene , and benzene derivatives ( table 1 , compounds 1418 and 2126 , ic50 2.23 m ) . compounds with a single fluorine atom in the ar region ( 23 and 26 ) exhibited 35-fold and 9-fold increase in potency , respectively , compared to 19 and 20 ( 23 vs 19 and 26 vs 20 ) , thereby confirming our previous observations of its requirement for antiplasmodial activity ( perez - silanes et al . , 2009 , mendoza et al . a good example of this observation is the comparison the ic50 values of previous non - fluorinated analogues ( * compounds ) with new apd ( see supplementary data table 2 , 14 vs * 7 and 15 vs * 8) . exploration of the ar region showed that 4-fluoro-1-naphthyl derivatives are 5-fold more active than 5-fluorobenzo[b]thiophenyl analogues ( 18 vs 22 ) and 1- to 3-fold more active than 4 fluoro-1-phenyl analogues ( 23 vs 26 and 22 vs 25 ) ( table 1 ) . interestingly , a particular study on - amino alcohols and their corresponding hydrophobic region must be highlighted due to its structural similarity with apd . ( 2012 ) reported loss of antiplasmodial activity in derivatives compared with their corresponding totarol analogues . this loss of activity has involved the structural replacement of the totarol scaffold by simpler aromatic systems , common in apd , such as phenyl and naphthyl . as a result the compounds exhibited ic50 values ranging between 5 and 100 m ( d10 strain , p. falciparum ) . unfortunately , the authors did not explore the addition of a halogen atom in the hydrophobic portion . it is noteworthy that electron - withdrawing groups at the para position on the phenyl ring such as -cf3 , -no2 , and -f were well tolerated and resulted in highly active compounds ( 21 , 22 , 23 , 24 , 25 , and 26 ) . by varying the position of the substituents from para to ortho ( nitro group as r2 and r1 as trifluoromethyl group ) this finding reinforced our preliminary observations that indicate the necessity of a hydrophobic group at the ortho position to improve activity ( mendoza et al . , 2011 ) . additionally , when the trifluoromethyl group is substituted at the para position and the nitro group is removed , the activity remains almost unchanged ( 14 ic50 = 1.32 m vs 16 ic50 = 1.68 m ) . the central amine also influences the activity as seen with aminopiperidine analogues that are more active than piperazine analogues ( 14 vs 18 and 21 vs 22 vs 23 ) . the same trend is observed when compound 22 is compared with an analogue previously published by our group where only piperazine was replaced ( mendoza et al . , 2011 ) . in relation to the exploration of arylamines , this finding is in agreement with the results reported by molyneaux et al . ( 2005 ) that show unsubstituted amines ( free nh ) have significant antiplasmodial activity in resistant strains compared to their substituted analogues . one feasible explanation is that unsubstituted nitrogen ( nh ) may interact easily with possible targets , as there is a greater hydrogen bond capacity . the cytotoxicity of the thirteen newly synthesized hydroxyl analogues ( 1426 ) was determined against vero cells using the mtt - assay ( mosmann , 1983 ) ( table 1 ) . the cytotoxic assays showed that analogues with aminopiperidine as the central amine are more cytotoxic than piperazine and tetrahydropyridine ( 14 vs 18 , 24 vs 25 , and 22 vs 23 vs 26 , respectively ) . ( 2012 ) and our group have also reported low cytotoxicity in - and -amino alcohols with aryl - piperazinyl groups . in general , with the exception of compound 19 , all tetrahydropyridine and piperazine derivatives showed negligible cytotoxicity in vero cells . in this context , an important criterion was the degree of selectivity of the apd that was expressed as selectivity index ( si ) ( table 1 ) , where a greater si value indicates increased selectivity for fcr-3 over vero cells . the most active compounds ( 22 , 23 , 24 , and 25 ) showed moderate to high degree of selectivity index ( 37 si 244 ) . a computational study was performed for the prediction of an adme profile of all hydroxyl analogues ( table 2 ) . topological polar surface area ( tpsa ) is a good descriptor of drug absorbance in the intestines , caco-2 monolayer penetration , and blood - brain barrier crossing ( ertl et al . , 2000 ) . tpsa was used to calculate the percentage of human intestinal absorption ( % abs ) according to the equation : % abs = 1090.345 tpsa , as shown by zhao et al . in addition , lipinski 's rule of five ( lipinski et al . , 1997 ) and the number of rotatable bonds ( n - rotb ) ( veber et al . , 2002 ) were also calculated in order to evaluate their druglikeness . from these parameters , all compounds exhibited a % abs ranging between 80 and 100% . only compounds 19 and 23 violated one lipinski 's parameter , 23 being at the limit ( alogps = 5 ) . therefore , the oral bioavailability of hydroxyl analogues could be considered interesting as agents for antimalarial therapy . studies to analyze the dna - damaging effect or genotoxicity of the most active compounds were performed . the nitro substituent often causes safety concerns due to its well - documented mutagenic and carcinogenic potential ( purohit and basu , 2000 ) . thus , the sos / umu test was included as a preliminary genotoxicity screening assay because of the high degree of agreement between the sos / umu test and the standardized ames test ( oecd guideline 471 ) ( reifferscheid and heil , 1996 ) . compounds 22 , 23 , 24 , and 25 were not considered genotoxic since the induction factor ( if ) was always lower than 2 at non - cytotoxic concentrations with or without s9 fraction ( supplementary data table 3 ) . it should be noted , however , that in the first screening assay all the concentrations of 22 were cytotoxic ( < 80% survival ) when tested in absence of metabolic activation and thus further testing at non - cytotoxic concentrations was needed to understand its genotoxic effects . a second screening test was performed at lower concentrations in order to have a larger range of non - cytotoxic concentrations ( supplementary data table 4 ) . again additionally , if a high degree of agreement between the sos / umu test and ames test was found ( reifferscheid and heil , 1996 ) , the sos / umu test was used for screening purposes and selecting the best candidates . for regulatory purposes , negative results should be further evaluated with the standardized ames test . along with the extensive sar and toxicological studies , in vivo antimalarial activity in the p. the criteria to select compounds was based on fcr-3 ic50 0.5 m , si > 35 , appropriate in silico bioavailability , and a negative genotoxicity test . thus , parasitemia reduction and mean survival days ( msd ) for chloroquine and promising apd compounds ( 22 , 23 , 24 , and 25 ) were evaluated at a unique oral dose ( 50 mg / kg 4 days ) ( table 3 ) . compound 22 displayed excellent parasitemia reduction ( 98 1% ) , and complete cure with all treated mice surviving through the entire 21-day period with no signs of toxicity ( msd > 35 ) . compounds 23 and 25 showed parasitemia reduction of 73 16% ( msd = 9 ) and 76 30% ( msd = 5 ) , respectively . despite its antiplasmodial activity ( fcr-3 ic50 = 0.36 m ) , compound 24 was inferior in terms of in vivo parasitemia reduction ( 17 8% ; msd = 8) . almost all previous studies on -to - amino alcohol have not carried out in vivo efficacy studies . apart from our group ( perez - silanes et al . , 2009 , mendoza et al . , 2011 ) , only bahamontes - rosa et al . ( lowes et al . , 2012 ) , have explored the in vivo efficacy of - amino alcohol with unsuccessful results ( parasitemia reduction 60% ) . thus , compounds 22 , 23 , and 25 appear as interesting compounds for future antimalarial programs , due to the agreement between in vitro and in vivo studies for these compounds . as previously mentioned , two important factors in developing apd as effective antimalarials are generating compounds with remarkable potency against both chloroquine sensitive and multidrug resistant strains of p. falciparum . in order to evaluate and validate the series quality ( mmv , 2008 ) , the ic50 values for the best compounds were independently evaluated and validated in two laboratories utilizing two different strains of p. falciparum . only three compounds with in vivo parasitemia reduction above 70% were tested against the d6 chloroquine sensitive ( but naturally less susceptible to mefloquine ) and c235 multidrug resistant strain ( resistant to mefloquine , chloroquine , and pyrimethamine ) ( table 4 ) . compounds 22 ( d6 ic50 = 0.11 m , c235 ic50 = 0.13 m ) and 23 ( d6 ic50 = 0.19 m , c235 ic50 = 0.28 m ) showed potent antimalarial activity in both strains . in contrast , compound 25 was less active against the multidrug resistant strain ( d6 ic50 = 0.49 m , c235 ic50 = 1.05 m ) . notably , like chloroquine ( see table 1 ) , these apd compounds may act mechanistically different from mefloquine , a representative - amino alcohol , because of their high activity against the mefloquine resistant strain , and therefore , would not be affected by the same mechanism of mefloquine resistance . in addition , the resistance indices ( ri , supplementary data table 7 ) indicate that compounds 22 and 23 were slightly more active in the chloroquine sensitive strain . compound 22 should be highlighted due to its in vitro submicromolar values , with equal potency against three different strains of p. falciparum ( d6 ic50 = 0.11 m , c235 ic50 = 0.13 m , and fcr-3 ic50 = 0.15 m ) . when exploring new compounds , it is crucial to determine the lack of activity or genotoxicity of intermediates and potential metabolites early in the drug discovery process . in the case of apd , commercial or synthesized intermediates play a critical role for two reasons : ( 1 ) the mannich reaction , a key step in our synthetic route , requires amines to obtain our - amino carbonyl intermediates ( 113 ) and thus , are an important fragment of the final compounds ( 1426 ) ; ( 2 ) as a possible product of human or mouse p450-mediated oxidative metabolism of apd , these intermediates can be potential metabolites . to explore the activity profile , intermediates of the most active compounds ( 22 , 23 , and 25 ) were tested against p. falciparum fcr-3 chloroquine resistant strain ( table 5 ) . intermediates am01 and am02 ( ic50 = 8.2 m and ic50 = 4.3 m , respectively ) showed low antiplasmodial activity compared to their final products . to explore the probability of obtaining the corresponding intermediates as potential metabolites , this approach allowed for a simple , fast , and inexpensive method of identifying the most probable site of metabolism ( som ) of the most active compounds and therefore , predicts p450-derived metabolites . as shown in fig . 2 , metasite predicted the p450 2c9- , p450 3a4- , and p450 2d6-catalyzed n - dealkylation of 22 , 23 , and 25 as the most probable biotransformation pathway . our predictions indicated that n - dealkylation would occur principally on the side chain -carbon hydrogen(s ) next to the amino aliphatic nitrogen for 22 and 25 , and next to the tertiary amine for 23 . in fact , our results were reinforced by experimental and theoretical data found where the metabolism of 4-aminopiperidine drugs were well studied ( sun and scott , 2011 ) . genotoxicity profiles of intermediates ( potential metabolites ) were performed using the sos / umu test as a screening test ( supplementary data table 5 ) . intermediates am01 and am02 were not considered genotoxic as the if was always lower than 2 at non - cytotoxic concentrations with or without s9 fraction . thus , it can be deduced that in vitro and in vivo antimalarial activity of compounds 22 , 23 , and 25 is associated with the entire molecule and not only to the amine scaffold intermediate . additionally , the viability to find an intermediate as a product of cyp biotransformation exists . genotoxicity assay of potential metabolites reinforce no genotoxic results observed with metabolic activation on apd . it should be noted that metasite and sos / umu approaches were used for screening purposes and initial studies . more in depth studies must be performed using standard assays such as the ames test and human or rat liver microsomal stability . based on previous in silico studies performed by our group ( mendoza et al . , 2011 ) , pm2 enzyme was proposed as a putative target for apd . pm2 , which is involved in the initial steps of hemoglobin degradation , plays a critical role in the intraerythrocytic cycle of the parasite . consequently , in recent years pm2 has sparked interest in the antimalarial community ( marvin and daniel , 2012 ) . docking studies showed a possible mode of union associated with good affinity values ( gibbs free energy ) . to confirm our predictions , eight compounds synthesized in this manuscript with antiplasmodial compounds 14 , 16 , 17 , 18 , 22 , 23 , 24 , and 25 ( tested at 5 m ) showed no significant inhibition of pm2 compared to the control ( supplementary data table 6 ) . interestingly , our findings were better understood when we compared our results with other hybrid studies ( computational and experimental approaches ) . friedman and caflisch ( 2009 ) performed high - throughput docking simulations using consensus - scoring methods to screen a database of 40,000 molecules resulting in 11 arylamino alcohols , halofantrine and 10 structurally related to halofantrine and lumefantrine , that were plasmepsin inhibitors ( pm1 , pm2 , and pm3 ) . important facts when analyzing and comparing our results include the following : ( 1 ) halofantrine shares the -amino alcohol moiety with apd ( fig . 1 ) ; ( 2 ) according to the proposed docking experiments , halofantrine may share the same theoretical binding mode as apd ( friedman and caflisch , 2009 , mendoza et al . , 2011 ) . this binding mode includes one hydrogen bond between the oxygen of the hydroxyl group and asp214 and electrostatic interactions between the nitrogen of the tertiary or secondary amine located near the charged asp34 ; ( 3 ) the unique major structural differences between apd and halofantrine ( or its derivatives ) are the aliphatic chains of the four carbon length bond to the tertiary amine that are not present in apd , and ( 4 ) the ic50 values reported by friedman and caflish ( 2009 ) for the 11 arylamino alcohols are considered medium to low ( 2 m ic50 100 m ) , when compared with other well - known plasmepsin inhibitors ( ic50 0.01 m ) ( marvin and daniel , 2012 ) . based on these results , we reinforce the hypothesis that the primary mechanism of action of arylamino alcohols such as apd , halofantrine , lumefantrine , and mefloquine is not through plasmepsin inhibition . therefore , in depth studies related to alternative mechanisms of action must be performed with apd . additionally , while computational methodologies are valuable tools , even well validated methodologies can produce false positives . ( 2009 ) designed and developed a rigorous in silico pipeline to observe pm2 inhibition , yet still obtained four false positives of thirty compounds tested . further , future apd could be useful for the antimalarial drug discovery community as decoys or negative controls for in silico high - throughput screening campaigns to identify pm2 inhibitors ( similar physicochemical properties , but structurally dissimilar to apd ) ( huang et al . , 2006 ) . the compounds with both in vivo and in vitro antiplasmodial activity against p. berghei and p. falciparum , respectively , were further tested against the hemozoin formation drug target pathway to understand the mechanism of action . ( 2011 ) was utilized for these three compounds ( 22 , 23 , and 25 ) . only compound 22 was found to inhibit -hematin formation with an ic50 of 80.7 1.7 m ( table 4 ) . the affinity of compound 22 for the -oxo dimer form of heme was shown through a binding curve with 22 . when compared to positive and negative control compound binding curves ( chloroquine and pyrimethamine , respectively ) , compound 22 exhibited affinity resembling that of chloroquine ( supplementary data fig . 2 ) , a known hemozoin inhibitor . to further explore the mechanism of action of compound 22 , target validation of the hemozoin formation pathway was conducted in a culture of parasites . examination of the three species of parasitic heme ( host ingested hemoglobin , intracellular free heme , and hemozoin ) revealed that the primary mode of action for compound 22 is not through hemozoin formation inhibition . despite a decline in parasite viability with increasing concentrations of compound , the levels of free heme and hemozoin remained constant . therefore , the hemozoin formation pathway is not the predominant mode of death when a culture of parasite is subjected to compound 22 . while our in vitro assay used has previously resulted in the highest hit rate and lowest false positive rates amongst other -hematin inhibition assays ( sandlin et al . , 2014 ) , there are still limits . this plate assay closely resembles the biological environment of the site of hemozoin formation , but target validation is vital to ensure that the in vitro results translate to a parasite culture . one hypothesis is that compound 22 inhibits hemozoin formation ; however , secondarily to a more potent mechanism of action , causing the levels of heme species to remain unchanged . a pharmacophore for arylamino alcohols is defined as the general structural requirements necessary for its antiplasmodial activity ( fig . 1 ) . this definition led us to ask : are apd like any of the historical arylamino alcohols ? do they share the same pharmacophore ? based on in vitro , in vivo , and target exploration results , we investigated the structural similarities and differences between apd and caa ( quinine , mefloquine , lumefantrine , and halofantrine ) using 3d pharmacophore models . first , we constructed and analyzed an in silico 3d pharmacophore model for the new apd . second , the resulting 3d pharmacophore model for apd was compared with the in silico 3d pharmacophore model of caa in order to identify common features between apd and caa . although two - dimensional ( 2d ) pharmacophore models could be inferred from fig . 1 , these models lack 3d arrangements ( 3d conditional patterns ) , which are important at the moment to study potential modes of union between ligands and macromolecules . two 3d pharmacophore models ( r and s configuration ) were generated for apd ( fig . 3a ) , resulting in similar features , but a different 3d arrangement of the naphthyl group . then , both models were aligned and the pharmacophoric features were merged into a unique model , creating a consensus pharmacophore ( fig . the final model consisted of four hpf , one pif , two arf , seven hbaf , and one hbdf . due to the absence of the trifluoromethyl group in compound 23 , a detailed summary of the pharmacophoric features for each compound is presented in supplementary data fig . 3b ) , consisting of four hpf , one pif , two arf , one hbdf , and one hbaf . two hpf were shown as optional since mefloquine and quinine do not share identical features with halofantrine and lumefantrine . additionally , one arf was shown as optional due to lumefantrine not sharing this feature with quinine , halofantrine , and mefloquine . the consensus pharmacophore models for apd and caa were aligned and the common features were extracted ( fig . 3b ) , including two hpf , one pif , one arf , one hbdf , and one hbaf . ( 1996 ) reported for the arylamino alcohol chemotype ( fig . 1 ) . specifically for apd , these common features are observed in the hydrophobic ( naphthyl and phenyl system ) and the amine region of the scaffold ; however , apd exhibited nine differences in comparison with caa ( two hpf , one arf , and six hbaf ) . these pharmacophore differences may explain the lack of inhibition with the pm2 enzyme and hemozoin pathway . thus , only a 40% ( 6/15 ) of similarity between apd and caa was observed at the pharmacophore level . this manuscript has shown the synthesis , racemic separation , in silico drug - likeness studies , in vitro evaluation against chloroquine sensitive ( f32 and d6 ) and multidrug resistant ( fcr-3 and c235 ) strains of p. falciparum , cytotoxicity ( vero ) , in silico metabolism studies , genotoxicity , in vivo efficacy in p. berghei mouse model , target exploration , and pharmacophore modeling of new apd . this work led to the identification of four promising compounds ( 22 , 23 , 24 , and 25 ) that exhibit values of antiplasmodial activity below 0.5 m ( fcr-3 ) , appropriate drug - likeness profile , adequate selectivity index ( 37 si 244 ) , and absence of genotoxicity . in vivo efficacy in p. notably , compound 22 displayed excellent parasitemia reduction ( 98 1% ) and complete cure with all treated mice surviving through the entire 21-day period with no signs of toxicity . additionally , compounds 22 and 23 showed potent antimalarial activity in chloroquine sensitive and multidrug resistant strains ( d6 ic50 0.19 m , c235 ic50 0.28 m ) . target exploration was performed in order to establish a possible mechanism of action ; however , both the pm2 enzyme and the hemozoin inhibition pathway were ruled out as primary targets for apd . this similarity consisted of two hpf , one pif , one arf , one hbdf , and one hbaf . this series of new apd showed not only promising in vitro and in vivo efficacy values , but also exhibited agreement between in vitro and in vivo studies . this last point is important due to the large number of potent antimalarial compounds reported in the literature that are only efficacious at in vitro and will no longer proceed along the drug discovery pipeline . apd are promising compounds as new antimalarial therapeutics not only for their particular unexplored chemotype , but also for their unknown mechanism of action , which we found to differ from chloroquine or classical amino alcohols . further optimization of the scaffold must be done through complementary sar , mechanistic , and pharmacology studies .

synthesis of new 1-aryl-3-substituted propanol derivatives followed by structure - activity relationship , in silico drug - likeness , cytotoxicity , genotoxicity , in silico metabolism , in silico pharmacophore modeling , and in vivo studies led to the identification of compounds 22 and 23 with significant in vitro antiplasmodial activity against drug sensitive ( d6 ic50 0.19 m ) and multidrug resistant ( fcr-3 ic50 0.40 m and c235 ic50 0.28 m ) strains of plasmodium falciparum . adequate selectivity index and absence of genotoxicity was also observed . notably , compound 22 displays excellent parasitemia reduction ( 98 1% ) , and complete cure with all treated mice surviving through the entire period with no signs of toxicity . one important factor is the agreement between in vitro potency and in vivo studies . target exploration was performed ; this chemotype series exhibits an alternative antimalarial mechanism .

Introduction Material and methods Results and discussion Conclusions Author's contributions

, 2015 ) , extensive studies on classical antimalarial chemotypes or drug repurposing are needed due to the high cost and time required in the drug discovery and development process ( andrews et al . one classic antimalarial chemotype present in potent antimalarial drugs , including quinine ( 1 ) , mefloquine ( 2 ) , lumefantrine ( 3 ) , and halofantrine ( 4 ) , is the arylamino alcohol ( - amino or -amino alcohol moiety ) . , 2006 ) , and smith and chibale et al . , 2011 , ( 2009 ) reported a wide range of antiplasmodial activity against the chloroquine sensitive p. falciparum strain d10 ; however , submicromolar values were not reached ( 6 m ic50 175 m ) . our research on -amino alcohols explored the structure - activity relationship ( sar ) of 1-aryl-3-susbtituted propanol derivatives ( apd ) with promising results ( perez - silanes et al . initial sar studies showed that all of the aryl - ketone derivatives were inactive against 3d7 , nf54 , and fcr-3 strains of p. falciparum ( perez - silanes et al . in contrast , apd were active against 3d7 ( 0.19 ic50 0.38 m ) ( perez - silanes et al . , 2009 ) , nf54 ( 1.3 ic50 8 m ) ( mendoza et al . , 2011 ) , and fcr-3 ( 0.5 ic50 10 m ) ( mendoza et al . , 2011 ) strains of p. falciparum . however , low parasitemia reduction ( 65% ) was observed in the in vivo plasmodium berghei ( p. berghei ) mouse model . previous , in silico studies proposed plasmepsin ii ( pm2 ) as a putative target for apd ( mendoza et al . thus , exploration and development of apd as antimalarials requires ( 1 ) expanding sar studies , ( 2 ) generating additional analogues with high potency against both chloroquine sensitive and multidrug resistant strains of p. falciparum , ( 3 ) improving parasitemia reduction in the p. berghei mouse model , ( 4 ) establishing a safe toxicological profile , and ( 5 ) exploring biological targets in p. falciparum . in this study , we expanded the chemical scope in the antimalarial framework by preparing new apd for testing their antiplasmodial activity in vitro against p. falciparum ( sensitive and resistant strains ) and in vivo against p. berghei . all of the synthesized final compounds were chemically characterized by thin layer chromatography ( tlc ) , melting point ( mp ) and proton and carbon nuclear magnetic resonance ( h nmr and c nmr ) spectra as well as by elemental microanalysis . the resulting mixture was heated at reflux for 12 h. naoh ( 2 m ) was then added ( 50 ml ) and the product was extracted with dcm ( 3 50 ml ) . the fast screening strategy was performed testing organic modifiers ( meoh , isopropanol ( i - pr ) , and diethylamine ( dea ) , all hplc grade in the co2 mobile phase and 5 chiral columns ( chiralpak ia , od , ad , oj and ic , 100 4.6 mm i.d . then , the overnight culture was diluted with fresh tga medium ( not supplemented with ampicillin ) and incubated for 2 h at 37 c , 155 rpm , in order to obtain a log - phase bacterial growth culture ( od600 between 0.15 and 0.4 ) . the test was performed in the absence and presence of an external metabolic activation system ( 10% of rat s9 mix , prepared from s9 sd rat liver aroclor kcl frozen , trinova , germany ) , in order to also determine the possible genotoxic effects of any metabolite . a chloroquine sensitive ( d6 , obtained from walter reed army institute of research ) and multidrug resistant ( c235 , obtained from walter reed army institute of research ) strain of p. falciparum were continuously cultured following modifications to the original trager and jensen method ( 1976 ) . the d6 and c235 strains were sorbitol synchronized to the ring stage , added to the plate at an initial 0.3% parasitemia level and 2% hematocrit , and incubated for 72 h. the parasites were then lysed with 10 mm edta , 100 mm tris - hcl ( ph 7.5 ) , 0.16% ( w / v ) saponin , and 1.6% ( v / v ) triton x-100 , mixed with sybr green-1 ( 1:10,000 dilution ) and incubated in the dark for 24 h. fluorescence was read using a synergy h4 hybrid plate reader ( biotek ) ( exc : 485 nm , em : 535 nm ) with concentration response curves generated using the sigmoidal dose response variable slope curve fit on graphpad prism v5.0 . the commercially available intermediate 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine ( am01 ) and the synthesized intermediate 4-(4-nitro-2-trifluoromethylphenyl)aminopiperidine ( am02 ) were tested against p. falciparum fcr-3 multidrug resistant strain using the methodology followed to evaluate final compounds ( 1426 ) . genotoxicity test sos / umu was performed on am01 and am02 using the protocol followed to evaluate final compounds ( 22 , 23 , 24 , and 25 ) . was used to predict human liver metabolism ( site of metabolism ) and potential metabolites using the p450 liver model that involves the three major liver isoforms ( cyp3a4 , cyp2c9 , and cyp2d6 ) . compounds dissolved in 100% dmso were added to the enzyme for a final concentration of 5 m ( final dmso concentration of 2% ) and incubated with enzyme for 5 min . from this stock solution , a 228-m hematin suspension was prepared in 2 m acetate buffer ( ph 4.8 ) and added to the plate for a final hematin concentration of 100 m . after a 6 h shaking incubation at 37 c , pyridine was added to the plate ( 5% v / v final concentration ) and was shaken an additional hour . for the apd model , the most active compounds were used as a training set ( 22 , 23 , 24 , and 25 ; fcr-3 ic50 0.5 m ) , which included both possible enantiomers ( r and s configurations ) for each compound . for the classical arylamino alcohols ( caa ) , a consensus model was generated using the four molecules ( quinine , mefloquine , lumefantrine , and halofantrine ) and respective stereoisomers . pharmacophore representation including hydrophobic features ( hpf ) , hydrogen bond donor features ( hbdf ) , hydrogen bond acceptor features ( hbaf ) , positive ionizable features ( pif ) , and aromatic ring features ( arf ) were projected on molecules . there were no significant potency differences between the compound 25 enantiomers and racemic mixture against p. falciparum f32 tanzania , chloroquine sensitive strain ( f32 ic50 0.50 m ) . the activity of thirteen newly synthesized hydroxyl analogues ( 1426 ) was determined against fcr-3 multidrug resistant strain ( resistant to chloroquine , cycloguanil , and pyrimethamine ) ( table 1 ) . chloroquine was used as a reference drug in all experiments for comparison ( fcr-3 ic50 = 0.13 m ) . compounds 17 , 18 , 21 , 22 , 23 , 24 , 25 , and 26 all resulted in submicromolar activity ( fcr-3 ic50 < 1 m ) with four of these below 0.5 m ( 22 , 23 , 24 and 25 ) . overall , incorporation of a single fluorine atom in the ar ( hydrophobic ) region provided potency across the different benzo[b]thiophene , naphthalene , and benzene derivatives ( table 1 , compounds 1418 and 2126 , ic50 2.23 m ) . compounds with a single fluorine atom in the ar region ( 23 and 26 ) exhibited 35-fold and 9-fold increase in potency , respectively , compared to 19 and 20 ( 23 vs 19 and 26 vs 20 ) , thereby confirming our previous observations of its requirement for antiplasmodial activity ( perez - silanes et al . in this context , an important criterion was the degree of selectivity of the apd that was expressed as selectivity index ( si ) ( table 1 ) , where a greater si value indicates increased selectivity for fcr-3 over vero cells . the most active compounds ( 22 , 23 , 24 , and 25 ) showed moderate to high degree of selectivity index ( 37 si 244 ) . compounds 22 , 23 , 24 , and 25 were not considered genotoxic since the induction factor ( if ) was always lower than 2 at non - cytotoxic concentrations with or without s9 fraction ( supplementary data table 3 ) . again additionally , if a high degree of agreement between the sos / umu test and ames test was found ( reifferscheid and heil , 1996 ) , the sos / umu test was used for screening purposes and selecting the best candidates . along with the extensive sar and toxicological studies , in vivo antimalarial activity in the p. the criteria to select compounds was based on fcr-3 ic50 0.5 m , si > 35 , appropriate in silico bioavailability , and a negative genotoxicity test . thus , parasitemia reduction and mean survival days ( msd ) for chloroquine and promising apd compounds ( 22 , 23 , 24 , and 25 ) were evaluated at a unique oral dose ( 50 mg / kg 4 days ) ( table 3 ) . compound 22 displayed excellent parasitemia reduction ( 98 1% ) , and complete cure with all treated mice surviving through the entire 21-day period with no signs of toxicity ( msd > 35 ) . compounds 23 and 25 showed parasitemia reduction of 73 16% ( msd = 9 ) and 76 30% ( msd = 5 ) , respectively . despite its antiplasmodial activity ( fcr-3 ic50 = 0.36 m ) , compound 24 was inferior in terms of in vivo parasitemia reduction ( 17 8% ; msd = 8) . , 2012 ) , have explored the in vivo efficacy of - amino alcohol with unsuccessful results ( parasitemia reduction 60% ) . thus , compounds 22 , 23 , and 25 appear as interesting compounds for future antimalarial programs , due to the agreement between in vitro and in vivo studies for these compounds . as previously mentioned , two important factors in developing apd as effective antimalarials are generating compounds with remarkable potency against both chloroquine sensitive and multidrug resistant strains of p. falciparum . in order to evaluate and validate the series quality ( mmv , 2008 ) , the ic50 values for the best compounds were independently evaluated and validated in two laboratories utilizing two different strains of p. falciparum . only three compounds with in vivo parasitemia reduction above 70% were tested against the d6 chloroquine sensitive ( but naturally less susceptible to mefloquine ) and c235 multidrug resistant strain ( resistant to mefloquine , chloroquine , and pyrimethamine ) ( table 4 ) . compounds 22 ( d6 ic50 = 0.11 m , c235 ic50 = 0.13 m ) and 23 ( d6 ic50 = 0.19 m , c235 ic50 = 0.28 m ) showed potent antimalarial activity in both strains . in contrast , compound 25 was less active against the multidrug resistant strain ( d6 ic50 = 0.49 m , c235 ic50 = 1.05 m ) . notably , like chloroquine ( see table 1 ) , these apd compounds may act mechanistically different from mefloquine , a representative - amino alcohol , because of their high activity against the mefloquine resistant strain , and therefore , would not be affected by the same mechanism of mefloquine resistance . in addition , the resistance indices ( ri , supplementary data table 7 ) indicate that compounds 22 and 23 were slightly more active in the chloroquine sensitive strain . compound 22 should be highlighted due to its in vitro submicromolar values , with equal potency against three different strains of p. falciparum ( d6 ic50 = 0.11 m , c235 ic50 = 0.13 m , and fcr-3 ic50 = 0.15 m ) . intermediates am01 and am02 ( ic50 = 8.2 m and ic50 = 4.3 m , respectively ) showed low antiplasmodial activity compared to their final products . our predictions indicated that n - dealkylation would occur principally on the side chain -carbon hydrogen(s ) next to the amino aliphatic nitrogen for 22 and 25 , and next to the tertiary amine for 23 . thus , it can be deduced that in vitro and in vivo antimalarial activity of compounds 22 , 23 , and 25 is associated with the entire molecule and not only to the amine scaffold intermediate . to confirm our predictions , eight compounds synthesized in this manuscript with antiplasmodial compounds 14 , 16 , 17 , 18 , 22 , 23 , 24 , and 25 ( tested at 5 m ) showed no significant inhibition of pm2 compared to the control ( supplementary data table 6 ) . this binding mode includes one hydrogen bond between the oxygen of the hydroxyl group and asp214 and electrostatic interactions between the nitrogen of the tertiary or secondary amine located near the charged asp34 ; ( 3 ) the unique major structural differences between apd and halofantrine ( or its derivatives ) are the aliphatic chains of the four carbon length bond to the tertiary amine that are not present in apd , and ( 4 ) the ic50 values reported by friedman and caflish ( 2009 ) for the 11 arylamino alcohols are considered medium to low ( 2 m ic50 100 m ) , when compared with other well - known plasmepsin inhibitors ( ic50 0.01 m ) ( marvin and daniel , 2012 ) . the compounds with both in vivo and in vitro antiplasmodial activity against p. berghei and p. falciparum , respectively , were further tested against the hemozoin formation drug target pathway to understand the mechanism of action . when compared to positive and negative control compound binding curves ( chloroquine and pyrimethamine , respectively ) , compound 22 exhibited affinity resembling that of chloroquine ( supplementary data fig . examination of the three species of parasitic heme ( host ingested hemoglobin , intracellular free heme , and hemozoin ) revealed that the primary mode of action for compound 22 is not through hemozoin formation inhibition . based on in vitro , in vivo , and target exploration results , we investigated the structural similarities and differences between apd and caa ( quinine , mefloquine , lumefantrine , and halofantrine ) using 3d pharmacophore models . due to the absence of the trifluoromethyl group in compound 23 , a detailed summary of the pharmacophoric features for each compound is presented in supplementary data fig . 3b ) , consisting of four hpf , one pif , two arf , one hbdf , and one hbaf . 3b ) , including two hpf , one pif , one arf , one hbdf , and one hbaf . specifically for apd , these common features are observed in the hydrophobic ( naphthyl and phenyl system ) and the amine region of the scaffold ; however , apd exhibited nine differences in comparison with caa ( two hpf , one arf , and six hbaf ) . this manuscript has shown the synthesis , racemic separation , in silico drug - likeness studies , in vitro evaluation against chloroquine sensitive ( f32 and d6 ) and multidrug resistant ( fcr-3 and c235 ) strains of p. falciparum , cytotoxicity ( vero ) , in silico metabolism studies , genotoxicity , in vivo efficacy in p. berghei mouse model , target exploration , and pharmacophore modeling of new apd . this work led to the identification of four promising compounds ( 22 , 23 , 24 , and 25 ) that exhibit values of antiplasmodial activity below 0.5 m ( fcr-3 ) , appropriate drug - likeness profile , adequate selectivity index ( 37 si 244 ) , and absence of genotoxicity . in vivo efficacy in p. notably , compound 22 displayed excellent parasitemia reduction ( 98 1% ) and complete cure with all treated mice surviving through the entire 21-day period with no signs of toxicity . additionally , compounds 22 and 23 showed potent antimalarial activity in chloroquine sensitive and multidrug resistant strains ( d6 ic50 0.19 m , c235 ic50 0.28 m ) . target exploration was performed in order to establish a possible mechanism of action ; however , both the pm2 enzyme and the hemozoin inhibition pathway were ruled out as primary targets for apd . this series of new apd showed not only promising in vitro and in vivo efficacy values , but also exhibited agreement between in vitro and in vivo studies . this last point is important due to the large number of potent antimalarial compounds reported in the literature that are only efficacious at in vitro and will no longer proceed along the drug discovery pipeline .

cardiovascular disease , particularly coronary heart disease ( chd ) , is one of the main causes of mortality in developed countries [ 1 , 2 ] . atherosclerosis is displayed when fat , cellular waste products , calcium , and other substances , particularly cholesterol , are deposited inside the artery , gradually reducing the inside diameter of the artery . subsequently , this may cause blockage of large- and medium - sized arteries [ 1 , 3 ] . hence epidemiological studies have demonstrated a strong association between plasma cholesterol concentration and risk of chd . statins ( in particular , monacolin k , mevinolin , or lovastatin ) form a class of molecules with a polyketide structure , a well - known inhibitor of hmg - coa reductase . they are produced by secondary metabolism of several fungi such as aspergillus terreus , penicillium citrinum , and monascus spp . . at present , statins are widely used as hypercholesterolemia drugs for reduction of plasma cholesterol level . since , the great effect of food on health has been accepted , utilization of complementary and alternative therapies ( natural treatments ) for a variety of chronic diseases are increasing in demand . red fermented rice or specifically , the monascus - fermented product ( mfp ) , is one of the popular remedies advertised as a therapy for hyperlipidemia . consumer spending on red yeast rice grew by about 80% from 2005 to 2008 in the united states , with the total sales of usd 20 million in 2008 [ 6 , 7 ] . in asia , particularly in china , taiwan , japan , and indonesia , mfps produced by monascus spp . have been used as dietary supplements and remedy for decades [ 8 , 9 ] . mfp contains at least 14 major chemical constituents which have chemical similarity to statin . it is believed that the cholesterol - lowering activity of mfp is mainly due to monacolin k ( mevinolin or lovastatin ) . however , the effect of mfp on blood cholesterol could not be fully explained by the action of monacolin k alone , instead , it might be the combined effect of monacolins and other substances in mfp . since red fermented rice has not yet been approved as medicinal by the food and drug administration ( fda ) , physicians may be limited in suggesting the guidelines of using this product by the patients . in this regard , generation of accurate information on the type and amount of monacolins present in mfp as a natural supplement is a complicated task . the main objective of this study was to compare the quality and quantity of monacolins present in mfps produced by m. purpureus ftc5391 with two commercial mfps available in the market . the fermented products by m. purpureus ftc5391 were prepared using solid and submerged fermentation techniques with various types of substrate for enhancement of monacolins synthesis . to maximize the extraction of monacolins from the mfp some important points for the improvement of methods for the detection of monacolins present in mfp were also identified in this study . mk ( mevinolin , lovastatin ) standard , trifluoroacetic acid ( tfa ) analytical grade , corn steep liquor , and monosodium glutamate were purchased from sigma ( st . louis , mo , usa ) . soybean , groundnut , coconut waste , gluten , pineapple waste , rice , and rice bran were purchased from local markets . the red yeast rice ( ryr ) capsule was obtained from cosway ( malaysia ) , while hypocol was purchased from wearnes biotech and medicals ( malaysia ) . yeast extract , malt extract , casamino acids , agar , standard czapek medium , potato dextrose agar ( pda ) and peptone were purchased from difco ( usa ) . other chemicals , including methanol and acetonitrile ( acn ) , ethyl acetate , isopropanol , butanol ( hplc grade ) , and ethanol ( analytical grade ) were obtained from merck ( germany ) . m. purpureus ftc5391 is a red - pigment - producing fungus isolated from fermented rice ( angkak ) and maintained at the culture collection in malaysian agricultural research and development institute ( mardi ) . m. purpureus ftc5391 was maintained after routine inoculation on pda and incubated at 30c for 7 days , by storage at 4c and refreshing monthly . for inoculum preparation , mycelia blocks ( 4 4 mm ) of m. purpureus were transferred into 100 ml of ymp ( 3 g / l yeast extract ; 3 g / l malt extract ; 5 g / l peptone ; 20 g / l glucose ) in a 250 ml flask . the flasks were incubated in rotary orbital shaker at 30c , agitated at 150 rpm for 4 days , and these cultures were used as a standard inoculum for all fermentations . the ability of m. purpureus ftc5391 in producing mk was tested on solid ( ssf ) and in submerged fermentations ( smf ) using different media , including : hiroi or sucrose medium ( sucrose ( 100 g / l ) ) ; glucose - glycerol - peptone ( ggp ) medium [ 13 , 14 ] ( glucose ( 30 g / l ) ) ; and , glucose - peptone - corn steep liquor ( gpc ) medium [ 15 , 16 ] ( glucose ( 60 smfs were carried out in 1000 ml erlenmeyer flasks containing 500 ml liquid medium . all media were autoclaved for 15 min at 121c after adjusting the ph at 6.0 . glucose was autoclaved separately to avoid the caramelization process and then was added to the related media under sterile conditions . the flasks were inoculated with 10% ( v / v ) inoculum and incubated in a rotary orbital shaker at different temperatures ( 2537c ) and different agitation rates ( 110250 rpm ) for 25 days . samples ( 5 ml ) were withdrawn at 48 h intervals for mk analysis . ssfs were carried out using different solid substrates , including soybean , ground nut , grated coconut waste , gluten , pineapple waste , rice , and rice bran . the substrates were ground and mixed with 1% ( w / v ) acetic acid . the 500 ml beaker containing 200 g of substrate was sterilized for 15 min at 121c . to start the fermentation , the beaker was inoculated with the standard inoculum ( 10% v / w ) and incubated at different temperatures ( 2537c ) until the color of media had completely changed to red , normally about 10 days . during the fermentation , the following solvents were used for the extraction process : ethanol , methanol , ethyl acetate , acn , isopropanol , ethyl acetate , and butanol . the extraction was conducted for 30 min , 1 h , 2 h , and 12 h at different temperatures ( 3780c ) . extraction of monacolin compoundsslightly different methods for extraction of monacolins were applied to solid and liquid samples of the prepared fermented products . solid fermented product was dried and finely ground into powder . the sample ( 0.5 g ) was mixed with 5 ml of ethanol / water solution ( 75 : 25 ) for 2 h at 60c under agitation , followed by centrifugation for 10 min at 3000 g . liquid fermented product was homogenized to break the mycelia cells . the homogenized sample ( 5 ml ) was extracted with 5 ml of 95% ethanol for 2 h at 60c under agitation and was subsequently centrifuged for 10 min at 3000 g . in both cases , the supernatant ( 1 ml ) was concentrated and dried under vacuum and then was resolved in 1 ml acn . the mixture was filtered through a membrane filter ( 0.45 m ) prior to hplc analysis . slightly different methods for extraction of monacolins were applied to solid and liquid samples of the prepared fermented products . the sample ( 0.5 g ) was mixed with 5 ml of ethanol / water solution ( 75 : 25 ) for 2 h at 60c under agitation , followed by centrifugation for 10 min at 3000 g . the homogenized sample ( 5 ml ) was extracted with 5 ml of 95% ethanol for 2 h at 60c under agitation and was subsequently centrifuged for 10 min at 3000 g . in both cases , the supernatant ( 1 ml ) was concentrated and dried under vacuum and then was resolved in 1 ml acn . the mixture was filtered through a membrane filter ( 0.45 m ) prior to hplc analysis . mk ( 10 mg ) was dissolved in pure solvents aforementioned or ethanol in water ( 75 : 25 v / v ) inside a 50 ml actinic volumetric flask . the solutions were diluted with solvents to obtain the standard solutions for the calibration curve in a concentration ranging from 0.2 to 20 ng / ml . individual solutions were filtered through a 0.45 m syringe membrane filter prior to injection ( 20 l ) into hplc . mk has two forms , lactone ( mkl ) and hydroxyl acid ( mka ) forms . for lactonization of mk , 1 ml of the extracted supernatant was mixed with 0.1 n naoh ( 1 ml ) and kept for 2 h at 30c . the mixture was concentrated and dried under vacuum , which was then resolved in 1 ml acn . the solution was filtered through a 0.45 m membrane syringe filter prior to analysis using hplc . usa ) was equipped with an on - line degasser , and an autosampler was used for detection of monacolin . the raw data were detected by 2996 pda , acquired and processed by a waters millennium32/empower software , chromatographic workstation loaded on an ibm computer . the column of waters symmetry c18 ( 150 mm 3.9 mm i.d . , 5 m ) was used as a stationary phase . a linear gradient of concentrated acn ( eluent a ) and 0.1% tfa ( eluent b ) was used as the mobile phase at a flow rate of 0.9 ml / min . the amount of eluent a was increased from 5 to 75% in the first 15 min , kept at 75% for 5 min , increased to 95% , and then reduced to 5% in another 10 min . the column temperature was set at 28c , and the injection volume was 20 l . the thermo - finnigan lcq classic was employed to analyze separation via liquid chromatography , coupled with electrospray ionization ( esi ) quadrupole ion - trap mass spectrometry . the chromatographic conditions of lc / pda / ms were modified from the conditions of lc / pda . the separations by lc / pda / ms were performed using an arrow - bore reversed - phase zorbax sb - c18 hplc column ( 2.1 mm 100 mm i.d . , 5 m , agilent scientific , calif , usa ) with a gradient elution consisting of acn ( eluent a ) and aqueous 0.2% acetic acid ( eluent b ) at a flow rate of 300 l / min . the hplc elution after pda was analyzed by the mass spectrometer ( ms ) using electrospray ionization ( esi ) . all analyses were performed using an esi interface with the following settings : positive ionization mode ; the capillary temperature was set at 250c ; spray voltage was set at 4.5 kv ; capillary voltage was set at 6 v ; sheath gas ( n2 ) flow was fixed at 30 au ; auxiliary gas ( n2 ) flow was fixed at 10 au . in the preliminary study , hypocholesterolemic activity in the fermented products using m. purpureus ftc5391 was analysed in vivo via animal testing . hypocholesterolemic activity was detected in all mfps tested , and it was assumed due to the existence of monacolins ( unpublished data ) . in this study , the existence of monacolins in similar mfps was evaluated using lc / pda / ms . basically , different derivatives of monacolins have similar structures , and consequently , similar uv spectra . in this study , different concentrations of mka and mkl standards were prepared and analyzed via lc / pda / ms . figure 1 shows the chromatogram and uv spectrum of the mk standard . the retention time ( rt ) of mkl and mka the uv spectrum of mkl and mka showed a pattern with three max at 232 , 239 , and 248 nm . the chromatograms of hypocol ( figure 2 ) and ryr ( figure 3 ) showed 12 and 3 extra peaks , respectively , with a similar uv spectrum pattern of mk . these extra peaks may be related to other derivatives of monacolin due to existence of similarities in their structures ( common polyketide portion , a hydroxy - hexahydro - naphthalene ring system , where different side chains are attached ) . the chromatogram results of all mfps produced using m. purpureus ftc5391 by two different methods of fermentation ( ssf and smf ) with various substrates showed that there was only one peak with the same rt as mk standard , while its uv spectrum was different . the uv spectrum of this peak showed the max at 228 nm and that was not similar with uv spectrum of mountain - like peak at max of 232 , 239 , and 248 nm ( figure 4 ) . this means that all mfps using m. purpureus ftc5391 , tested in this study , did not contain monacolin derivatives . the uv spectrum peak at rt of 11.917 min was similar to yellow pigment spectrum with max pattern at 237 , 280 , and 332 nm ( figure 4 ) . lc / ms is one of the informative methods for the detection and identification of the bioactive molecules . recently , rp - hplc has been widely applied for the determination of secondary metabolites in mfp such as pigments , monacolins , gaba , and several other components [ 8 , 9 , 1719 ] . adjustment and selection of the mobile phase is one of most important stages of analyses in the rp - hplc . although a large variety of organic solvents can be used in reversed phase chromatography , in practice only a few are routinely employed . acetonitrile and methanol are the most commonly used solvents in this case , since both have low viscosity ( even when mixed with aqueous solutions ) and are uv transparent . reverse phase separations are most often performed at low ph values , generally between ph 2 to 4 . the low ph results in good solubility of the sample components and ion suppression , not only of acidic groups on the sample molecules , but also of residual silanol groups on the silica matrix . acids such as trifluoroacetic acid , heptafluorobutyric acid , and orthophosphoric acid in the concentration range of 0.050.1% would be suitable in this type of separation . hence , trifluoroacetic acid ( 0.1% ) and concentrated acetonitrile were used in this study as the mobile phase . lactonization or alkalization of monacolins was performed by mixing the sample with naoh before applying to lc / pda system . alkalization of mk standard caused a shift in the rrt from 21 min to 17 min without changing the uv spectrum , indicating that both peaks have similar molecular structures ( figure 1 ) , where it was in agreement with the previous report . comparison between two chromatograms of pre- and postalkalized hypocol , ryr , and mfp ftc5391 extracts ( figures 2 , 3 and 4 , resp . ) revealed that some peaks disappeared , while some new peaks appeared . however , the similarity of uv spectra of the missed peaks and those with the increased peak areas was high . although the typical chromatogram for mfp using m. purpureus ftc5391 extract before and after lactonization did not change ( figure 4 ) , the comparison between hypocol and ryr chromatograms and their uv spectra before and after lactonization showed some differences ( figures 2 and 3 ) . the ryr chromatogram ( figure 3 ) showed a large peak in the rt of 21 min before lactonization , this peak shifted to 17 min after lactonization with similar spectrum of mkl . after the shifted of large peak , a small peak at the rt of 21 min appeared with different uv spectrum of mkl . this peak showed a similar uv spectrum with the peak for mfp using m. purpureus ftc5391 extract at rt of 21 min . in addition , the hypocol chromatogram demonstrated a small peak in the rt of 17 min that grew up after lactonization . these results suggested that there were two different peaks belonging to different compounds , where the small peak was masked by the large peak in the same rt . additional mk standard solution in mfp using m. purpureus ftc5391 extract was employed to confirm the existence of mk . figure 5 shows that the addition of mk standard reproduced the peak at rt of 21 min in the mfp chromatogram . moreover , there was a significant difference in comparison between the uv spectra related to peaks at rt of 21 min in pre- and postmixing of the mk standard solution with mfp extract . the premixing uv spectrum associated with the peak at rt of 21 min showed max at 228 nm , while the postmixing uv spectrum transformed to the mk spectrum shape with three max at 232 , 239 , and 248 nm . the chromatogram of mfp using m. purpureus ftc5391 extract blended with mk standard solution after lactonization showed the appearance of a peak at rt 17 , where the peak area at rt 21 decreased and its uv spectrum also changed . these results doubly confirmed the existence of two different coincided peaks in the same rt , those concealed each other . therefore , it was concluded that all mfps produced using ftc5391 m. purpureus ftc5391 , tested in this study , did not contain mk . it is interesting to note that the conversion of monacolins from the lactone form to acid form is a special clue for the development of simple and rapid method for monacolins detection via lc / pda / ms . in fact , rt evaluation alone is not enough for the identification of bioactive compounds by the lc / pda method . thus , this method should be utilized with the examination of other special properties of the target molecule simultaneously . in these cases , where there are two or more compounds with the same rt in the sample , the peaks cover each other and the compound with the higher amount may dominate and mask the other molecules . for a case where the small peaks were related to monacolin , a step of lactonization of monacolins must be added in the detection procedure of lc / pda / ms to enable identification . for further identification of the existence of monacolins in the samples and to complete the monacolins identification , all samples and standard solutions were traced by hyphenated instrumentation of lc - ms . the quadruple ion trap mass analyzer serves to store , fragment in , and select ions for detection . therefore , the total ion current ( tic ) chromatogram , mass , uv spectra , and selective ion chromatograms ( sic ) for each peak were evaluated , one by one . the results confirmed the existence of mkl , mka , mja , mj , mxa , mla , mx , mka , ml , p1 , mma , mm , and dmk in hypocol ; mkl , mm , and dmk in ryr . the molecular ion chromatograms and mass spectra patterns for mkl and mka std by the lc / pda / ms showed the predominant peaks at rt around 15.82 min and 11.82 min ; max at 245 nm ; mass spectrum 405 ( m + 1 ) and 423 ( m + 1 ) , respectively . the lc / pda / ms chromatograms of standards ( mkl and mka ) in comparison to mfp using m. purpureus ftc5391 , hypocol , and ryr chromatograms revealed that the hypocol included both mkl and mka , whereas ryr included only mkl , and the mfp using m. purpureus ftc5391 tested had no monacolins , not even mk . figures 6 and 7 show the differences between mass spectra of mk standard and mfp using m. purpureus ftc5391 compound with similar rt of mk . production of monacolins , secondary metabolites , is influenced by physicochemical factors of the fermentation conditions and is strain - dependent based on individual abilities of the microorganisms [ 2125 ] . therefore , manipulation of the fermentation conditions ( temperature , aeration , nutritional factors , as well as fermentation method ) is necessary to induce or enhance the ability of m. purpureus strains to secrete monacolins . results from this study indicated that the use of two types of fermentation methods ( smf and ssf ) with various medium formulations and fermentation conditions did not induce monacolins production by m. purpureus ftc5391 . comparison between hplc chromatogram profiles of hypocol and ryr revealed that the uv spectra and the number of chromatogram peaks were not similar even though both extracts were derived from mfps using m. purpureus ftc5391 ( figures 2 and 3 ) . hypocol chromatogram showed the existence of mkl , mka , and several other peaks with mk - like uv spectra , while the ryr chromatogram revealed just the existence of mkl and a few numbers of peaks with mk - like uv spectra . since the supplements tested in this study were produced by the different companies under different fermentation conditions using different strains of m. purpureus , some of the metabolites or all of them may not be existed in the products . this is in agreement with the results for the analysis of citrinin ; and the lactone acid forms of mk in red mold rice . mk is soluble in methanol , ethanol , acetone , chloroform and benzene but not in n - hexane and petroleum ether . moreover , ethyl acetate has been used for the extraction of mk . the ability of several polar and nonpolar organic solvents ( log p values : 0.76 to 0.8 ) to dissolve mk for the enhancement of the extraction of mk from hypocol the following sequence for solubility strength of solvents was achieved to dissolve the mk : methanol > ethanol > isopropanol > acetonitrile > ethyl acetate > butanol the peaks pattern ( mk - like - spectra ) from the hplc chromatograms was as follows ( the numbers in parentheses indicate the numbers of peaks ) : methanol ( 18 ) > ethanol ( 17 ) > acetonitrile ( 15 ) > isopropanol ( 14 ) > butanol ( 12 ) > ethyl acetate(9 ) methanol and ethanol were identified as the preferred solvents for monacolins extraction due to their ability to extract the highest amount of mkl and the highest numbers of monacolin derivatives . in accordance with these results , lee et al . also reported that ethanol and methanol were the preferred extraction solvents for mkl , while ethanol was suggested as the best solvent to extract citrinin , mkl , and mka . methanolic and ethanolic hypocol extracts included about 17 and 18 peaks , respectively , which were similar to the uv spectrum of mkl ( mountain - like peak at max of 232 , 239 , and 248 nm ) . the solvents that could dissolve the mk were in a range of semipolar to nonpolar . in spite of no clear correlation between log p values of organic solvents and the solubility of monacolins , results of this study indicated that the nonpolar solvents were the preferred solvents for monacolins extraction . the highest extraction efficiency was obtained by methanol ( log p value of 0.76 ) . moreover , mixing a proper quantity of water to the water miscible solvents or powder ( solid ) samples increased the dissolving ability of the target compounds . the diluted solvent could diffuse better in the sample and , consequently , promoted the extraction of monacolins from the base material . therefore , methanol , ethanol , and isopropanol , as water miscible solvents , were preferred for monacolins extraction from monascus - fermented product prepared through ssf . one of the effective methods for improving the extraction of thermostable compounds is heating . since mk is a thermostable molecule ( mp = 157159c ) , it was predictable that heating enhanced the amount of mk extraction . the performance of mk extraction was also evaluated under different temperatures ( 37c80c ) for 30 min , 1 h , 2 h , and 12 h. the results showed that the extraction performance was increased up to 60c for 2 h and decreased beyond this temperature . extension of the extraction time beyond 2 h did not further improve the extraction performance of the target compound . the optimum conditions for mk extraction was a mixture of sample with ethanol and water ( 75 : 25 ) at 60c for 2 h under shaking conditions for ssf and a mixture of concentrated ethanol with sample ( 1 : 1 v / v ) for the smf under shaking conditions at 60c for 2 h. results from this study have demonstrated the uniqueness of hplc chromatograms and uv spectrum profiles for the identification and quantification of monacolins in the fermented products of m. purpureus . monacolins lactonization was found to be the important property of monacolins that can be used to solve the problem of false - positive results in monacolin identification and quantification using lc / pda / ms . the methanolic extraction of mfp using m. purpureus ftc5391 pulled 18 compounds with a similar uv spectrum to mk was observed in this study , though the existence of 14 derivatives of monacolins has been reported to date . using this improved technique , monacolin was not detected in all mfps tested in this study ( produced by m. purpureus ftc5391 using different fermentation techniques and substrates ) . the hypocholesterolemic activity of the mfps using m. purpureus ftc5391 may be due to the present of other compounds aside from monacolins with hypocholesterolemic effects . future study is necessary to identify the substance(s ) that are responsible for this activity and the underlying molecular mechanisms involved .

monacolins , as natural statins , form a class of fungal secondary metabolites and act as the specific inhibitors of hmg - coa reductase . the interest in using the fermented products as the natural source of monacolins , instead of statin drugs , is increasing enormously with its increasing demand . in this study , the fermented products were produced by monascus purpureus ftc5391 using submerged and solid state fermentations . two commercial monascus - fermented products were also evaluated for comparison . improved methods of monacolins extraction and identification were developed for the assessment of monacolins in the fermented products . methanol and ethanol were found to be the most favorable solvents for monacolins extraction due to their ability to extract higher amount of monacolin k and higher numbers of monacolin derivatives . problem related to false - positive results during monacolins identification was solved by adding monacolin lactonization step in the assessment method . using this improved method , monacolin derivatives were not detected in all monascus - fermented products tested in this study , suggesting that their hypocholesterolemic effects may be due to other compounds other than monacolins .

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

cardiovascular disease , particularly coronary heart disease ( chd ) , is one of the main causes of mortality in developed countries [ 1 , 2 ] . atherosclerosis is displayed when fat , cellular waste products , calcium , and other substances , particularly cholesterol , are deposited inside the artery , gradually reducing the inside diameter of the artery . subsequently , this may cause blockage of large- and medium - sized arteries [ 1 , 3 ] . hence epidemiological studies have demonstrated a strong association between plasma cholesterol concentration and risk of chd . statins ( in particular , monacolin k , mevinolin , or lovastatin ) form a class of molecules with a polyketide structure , a well - known inhibitor of hmg - coa reductase . they are produced by secondary metabolism of several fungi such as aspergillus terreus , penicillium citrinum , and monascus spp . since , the great effect of food on health has been accepted , utilization of complementary and alternative therapies ( natural treatments ) for a variety of chronic diseases are increasing in demand . red fermented rice or specifically , the monascus - fermented product ( mfp ) , is one of the popular remedies advertised as a therapy for hyperlipidemia . consumer spending on red yeast rice grew by about 80% from 2005 to 2008 in the united states , with the total sales of usd 20 million in 2008 [ 6 , 7 ] . in asia , particularly in china , taiwan , japan , and indonesia , mfps produced by monascus spp . it is believed that the cholesterol - lowering activity of mfp is mainly due to monacolin k ( mevinolin or lovastatin ) . however , the effect of mfp on blood cholesterol could not be fully explained by the action of monacolin k alone , instead , it might be the combined effect of monacolins and other substances in mfp . since red fermented rice has not yet been approved as medicinal by the food and drug administration ( fda ) , physicians may be limited in suggesting the guidelines of using this product by the patients . in this regard , generation of accurate information on the type and amount of monacolins present in mfp as a natural supplement is a complicated task . the main objective of this study was to compare the quality and quantity of monacolins present in mfps produced by m. purpureus ftc5391 with two commercial mfps available in the market . the fermented products by m. purpureus ftc5391 were prepared using solid and submerged fermentation techniques with various types of substrate for enhancement of monacolins synthesis . to maximize the extraction of monacolins from the mfp some important points for the improvement of methods for the detection of monacolins present in mfp were also identified in this study . mk ( mevinolin , lovastatin ) standard , trifluoroacetic acid ( tfa ) analytical grade , corn steep liquor , and monosodium glutamate were purchased from sigma ( st . other chemicals , including methanol and acetonitrile ( acn ) , ethyl acetate , isopropanol , butanol ( hplc grade ) , and ethanol ( analytical grade ) were obtained from merck ( germany ) . m. purpureus ftc5391 is a red - pigment - producing fungus isolated from fermented rice ( angkak ) and maintained at the culture collection in malaysian agricultural research and development institute ( mardi ) . m. purpureus ftc5391 was maintained after routine inoculation on pda and incubated at 30c for 7 days , by storage at 4c and refreshing monthly . the flasks were incubated in rotary orbital shaker at 30c , agitated at 150 rpm for 4 days , and these cultures were used as a standard inoculum for all fermentations . the ability of m. purpureus ftc5391 in producing mk was tested on solid ( ssf ) and in submerged fermentations ( smf ) using different media , including : hiroi or sucrose medium ( sucrose ( 100 g / l ) ) ; glucose - glycerol - peptone ( ggp ) medium [ 13 , 14 ] ( glucose ( 30 g / l ) ) ; and , glucose - peptone - corn steep liquor ( gpc ) medium [ 15 , 16 ] ( glucose ( 60 smfs were carried out in 1000 ml erlenmeyer flasks containing 500 ml liquid medium . the flasks were inoculated with 10% ( v / v ) inoculum and incubated in a rotary orbital shaker at different temperatures ( 2537c ) and different agitation rates ( 110250 rpm ) for 25 days . samples ( 5 ml ) were withdrawn at 48 h intervals for mk analysis . the substrates were ground and mixed with 1% ( w / v ) acetic acid . to start the fermentation , the beaker was inoculated with the standard inoculum ( 10% v / w ) and incubated at different temperatures ( 2537c ) until the color of media had completely changed to red , normally about 10 days . during the fermentation , the following solvents were used for the extraction process : ethanol , methanol , ethyl acetate , acn , isopropanol , ethyl acetate , and butanol . extraction of monacolin compoundsslightly different methods for extraction of monacolins were applied to solid and liquid samples of the prepared fermented products . in both cases , the supernatant ( 1 ml ) was concentrated and dried under vacuum and then was resolved in 1 ml acn . slightly different methods for extraction of monacolins were applied to solid and liquid samples of the prepared fermented products . the sample ( 0.5 g ) was mixed with 5 ml of ethanol / water solution ( 75 : 25 ) for 2 h at 60c under agitation , followed by centrifugation for 10 min at 3000 g . in both cases , the supernatant ( 1 ml ) was concentrated and dried under vacuum and then was resolved in 1 ml acn . the solutions were diluted with solvents to obtain the standard solutions for the calibration curve in a concentration ranging from 0.2 to 20 ng / ml . usa ) was equipped with an on - line degasser , and an autosampler was used for detection of monacolin . a linear gradient of concentrated acn ( eluent a ) and 0.1% tfa ( eluent b ) was used as the mobile phase at a flow rate of 0.9 ml / min . the amount of eluent a was increased from 5 to 75% in the first 15 min , kept at 75% for 5 min , increased to 95% , and then reduced to 5% in another 10 min . the chromatographic conditions of lc / pda / ms were modified from the conditions of lc / pda . in the preliminary study , hypocholesterolemic activity in the fermented products using m. purpureus ftc5391 was analysed in vivo via animal testing . hypocholesterolemic activity was detected in all mfps tested , and it was assumed due to the existence of monacolins ( unpublished data ) . in this study , the existence of monacolins in similar mfps was evaluated using lc / pda / ms . basically , different derivatives of monacolins have similar structures , and consequently , similar uv spectra . in this study , different concentrations of mka and mkl standards were prepared and analyzed via lc / pda / ms . these extra peaks may be related to other derivatives of monacolin due to existence of similarities in their structures ( common polyketide portion , a hydroxy - hexahydro - naphthalene ring system , where different side chains are attached ) . the chromatogram results of all mfps produced using m. purpureus ftc5391 by two different methods of fermentation ( ssf and smf ) with various substrates showed that there was only one peak with the same rt as mk standard , while its uv spectrum was different . this means that all mfps using m. purpureus ftc5391 , tested in this study , did not contain monacolin derivatives . lc / ms is one of the informative methods for the detection and identification of the bioactive molecules . recently , rp - hplc has been widely applied for the determination of secondary metabolites in mfp such as pigments , monacolins , gaba , and several other components [ 8 , 9 , 1719 ] . adjustment and selection of the mobile phase is one of most important stages of analyses in the rp - hplc . acetonitrile and methanol are the most commonly used solvents in this case , since both have low viscosity ( even when mixed with aqueous solutions ) and are uv transparent . acids such as trifluoroacetic acid , heptafluorobutyric acid , and orthophosphoric acid in the concentration range of 0.050.1% would be suitable in this type of separation . hence , trifluoroacetic acid ( 0.1% ) and concentrated acetonitrile were used in this study as the mobile phase . lactonization or alkalization of monacolins was performed by mixing the sample with naoh before applying to lc / pda system . alkalization of mk standard caused a shift in the rrt from 21 min to 17 min without changing the uv spectrum , indicating that both peaks have similar molecular structures ( figure 1 ) , where it was in agreement with the previous report . however , the similarity of uv spectra of the missed peaks and those with the increased peak areas was high . although the typical chromatogram for mfp using m. purpureus ftc5391 extract before and after lactonization did not change ( figure 4 ) , the comparison between hypocol and ryr chromatograms and their uv spectra before and after lactonization showed some differences ( figures 2 and 3 ) . the ryr chromatogram ( figure 3 ) showed a large peak in the rt of 21 min before lactonization , this peak shifted to 17 min after lactonization with similar spectrum of mkl . after the shifted of large peak , a small peak at the rt of 21 min appeared with different uv spectrum of mkl . this peak showed a similar uv spectrum with the peak for mfp using m. purpureus ftc5391 extract at rt of 21 min . in addition , the hypocol chromatogram demonstrated a small peak in the rt of 17 min that grew up after lactonization . these results suggested that there were two different peaks belonging to different compounds , where the small peak was masked by the large peak in the same rt . additional mk standard solution in mfp using m. purpureus ftc5391 extract was employed to confirm the existence of mk . figure 5 shows that the addition of mk standard reproduced the peak at rt of 21 min in the mfp chromatogram . moreover , there was a significant difference in comparison between the uv spectra related to peaks at rt of 21 min in pre- and postmixing of the mk standard solution with mfp extract . the chromatogram of mfp using m. purpureus ftc5391 extract blended with mk standard solution after lactonization showed the appearance of a peak at rt 17 , where the peak area at rt 21 decreased and its uv spectrum also changed . these results doubly confirmed the existence of two different coincided peaks in the same rt , those concealed each other . therefore , it was concluded that all mfps produced using ftc5391 m. purpureus ftc5391 , tested in this study , did not contain mk . it is interesting to note that the conversion of monacolins from the lactone form to acid form is a special clue for the development of simple and rapid method for monacolins detection via lc / pda / ms . in fact , rt evaluation alone is not enough for the identification of bioactive compounds by the lc / pda method . thus , this method should be utilized with the examination of other special properties of the target molecule simultaneously . in these cases , where there are two or more compounds with the same rt in the sample , the peaks cover each other and the compound with the higher amount may dominate and mask the other molecules . for a case where the small peaks were related to monacolin , a step of lactonization of monacolins must be added in the detection procedure of lc / pda / ms to enable identification . for further identification of the existence of monacolins in the samples and to complete the monacolins identification , all samples and standard solutions were traced by hyphenated instrumentation of lc - ms . therefore , the total ion current ( tic ) chromatogram , mass , uv spectra , and selective ion chromatograms ( sic ) for each peak were evaluated , one by one . the lc / pda / ms chromatograms of standards ( mkl and mka ) in comparison to mfp using m. purpureus ftc5391 , hypocol , and ryr chromatograms revealed that the hypocol included both mkl and mka , whereas ryr included only mkl , and the mfp using m. purpureus ftc5391 tested had no monacolins , not even mk . figures 6 and 7 show the differences between mass spectra of mk standard and mfp using m. purpureus ftc5391 compound with similar rt of mk . production of monacolins , secondary metabolites , is influenced by physicochemical factors of the fermentation conditions and is strain - dependent based on individual abilities of the microorganisms [ 2125 ] . therefore , manipulation of the fermentation conditions ( temperature , aeration , nutritional factors , as well as fermentation method ) is necessary to induce or enhance the ability of m. purpureus strains to secrete monacolins . results from this study indicated that the use of two types of fermentation methods ( smf and ssf ) with various medium formulations and fermentation conditions did not induce monacolins production by m. purpureus ftc5391 . comparison between hplc chromatogram profiles of hypocol and ryr revealed that the uv spectra and the number of chromatogram peaks were not similar even though both extracts were derived from mfps using m. purpureus ftc5391 ( figures 2 and 3 ) . hypocol chromatogram showed the existence of mkl , mka , and several other peaks with mk - like uv spectra , while the ryr chromatogram revealed just the existence of mkl and a few numbers of peaks with mk - like uv spectra . since the supplements tested in this study were produced by the different companies under different fermentation conditions using different strains of m. purpureus , some of the metabolites or all of them may not be existed in the products . this is in agreement with the results for the analysis of citrinin ; and the lactone acid forms of mk in red mold rice . moreover , ethyl acetate has been used for the extraction of mk . the ability of several polar and nonpolar organic solvents ( log p values : 0.76 to 0.8 ) to dissolve mk for the enhancement of the extraction of mk from hypocol the following sequence for solubility strength of solvents was achieved to dissolve the mk : methanol > ethanol > isopropanol > acetonitrile > ethyl acetate > butanol the peaks pattern ( mk - like - spectra ) from the hplc chromatograms was as follows ( the numbers in parentheses indicate the numbers of peaks ) : methanol ( 18 ) > ethanol ( 17 ) > acetonitrile ( 15 ) > isopropanol ( 14 ) > butanol ( 12 ) > ethyl acetate(9 ) methanol and ethanol were identified as the preferred solvents for monacolins extraction due to their ability to extract the highest amount of mkl and the highest numbers of monacolin derivatives . also reported that ethanol and methanol were the preferred extraction solvents for mkl , while ethanol was suggested as the best solvent to extract citrinin , mkl , and mka . methanolic and ethanolic hypocol extracts included about 17 and 18 peaks , respectively , which were similar to the uv spectrum of mkl ( mountain - like peak at max of 232 , 239 , and 248 nm ) . the solvents that could dissolve the mk were in a range of semipolar to nonpolar . in spite of no clear correlation between log p values of organic solvents and the solubility of monacolins , results of this study indicated that the nonpolar solvents were the preferred solvents for monacolins extraction . the highest extraction efficiency was obtained by methanol ( log p value of 0.76 ) . the diluted solvent could diffuse better in the sample and , consequently , promoted the extraction of monacolins from the base material . therefore , methanol , ethanol , and isopropanol , as water miscible solvents , were preferred for monacolins extraction from monascus - fermented product prepared through ssf . since mk is a thermostable molecule ( mp = 157159c ) , it was predictable that heating enhanced the amount of mk extraction . the performance of mk extraction was also evaluated under different temperatures ( 37c80c ) for 30 min , 1 h , 2 h , and 12 h. the results showed that the extraction performance was increased up to 60c for 2 h and decreased beyond this temperature . the optimum conditions for mk extraction was a mixture of sample with ethanol and water ( 75 : 25 ) at 60c for 2 h under shaking conditions for ssf and a mixture of concentrated ethanol with sample ( 1 : 1 v / v ) for the smf under shaking conditions at 60c for 2 h. results from this study have demonstrated the uniqueness of hplc chromatograms and uv spectrum profiles for the identification and quantification of monacolins in the fermented products of m. purpureus . monacolins lactonization was found to be the important property of monacolins that can be used to solve the problem of false - positive results in monacolin identification and quantification using lc / pda / ms . the methanolic extraction of mfp using m. purpureus ftc5391 pulled 18 compounds with a similar uv spectrum to mk was observed in this study , though the existence of 14 derivatives of monacolins has been reported to date . using this improved technique , monacolin was not detected in all mfps tested in this study ( produced by m. purpureus ftc5391 using different fermentation techniques and substrates ) . the hypocholesterolemic activity of the mfps using m. purpureus ftc5391 may be due to the present of other compounds aside from monacolins with hypocholesterolemic effects .

in the united states , 34% of adults aged 20 and over are considered overweight , 34% obese , and 6% extremely obese . it is estimated that approximately two - thirds of overweight and obese individuals are currently trying to lose weight [ 2 , 3 ] . despite the fact that losing weight and maintaining weight loss can be difficult , research shows that 1720% of overweight or obese individuals are able to lose weight and maintain their weight loss for at least 1 year [ 4 , 5 ] . a sustained reduction of 10% of initial body weight is generally considered a success for clinical and research purposes , as this amount , even for very obese persons , can result in numerous health and economic benefits , such as an increase in life expectancy and a reduction in comorbidities such as diabetes , hypertension , and coronary heart disease , along with associated medical costs [ 5 , 6 ] . thus , obtaining better understanding of factors that influence successful weight loss maintenance ( wlm ) is important and could lead to the development of more effective intervention strategies . the national weight control registry ( nwcr ) has provided important information regarding correlates of successful weight loss maintenance ( defined as having lost at least 13.6 kg ( 30 pounds ) and having kept it off for a minimum of one year ) . the nwcr is a group of over 3,000 individuals who have lost an average of 30 kg and maintained this loss for an average duration of 5.5 years . data from the nwcr indicates that successful weight loss maintainers have high physical activity levels , eat a diet low in fat and high in carbohydrate , and regularly self - monitor their weight , while higher levels of depression , disinhibition , and binge eating increase the risk of weight regain , suggesting that binge eating may be a behavior important to successful wlm . although considerable research examining the relationship between binge eating and weight loss has been conducted [ 711 ] , there is a paucity of research on the relationship between binge eating and prolonged wlm . of interest , rates of binge eating in nwcr participants are comparable to those observed in community samples , with 8% of members reporting 4 or more binges per month . beyond this observational study , empirical evidence on the relationship between binge eating and most studies have not found binge eating to be a strong predictor of weight loss success [ 711 ] . however , one study found that binge eaters who abstained from binging earlier in the program had significantly greater weight loss than those who continued binge eating and several other studies have found that individuals who engage in binge eating regain weight more rapidly after treatment compared to nonbingers [ 911 ] . it has also been documented that participants who binge eat are more likely to drop out of treatment programs [ 7 , 911 ] , which may mask potential relationships that exist between binge eating and weight loss outcomes . although there is limited support for binge eating as a predictor of weight loss , binge eating behavior may be negatively associated with wlm , based on evidence from the studies cited previously , along with the evidence that successful weight maintainers report low rates of binge eating and individuals who binge eat have been shown to gain more weight over time . the diagnostic and statistical manual of mental disorders 5th edition ( dsm-5 ) now includes binge eating disorder as a psychiatric illness in recognition of the psychological distress and impaired functioning that is associated with this behavior . a binge eating episode is defined as consumption of a subjectively large amount of food and sense of loss of control over eating during this episode . to meet the dsm-5 's criteria for bed , an individual must engage in a binge eating episode at least 1 time per week for 3 months . furthermore , the dsm-5 denotes classifications for severity of binge eating : if 13 binge episodes occur weekly , this is classified as mild binge eating disorder . bed of moderate severity occurs at a frequency of 47 episodes per week , severe at a frequency of 813 episodes per week , and extreme if 14 or more episodes occur weekly . the prevalence of bed in individuals seeking treatment for obesity has been reported to be as high as 25% , compared to estimates of 25% from community samples . although binge eating behavior has been studied extensively , controversy exists about how to best operationalize the disorder in a research setting . many studies have measured binge eating at a sole time point ( e.g. , [ 7 , 14 ] ) while few studies have examined binge eating over time [ 17 , 18 ] . this is important because it is possible that binge eating behavior fluctuates over time and a single time point may not capture all experiencing symptoms . though data has been used to assert that binge eating behaviors remain stable , other research shows that certain disordered eating behaviors , like binge eating , fluctuate over time . though hawkins and clement reported temporal stability of binge eating ( test retest = 0.88 ) , their sample consisted of undergraduates , most of whom were of normal weight , and the binge eating scale utilized had only moderate internal consistency ( 0.68 ) . moreover , the period of assessment for stability was five weeks and it is possible that cycles of binge eating exceed five weeks . consequently , it may be important to measure binge eating at multiple time points in order to more accurately assess the relationship between binge eating and weight change , particularly among adults who are trying to maintain a clinically meaningful weight loss . an important and potentially confounding factor to consider when studying binge eating is depression . though depression and binge eating are not interchangeable , it has been suggested that there is a relationship between the two . a number of previous studies have found depression to be associated with both binge eating [ 9 , 2022 ] as well as with weight status [ 23 , 24 ] . more recently , negative affective states have been shown to precede binge eating in overweight and obese adults . thus , measuring and attempting to prevent depression score from confounding the relationship between binge eating behavior and weight loss maintenance may be advisable . the current study addresses a gap in the literature by examining the relationship between binge eating behavior and wlm among adults who recently lost at least 10% of their body weight and enrolled in a study to test the effect of an intervention on wlm success . because binge eating may fluctuate over time and this may influence wlm , binge eating behavior was assessed at multiple time points . binge eating behavior was measured at baseline , 12 months , and 24 months , allowing for both cross - sectional and longitudinal approaches to categorizing this behavior . we hypothesize that ( h1 ) individuals who report binge eating behaviors at baseline will have a greater rate of weight regain over the 24-month trial compared to individuals who do not report binge eating at baseline . additionally , accounting for binge eating status at multiple time points , taking a longitudinal approach to this construct , will be informative beyond the use of baseline binge eating status alone . specifically , we hypothesize that ( h2 ) participants who report binge eating at multiple time points will have a greater rate of weight regain over the 24-month trial relative to participants who report no binge eating or less consistent binge eating ( reporting binge eating at one out of 3 time points ) . because of its potential role in the relationship between binge eating and weight regain , depression was included in analyses . keep it off ( kio ) participants are members of the minnesota - based healthpartners managed - care organization who intentionally lost at least 10% of their body weight during the previous year . briefly , a participant 's progression through the study was as follows : investigators recruited participants who had lost at least 10% of their body weight in the past year , interested individuals were telephone - screened for eligibility ( n = 875 ) , and those who met inclusion criteria were enrolled and randomized to one of two weight loss maintenance groups ( n = 419 ) and then received either the self - directed or guided weight loss maintenance intervention . participants in the self - directed intervention received less education about weight loss maintenance ( two 20-minute phone calls and a self - monitoring logbook to keep track of progress ) compared to the guided intervention ( twenty - four 20-minute phone calls plus followups along with the self - monitoring logbook which they were required to submit at regular intervals to intervention staff ) . during eligibility screening , participants were asked to describe how ( e.g. , weight loss program or decreasing caloric intake on their own ) and why they purposefully lost weight . inclusion criteria for participation in the study were as follows : 19 to 70 years old , bmi > 20.5 kg / m , and the capacity to communicate with research staff by telephone . participants could not have a history of anorexia nervosa , previous bariatric surgery , recent diagnosis of a non - skin cancer or congestive heart failure , and/or current participation in another phone - based weight loss program or in another weight - management study . the kio intervention and primary outcomes are described in detail elsewhere [ 26 , 27 ] . weight and height were measured at baseline , 12 months , and 24 months during in - person visits with subjects wearing light clothes and without shoes ( seca 770 medical scale ; seca 214 portable height rod ) . body mass index ( bmi ) was calculated from this measure in addition to an in - person height measurement . binge eating behavior was self - reported via survey at baseline , 12 months , and 24 months during in - person visits and defined using three items from the eating disorder diagnostic scale ( edds ) : ( 1 ) eating what other people would regard as an unusually large amount of food have there been times when you felt that you have eaten what other people would regard as an unusually large amount of food ( e.g. , a quart of ice cream ) given the circumstances ? ( response options = yes / no ) ; ( 2 ) a perceived loss of control during these episodes during the times when you ate an unusually large amount of food , did you experience a loss of control ( feel you could not stop eating or control what or how much you were eating ) ? ( response options = yes / no ) ; and ( 3 ) the frequency of binge episodes how many times per week on average over the past 6 months have you eaten an unusually large amount of food and experienced a loss of control ? ( response options = 0 , 1 , 2, 13 , 14 + ) . the edds has good psychometric properties : internal consistency : alpha = 0.89 ; test - retest reliability : r = 0.87 [ 28 , 29 ] . at baseline and at 12 and 24 months , each participant was categorized as a binge eater if they reported eating a large amount of food and feeling a loss of control over eating at least once per week on average over the past 6 months . in cases of missing data , if it could not be verified that the participant self - reported this behavior at least once a week , in accordance with dsm-5 criteria , they were labeled as missing or not experiencing binge eating depending upon the other information available . for example , some participants may have reported eating a large amount and skipped the question about experiencing a loss of control but reported a frequency of an average of > 1 times per week ; these participants were classified as engaging in binge eating because they reported a frequency specified by the dsm-5 , and the final question clearly states episodes where both a large amount and a loss of control were experienced . binge eating behavior was categorized as being present or absent at each time point ( baseline , 12 months , and 24 months ) . for h1 , we were interested in how binge eating behavior at baseline was associated with rate of weight regain over two years . for h2 , we were interested in how the consistency of binge eating behavior at multiple time points ( e.g. , an individual may not have reported binge eating at baseline but may have reported binge eating at 12 months and/or 24 months ) was associated with rate of weight regain over two years . all analyses pertaining to this hypothesis used the aforementioned definition of binge eating behavior and applied it to baseline only . assessment of binge based on assessments at multiple time points may provide different estimates of the occurrence of this behavior relative to a single point in time , such as baseline . for this reason , a variable was created to assess whether participants met criteria for binge eating behavior at any data collection time point throughout the two - year study . this is a dichotomous variable if a participant met the aforementioned criteria for binge eating at baseline or 12 months or 24 months , they would be classified as having binge eating behavior . as shown in table 2 , nearly a third of the sample self - reported binge eating behavior during at least one time point during the two years of the study . to address h2 , a cumulative binge score assessing recurrent binge eating was created ; this score is the sum of self - reported binge eating behavior at each time point with a minimum of 0 indicating no binge eating at any time point and a maximum of 3 indicating a participant self - reporting binge eating at all three time points ( baseline , 12 months , and 24 months ) . a three - level categorical variable was then created for individuals reporting no binge eating at any time points , binge eating behaviors at one time point , or binge eating behaviors at 2 or more time points . since a score of 2 meant reporting binge eating more often than not ( 2 out of 3 possible time points ) , we classified them as consistent binge eaters and those with a score of 1 as inconsistent binge eaters . based on the new dsm-5 definitions for binge eating severity , severity of binge eating was categorized for each of the time points at which a person was classified as a binge eater . as discussed , if 13 binge episodes occur weekly , this is classified as mild binge eating disorder . bed of moderate severity occurs at a frequency of 47 episodes per week , severe at a frequency of 813 episodes per week , and extreme if 14 or more episodes occur weekly . the 11-item short form of the center for epidemiological studies depression ( ces - d ) symptoms scale was used to measure depression via in - person survey at baseline , 12 months , and 24 months . this version of the ces - d has been shown to have high internal consistency and correlates strongly with the original 20-item ces - d scale that is broadly accepted by researchers and clinicians . a participant 's score on the ces - d is the sum of responses to 11 items ( e.g. , i felt sad ; rated on a scale of rarely or none , some , moderate , and all ) regarding the amount of time at which they felt they agreed with the statement in the past week , with two items reverse - coded . a score of 9 or higher on the 11-item scale corresponds with a score of 16 or higher on the 20-item scale and represents clinically significant symptoms of depression . measures of central tendency and dispersion were calculated for demographic variables , depression , binge eating , and body weight for the study sample and then stratified by baseline and by consistency of binge eating behavior . t - tests and analysis of variance compared means between groups for continuous variables , and chi - square tests compared proportions between groups for categorical variables . hypotheses 1 and 2 were tested by estimating mixed models to predict three weight measures per person from two key predictors and their interaction : baseline binge eating status / consistency of binge eating status and the time at which weight was measured ( baseline , 12 m , and 24 m ) . main effects for treatment group assignment , depression , age at baseline , and gender were included as covariates , as well as the treatment by time interaction to control for intervention efficacy . interactions between binge eating and depression and between treatment group , time , and binge status were included in preliminary models to ensure that the effects of primary interest were not modified by other modeled covariates . the nonsignificant interaction between binge eating and depression indicated that participants that were more depressed did not show a significantly different relationship between binge eating and weight over the two years compared to participants who were less depressed . study participant was the unit of analysis in these models , with repeated weight observations nested within participants . two random effects , the weight intercept and the time slope , were estimated for each participant with the remaining parameters treated as fixed . the prediction that binge eaters would regain weight at a faster rate over the two - year period would be most strongly supported by a significant interaction between binge status and time . the key difference between the h1 and h2 models was that binge eating behavior had two values ( i.e. , not a binge eater at baseline and binge eater at baseline ) in the h1 model but three ( i.e. , not a binge eater at any time , inconsistent binge eater , and consistent binge eater ) in the h2 model . all statistical analyses were completed using statistical analysis system ( sas ) version 9.3 and the statistical package for the social sciences ( spss ) version 20 [ 32 , 33 ] . the majority of participants were caucasian , married , employed , nonsmoking females with an average age of 46.5 years . the average bmi was 28.5 kg / m , classifying most participants as overweight despite recent weight loss of at least 10% of body weight . prevalence of self - reported binge eating is presented in table 2 for baseline , 12 months , 24 months , and any of the three time points . binge eating behavior was self - reported by about one - fifth of the sample at baseline and 12 months ; the proportion of the sample reporting binge eating behavior was about one out of six at 24 months . as shown in table 2 the majority ( three - quarters or greater ) of participants reported binge eating episodes that would be classified as mild , occurring between 1 and 3 times per week . binge eating behavior was missing for 28 participants at baseline ( 6.7% of the sample ) , 76 participants at 12 months ( 18.1% of the sample ) , and 80 participants at 24 months ( 19.1% of the sample ) . table 3 presents demographic and descriptive statistics for the study sample , stratified by binge eating behavior at baseline and by binge eating behavior consistency ( no binge , inconsistent binge , or consistent binge ) . significant differences observed between those not reporting binge eating behaviors and those reporting binge eating behaviors at baseline included weight , bmi ( calculated using weight ) , baseline depression , and average depression score , all of which were higher in those reporting binge eating behavior . bmi was not significantly different between genders at any time point but was significantly , but weakly positively , associated with both baseline depression and average depression at each time point ( p < 0.05 ; r - values : 0.1090.217 ) . stratifying by binge consistency shows that approximately 69.9% of participants reported no binge eating behavior , 17.7% reported inconsistent binge eating behavior ( reported binge eating at one time point ) , and 12.4% were consistent binge eaters ( reported binge eating at two or three time points ) . similar to the comparisons made by baseline binge status , these categorizations are associated with significant differences in baseline bmi and depression scores . baseline depression score was not significantly different between groups of binge eating consistency in this analysis ; however age was such that those reporting no and consistent binging were significantly older than those reporting inconsistent binging . figure 1 presents descriptive results of weight change over 24 months for ( a ) those who reported presence or absence of binge eating at baseline ( no baseline binge eating corresponded with an average gain of 7.7 pounds ( 3.5 kg ) ; baseline binge eating corresponded with an average regain of 11.7 pounds ( 5.3 kg ) ) and ( b ) those who reported no binge eating , inconsistent binge eating , or consistent binge eating ( average regains of 7.0 pounds ( 3.2 kg ) , 12.2 pounds ( 5.5 kg ) , and 13.4 pounds ( 6.1 kg ) , resp . ) , supporting the hypothesis that binge eating is associated with more weight regain . figure 2 displays the model - estimated weight gain trajectories by ( a ) baseline binge eating behavior and ( b ) binge consistency . in both graphs , those that report binge eating are heavier at baseline , corroborating significant differences reported in table 3 . in figure 2(a ) , individuals who report no binge eating behavior at baseline gain roughly 3.8 pounds ( 1.7 kg ) per year while individuals who report binge eating behavior at baseline gain about 5.7 pounds ( 2.6 kg ) per year ( p value for time < 0.001 ) . the difference in rate of regain was in the predicted direction but did not reach conventional levels of statistical significance ( p < 0.08 , pseudo - r = 0.545 ) . as for the additional parameters that were estimated , the intervention by time interaction was statistically significant ( p = 0.011 ) , which is consistent with the primary outcome paper result that participants in the guided versus self - directed intervention arm regained weight at a slower rate over the 24-month followup . baseline depression and gender were significant main effects ( p = 0.006 and < 0.001 , resp . ) meaning that more depressed people weighed more than less depressed people and women weighed less than men . the yearly rate of regain for participants that did not meet criteria for binge eating at any of the three time points was 3.6 pounds ( 1.6 kg ) on average , for those reporting binge eating at one time point was 6.1 pounds ( 2.8 kg ) on average , and for those reporting binge eating at 2 or 3 time points was 6.5 pounds ( 3.0 kg ) on average . in contrast to the first model that only considered binge eating status at baseline , this model reveals that participants who did not report binge eating , reported inconsistent binge eating , and reported consistent binge eating across multiple time points had significantly different weight regain trajectories over the 2 year study period ( p = 0.013 , pseudo - r = 0.521 ) . the effect appears to be driven by the two binge groups relative to the group reporting no binge eating . weight and bmi trajectories followed similar patterns and significance levels of terms were very close to the reported p values . this is one of the first studies to assess the relationship between binge eating behaviors and weight loss maintenance . participants in this study had lost at least 10% of their body weight in the past year and were randomized to either a self - directed program or a guided intervention designed to help maintain weight loss . results show that binge eating behavior is associated with greater weight regain independent of the effect of the wlm intervention . numerous individuals reported binge eating at baseline and reported no binge eating at future evaluations and vice versa . in response to this observation , data were modeled separately using baseline binge eating behavior and the measure of binge eating consistency to examine how results might differ according to whether only baseline binge eating is considered or a more longitudinal proxy for binge eating behavior is used . concerning our first hypothesis , when considering only baseline binge eating , rate of weight regain was not statistically different ( p = 0.077 ) between groups , though differences in rates were in the hypothesized directions as depicted by figure 2(a ) . when using the newly created variable to address binge eating behavior consistency throughout the study , differences in rate of weight change were more pronounced and were statistically significantly different ( p = 0.013 ) , supporting our second hypothesis . this could be due to increased power to detect an effect or a truly increased rate of weight regain when taking binge behavior at any time point into account . both baseline and average depression scores were significantly related to weight change over 2 years . the effect of the treatment group on weight regain over time was also substantial , as those randomized to the self - directed group regained significantly more weight by the 24-month followup compared to those in the guided maintenance intervention . however , no significant interaction was found between depression and binge eating , or binge eating , treatment group , and time , as binge eating was equally detrimental for those in both treatment groups . this may be a result of the fact that the intervention focused more broadly on weight loss maintenance strategies and did not specifically target binge eating behaviors . first , because the primary goal of the keep it off study was to evaluate the effectiveness of a wlm intervention , data may not have been collected in a manner that would most accurately assess binge eating behavior . three questions were used from the eating disorder diagnostic scale along with guidance from newly defined dsm-5 criteria to determine whether an individual self - reported engaging in binge eating behavior . the edds is subject to limitations inherent in any self - report measure : retrospective recall bias , memory ( especially over a 6-month period ) , and distortion or inaccurate reporting due to desire to please the experimenter or social desirability . however , in one study self - report has shown to have adequate validity compared with the gold standard interview method for assessing binge eating disorder behaviors . another limitation includes inability to assess directionality of binge eating and weight change due to impracticably small numbers of participants grouped according to the direction of change in binge status . trends were in the expected direction , as individuals who reported binging at the end but not at the beginning of a year gained more weight than those reporting binging at the beginning but not at the end of the year . binge cycles using clinical populations . finally , the sample recruited for this study was not representative , as participants were mostly caucasian ( 86.87% ) and female ( 81.62% ) . previous research shows that , unlike other eating disorders , bed is distributed fairly equally among women and men . research also shows that differences exist in the rates and severity of binge eating , as well as in the psychological correlates of binge behaviors by ethnicity . it would be advisable for future research on binge eating to include greater proportions of male subjects and ethnic minorities . results from this study demonstrate primarily that individuals reporting engaging in binge eating behaviors regain weight at a faster rate than those who do not report binge eating behaviors . for future studies , assessing binge eating behavior at multiple time points may be helpful in examining the magnitude and change in binge eating behaviors over time . this may capture those individuals who do not meet the full criteria for bed , but who engage in a level of subclinical binge eating behaviors that could affect health outcomes . based on the findings presented here , binge eating behaviors affect a substantial percentage of individuals that have lost weight and are trying to prevent weight regain . binge eating behaviors were shown , in this study , to significantly affect the amount and rate of weight regained over a two - year period . as mentioned , little attention has been paid to the role of binge eating in weight loss maintenance . work has been done looking at different treatment modalities for individuals experiencing binge eating and suggests that cognitive behavioral therapy ( cbt ) works to decrease symptomology but does not substantially reduce body weight while standard weight loss therapy is more successful in terms of amount of weight loss during the active treatment phase [ 3538 ] . however , as time progresses after treatment , recurrence of binge eating symptoms may contribute to weight regain , which could be one factor contributing to nonsignificant weight differences between treatment groups at followup . additionally , interpersonal therapy ( ipt ) has been studied in treating bed and found to reduce symptoms and performed significantly better than behavioral weight loss . developing interventions that offer more tailored support is essential in moving forward to promote weight loss maintenance in the appreciable portion of individuals who have lost weight and experience binge eating behavior .

objective . to investigate the relationship between binge eating behavior and weight loss maintenance over a two - year period in adults . design . secondary data analysis using the keep it off study , a randomized trial evaluating an intervention to promote weight loss maintenance . participants . 419 men and women ( ages : 20 to 70 y ; bmi : 2044 kg / m2 ) who had intentionally lost 10% of their weight during the previous year . measurements . body weight was measured and binge eating behavior over the past 6 months was reported at baseline , 12 months and 24 months . height was measured at baseline . results . prevalence of binge eating at baseline was 19.4% ( n = 76 ) . prevalence of binge eating at any time point was 30.1% ( n = 126 ) . although rate of weight regain did not differ significantly between those who did or did not report binge eating at baseline , binge eating behavior across the study period ( additive value of presence or absence at each time point ) was significantly associated with different rates of weight regain . conclusion . tailoring weight loss maintenance interventions to address binge eating behavior is warranted given the prevalence and the different rates of weight regain experienced by those reporting this behavior .

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

despite the fact that losing weight and maintaining weight loss can be difficult , research shows that 1720% of overweight or obese individuals are able to lose weight and maintain their weight loss for at least 1 year [ 4 , 5 ] . a sustained reduction of 10% of initial body weight is generally considered a success for clinical and research purposes , as this amount , even for very obese persons , can result in numerous health and economic benefits , such as an increase in life expectancy and a reduction in comorbidities such as diabetes , hypertension , and coronary heart disease , along with associated medical costs [ 5 , 6 ] . thus , obtaining better understanding of factors that influence successful weight loss maintenance ( wlm ) is important and could lead to the development of more effective intervention strategies . the national weight control registry ( nwcr ) has provided important information regarding correlates of successful weight loss maintenance ( defined as having lost at least 13.6 kg ( 30 pounds ) and having kept it off for a minimum of one year ) . data from the nwcr indicates that successful weight loss maintainers have high physical activity levels , eat a diet low in fat and high in carbohydrate , and regularly self - monitor their weight , while higher levels of depression , disinhibition , and binge eating increase the risk of weight regain , suggesting that binge eating may be a behavior important to successful wlm . although considerable research examining the relationship between binge eating and weight loss has been conducted [ 711 ] , there is a paucity of research on the relationship between binge eating and prolonged wlm . of interest , rates of binge eating in nwcr participants are comparable to those observed in community samples , with 8% of members reporting 4 or more binges per month . beyond this observational study , empirical evidence on the relationship between binge eating and most studies have not found binge eating to be a strong predictor of weight loss success [ 711 ] . however , one study found that binge eaters who abstained from binging earlier in the program had significantly greater weight loss than those who continued binge eating and several other studies have found that individuals who engage in binge eating regain weight more rapidly after treatment compared to nonbingers [ 911 ] . it has also been documented that participants who binge eat are more likely to drop out of treatment programs [ 7 , 911 ] , which may mask potential relationships that exist between binge eating and weight loss outcomes . although there is limited support for binge eating as a predictor of weight loss , binge eating behavior may be negatively associated with wlm , based on evidence from the studies cited previously , along with the evidence that successful weight maintainers report low rates of binge eating and individuals who binge eat have been shown to gain more weight over time . the diagnostic and statistical manual of mental disorders 5th edition ( dsm-5 ) now includes binge eating disorder as a psychiatric illness in recognition of the psychological distress and impaired functioning that is associated with this behavior . furthermore , the dsm-5 denotes classifications for severity of binge eating : if 13 binge episodes occur weekly , this is classified as mild binge eating disorder . the prevalence of bed in individuals seeking treatment for obesity has been reported to be as high as 25% , compared to estimates of 25% from community samples . although binge eating behavior has been studied extensively , controversy exists about how to best operationalize the disorder in a research setting . many studies have measured binge eating at a sole time point ( e.g. this is important because it is possible that binge eating behavior fluctuates over time and a single time point may not capture all experiencing symptoms . though hawkins and clement reported temporal stability of binge eating ( test retest = 0.88 ) , their sample consisted of undergraduates , most of whom were of normal weight , and the binge eating scale utilized had only moderate internal consistency ( 0.68 ) . moreover , the period of assessment for stability was five weeks and it is possible that cycles of binge eating exceed five weeks . consequently , it may be important to measure binge eating at multiple time points in order to more accurately assess the relationship between binge eating and weight change , particularly among adults who are trying to maintain a clinically meaningful weight loss . though depression and binge eating are not interchangeable , it has been suggested that there is a relationship between the two . a number of previous studies have found depression to be associated with both binge eating [ 9 , 2022 ] as well as with weight status [ 23 , 24 ] . thus , measuring and attempting to prevent depression score from confounding the relationship between binge eating behavior and weight loss maintenance may be advisable . the current study addresses a gap in the literature by examining the relationship between binge eating behavior and wlm among adults who recently lost at least 10% of their body weight and enrolled in a study to test the effect of an intervention on wlm success . because binge eating may fluctuate over time and this may influence wlm , binge eating behavior was assessed at multiple time points . binge eating behavior was measured at baseline , 12 months , and 24 months , allowing for both cross - sectional and longitudinal approaches to categorizing this behavior . we hypothesize that ( h1 ) individuals who report binge eating behaviors at baseline will have a greater rate of weight regain over the 24-month trial compared to individuals who do not report binge eating at baseline . specifically , we hypothesize that ( h2 ) participants who report binge eating at multiple time points will have a greater rate of weight regain over the 24-month trial relative to participants who report no binge eating or less consistent binge eating ( reporting binge eating at one out of 3 time points ) . because of its potential role in the relationship between binge eating and weight regain , depression was included in analyses . keep it off ( kio ) participants are members of the minnesota - based healthpartners managed - care organization who intentionally lost at least 10% of their body weight during the previous year . briefly , a participant 's progression through the study was as follows : investigators recruited participants who had lost at least 10% of their body weight in the past year , interested individuals were telephone - screened for eligibility ( n = 875 ) , and those who met inclusion criteria were enrolled and randomized to one of two weight loss maintenance groups ( n = 419 ) and then received either the self - directed or guided weight loss maintenance intervention . participants in the self - directed intervention received less education about weight loss maintenance ( two 20-minute phone calls and a self - monitoring logbook to keep track of progress ) compared to the guided intervention ( twenty - four 20-minute phone calls plus followups along with the self - monitoring logbook which they were required to submit at regular intervals to intervention staff ) . inclusion criteria for participation in the study were as follows : 19 to 70 years old , bmi > 20.5 kg / m , and the capacity to communicate with research staff by telephone . weight and height were measured at baseline , 12 months , and 24 months during in - person visits with subjects wearing light clothes and without shoes ( seca 770 medical scale ; seca 214 portable height rod ) . binge eating behavior was self - reported via survey at baseline , 12 months , and 24 months during in - person visits and defined using three items from the eating disorder diagnostic scale ( edds ) : ( 1 ) eating what other people would regard as an unusually large amount of food have there been times when you felt that you have eaten what other people would regard as an unusually large amount of food ( e.g. , a quart of ice cream ) given the circumstances ? ( response options = yes / no ) ; and ( 3 ) the frequency of binge episodes how many times per week on average over the past 6 months have you eaten an unusually large amount of food and experienced a loss of control ? at baseline and at 12 and 24 months , each participant was categorized as a binge eater if they reported eating a large amount of food and feeling a loss of control over eating at least once per week on average over the past 6 months . in cases of missing data , if it could not be verified that the participant self - reported this behavior at least once a week , in accordance with dsm-5 criteria , they were labeled as missing or not experiencing binge eating depending upon the other information available . for example , some participants may have reported eating a large amount and skipped the question about experiencing a loss of control but reported a frequency of an average of > 1 times per week ; these participants were classified as engaging in binge eating because they reported a frequency specified by the dsm-5 , and the final question clearly states episodes where both a large amount and a loss of control were experienced . binge eating behavior was categorized as being present or absent at each time point ( baseline , 12 months , and 24 months ) . for h1 , we were interested in how binge eating behavior at baseline was associated with rate of weight regain over two years . for h2 , we were interested in how the consistency of binge eating behavior at multiple time points ( e.g. , an individual may not have reported binge eating at baseline but may have reported binge eating at 12 months and/or 24 months ) was associated with rate of weight regain over two years . all analyses pertaining to this hypothesis used the aforementioned definition of binge eating behavior and applied it to baseline only . assessment of binge based on assessments at multiple time points may provide different estimates of the occurrence of this behavior relative to a single point in time , such as baseline . for this reason , a variable was created to assess whether participants met criteria for binge eating behavior at any data collection time point throughout the two - year study . this is a dichotomous variable if a participant met the aforementioned criteria for binge eating at baseline or 12 months or 24 months , they would be classified as having binge eating behavior . as shown in table 2 , nearly a third of the sample self - reported binge eating behavior during at least one time point during the two years of the study . to address h2 , a cumulative binge score assessing recurrent binge eating was created ; this score is the sum of self - reported binge eating behavior at each time point with a minimum of 0 indicating no binge eating at any time point and a maximum of 3 indicating a participant self - reporting binge eating at all three time points ( baseline , 12 months , and 24 months ) . a three - level categorical variable was then created for individuals reporting no binge eating at any time points , binge eating behaviors at one time point , or binge eating behaviors at 2 or more time points . based on the new dsm-5 definitions for binge eating severity , severity of binge eating was categorized for each of the time points at which a person was classified as a binge eater . the 11-item short form of the center for epidemiological studies depression ( ces - d ) symptoms scale was used to measure depression via in - person survey at baseline , 12 months , and 24 months . measures of central tendency and dispersion were calculated for demographic variables , depression , binge eating , and body weight for the study sample and then stratified by baseline and by consistency of binge eating behavior . hypotheses 1 and 2 were tested by estimating mixed models to predict three weight measures per person from two key predictors and their interaction : baseline binge eating status / consistency of binge eating status and the time at which weight was measured ( baseline , 12 m , and 24 m ) . main effects for treatment group assignment , depression , age at baseline , and gender were included as covariates , as well as the treatment by time interaction to control for intervention efficacy . interactions between binge eating and depression and between treatment group , time , and binge status were included in preliminary models to ensure that the effects of primary interest were not modified by other modeled covariates . the nonsignificant interaction between binge eating and depression indicated that participants that were more depressed did not show a significantly different relationship between binge eating and weight over the two years compared to participants who were less depressed . the prediction that binge eaters would regain weight at a faster rate over the two - year period would be most strongly supported by a significant interaction between binge status and time . the key difference between the h1 and h2 models was that binge eating behavior had two values ( i.e. , not a binge eater at baseline and binge eater at baseline ) in the h1 model but three ( i.e. the average bmi was 28.5 kg / m , classifying most participants as overweight despite recent weight loss of at least 10% of body weight . prevalence of self - reported binge eating is presented in table 2 for baseline , 12 months , 24 months , and any of the three time points . binge eating behavior was self - reported by about one - fifth of the sample at baseline and 12 months ; the proportion of the sample reporting binge eating behavior was about one out of six at 24 months . binge eating behavior was missing for 28 participants at baseline ( 6.7% of the sample ) , 76 participants at 12 months ( 18.1% of the sample ) , and 80 participants at 24 months ( 19.1% of the sample ) . table 3 presents demographic and descriptive statistics for the study sample , stratified by binge eating behavior at baseline and by binge eating behavior consistency ( no binge , inconsistent binge , or consistent binge ) . significant differences observed between those not reporting binge eating behaviors and those reporting binge eating behaviors at baseline included weight , bmi ( calculated using weight ) , baseline depression , and average depression score , all of which were higher in those reporting binge eating behavior . bmi was not significantly different between genders at any time point but was significantly , but weakly positively , associated with both baseline depression and average depression at each time point ( p < 0.05 ; r - values : 0.1090.217 ) . stratifying by binge consistency shows that approximately 69.9% of participants reported no binge eating behavior , 17.7% reported inconsistent binge eating behavior ( reported binge eating at one time point ) , and 12.4% were consistent binge eaters ( reported binge eating at two or three time points ) . baseline depression score was not significantly different between groups of binge eating consistency in this analysis ; however age was such that those reporting no and consistent binging were significantly older than those reporting inconsistent binging . figure 1 presents descriptive results of weight change over 24 months for ( a ) those who reported presence or absence of binge eating at baseline ( no baseline binge eating corresponded with an average gain of 7.7 pounds ( 3.5 kg ) ; baseline binge eating corresponded with an average regain of 11.7 pounds ( 5.3 kg ) ) and ( b ) those who reported no binge eating , inconsistent binge eating , or consistent binge eating ( average regains of 7.0 pounds ( 3.2 kg ) , 12.2 pounds ( 5.5 kg ) , and 13.4 pounds ( 6.1 kg ) , resp . ) , supporting the hypothesis that binge eating is associated with more weight regain . figure 2 displays the model - estimated weight gain trajectories by ( a ) baseline binge eating behavior and ( b ) binge consistency . in both graphs , those that report binge eating are heavier at baseline , corroborating significant differences reported in table 3 . in figure 2(a ) , individuals who report no binge eating behavior at baseline gain roughly 3.8 pounds ( 1.7 kg ) per year while individuals who report binge eating behavior at baseline gain about 5.7 pounds ( 2.6 kg ) per year ( p value for time < 0.001 ) . the difference in rate of regain was in the predicted direction but did not reach conventional levels of statistical significance ( p < 0.08 , pseudo - r = 0.545 ) . the yearly rate of regain for participants that did not meet criteria for binge eating at any of the three time points was 3.6 pounds ( 1.6 kg ) on average , for those reporting binge eating at one time point was 6.1 pounds ( 2.8 kg ) on average , and for those reporting binge eating at 2 or 3 time points was 6.5 pounds ( 3.0 kg ) on average . in contrast to the first model that only considered binge eating status at baseline , this model reveals that participants who did not report binge eating , reported inconsistent binge eating , and reported consistent binge eating across multiple time points had significantly different weight regain trajectories over the 2 year study period ( p = 0.013 , pseudo - r = 0.521 ) . this is one of the first studies to assess the relationship between binge eating behaviors and weight loss maintenance . participants in this study had lost at least 10% of their body weight in the past year and were randomized to either a self - directed program or a guided intervention designed to help maintain weight loss . results show that binge eating behavior is associated with greater weight regain independent of the effect of the wlm intervention . numerous individuals reported binge eating at baseline and reported no binge eating at future evaluations and vice versa . in response to this observation , data were modeled separately using baseline binge eating behavior and the measure of binge eating consistency to examine how results might differ according to whether only baseline binge eating is considered or a more longitudinal proxy for binge eating behavior is used . concerning our first hypothesis , when considering only baseline binge eating , rate of weight regain was not statistically different ( p = 0.077 ) between groups , though differences in rates were in the hypothesized directions as depicted by figure 2(a ) . when using the newly created variable to address binge eating behavior consistency throughout the study , differences in rate of weight change were more pronounced and were statistically significantly different ( p = 0.013 ) , supporting our second hypothesis . this could be due to increased power to detect an effect or a truly increased rate of weight regain when taking binge behavior at any time point into account . however , no significant interaction was found between depression and binge eating , or binge eating , treatment group , and time , as binge eating was equally detrimental for those in both treatment groups . this may be a result of the fact that the intervention focused more broadly on weight loss maintenance strategies and did not specifically target binge eating behaviors . first , because the primary goal of the keep it off study was to evaluate the effectiveness of a wlm intervention , data may not have been collected in a manner that would most accurately assess binge eating behavior . three questions were used from the eating disorder diagnostic scale along with guidance from newly defined dsm-5 criteria to determine whether an individual self - reported engaging in binge eating behavior . another limitation includes inability to assess directionality of binge eating and weight change due to impracticably small numbers of participants grouped according to the direction of change in binge status . research also shows that differences exist in the rates and severity of binge eating , as well as in the psychological correlates of binge behaviors by ethnicity . results from this study demonstrate primarily that individuals reporting engaging in binge eating behaviors regain weight at a faster rate than those who do not report binge eating behaviors . for future studies , assessing binge eating behavior at multiple time points may be helpful in examining the magnitude and change in binge eating behaviors over time . based on the findings presented here , binge eating behaviors affect a substantial percentage of individuals that have lost weight and are trying to prevent weight regain . binge eating behaviors were shown , in this study , to significantly affect the amount and rate of weight regained over a two - year period . as mentioned , little attention has been paid to the role of binge eating in weight loss maintenance . work has been done looking at different treatment modalities for individuals experiencing binge eating and suggests that cognitive behavioral therapy ( cbt ) works to decrease symptomology but does not substantially reduce body weight while standard weight loss therapy is more successful in terms of amount of weight loss during the active treatment phase [ 3538 ] . however , as time progresses after treatment , recurrence of binge eating symptoms may contribute to weight regain , which could be one factor contributing to nonsignificant weight differences between treatment groups at followup . developing interventions that offer more tailored support is essential in moving forward to promote weight loss maintenance in the appreciable portion of individuals who have lost weight and experience binge eating behavior .

a challenge in medicinal chemistry is the appropriate evaluation of structure activity relations . if the assays fail to fully capture the critical features determining biological activity , then the impact from the synthetic effort is correspondingly diminished . the traditional model of drug action is of a ligand binding to a drug target , with response linked to target occupancy . the profound progress in our understanding of cellular biochemistry now affords detailed , albeit still developing , insights into the complexity of regulation of individual drug targets . incorporating these insights into the evaluation of structural analogues activity relationships ( csars ) , is illustrated here for a particular therapeutic target , protein kinase c ( pkc ) , which displays complex regulation in response to ligands directed at its regulatory c1 domain . we show that a series of ligands for the regulatory domain of pkc are not equivalent but can be distinguished by the isoelectric focusing signatures of pkc that they induce , as detected by a capillary isoelectric focusing immunoassay system . in this system , proteins and their phosphorylated isoforms are separated by charge , followed by target specific antibody probing and chemiluminescence detection . multiple phosphorylation isoforms can be simultaneously separated , detected , and quantified allowing fine dissection of molecular signaling events . pkc plays a central role in cellular signaling , responding to the lipophilic second messenger sn-1,2-diacylglycerol ( dag ) , and is a validated therapeutic target for cancer and a range of other conditions . dag , which is generated as one of the products of phosphoinositide turnover in response to activation of a wide variety of cellular receptors , binds to the c1 domains of pkc . the c1 domains function as hydrophobic switches . they possess a hydrophobic surface interrupted by a hydrophilic cleft . the dag , ultrapotent surrogates such as the phorbol esters , or synthetic ligands such as dag - lactones or benzolactams insert into this hydrophilic cleft , completing the hydrophobic surface as well as providing additional hydrophobic structural elements . driven by this increase in hydrophobicity , the c1 domain ligand complex associates with the membrane , bringing about conformational change in the pkc leading to enzymatic activation and a shift in its subcellular localization . both the pattern of membrane localization and the kinetics of the shift in localization markedly depend on the structure of the ligand . more lipophilic ligands , such as phorbol 12-myristate 13-acetate ( pma ) , initially cause pkc to move to the plasma membrane , after which it slowly shifts in part to internal membranes . more hydrophilic ligands , such as phorbol 12,13-dibutyrate , in contrast , cause the initial localization to the internal membranes . the pattern of localization , moreover , correlates in part with the pattern of biological response . thus , the tumor promoting derivative 12-deoxyphorbol 13-tetradecanoate behaves like pma , whereas the antipromoting derivative 12-deoxyphorbol 13-phenylacetate acts like phorbol 12,13-dibutyrate . marquez and co - workers evaluated , in a range of biological systems , combinatorial libraries of dag - lactones that only varied in their hydrophobic substituents . zip codes to membrane subdomains , at a level of resolution beyond that revealed by imaging of gfp - tagged pkc constructs . a further critical level of regulation for pkc is by phosphorylation ( figure 1 ) , where phosphorylation at serine / threonine sites in the activation loop , the turn motif , and the hydrophobic motif of the kinase domain are required for rendering the enzyme capable of being activated upon binding of ligands to its c1 regulatory domain , as well as controlling its stability within the cell . additional regulation , although not as well understood , is exerted by phosphorylation of tyrosine residues . in the case of pkc , different tyrosine residues are required for the function of pkc for different biological end points . thus , mutation to phenylalanine of tyrosine residues at positions 64 and 187 of pkc blocks its contribution to apoptosis of c6 glioma cells in response to etoposide , whereas mutation of residues at positions 52 , 64 , and 155 enhanced apoptosis of the c6 cells in response to sindbis virus . additionally , phosphorylation at tyrosine 311 has been reported to change its selectivity for the substrate cardiac troponin i. multiple additional phosphorylation sites on pkc have been characterized ( figure 1 ) . ser 299 is of particular interest in that it has been suggested that this site provides a marker of the enzymatically active pkc , as distinct from the enzyme simply being in a state capable of being activated by ligands . for most of the sites of phosphorylation , however , neither is their regulatory impact known nor are reagents available for assessing their phosphorylation . sites of phosphorylation on pkc. sites of phosphorylation on ser / thr and on tyr of human pkc are indicated ( numbering for the mouse / rat isoforms is in italic ) . the pattern of pkc phosphorylation will necessarily reflect an integrated measure of ligand binding , conformational change , and colocalization with kinases such as pdk1 , with tyrosine kinases such as abl or src , or with protein phosphatases such as phlpp . in elegant studies using fret . showed marked differences in the level of phosphatase activity as a function of the particular membrane compartment . furthermore , the pattern of phosphorylation itself influences localization of pkc. in the present study , we direct particular attention to three pkc ligands with different biology . although pkc isoforms are activated by binding of diacylglycerol , phorbol esters , and related ligands to their c1 domains , downstream consequences are not necessarily the same . bryostatin 1 is a complex macrocyclic lactone that binds with high affinity to the same site on the c1 domain as do the phorbol esters , leading to enzymatic activation . paradoxically , however , bryostatin 1 fails to induce many of the responses typical of the phorbol esters and , when added in combination with the phorbol ester , suppresses the phorbol ester response . of particular note , whereas the phorbol esters represent the paradigm for tumor promotion , bryostatin 1 fails to be tumor promoting and indeed suppresses the tumor promotion induced by phorbol ester . given the extensive involvement of pkc in cancer , bryostatin 1 is being extensively evaluated in cancer clinical trials . exciting advances in the chemistry of the bryostatins are now making it possible to explore which structural features confer their unusual pattern of response . the bryostatin derivative merle 23 has been of particular interest . in the u937 human leukemia cell line , the phorbol ester pma induces attachment and inhibits proliferation ; bryostatin 1 shows little effect in either assay and suppresses the action of pma ; the bryostatin derivative merle 23 acts like pma , highlighting the structural features that distinguish merle 23 from bryostatin 1 . in the lncap human prostate cancer cell line , pma induces tumor necrosis factor ( tnf ) secretion and inhibits cell growth ; bryostatin 1 again shows little effect on either end point ; merle 23 , in contrast to its behavior in the u937 cells , now acts more like bryostatin 1 , showing reduced induction of tnf secretion and little inhibition of cell growth . detailed analysis of its actions in the lncap cells , however , shows that merle 23 may be more or less pma - like in this system , depending on the specific response . single measures of ligand interaction with pkc , such as in vitro ligand binding or enzymatic activation , while a clear first level of characterization , necessarily can not capture this degree of complexity found in the intact cell . chromatographic fractionation of stimulated pkc by fplc yields a broad profile , with different functional characteristics in different portions of the profile , but lacks resolution . western blotting of pkc with an ppkcy311 antibody reveals that bryostatin 1 fails to induce phosphorylation at this site in lncap cells , unlike pma , but addresses neither the absolute level of substitution at this site nor the status at the other sites of phosphorylation . here , we explore the potential of high resolution isoelectric focusing with immunoassay detection to provide a signature of the pattern of phosphorylation ( or other charge altering modifications ) of pkc as a function of ligand . charge - based simple western technology is a capillary - based isoelectic focusing ( ief ) immunoassay system , which employs high - resolution ief separation of proteins by charge followed by target - specific immune - probing to simultaneously detect and quantify multiple protein phosphorylation ( or other post - translational modification ) isoforms . the charge - based simple western apparatus and associated software provide a robust platform for sample evaluation and have the potential to detect signals that are not accessible by conventional western blot . analyzing a series of ligands for pkc that differ in the patterns of response that they induce in the lncap human prostate cell line , we show that these ligands likewise induce different isoelectric focusing signatures for pkc. the concept of function - oriented synthesis is that the modern medicinal chemist seeks to capture the relevant functional activity of a lead compound while reducing synthetic complexity . for pkc , the isoelectric focusing signature provides an integrated window into the complex consequences of ligand binding and localization in the intact cell , as reflected in its pattern of regulatory phosphorylations or other modifications . this more detailed picture provides an additional layer of insight into contextual structure activity relationships ( csars ) for pkc ligands . such a signature has value even if , like a gene expression microarray , the individual elements contributing to the signature remain to be dissected . analysis by immunoblotting of pkc and pkc in the lncap cells upon treatment for 30 min with pma , bryostatin 1 , and merle 23 showed only modest differences ( figure 2 ) , consistent with our findings as reported previously . with all three ligands , pkc showed a limited increase in staining for pt638 , a regulatory phosphorylation in the turn motif . pkc displayed a reduction in mobility together with phosphorylation detected with an antibody directed at ps299 , a site associated with pkc activation . as shown both by the similar intensities of the bands detected with the pkc antibody and by the lack of appearance of lower molecular weight forms ( data not shown ) , proteolytic degradation of pkc was not evident under these conditions . in contrast to the similar effects of pma , bryostatin 1 , and merle 23 on phosphorylation of pkc at s299 , the three ligands had differential effects , as revealed by staining with an antibody directed at py311 , as previously reported . strong staining was seen with pma , very little with bryostatin 1 , and intermediate staining with merle 23 . similar levels of erk1/2 phosphorylation were observed with all three ligands , confirming that all three ligands were being used at effective concentrations . because of different affinities of the different antibodies , the absolute levels of modification as detected by the various antibodies could not be compared . lncap cells were treated with pma , bryostatin 1 , or merle 23 ( 1000 nm ) for 30 min . total cell lysates were prepared and evaluated by western blotting with the indicated antibodies . by use of antibody directed against total pkc , a complex pattern of peaks was already evident for the dmso control ( figure 3a ) . in the illustrated experiment , 38.8% of total pkc had a pi of 6.66 ( peak 18 ) , 19.2% and 17.35% had pi s of 6.31 and 6.94 , respectively ( peaks 13 and 19 ) and 10.1% had a pi of 6.61 ( peak 17 ) . as discussed below ( see figure 6 ) , peak 18 is detected with antibodies directed against the phosphorylated activation loop ( t505 ) and turn motifs ( s643 ) , and , based on its abundance as the major pkc peak in the absence of stimulation , it is plausibly the triphosphorylated pkc described as being the mature protein capable of activation ( there was not a suitable antibody for the phosphorylated hydrophobic motif of pkc ) . peak 19 is also phosphorylated at the activation loop and turn motifs . because it has a higher pi ( is less phosphorylated ) than peak 18 , it is possible that it is lacking phosphorylation on the hydrophobic motif . peak 13 , based on its pi , is more highly phosphorylated than is peak 18 and is detected with the antibody for ppkcs299 . it is most like the tetraphosphorylated pkc containing the activation loop , turn , and hydrophobic motif phosphorylations . this is not so clear in figure 6 but is supported by other experiments in which peak 13 is more prominent . in any case , as emphasized in our discussion regarding figure 6 , considerable additional work will be required before the modifications responsible for the various peaks are fully identified . complex pattern of pkc modification measured by the charge - based simple western system . lncap cells were treated with pma , bryostatin 1 , or merle 23 ( 1000 nm ) for 30 min . total cell lysates were prepared as described in experimental section and evaluated by the charge - based simple western system using total pkc antibody ( from santa cruz ) ( a ) or ppkcs299 antibody ( b ) . peaks with different isoelectric focusing points ( pi ) were numbered for easier comparison between runs . upon pma treatment , there was a dramatic reduction in most of these baseline peaks and the emergence of a highly complex pattern at lower pi , consistent with phosphorylation of pkc. although an overall similar pattern was observed upon treatment with bryostatin 1 or merle 23 as was seen with pma , closer examination revealed both the absence of some bands and the emergence of others ( figure 3a ) . for identification , the pattern of response was reproducible between different runs for the different treatments , but minor shifts in the pi values for the different peaks were observed and the region around peaks 810 showed more variability . the above pattern was detected with an antibody that detects total pkc independent of its state of phosphorylation . we similarly compared the patterns using the ppkcs299 antibody , which has been described as being selective for the active enzyme ( figure 3b ) . here , the pattern is somewhat simplified , representing a subset of the bands detected with the antibody to total pkc , but the multiplicity of peaks is still dramatic . comparison between the patterns with pma , bryostatin 1 , and merle 23 reveals appreciable differences , with bryostatin 1 treatment leading to a relative deficiency in the more acidic bands seen with pma , e.g. , peak 3 , and with merle 23 yielding a pattern intermediate between that of bryostatin 1 and pma . differences in the relative abundance of specific peaks within the profile were also evident . thus , peak 6 in the pma treatment profile was markedly diminished upon bryostatin 1 treatment whereas peak 7 was elevated . conceptually important , all of these peaks were being detected with the ppkcs299 antibody , so this single antibody was detecting a multiplicity of different modification states of activated pkc , which presumably are not functionally equivalent but which all are combined into a single band on immunoblotting and interpreted as activated pkc. as discussed in more detail later , we do not know the specific modifications accounting for most elements of the isoelectric focusing profiles in the presence of pma , bryostatin 1 , or merle 23 . all the peaks detected with ppkcs299 would be expected to contain this phosphorylation and most presumably would contain the phosphorylations at the activation loop ( t505 ) , the turn motif ( s643 ) , and the hydrophobic motif ( s662 ) . profiles were dependent not only on the ligand but also on the time of treatment . with time , not only is the absolute level of the pkc signal diminished but shifts in the pattern are also observed ( figure 4 ) . this was most evident with pma and least evident with bryostatin 1 . the pattern of pkc phosphorylation changes with time . lncap cells were treated with pma , bryostatin 1 , or merle 23 ( 1000 nm ) for 30 or 150 min . total cell lysates were prepared and evaluated by the charge - based simple western system using total pkc antibody . asterisks denote that peaks 9 and 10 show variability compared to peaks labeled 9 and 10 in figure 3 . elsewhere , we have described differences in the extents to which different phorbol esters and related derivatives inhibit proliferation in lncap cells or induce tumor necrosis factor secretion , an important mediator of the inhibitory response . similarly , tested at concentrations at or above those giving maximal response for these end points , these ligands had different capacities to induce phosphorylation of pkc at s299 or y311 ( figure 5a ) . we therefore examined this series of compounds for their effects on the pkc signature in the lncap cells , as detected with the ppkcs299 antibody ( figure 5b ) and total pkc antibody ( figure 5c ) . levels of individual peaks for the various ligands were expressed as percent of the total pma signal observed in the case of the ppkcs299 antibody ( to better compare the level of signal activation by different compounds ) or as percent of the total signal of that sample in the case of total pkc antibody and presented as a heat map ( figure 5b and figure 5c ) . the average values sem of all points on the heat map are presented in supporting information ( supplementary data 1a and 1b ) . a partial correlation with lipophilicity was observed as well as marked differences among ligands . lncap cells were treated with different pkc activators ( 10 000 nm for phorbol 13-acetate and prostratin , 1000 nm for others ) for 150 min . total cell lysates were prepared and evaluated by western blotting ( a ) , by the charge - based simple western system using ppkcs299 antibody ( b ) , or by the charge - based simple western system using total pkc antibody ( c ) . ( a ) immunoblot analysis of total cell lysates using the indicated antibodies . a representative image of three independent experiments is shown . for analysis using the charge - based simple western system the areas under peaks were calculated and expressed as % of the total peaks for pma in the same set of runs ( b ) or % of total peaks in the sample ( c ) . the heat map represents the average % values for each detected peak ( n = 3 except for phorbol 13-acetate , phorbol 12,13-diacetate , phorbol 12,13-dibenzoate , and phorbol 13-decanoate where n = 2 ) . although deconvolution of the various pkc modifications responsible for the multiplicity of peaks will be a highly laborious undertaking beyond the scope of the present analysis , it is possible to begin to approach this question using those phospho - specific antibodies that are available ( and sensitive enough to detect the responses ) . this is illustrated for the dmso control sample , detected with another antibody directed against total pkc ( antibody from bd biosciences ) , as well as those directed against ppkct505 , ppkcs643 , and ppkcs299 ( figure 6 ) . the peak profiles detected using the two different total pkc antibodies are very similar ( figure 3a , figure 6 , supporting information figure 2 ) . peaks 18 and 19 detected with the total pkc antibody also show phosphorylation on sites t505 and s643 ( 81.35% of total pkc is phosphorylated on t505 and s643 ) , whereas about 12.53% of total pkc is phosphorylated at s299 under these basal conditions in this experiment ( peaks 12 , 13 , and 17 ) ( figure 6 ) . the slide also illustrates that the level of basal pkc phosphorylation at s299 showed some variation with different batches of cells ( compare figure 3a and figure 6 ) . importantly , the figure illustrates that the relative proportions of the peaks with the antibodies directed against total pkc permit an estimate of the relative proportions of the modified peaks , whereas their relative contributions could not be inferred from the absolute signal strengths with the various antibodies . unfortunately , the sensitivities of the antibodies directed against t505 and s643 were not adequate for analysis once the pkc profile was spread over the multiple bands upon phorbol ester treatment . detection of changes in pkc phosphorylation by the charge - based simple western system using different pkc antibodies . the total cell lysate of dmso treated lncap cells used in figure 3 was analyzed by the charge - based simple western system using the indicated antibodies . single experiments were performed with total pkc , ppkct505 , and ppkcs643 . to evaluate the role of pkc activity in the subsequent phosphorylation changes , we examined the effect of the general pkc inhibitor g6983 ( 3000 nm ) on the phosphorylation response . under these conditions , this concentration of g6983 largely but not entirely blocked the pma stimulated pkc activity , as revealed by immunoblotting ( figure 7a ) , with a reduction in erk1/2 phosphorylation and ppkcs299 staining . by charge - based simple western analysis , treatment with g6983 alone had no effect on the pattern of peaks ( figure 7b ) . in contrast , a marked reduction in the overall magnitude of the profile from the pma stimulated cells was observed upon detection with the ppkcs299 antibody with the residual bands predominantly at higher pi . lncap cells were treated with pma ( 1000 nm ) with or without pretreatment with pkc inhibitor ( g6983 , 3000 nm for 30 min ) . the total cell lysates were examined by immunoblotting ( a ) and by the charge - based simple western system using ppkcs299 antibody ( b ) . data presented are representative of four ( a ) or two ( b ) independent experiments . asterisks in ( b ) denote that peaks 9 and 10 show variability compared to the peaks labeled 9 and 10 in figure 3 we have described earlier that treatment with pma or bryostatin 1 causes different distribution between a nuclear enriched fraction and the cytoplasm . we confirm here that pma treatment causes enhanced phosphorylation on y311 of pkc and this species is predominantly found in the nuclear enriched fraction ( figure 8a ) . likewise , the ps299 signal upon pma treatment is predominantly in the nuclear enriched fraction whereas that upon bryostatin 1 treatment is predominantly in the cytoplasmic fraction ( figure 8a ) . analysis by the charge - based simple western system emphasizes the difference in the profile of pkc between these two fractions , with the nuclear enriched fraction enriched in the lower pi forms upon pma treatment , which is not observed with bryo 1 treatment ( figure 8b ) . the ppkcy311 antibody did not yield a sufficiently strong signal for analysis of the multiple bands generated upon phorbol ester treatment . although we do not know if phosphorylation at s299 and at y311 are found in some of the same peaks , it seems highly likely that they would be at least partially coincident because y311 phosphorylation is associated with pkc that is active and s299 phosphorylation presumably reflects the active conformation of the pkc exposing the hinge region , where s299 is located . lncap cells were treated for 60 min with pma , bryostatin 1 ( 1000 nm ) , or dmso as control . pkc phosphorylation in the total cell lysates , the cytoplasmic fraction , and the nuclear fraction was detected by immunoblotting ( a ) and by the charge - based simple western system using the ppkcs299 antibody ( b ) . asterisks in ( b ) denote that peaks 9 and 10 show variability compared to the peaks labeled 9 and 10 in figure 3 in marked contrast to the complex pattern of modification of pkc upon ligand addition , analysis of pkc by the charge - based simple western system showed only minor changes . treatment with pma , bryostatin 1 , or merle 23 caused similar , quantitatively minor changes with the appearance of three bands at lower pi ( figure 9 ) . the same simple pattern was detected with the pkc antibody from epitomics ( data not shown ) . simple phosphorylation pattern of pkc measured by the charge - based simple western system . the total cell lysates used in figure 3 were evaluated by the charge - based simple western system using pkc antibody ( santa cruz ) . our findings have impact at multiple levels . we find that different c1 domain - targeted pkc ligands have qualitatively different effects on pkc phosphorylation ( or other modifications affecting the isoelectric point ) . the isoelectric focusing signature can thus be used as a guide to synthesis , reporting how congeners may retain or diverge in their pattern of action , as reflected through this signature . the charge - based simple western resolution of pkc isoelectric states revealed both the great complexity and extensiveness of pkc modification . finally , our analysis of pkc provided an initial glimpse into the methodological issues and exciting potential of the approach . it is essential to emphasize that the isoelectric focusing patterns obtained yield a signature for pkc modification . individual peaks within the pattern may represent several combinations of phosphorylation events , provided that each combination results in the same isoelectric point for the protein . indeed , although the individual peaks are interpreted for convenience as representing different phosphorylation states of pkc , the results could also incorporate any changes in isoelectric point arising through other mechanisms . although degradation can not be excluded as a contributor to such complexity , we failed to see significant degradation at these early times of treatment upon size fractionation on sds polyacrylamide gels , either as a decrease in the signal at the size of the intact protein or as the appearance of lower molecular weight bands under our detection conditions . in any case , while we have developed some insights through the use of antibodies of different specificities , full deconvolution of the identities of the combinations of underlying sites and types of modifications constitutes a formidable future challenge . just as patterns of gene expression provide a signature of the systems biology of a cell or tissue , with different signatures being associated with different underlying perturbations of cellular control , so the pattern of modification of protein kinase c provides a signature for the integrated consequences of activation , of autophosphorylation , of heterophosphorylation by both serine / threonine specific kinases and tyrosine kinases , and of dephosphorylation by phosphatases . these in turn will have been influenced not only by pkc activation but also by changes in its subcellular localization and interaction with anchoring proteins as well as the kinetics of these changes . speculatively , a further potential element may be intermediate conformational states of pkc . in the case of pkc ii , recent elegant crystallographic studies indicate that one of the twin c1 domains , the c1b domain , is involved in intramolecular contacts with the kinase domain . activity relations for the two c1 domains , and occupancy of just one c1 domain may yield a different conformation for pkc than does occupancy of both . the lncap cells show very complicated behavior in response to treatment with phorbol esters or related derivatives . in response to the typical phorbol ester phorbol 12-myristate 13-acetate ( pma ) this response reflects both stimulation of secretion of tumor necrosis factor and modulation of downstream signaling . like pma , bryostatin 1 binds to the c1 domains of pkc and leads to pkc activation . paradoxically , however , bryostatin 1 fails to induce many of the responses typically induced by the phorbol esters , and for those responses that it fails to induce , it inhibits response to the phorbol ester upon cotreatment . correspondingly , in the lncap cells bryostatin 1 fails to inhibit cell growth and induces only minimal tumor necrosis factor secretion . pkc appears to be the primary pkc isoform determining the outcome in this system . this is consistent with our finding that the expression level of pkc is at least 20 times higher than other pkc isoforms in lncap cells . bryostatin 1 is of great interest as a therapeutic agent for cancer and dementia , as reflected in multiple clinical trials . because of the formidable obstacles to supply of this complex natural product , there has been intense interest in the development of synthetic congeners , retaining the same pattern of biological activity as bryostatin 1 while eliminating those structural features that complicate synthesis but are unnecessary for activity . using the lncap cell system as a potential cellular model for evaluating such congeners , we found that the simplified synthetic bryostatin 1 derivative merle 23 appeared to act like bryostatin 1 in this system , failing to inhibit proliferation and stimulating only a low level of tumor necrosis factor secretion . further examination of the behavior of merle 23 in the lncap cells , however , revealed appreciable differences between it and bryostatin 1 . for example , addition of a proteosome inhibitor shifted the behavior of merle 23 to that of pma , whereas the response to bryostatin 1 remained largely the same . in further studies , we showed that a series of other phorbol esters and indolactams likewise did not all behave like pma but showed variable extents of lesser response , in partial similarity to bryostatin 1 . analysis in the lncap cells of compounds drawn from this series thus seemed well suited for detecting potential differences in pkc phosphorylation in response to different ligands . consistent with the different patterns of biological response , we observed that different ligands indeed caused different signatures of modification of pkc ( as summarized in figure 5b ) . consistent with the response to merle 23 not fully resembling that to bryostatin 1 , we observed an intermediate pattern of modification . it was also clear that , considering all of the ligands , the patterns could not be simply ordered in parallel with their extents of induction of tumor necrosis factor or inhibition of proliferation . for example , indolactam v was among the most effective for growth inhibition of the lncap cells whereas sapintoxin d was among the least effective . conversely , phorbol 13-decanoate and phorbol 12-myristate 13-acetate showed similar maximal levels of growth inhibition . we suggest that examination of pkc isoelectric focusing signatures for mimicry of the pattern observed for bryostatin 1 may afford a stringent test for synthetic bryostatin derivatives that capture its unique pattern of biological response . one striking aspect of the difference between the effects of pma and bryostatin 1 was the presence upon pma treatment of the more acidic pkc bands , as detected with the ppkcs299 antibody , in the nuclear extract fraction . while this nuclear extract fraction was not characterized in detail and thus could also include some other cellular elements , it showed dramatic contrast between the effects of pma and bryostatin 1 . we have speculated that this difference between pma and bryostatin 1 may be an important contributor to the differential action of these two agents . although our goal was to compare signatures of phosphorylation ( plus other modifications of isoelectric point ) rather than to identify the particular sets of phosphorylated residues represented by each peak , we were able to develop some insights by comparison of the patterns detected with antibodies directed against total pkc and against pkc phosphorylated at s299 , t505 , and ser643 . ps299 has been described as a marker of pkc activation in response to ligand binding . pt505 represents phosphorylation of the activation loop , leading to pkc being converted to a more active state , and ps643 represents phosphorylation of the turn motif . we could detect and quantify a basal level of s299 phorphorylation in the absence of external activation . while not characterized in detail , this basal s299 phosphorylated pkc displayed a lower pi consistent with additional phosphorylations , presumably those at the activation loop , the turn motif , and the hydrophobic motif . although y311 phosphorylation would have been of particular interest to evaluate , the quality of py311 antibodies was not sufficient for analysis with the charge - based simple western system . although the focus of our analyses was pkc , since this residue has been centrally implicated in the unique patterns of response to bryostatin 1 both in lncap cells and in other systems , we conducted an initial examination of the response of pkc , one of the other functionally important pkc isoforms in the lncap cells . in comparison with pkc , a similar situation was observed for pkd1 , a kinase that is positionally regulated through its intrinsic c1 domains as well as activated by phosphorylation by pkc isoforms . the complexity of pkc phosphorylation is yet another example of its exceptional behavior among pkc isoforms , along with its differential localization by different ligands and the function of its c2 domain as a novel phosphotyrosine binding motif . one potential basis for its complexity of phosphorylation could be through complex formation of pkc with other kinases . through its c2 domain it can bind to phosphotyrosine residues on other kinases ; through its own phosphotyrosine groups it could interact with kinases possessing sh2 domains ; through either mechanism it further could interact with adapter proteins bringing it into proximity with other kinases . the particular value of the isoelectric focusing signatures enabled by the charge - based simple western system for analysis of contextual structure activity relationships probably lies in targets such as pkc , which reflect such complexity . the current analysis of pkc response to ligands using the charge - based simple western system represents an early stage in the evaluation of the utility of this approach . signatures such as observed in the treated cellular systems could be extended to define signatures in dissected tumor samples , reporting on signaling pathway regulation in the tissue sample . modifications , such as the use of a validated detection tag , which could be coupled to any target of interest , would facilitate its generalized application to a wide range of targets , without needing to identify and validate specific antibodies for each target . an efficient system for subsequent identification of the specific sites of phosphorylation represented would move the analysis to the next step from response signature toward dissected phosphorylation pathway . nonetheless , the methodology has already proven to be highly informative for our understanding of pkc pharmacology and regulation . all phorbol esters were purchased from lc laboratories ( woburn , ma ) unless otherwise specified ( purity for all > 99% ) . sapintoxin d ( purity > 98% by hplc ) was from enzo life sciences international inc . bryostatin 1 was provided by the developmental therapeutics program , nci ( frederick , md ) . bryostatin 1 was judged to be greater than or equal to 95% purity , as determined by h and c nmr , as well as hplc analysis . merle 23 was isolated as single observable tlc spot in 40% etoac / hexanes , appeared as a single peak by reverse phase hplc using a waters 4.5 m 150 m c18 column and was judged to be > 95% pure by 500 mhz h nmr and 125 mhz c nmr . the lncap human prostate cancer cell line , fetal bovine serum ( fbs ) , and rpm1 - 1640 medium were obtained from atcc ( manassas , va ) . precast 10% sds gels and pbs were from invitrogen ( carlsbad , ca ) . the primary antibody against pkc ( sc-208 ) was from santa cruz biotechnology ( santa cruz , ca ) and that against pkc was from santa cruz biotechnology ( sc-937 ) or bd biosciences ( san jose , ca ) . the primary antibodies against phosphorylated pkc ( ppkcy311 , ppkct505 , ppkcs643 ) and phosphorylated pkc ( ppkct638 ) , perk1/2 ( no . those against pkc ( no . 1510 - 1 ) , pkc ( no . 2222 - 1 ) , and phosphorylated pkc ( ppkcs299 and another antibody against ppkcy311 ) were from epitomics ( burlingame , ca ) . the mouse monoclonal antibody against -actin was from sigma ( st . louis , mo ) . the horseradish peroxidase conjugated secondary anti - rabbit antibodies , the nonfat dry milk , tween-20 , and the triton x-100 solution were from bio - rad ( hercules , ca ) . the ecl ( electrochemiluminescence ) reagent and the films were from ge healthcare ( piscataway , nj ) . the following reagents were from proteinsimple ( santa clara , ca ) : 1 fluorescent pi standard 6.4 , 7.0 and ladder 4 ( no . 040 - 031 , and no . 040 - 647 , respectively ) , 1 g2 premix 5 - 8 ampholyte ( no . 040 - 973 ) , luminol ( no . the secondary goat anti - rabbit antibody ( no . 111 - 035 - 144 ) and donkey anti - mouse antibody ( no . 715 - 035 - 150 ) used for charge - based simple western analysis were from jackson immunoresearch ( west grove , pa ) . cell culture , treatment of cells , preparation of total cells lysates and nuclear extracts , and the western blotting were performed as described earlier . except for cell fractionation samples , cells were lysed with ripa buffer ( 20 mm hepes , ph 7.5 , 150 mm nacl , 1% np 40 alternative , 0.25% sodium deoxycholate ) containing phosphatase and protease inhibitors ( emd millipore catalog no . cell lysates ( approximately 20 ng of protein ) were mixed with 1 g2 premix 5 - 8 ampholyte , and 1 fluorescent pi standard 6.4 , 7.0 , and ladder 4 before being loaded into the nanopro1000 system ( proteinsimple , santa clara , ca ) for analysis . during isoelectric focusing electrophoresis , proteins were separated by charge and concentrated at their respective isoelectric focusing points in the capillaries . separated proteins were immobilized on the capillary wall using uv light , followed by immunoprobing with the indicated primary antibodies and hrp - conjugated goat anti - rabbit ( 1:100 diluted ) or donkey anti - mouse ( 1:100 diluted ) secondary antibody . the primary antibodies used in the study were the following : ppkcs299 ( 1:50 diluted ) , pkc ( 1:100 diluted , santa - cruz sc-937 ) , pkc ( 1:50 diluted , santa - cruz sc-208 ) , pkc ( 1:100 diluted , bd biosciences no . luminol and peroxide were added to generate chemiluminescence , which was captured by a ccd camera . the digital image was analyzed by compass software ( proteinsimple , santa clara , ca ) . the software calculates the height and area under the curve ( auc ) of the separated / individual peaks . for quantitative analysis of pkc signals the auc value of peaks was expressed as % of total auc ( sum of auc of all peaks for that sample ) . for ppkcs299 signals the auc of individual peaks was expressed as % of total auc of the pma treated sample from the same set of runs to reflect the differences in pkc activation induced by the different treatments .

protein kinase c ( pkc ) , a validated therapeutic target for cancer chemotherapy , provides a paradigm for assessing structure activity relations , where ligand binding has multiple consequences for a target . for pkc , ligand binding controls not only pkc activation and multiple phosphorylations but also subcellular localization , affecting subsequent signaling . using a capillary isoelectric focusing immunoassay system , we could visualize a high resolution isoelectric focusing signature of pkc upon stimulation by ligands of the phorbol ester and bryostatin classes . derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures . consistent with different patterns of pkc localization as one factor linked to these different signatures , we found different signatures for activated pkc from the nuclear and non - nuclear fractions . we conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development .

Introduction Results Discussion Experimental Section

a challenge in medicinal chemistry is the appropriate evaluation of structure activity relations . the traditional model of drug action is of a ligand binding to a drug target , with response linked to target occupancy . incorporating these insights into the evaluation of structural analogues activity relationships ( csars ) , is illustrated here for a particular therapeutic target , protein kinase c ( pkc ) , which displays complex regulation in response to ligands directed at its regulatory c1 domain . we show that a series of ligands for the regulatory domain of pkc are not equivalent but can be distinguished by the isoelectric focusing signatures of pkc that they induce , as detected by a capillary isoelectric focusing immunoassay system . pkc plays a central role in cellular signaling , responding to the lipophilic second messenger sn-1,2-diacylglycerol ( dag ) , and is a validated therapeutic target for cancer and a range of other conditions . dag , which is generated as one of the products of phosphoinositide turnover in response to activation of a wide variety of cellular receptors , binds to the c1 domains of pkc . driven by this increase in hydrophobicity , the c1 domain ligand complex associates with the membrane , bringing about conformational change in the pkc leading to enzymatic activation and a shift in its subcellular localization . the pattern of localization , moreover , correlates in part with the pattern of biological response . a further critical level of regulation for pkc is by phosphorylation ( figure 1 ) , where phosphorylation at serine / threonine sites in the activation loop , the turn motif , and the hydrophobic motif of the kinase domain are required for rendering the enzyme capable of being activated upon binding of ligands to its c1 regulatory domain , as well as controlling its stability within the cell . in the case of pkc , different tyrosine residues are required for the function of pkc for different biological end points . thus , mutation to phenylalanine of tyrosine residues at positions 64 and 187 of pkc blocks its contribution to apoptosis of c6 glioma cells in response to etoposide , whereas mutation of residues at positions 52 , 64 , and 155 enhanced apoptosis of the c6 cells in response to sindbis virus . ser 299 is of particular interest in that it has been suggested that this site provides a marker of the enzymatically active pkc , as distinct from the enzyme simply being in a state capable of being activated by ligands . for most of the sites of phosphorylation , however , neither is their regulatory impact known nor are reagents available for assessing their phosphorylation . the pattern of pkc phosphorylation will necessarily reflect an integrated measure of ligand binding , conformational change , and colocalization with kinases such as pdk1 , with tyrosine kinases such as abl or src , or with protein phosphatases such as phlpp . in the present study , we direct particular attention to three pkc ligands with different biology . paradoxically , however , bryostatin 1 fails to induce many of the responses typical of the phorbol esters and , when added in combination with the phorbol ester , suppresses the phorbol ester response . of particular note , whereas the phorbol esters represent the paradigm for tumor promotion , bryostatin 1 fails to be tumor promoting and indeed suppresses the tumor promotion induced by phorbol ester . in the u937 human leukemia cell line , the phorbol ester pma induces attachment and inhibits proliferation ; bryostatin 1 shows little effect in either assay and suppresses the action of pma ; the bryostatin derivative merle 23 acts like pma , highlighting the structural features that distinguish merle 23 from bryostatin 1 . single measures of ligand interaction with pkc , such as in vitro ligand binding or enzymatic activation , while a clear first level of characterization , necessarily can not capture this degree of complexity found in the intact cell . chromatographic fractionation of stimulated pkc by fplc yields a broad profile , with different functional characteristics in different portions of the profile , but lacks resolution . western blotting of pkc with an ppkcy311 antibody reveals that bryostatin 1 fails to induce phosphorylation at this site in lncap cells , unlike pma , but addresses neither the absolute level of substitution at this site nor the status at the other sites of phosphorylation . here , we explore the potential of high resolution isoelectric focusing with immunoassay detection to provide a signature of the pattern of phosphorylation ( or other charge altering modifications ) of pkc as a function of ligand . charge - based simple western technology is a capillary - based isoelectic focusing ( ief ) immunoassay system , which employs high - resolution ief separation of proteins by charge followed by target - specific immune - probing to simultaneously detect and quantify multiple protein phosphorylation ( or other post - translational modification ) isoforms . the charge - based simple western apparatus and associated software provide a robust platform for sample evaluation and have the potential to detect signals that are not accessible by conventional western blot . analyzing a series of ligands for pkc that differ in the patterns of response that they induce in the lncap human prostate cell line , we show that these ligands likewise induce different isoelectric focusing signatures for pkc. for pkc , the isoelectric focusing signature provides an integrated window into the complex consequences of ligand binding and localization in the intact cell , as reflected in its pattern of regulatory phosphorylations or other modifications . this more detailed picture provides an additional layer of insight into contextual structure activity relationships ( csars ) for pkc ligands . analysis by immunoblotting of pkc and pkc in the lncap cells upon treatment for 30 min with pma , bryostatin 1 , and merle 23 showed only modest differences ( figure 2 ) , consistent with our findings as reported previously . pkc displayed a reduction in mobility together with phosphorylation detected with an antibody directed at ps299 , a site associated with pkc activation . as shown both by the similar intensities of the bands detected with the pkc antibody and by the lack of appearance of lower molecular weight forms ( data not shown ) , proteolytic degradation of pkc was not evident under these conditions . by use of antibody directed against total pkc , a complex pattern of peaks was already evident for the dmso control ( figure 3a ) . as discussed below ( see figure 6 ) , peak 18 is detected with antibodies directed against the phosphorylated activation loop ( t505 ) and turn motifs ( s643 ) , and , based on its abundance as the major pkc peak in the absence of stimulation , it is plausibly the triphosphorylated pkc described as being the mature protein capable of activation ( there was not a suitable antibody for the phosphorylated hydrophobic motif of pkc ) . peaks with different isoelectric focusing points ( pi ) were numbered for easier comparison between runs . upon pma treatment , there was a dramatic reduction in most of these baseline peaks and the emergence of a highly complex pattern at lower pi , consistent with phosphorylation of pkc. here , the pattern is somewhat simplified , representing a subset of the bands detected with the antibody to total pkc , but the multiplicity of peaks is still dramatic . conceptually important , all of these peaks were being detected with the ppkcs299 antibody , so this single antibody was detecting a multiplicity of different modification states of activated pkc , which presumably are not functionally equivalent but which all are combined into a single band on immunoblotting and interpreted as activated pkc. as discussed in more detail later , we do not know the specific modifications accounting for most elements of the isoelectric focusing profiles in the presence of pma , bryostatin 1 , or merle 23 . profiles were dependent not only on the ligand but also on the time of treatment . with time , not only is the absolute level of the pkc signal diminished but shifts in the pattern are also observed ( figure 4 ) . elsewhere , we have described differences in the extents to which different phorbol esters and related derivatives inhibit proliferation in lncap cells or induce tumor necrosis factor secretion , an important mediator of the inhibitory response . the slide also illustrates that the level of basal pkc phosphorylation at s299 showed some variation with different batches of cells ( compare figure 3a and figure 6 ) . importantly , the figure illustrates that the relative proportions of the peaks with the antibodies directed against total pkc permit an estimate of the relative proportions of the modified peaks , whereas their relative contributions could not be inferred from the absolute signal strengths with the various antibodies . unfortunately , the sensitivities of the antibodies directed against t505 and s643 were not adequate for analysis once the pkc profile was spread over the multiple bands upon phorbol ester treatment . to evaluate the role of pkc activity in the subsequent phosphorylation changes , we examined the effect of the general pkc inhibitor g6983 ( 3000 nm ) on the phosphorylation response . in contrast , a marked reduction in the overall magnitude of the profile from the pma stimulated cells was observed upon detection with the ppkcs299 antibody with the residual bands predominantly at higher pi . we confirm here that pma treatment causes enhanced phosphorylation on y311 of pkc and this species is predominantly found in the nuclear enriched fraction ( figure 8a ) . analysis by the charge - based simple western system emphasizes the difference in the profile of pkc between these two fractions , with the nuclear enriched fraction enriched in the lower pi forms upon pma treatment , which is not observed with bryo 1 treatment ( figure 8b ) . the ppkcy311 antibody did not yield a sufficiently strong signal for analysis of the multiple bands generated upon phorbol ester treatment . although we do not know if phosphorylation at s299 and at y311 are found in some of the same peaks , it seems highly likely that they would be at least partially coincident because y311 phosphorylation is associated with pkc that is active and s299 phosphorylation presumably reflects the active conformation of the pkc exposing the hinge region , where s299 is located . pkc phosphorylation in the total cell lysates , the cytoplasmic fraction , and the nuclear fraction was detected by immunoblotting ( a ) and by the charge - based simple western system using the ppkcs299 antibody ( b ) . asterisks in ( b ) denote that peaks 9 and 10 show variability compared to the peaks labeled 9 and 10 in figure 3 in marked contrast to the complex pattern of modification of pkc upon ligand addition , analysis of pkc by the charge - based simple western system showed only minor changes . the isoelectric focusing signature can thus be used as a guide to synthesis , reporting how congeners may retain or diverge in their pattern of action , as reflected through this signature . finally , our analysis of pkc provided an initial glimpse into the methodological issues and exciting potential of the approach . it is essential to emphasize that the isoelectric focusing patterns obtained yield a signature for pkc modification . indeed , although the individual peaks are interpreted for convenience as representing different phosphorylation states of pkc , the results could also incorporate any changes in isoelectric point arising through other mechanisms . although degradation can not be excluded as a contributor to such complexity , we failed to see significant degradation at these early times of treatment upon size fractionation on sds polyacrylamide gels , either as a decrease in the signal at the size of the intact protein or as the appearance of lower molecular weight bands under our detection conditions . just as patterns of gene expression provide a signature of the systems biology of a cell or tissue , with different signatures being associated with different underlying perturbations of cellular control , so the pattern of modification of protein kinase c provides a signature for the integrated consequences of activation , of autophosphorylation , of heterophosphorylation by both serine / threonine specific kinases and tyrosine kinases , and of dephosphorylation by phosphatases . these in turn will have been influenced not only by pkc activation but also by changes in its subcellular localization and interaction with anchoring proteins as well as the kinetics of these changes . speculatively , a further potential element may be intermediate conformational states of pkc . in the case of pkc ii , recent elegant crystallographic studies indicate that one of the twin c1 domains , the c1b domain , is involved in intramolecular contacts with the kinase domain . activity relations for the two c1 domains , and occupancy of just one c1 domain may yield a different conformation for pkc than does occupancy of both . like pma , bryostatin 1 binds to the c1 domains of pkc and leads to pkc activation . paradoxically , however , bryostatin 1 fails to induce many of the responses typically induced by the phorbol esters , and for those responses that it fails to induce , it inhibits response to the phorbol ester upon cotreatment . this is consistent with our finding that the expression level of pkc is at least 20 times higher than other pkc isoforms in lncap cells . because of the formidable obstacles to supply of this complex natural product , there has been intense interest in the development of synthetic congeners , retaining the same pattern of biological activity as bryostatin 1 while eliminating those structural features that complicate synthesis but are unnecessary for activity . using the lncap cell system as a potential cellular model for evaluating such congeners , we found that the simplified synthetic bryostatin 1 derivative merle 23 appeared to act like bryostatin 1 in this system , failing to inhibit proliferation and stimulating only a low level of tumor necrosis factor secretion . further examination of the behavior of merle 23 in the lncap cells , however , revealed appreciable differences between it and bryostatin 1 . consistent with the different patterns of biological response , we observed that different ligands indeed caused different signatures of modification of pkc ( as summarized in figure 5b ) . consistent with the response to merle 23 not fully resembling that to bryostatin 1 , we observed an intermediate pattern of modification . we suggest that examination of pkc isoelectric focusing signatures for mimicry of the pattern observed for bryostatin 1 may afford a stringent test for synthetic bryostatin derivatives that capture its unique pattern of biological response . one striking aspect of the difference between the effects of pma and bryostatin 1 was the presence upon pma treatment of the more acidic pkc bands , as detected with the ppkcs299 antibody , in the nuclear extract fraction . while this nuclear extract fraction was not characterized in detail and thus could also include some other cellular elements , it showed dramatic contrast between the effects of pma and bryostatin 1 . although our goal was to compare signatures of phosphorylation ( plus other modifications of isoelectric point ) rather than to identify the particular sets of phosphorylated residues represented by each peak , we were able to develop some insights by comparison of the patterns detected with antibodies directed against total pkc and against pkc phosphorylated at s299 , t505 , and ser643 . ps299 has been described as a marker of pkc activation in response to ligand binding . although the focus of our analyses was pkc , since this residue has been centrally implicated in the unique patterns of response to bryostatin 1 both in lncap cells and in other systems , we conducted an initial examination of the response of pkc , one of the other functionally important pkc isoforms in the lncap cells . in comparison with pkc , a similar situation was observed for pkd1 , a kinase that is positionally regulated through its intrinsic c1 domains as well as activated by phosphorylation by pkc isoforms . the particular value of the isoelectric focusing signatures enabled by the charge - based simple western system for analysis of contextual structure activity relationships probably lies in targets such as pkc , which reflect such complexity . the current analysis of pkc response to ligands using the charge - based simple western system represents an early stage in the evaluation of the utility of this approach . merle 23 was isolated as single observable tlc spot in 40% etoac / hexanes , appeared as a single peak by reverse phase hplc using a waters 4.5 m 150 m c18 column and was judged to be > 95% pure by 500 mhz h nmr and 125 mhz c nmr . 2222 - 1 ) , and phosphorylated pkc ( ppkcs299 and another antibody against ppkcy311 ) were from epitomics ( burlingame , ca ) . for ppkcs299 signals the auc of individual peaks was expressed as % of total auc of the pma treated sample from the same set of runs to reflect the differences in pkc activation induced by the different treatments .

cementum is a mineralized tissue with primary function to insert the ligament fibers on the root surface and releasing oclusal forces to the surrounding alveolar bone . there is considerable interest in observing the changes that occur on the cementum surface inside periodontal pockets as a result of periodontal disease . periodontitis - affected root surfaces are hypermineralized and contaminated with endotoxins and other biologically active substances . it has been suggested that endotoxin present in the cement could impair periodontal healing and should be removed to promote a more biologically acceptable surface than the one obtained only after scaling and root planning . the most important event in the reattachment of the connective tissue is related to the adhesion of blood elements to the collagen present at the root surface , which retards the apical migration of the sulcular epithelium into the pocket . ephitelial downgrowth is exacerbated by the strong adhesion of bacterial products and endotoxins , especially originated from gram negative bacteria . these bacterial compounds have affinity to mineral structures such as the tooth , promoting a constant aggression on the periodontal tissue , causing loss of tissue support until the loss of the tooth . endotoxins as bacterial cell wall lipopolysaccharides remain active even after bacterial death , not being removed by scaling and root planning . these bacteria product are found bound to the smear layer produced by the action of curettes , ultrasonic and rotary instruments . in an attempt to remove smear layer and demineralize the contaminated root surface , different approaches has been studied as the use of chemical agents and laser . special attention has been focused on the use of chemical agents as a mean to obtain adequate preparation of the root to development of new connective attachment . certain acids , especially citric acid has been used to clean the root surface by demineralization and , recently , some authors showed that demineralization of exposed dentin can significantly increase the reattachment of connective tissue to the root surface . previous studies on tissue regeneration have used tetracycline hydrochloride hcl to clean the root surface because its bactericidal and demineralizing effect . the parameters for root conditioning with tetracycline hcl were established by ishi , et al . however , the search for a less acid substance to avoid tissue necrosis and probably provide better conditions for clot adhesion to the root surface , showed promising results employing ethylenediaminetetraacetic acid ( edta ) the use of edta gel as a root surface conditioning agent negatively affected the outcome of root coverage . edta might have inhibited blood element adsorption and adhesion to the dentin surface because of a possible incomplete removal of the gel from the root surface . in addition , edta is a calcium chelator ; therefore , its residues may have inhibited or retarded coagulation events . citric acid is a substance such capable of removing smear layer and opening dentinal tubules . however , citric acid 's low ph may induce cytotoxic effects when in contact with connective tissue . in an attempt to find a substance that could efficiently remove smear layer and expose collagen fibers , leite , et al . however , this substance has not been tested before and the application parameters are not established yet . in order to establish the application parameters for sodium citrate and ascertain that this substance is capable of smear layer removal , we suggested conducting this study . in addition , as citric acid presented better results than edta for clot stabilization and as citric acid combined with platelet - derived growth factor - bb showed better results than edta and tetracycline hydrochloride on attachment of periodontal ligament cells on root surfaces , we proposed to test if a lower concentration of citric acid applied by a shorter time is capable of removing smear layer and exposing collagen fiber . the aim of this study was to establish concentrations , times and modes of ideal applications of citric acid and sodium citrate in removing smear layer and exposure of collagen fibers . a total of 124 periodontally involved human teeth were obtained at the oral and maxillofacial surgery and periodontics clinics at the araraquara school of dentistry , unesp - univ . the samples were prepared according to previous published data and briefly described as follows . for sample preparation the buccal and lingual root surfaces of each tooth were used . two parallel grooves with approximately 0.8 mm deep were made using a high speed cylindrical bur ( # 3099 - 1.6 mm ) ( kg sorensen , medical burs , cotia , sp , brazil ) under copious irrigation . one groove was made at the cementoenamel junction and another one approximately 3 mm distant from the first , in the apical direction . the same bur was used to remove the surface layer of the root between the two grooves . in order to create a smear layer , 50 apical to cervical strokes were performed using a sharp # 5 - 6 gracey curette ( hu - friedy , hu - friedy , chicago , il , usa ) . a total of 495 samples measuring about 2x3 mm were obtained cutting teeth with a flexible double faced diamond disc ( # 7020 - 0.22 mm - thickness : 0.15 mm)(kg sorensen , medical burs , cotia , sp , brazil ) at low speed . the citric acid was applied in five concentrations 0.5 , 1 , 2 , 15 and 25% ( five groups n=45 samples each ) . the sodium citrate was also divided into five groups with the following concentrations 3 , 10 , 20 , 30 and 40% ( n=45 each ) . five different concentrations were tested in order to determine if lower concentrations , and consequently less aggressive substances , could be effective in removing smear layer and exposing collagen fibers . in the same way , different modes and times of application were evaluated in an attempt to reduce the contact of the substances with the periodontal cells . the groups were divided into subgroups according to the mode and time of application solutions . three modes of application were used ( n=15 each ) : 1 ) simple positioning of a small cotton pellet embedded in solution ( passive application ) ; 2 ) brushing application with a soft brush ( disposable brush tips 2 , 3 m espe , seefeld , germany ) ; and 3 ) burnishing application ( friction ) with a small cotton pellet . each of these three subgroups was further divided into the three application periods of 1 , 2 , or 3 min ( n=5 each ) . samples were dehydrated in an increasingly graded series of ethanol : 30 , 50 , 70 , 80 , 95 and 100% . then , the samples were dried overnight in a dehydration jar ( corning , corning life sciences , so paulo , sp , brazil ) , mounted on metallic stubs ( senai , so paulo , sp , brazil ) and sputter - coated with a thin 25 nm layer of 99.99% pure gold ( balt - tec scd-050 , balt - tec , gnathole farm , kettleshulme , high peak , cheshire , uk ) . two micrographs were obtained from the center area of each sample with 1,500x and 3,500x magnifications , using a scanning electron microscope operated at an accelerating voltage of 20 kv ( jeol t330 a ; jeol ltd . , peabody , ma , usa ) . the micrographs were evaluated according to a root surface modification index adapted for this study . a previously calibrated ( kappa score=0.93 ) and experienced examiner evaluated three times each image , with an interval of 15 days between each evaluation . the score attributed to each sample was the most prevalent score in the three evaluations . the adapted index used for this study consisted of eight scores as shown in figure 1 . root modification index . e ) score 5 . smear layer covering the surface , formed by chemical dissolution of dentinal surface . f ) score 6 . a uniform smear layer covering dentin surface with some signs of tubule openings . dentin surface covered by uniform a smear layer , with no signs of dentinal tubule opening . rough smear layer covering dentin surface the non - parametric analysis of variance ( kruskal - wallis test ) was applied to independently evaluate the effect of the three dependent variables : solution concentration , mode of application and period . the level of significance adopted was 5% . if p0.05 , dunn 's multiple comparison post hoc test was applied to detect statistically significant differences among groups . statistical analysis was made with a computer software ( graphpad prism 5.00 ; graphpad software inc . the samples were prepared according to previous published data and briefly described as follows . for sample preparation two parallel grooves with approximately 0.8 mm deep were made using a high speed cylindrical bur ( # 3099 - 1.6 mm ) ( kg sorensen , medical burs , cotia , sp , brazil ) under copious irrigation . one groove was made at the cementoenamel junction and another one approximately 3 mm distant from the first , in the apical direction . the same bur was used to remove the surface layer of the root between the two grooves . in order to create a smear layer , 50 apical to cervical strokes were performed using a sharp # 5 - 6 gracey curette ( hu - friedy , hu - friedy , chicago , il , usa ) . a total of 495 samples measuring about 2x3 mm were obtained cutting teeth with a flexible double faced diamond disc ( # 7020 - 0.22 mm - thickness : 0.15 mm)(kg sorensen , medical burs , cotia , sp , brazil ) at low speed . the citric acid was applied in five concentrations 0.5 , 1 , 2 , 15 and 25% ( five groups n=45 samples each ) . the sodium citrate was also divided into five groups with the following concentrations 3 , 10 , 20 , 30 and 40% ( n=45 each ) . five different concentrations were tested in order to determine if lower concentrations , and consequently less aggressive substances , could be effective in removing smear layer and exposing collagen fibers . in the same way , different modes and times of application were evaluated in an attempt to reduce the contact of the substances with the periodontal cells . the groups were divided into subgroups according to the mode and time of application solutions . three modes of application were used ( n=15 each ) : 1 ) simple positioning of a small cotton pellet embedded in solution ( passive application ) ; 2 ) brushing application with a soft brush ( disposable brush tips 2 , 3 m espe , seefeld , germany ) ; and 3 ) burnishing application ( friction ) with a small cotton pellet . each of these three subgroups was further divided into the three application periods of 1 , 2 , or 3 min ( n=5 each ) . samples were dehydrated in an increasingly graded series of ethanol : 30 , 50 , 70 , 80 , 95 and 100% . then , the samples were dried overnight in a dehydration jar ( corning , corning life sciences , so paulo , sp , brazil ) , mounted on metallic stubs ( senai , so paulo , sp , brazil ) and sputter - coated with a thin 25 nm layer of 99.99% pure gold ( balt - tec scd-050 , balt - tec , gnathole farm , kettleshulme , high peak , cheshire , uk ) . two micrographs were obtained from the center area of each sample with 1,500x and 3,500x magnifications , using a scanning electron microscope operated at an accelerating voltage of 20 kv ( jeol t330 a ; jeol ltd . , peabody , ma , usa ) . the micrographs were evaluated according to a root surface modification index adapted for this study . a previously calibrated ( kappa score=0.93 ) and experienced examiner evaluated three times each image , with an interval of 15 days between each evaluation . the score attributed to each sample was the most prevalent score in the three evaluations . the adapted index used for this study consisted of eight scores as shown in figure 1 . root modification index . e ) score 5 . smear layer covering the surface , formed by chemical dissolution of dentinal surface . f ) score 6 . a uniform smear layer covering dentin surface with some signs of tubule openings . dentin surface covered by uniform a smear layer , with no signs of dentinal tubule opening . the non - parametric analysis of variance ( kruskal - wallis test ) was applied to independently evaluate the effect of the three dependent variables : solution concentration , mode of application and period . the level of significance adopted was 5% . if p0.05 , dunn 's multiple comparison post hoc test was applied to detect statistically significant differences among groups . statistical analysis was made with a computer software ( graphpad prism 5.00 ; graphpad software inc . citric acid was evaluated at concentrations of 0.5 , 1 , 2 , 15 and 25% . all concentrations were used for 1 , 2 and 3 min under the passive , brushing or burnishing forms . statistically significant differences were found between control group and the other concentrations used ( table 1 ) . even without statistical significance , the concentration of 25% and 15% showed less variation in score values ( figure 2 ) , the same way that the concentration of 25% presented more samples with score 1 ( table 1 ) . score 1 represents complete smear layer removal with dentin collagen fibers exposure and complete opened dentin tubules , without smear layer on root surface . sample distribution for the citric acid concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . p=0.05 * within the same category , scores with the same letter are not statistically different . sd= standard deviation median with range of the scores obtained for the five concentrations of citric acid and the control group . concentrations with the same symbol are not statistically different the comparison of the different periods of application of citric acid , showed no differences among the groups ( figure 3 ) . furthermore , table 1 shows a slight tendency of the 3-min application to be more effective in exposing collagen fibers ( score 1 ) . median with range of the scores obtained for the three application periods of the citric acid . application periods with the same symbol are not statistically different the same way , the evaluation of the different modes of application showed no significant difference between groups ( figure 4 ) . however , application by brushing seems to have favored the production of samples with more exposure of collagen fibers as seen in table 1 . median with range of the scores obtained for the three application methods of the citric acid . application methods with the same symbol are not statistically different the effect of sodium citrate in smear layer removal was evaluated in five different concentrations and in a control group . among the concentrations evaluated , the 3% showed less effective results than the others and significant statistical difference was observed . the concentrations of 10 , 20 , 30 and 40% showed no statistically significant differences among them ( figure 5 ) . table 2 shows the median values and standard deviation ( sd ) for the concentrations , periods of applications and modes of application . median with range of the scores obtained for the five concentrations of sodium citrate and the control group . concentrations with the same symbol are not statistically different sample distribution for the sodium citrate concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . p=0.05 * within the same category , scores with the same letter are not statistically different . sd= standard deviation the analysis of the application periods showed significant differences among the groups . even though , the results for 2 and 3 min were statistically the same , the 3-min application seems to have been slightly better than others ( figure 6 ) . median with range of the scores obtained for the three application periods of the sodium citrate . application periods with the same symbol are not statistically different regarding the mode of application , more favorable results were obtained with application by brushing or by burnishing than the passive application ( figure 7 ) . a statistically significant difference was observed among the modes of application and a slight tendency of best results can be seen with the application by burnishing ( table 2 ) . median with range of the scores obtained for the three application methods of the sodium citrate . citric acid was evaluated at concentrations of 0.5 , 1 , 2 , 15 and 25% . all concentrations were used for 1 , 2 and 3 min under the passive , brushing or burnishing forms . statistically significant differences were found between control group and the other concentrations used ( table 1 ) . even without statistical significance , the concentration of 25% and 15% showed less variation in score values ( figure 2 ) , the same way that the concentration of 25% presented more samples with score 1 ( table 1 ) . score 1 represents complete smear layer removal with dentin collagen fibers exposure and complete opened dentin tubules , without smear layer on root surface . sample distribution for the citric acid concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . p=0.05 * within the same category , scores with the same letter are not statistically different . sd= standard deviation median with range of the scores obtained for the five concentrations of citric acid and the control group . concentrations with the same symbol are not statistically different the comparison of the different periods of application of citric acid , showed no differences among the groups ( figure 3 ) . furthermore , table 1 shows a slight tendency of the 3-min application to be more effective in exposing collagen fibers ( score 1 ) . median with range of the scores obtained for the three application periods of the citric acid . application periods with the same symbol are not statistically different the same way , the evaluation of the different modes of application showed no significant difference between groups ( figure 4 ) . however , application by brushing seems to have favored the production of samples with more exposure of collagen fibers as seen in table 1 . median with range of the scores obtained for the three application methods of the citric acid . the effect of sodium citrate in smear layer removal was evaluated in five different concentrations and in a control group . among the concentrations evaluated , the 3% showed less effective results than the others and significant statistical difference was observed . the concentrations of 10 , 20 , 30 and 40% showed no statistically significant differences among them ( figure 5 ) . table 2 shows the median values and standard deviation ( sd ) for the concentrations , periods of applications and modes of application . median with range of the scores obtained for the five concentrations of sodium citrate and the control group . concentrations with the same symbol are not statistically different sample distribution for the sodium citrate concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . p=0.05 * within the same category , scores with the same letter are not statistically different . sd= standard deviation the analysis of the application periods showed significant differences among the groups . even though , the results for 2 and 3 min were statistically the same , the 3-min application seems to have been slightly better than others ( figure 6 ) . median with range of the scores obtained for the three application periods of the sodium citrate . application periods with the same symbol are not statistically different regarding the mode of application , more favorable results were obtained with application by brushing or by burnishing than the passive application ( figure 7 ) . a statistically significant difference was observed among the modes of application and a slight tendency of best results can be seen with the application by burnishing ( table 2 ) . median with range of the scores obtained for the three application methods of the sodium citrate . with the methodology used in this study it is possible to evaluate the morphological aspect of the conditioned root surface . the micrographs taken at 1,500 and 3,500x has excellent definition and quality for observation of the root surface . the sem micrographs should represent the evaluated sample , but it is not possible to take a micrograph of the entire sample . to solve this difficulty , the visualization was directed to the center of the sample and all the micrographs were taken from the center of the sample . larger samples take longer to be sputter - coated and are difficult to be obtained . these dimensions were obtained after sample reduction from the coronal root third and comprise approximately the measure between the cementoenamel junction and the furcation entrance in molars . despite the limitations presented , the methodology in this study has been extensively used before and is accepted as a method to evaluate root conditioning and blood cell adhesion with in vitro studies . the use of different concentrations , application modes and times was efficient in determine and standardize the parameters of application of a chemical substances and allows the use of the obtained parameters in other studies such as studies to evaluate blood cell adhesion and periodontal ligament cell attachment to the root surface . the rationale for this study is that the evident contamination of the cementum and dentin with bacterial toxins could impair periodontal healing . these products may be responsible for a marked progressive and irreversible destruction of the periodontal structures of support . scaling and root planning can be accomplished by manual instruments , rotary instruments , sonic and ultrasonic instruments . such methods often do not appear to be totally effective in removing mineralized debris of the root surface . furthermore , root instrumentation causes grooves and results in a root surface covered with smear layer , which contains remnants of dental calculus , contaminated cementum , bacterial endotoxin and subgingival plaque . over the past decade a number of alternatives have been presented as compensation for the limitations inherent to the mechanical therapy . in vitro and in vivo studies have emphasized the conditioning of the root surface using different methods as an adjuvant treatment to scaling and root planning in regenerative procedures . several chemical agents have been proposed including citric acid , edta , tetracycline hydrochloride among others . an important factor that should be considered and is often neglected is the mode of application , time , composition and concentration of the conditioning agent in addition to the ph of the substance used . root surfaces exposed to biofilm are hypermineralized and therefore more difficult to be both mechanically and chemically decontaminated , and thus , low ph substances were proposed to aid smear layer removal . with respect to ph , it was possible to demonstrate that citric acid which possesses a low ph , causes cell death when in contact with periodontal ligament ( pdl ) cells and thus could delay cell proliferation and repair of the area . because of this necrotizing effect , the use of 24% edta at neutral ph was proposed as an alternative for low ph substances . this way , studies were carried out to compare different formulations , concentrations , modes and length / duration of the application . most studies have shown that edta is a substance that provides smear layer removal and exposure of collagen fibers . however , researches on clot adherence to root surface bioengineered with edta obtained worse results when compared to citric acid and tetracycline hydrochloride . this may be because edta is an anticoagulant or even because it removes the calcium ions from the surface of root dentin that are important in blood clotting cascade . considering these results , a new substance was proposed in the present study - sodium citrate - which has the same principle of edta , but is more biocompatible and is also currently used in blood collection bags . it was also evaluated the citric acid different concentrations in order to ascertain the optimal parameters for its use . actually , the problem of this substance is its low ph and its high power to demineralization . this way , in the present study lower concentrations with consequently higher ph were evaluated . this high demineralization power can be observed after analysis of the sem micrographs , in which we found that almost all groups of citric acid concentrations of 15 , 20 , 25 and 30% can cause chemical dissolution of tooth surface ( score 5 ) with collagen destruction . these results disagrees with the majority of studies published , not being compatible with the principle of root conditioning , which requires the exposure of dentin collagen fibers for adhesion of the clot on the tooth surface . thus , these results suggest that the concentration should be decreased so that the smear layer could be effectively removed but without destroying the collagen fibers network . this way , the citric acid concentration was reduced to 0.5 , 1.0 and 2.0% to try to solve the problem , but no difference was observed . citric acid even at lower concentrations produced excessive demineralization not having a standard of performance ( table 1 ) . despite being more biocompatible , sodium citrate was not able to expose collagen fibers of the root surface ( table 1 ) even in the group with best results , which was vigorous application for 3 min ( graphs 5 and 6 ) . within the limitations of the methodology , it can be concluded that , despite the lack of statistical significance , the best results for collagen exposure using citric acid were obtained with brushing application at 25% for 3 min . sodium citrate was not able to adequately remove smear layer an expose collagen fibers , so it is not indicated for root conditioning .

objectivethe aim of this study was to establish the parameters of concentration , time and mode of application of citric acid and sodium citrate in relation to root conditioning . material and methodsa total of 495 samples were obtained and equally distributed among 11 groups ( 5 for testing different concentrations of citric acid , 5 for testing different concentrations of sodium citrate and 1 control group ) . after laboratorial processing , the samples were analyzed under scanning electron microscopy . a previously calibrated and blind examiner evaluated micrographs of the samples . non - parametric statistical analysis was performed to analyze the data obtained . resultsbrushing 25% citric acid for 3 min , promoted greater exposure of collagen fibers in comparison with the brushing of 1% citric acid for 1 minute and its topical application at 1% for 3 min . sodium citrate exposed collagen fibers in a few number of samples . conclusiondespite the lack of statistical significance , better results for collagen exposure were obtained with brushing application of 25% citric acid for 3 min than with other application parameter . sodium citrate produced a few number of samples with collagen exposure , so it is not indicated for root conditioning .

INTRODUCTION MATERIAL AND METHODS Sample preparation SEM evaluation Statistical analysis RESULTS Citric acid Sodium citrate DISCUSSION CONCLUSION

the most important event in the reattachment of the connective tissue is related to the adhesion of blood elements to the collagen present at the root surface , which retards the apical migration of the sulcular epithelium into the pocket . special attention has been focused on the use of chemical agents as a mean to obtain adequate preparation of the root to development of new connective attachment . the parameters for root conditioning with tetracycline hcl were established by ishi , et al . however , the search for a less acid substance to avoid tissue necrosis and probably provide better conditions for clot adhesion to the root surface , showed promising results employing ethylenediaminetetraacetic acid ( edta ) the use of edta gel as a root surface conditioning agent negatively affected the outcome of root coverage . edta might have inhibited blood element adsorption and adhesion to the dentin surface because of a possible incomplete removal of the gel from the root surface . however , citric acid 's low ph may induce cytotoxic effects when in contact with connective tissue . in an attempt to find a substance that could efficiently remove smear layer and expose collagen fibers , leite , et al . in order to establish the application parameters for sodium citrate and ascertain that this substance is capable of smear layer removal , we suggested conducting this study . in addition , as citric acid presented better results than edta for clot stabilization and as citric acid combined with platelet - derived growth factor - bb showed better results than edta and tetracycline hydrochloride on attachment of periodontal ligament cells on root surfaces , we proposed to test if a lower concentration of citric acid applied by a shorter time is capable of removing smear layer and exposing collagen fiber . the aim of this study was to establish concentrations , times and modes of ideal applications of citric acid and sodium citrate in removing smear layer and exposure of collagen fibers . a total of 124 periodontally involved human teeth were obtained at the oral and maxillofacial surgery and periodontics clinics at the araraquara school of dentistry , unesp - univ . the samples were prepared according to previous published data and briefly described as follows . a total of 495 samples measuring about 2x3 mm were obtained cutting teeth with a flexible double faced diamond disc ( # 7020 - 0.22 mm - thickness : 0.15 mm)(kg sorensen , medical burs , cotia , sp , brazil ) at low speed . the citric acid was applied in five concentrations 0.5 , 1 , 2 , 15 and 25% ( five groups n=45 samples each ) . the sodium citrate was also divided into five groups with the following concentrations 3 , 10 , 20 , 30 and 40% ( n=45 each ) . five different concentrations were tested in order to determine if lower concentrations , and consequently less aggressive substances , could be effective in removing smear layer and exposing collagen fibers . in the same way , different modes and times of application were evaluated in an attempt to reduce the contact of the substances with the periodontal cells . the groups were divided into subgroups according to the mode and time of application solutions . three modes of application were used ( n=15 each ) : 1 ) simple positioning of a small cotton pellet embedded in solution ( passive application ) ; 2 ) brushing application with a soft brush ( disposable brush tips 2 , 3 m espe , seefeld , germany ) ; and 3 ) burnishing application ( friction ) with a small cotton pellet . each of these three subgroups was further divided into the three application periods of 1 , 2 , or 3 min ( n=5 each ) . samples were dehydrated in an increasingly graded series of ethanol : 30 , 50 , 70 , 80 , 95 and 100% . then , the samples were dried overnight in a dehydration jar ( corning , corning life sciences , so paulo , sp , brazil ) , mounted on metallic stubs ( senai , so paulo , sp , brazil ) and sputter - coated with a thin 25 nm layer of 99.99% pure gold ( balt - tec scd-050 , balt - tec , gnathole farm , kettleshulme , high peak , cheshire , uk ) . two micrographs were obtained from the center area of each sample with 1,500x and 3,500x magnifications , using a scanning electron microscope operated at an accelerating voltage of 20 kv ( jeol t330 a ; jeol ltd . a previously calibrated ( kappa score=0.93 ) and experienced examiner evaluated three times each image , with an interval of 15 days between each evaluation . the adapted index used for this study consisted of eight scores as shown in figure 1 . rough smear layer covering dentin surface the non - parametric analysis of variance ( kruskal - wallis test ) was applied to independently evaluate the effect of the three dependent variables : solution concentration , mode of application and period . statistical analysis was made with a computer software ( graphpad prism 5.00 ; graphpad software inc . the samples were prepared according to previous published data and briefly described as follows . the same bur was used to remove the surface layer of the root between the two grooves . a total of 495 samples measuring about 2x3 mm were obtained cutting teeth with a flexible double faced diamond disc ( # 7020 - 0.22 mm - thickness : 0.15 mm)(kg sorensen , medical burs , cotia , sp , brazil ) at low speed . the citric acid was applied in five concentrations 0.5 , 1 , 2 , 15 and 25% ( five groups n=45 samples each ) . the sodium citrate was also divided into five groups with the following concentrations 3 , 10 , 20 , 30 and 40% ( n=45 each ) . five different concentrations were tested in order to determine if lower concentrations , and consequently less aggressive substances , could be effective in removing smear layer and exposing collagen fibers . in the same way , different modes and times of application were evaluated in an attempt to reduce the contact of the substances with the periodontal cells . three modes of application were used ( n=15 each ) : 1 ) simple positioning of a small cotton pellet embedded in solution ( passive application ) ; 2 ) brushing application with a soft brush ( disposable brush tips 2 , 3 m espe , seefeld , germany ) ; and 3 ) burnishing application ( friction ) with a small cotton pellet . then , the samples were dried overnight in a dehydration jar ( corning , corning life sciences , so paulo , sp , brazil ) , mounted on metallic stubs ( senai , so paulo , sp , brazil ) and sputter - coated with a thin 25 nm layer of 99.99% pure gold ( balt - tec scd-050 , balt - tec , gnathole farm , kettleshulme , high peak , cheshire , uk ) . two micrographs were obtained from the center area of each sample with 1,500x and 3,500x magnifications , using a scanning electron microscope operated at an accelerating voltage of 20 kv ( jeol t330 a ; jeol ltd . a previously calibrated ( kappa score=0.93 ) and experienced examiner evaluated three times each image , with an interval of 15 days between each evaluation . the adapted index used for this study consisted of eight scores as shown in figure 1 . the non - parametric analysis of variance ( kruskal - wallis test ) was applied to independently evaluate the effect of the three dependent variables : solution concentration , mode of application and period . statistical analysis was made with a computer software ( graphpad prism 5.00 ; graphpad software inc . citric acid was evaluated at concentrations of 0.5 , 1 , 2 , 15 and 25% . all concentrations were used for 1 , 2 and 3 min under the passive , brushing or burnishing forms . statistically significant differences were found between control group and the other concentrations used ( table 1 ) . even without statistical significance , the concentration of 25% and 15% showed less variation in score values ( figure 2 ) , the same way that the concentration of 25% presented more samples with score 1 ( table 1 ) . sample distribution for the citric acid concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . sd= standard deviation median with range of the scores obtained for the five concentrations of citric acid and the control group . concentrations with the same symbol are not statistically different the comparison of the different periods of application of citric acid , showed no differences among the groups ( figure 3 ) . furthermore , table 1 shows a slight tendency of the 3-min application to be more effective in exposing collagen fibers ( score 1 ) . median with range of the scores obtained for the three application periods of the citric acid . application periods with the same symbol are not statistically different the same way , the evaluation of the different modes of application showed no significant difference between groups ( figure 4 ) . however , application by brushing seems to have favored the production of samples with more exposure of collagen fibers as seen in table 1 . median with range of the scores obtained for the three application methods of the citric acid . application methods with the same symbol are not statistically different the effect of sodium citrate in smear layer removal was evaluated in five different concentrations and in a control group . among the concentrations evaluated , the 3% showed less effective results than the others and significant statistical difference was observed . table 2 shows the median values and standard deviation ( sd ) for the concentrations , periods of applications and modes of application . median with range of the scores obtained for the five concentrations of sodium citrate and the control group . concentrations with the same symbol are not statistically different sample distribution for the sodium citrate concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . sd= standard deviation the analysis of the application periods showed significant differences among the groups . even though , the results for 2 and 3 min were statistically the same , the 3-min application seems to have been slightly better than others ( figure 6 ) . median with range of the scores obtained for the three application periods of the sodium citrate . application periods with the same symbol are not statistically different regarding the mode of application , more favorable results were obtained with application by brushing or by burnishing than the passive application ( figure 7 ) . a statistically significant difference was observed among the modes of application and a slight tendency of best results can be seen with the application by burnishing ( table 2 ) . median with range of the scores obtained for the three application methods of the sodium citrate . citric acid was evaluated at concentrations of 0.5 , 1 , 2 , 15 and 25% . all concentrations were used for 1 , 2 and 3 min under the passive , brushing or burnishing forms . even without statistical significance , the concentration of 25% and 15% showed less variation in score values ( figure 2 ) , the same way that the concentration of 25% presented more samples with score 1 ( table 1 ) . sample distribution for the citric acid concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . p=0.05 * within the same category , scores with the same letter are not statistically different . sd= standard deviation median with range of the scores obtained for the five concentrations of citric acid and the control group . concentrations with the same symbol are not statistically different the comparison of the different periods of application of citric acid , showed no differences among the groups ( figure 3 ) . furthermore , table 1 shows a slight tendency of the 3-min application to be more effective in exposing collagen fibers ( score 1 ) . median with range of the scores obtained for the three application periods of the citric acid . application periods with the same symbol are not statistically different the same way , the evaluation of the different modes of application showed no significant difference between groups ( figure 4 ) . however , application by brushing seems to have favored the production of samples with more exposure of collagen fibers as seen in table 1 . median with range of the scores obtained for the three application methods of the citric acid . the effect of sodium citrate in smear layer removal was evaluated in five different concentrations and in a control group . table 2 shows the median values and standard deviation ( sd ) for the concentrations , periods of applications and modes of application . median with range of the scores obtained for the five concentrations of sodium citrate and the control group . concentrations with the same symbol are not statistically different sample distribution for the sodium citrate concentrations , periods and modes of application kruskal - wallis test and dunn 's multiple comparison post hoc test . sd= standard deviation the analysis of the application periods showed significant differences among the groups . even though , the results for 2 and 3 min were statistically the same , the 3-min application seems to have been slightly better than others ( figure 6 ) . median with range of the scores obtained for the three application periods of the sodium citrate . application periods with the same symbol are not statistically different regarding the mode of application , more favorable results were obtained with application by brushing or by burnishing than the passive application ( figure 7 ) . a statistically significant difference was observed among the modes of application and a slight tendency of best results can be seen with the application by burnishing ( table 2 ) . median with range of the scores obtained for the three application methods of the sodium citrate . with the methodology used in this study it is possible to evaluate the morphological aspect of the conditioned root surface . the micrographs taken at 1,500 and 3,500x has excellent definition and quality for observation of the root surface . the sem micrographs should represent the evaluated sample , but it is not possible to take a micrograph of the entire sample . to solve this difficulty , the visualization was directed to the center of the sample and all the micrographs were taken from the center of the sample . these dimensions were obtained after sample reduction from the coronal root third and comprise approximately the measure between the cementoenamel junction and the furcation entrance in molars . despite the limitations presented , the methodology in this study has been extensively used before and is accepted as a method to evaluate root conditioning and blood cell adhesion with in vitro studies . the use of different concentrations , application modes and times was efficient in determine and standardize the parameters of application of a chemical substances and allows the use of the obtained parameters in other studies such as studies to evaluate blood cell adhesion and periodontal ligament cell attachment to the root surface . the rationale for this study is that the evident contamination of the cementum and dentin with bacterial toxins could impair periodontal healing . these products may be responsible for a marked progressive and irreversible destruction of the periodontal structures of support . over the past decade a number of alternatives have been presented as compensation for the limitations inherent to the mechanical therapy . several chemical agents have been proposed including citric acid , edta , tetracycline hydrochloride among others . an important factor that should be considered and is often neglected is the mode of application , time , composition and concentration of the conditioning agent in addition to the ph of the substance used . with respect to ph , it was possible to demonstrate that citric acid which possesses a low ph , causes cell death when in contact with periodontal ligament ( pdl ) cells and thus could delay cell proliferation and repair of the area . because of this necrotizing effect , the use of 24% edta at neutral ph was proposed as an alternative for low ph substances . this way , studies were carried out to compare different formulations , concentrations , modes and length / duration of the application . most studies have shown that edta is a substance that provides smear layer removal and exposure of collagen fibers . however , researches on clot adherence to root surface bioengineered with edta obtained worse results when compared to citric acid and tetracycline hydrochloride . considering these results , a new substance was proposed in the present study - sodium citrate - which has the same principle of edta , but is more biocompatible and is also currently used in blood collection bags . it was also evaluated the citric acid different concentrations in order to ascertain the optimal parameters for its use . actually , the problem of this substance is its low ph and its high power to demineralization . this high demineralization power can be observed after analysis of the sem micrographs , in which we found that almost all groups of citric acid concentrations of 15 , 20 , 25 and 30% can cause chemical dissolution of tooth surface ( score 5 ) with collagen destruction . these results disagrees with the majority of studies published , not being compatible with the principle of root conditioning , which requires the exposure of dentin collagen fibers for adhesion of the clot on the tooth surface . thus , these results suggest that the concentration should be decreased so that the smear layer could be effectively removed but without destroying the collagen fibers network . this way , the citric acid concentration was reduced to 0.5 , 1.0 and 2.0% to try to solve the problem , but no difference was observed . despite being more biocompatible , sodium citrate was not able to expose collagen fibers of the root surface ( table 1 ) even in the group with best results , which was vigorous application for 3 min ( graphs 5 and 6 ) . within the limitations of the methodology , it can be concluded that , despite the lack of statistical significance , the best results for collagen exposure using citric acid were obtained with brushing application at 25% for 3 min . sodium citrate was not able to adequately remove smear layer an expose collagen fibers , so it is not indicated for root conditioning .

sudden sensorineural hearing loss ( ssnhl ) is defined as a hearing loss with a rapid onset in <3 days , and the level of the hearing loss is more than 30 db in at least three contiguous frequencies . in the united states , some case studies have shown that ssnhl typically occurs between 46 and 49 years of age . it has been reported that 6.6% of patients with ssnhl were under 18 years of age , 3.5% under 14 years , and only 1.2% under 9 years . however , a series of factors , including viral infections , microcirculatory disorders , autoimmune disorders , and labyrinthine hemorrhage , have been proposed as causative factors . in adults , the most possible causes of ssnhl might be microcirculatory disorders , whereas in children , viral infections could be a crucial causative factor . in the present study , we systematically analyzed the clinical and audiological characteristics , laboratory examinations , and prognostic factors of 136 chinese cssnhl patients . this retrospective study was performed at department of otolaryngology - head and neck surgery and institute of otolaryngology at chinese pla general hospital in beijing . one hundred and thirty - six hospitalized patients ( 151 ears ) between july 2008 and august 2015 in our department have been included in this study . written informed consents this inclusion criteria of cssnhl patients were : ( 1 ) diagnosed with ssnhl according to the criteria defined in clinical practice guideline : sudden hearing loss ; ( 2 ) aged between 2 and 18 years ; ( 3 ) had a complete audiological and other related inspection report ; and ( 4 ) hospitalized at our department for diagnosis and treatment during the 7-year period from july 2008 to august 2015 . exclusion criteria of cssnhl patients were : ( 1 ) aged under 2 years , because these patients were difficult to distinguish the acquired hearing loss from congenital hearing loss ; ( 2 ) had ear disorders not related to sudden onset hearing loss , including middle ear disease , retrocochlear disorders , auditory neuropathy , and large vestibular aqueduct syndrome ; ( 3 ) had systemic diseases ; ( 4 ) nonorganic hearing loss ; or ( 5 ) genetic or congenital deafness confirmed by genetic screening or neonatal hearing screening . all patients underwent a physical examination of the eardrum , and a neurological examination of the cranial nerves iii , iv , v , vii , and x. audiometry was performed with methods most suitable for the age of the patients . most patients were tested with pure - tone audiometry according to iso 8253 - 1 . moreover , tympanometry , auditory brainstem response ( abr ) , and distortion product otoacoustic emissions ( dpoaes ) were performed . the presence of waves i , iii , and v of abr were used in the analysis . the absolute latencies of abr waves were not analyzed because of the possibility of a latency shift caused by incomplete maturation in children . routine blood tests were performed . viral antibodies against cytomegalovirus ( cmv ) , rubella virus , and herpes simplex virus were detected . the hearing thresholds at 0.50 , 1.00 , 2.00 , and 4.00 khz were measured . the degrees of hearing loss were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hearing loss ( > 80 db hl ) according to the documentation on prevention of blindness and deafness : grades of hearing impairment from the world health organization . five types of audiogram configurations were defined based on the pattern of hearing loss : ascending ( the average threshold of 0.250.50 khz was 20 db higher than the median threshold of 4.008.00 khz ) , descending ( the average threshold of 4.008.00 khz was 20 db higher than the average threshold of 0.250.50 khz ) , flat ( similar threshold observed across the entire frequency range and hearing threshold not exceeding 80 db hl ) , profound ( similar threshold observed across the entire frequency range and hearing threshold over 80 db hl ) , and concave or convex type ( average hearing loss on the mid - tone frequency was 20 db higher than low- and high - frequencies ) . the patients were classified into four groups according to prognosis evaluation : complete recovery , partial recovery , slight recovery , and no - recovery . the complete recovery was defined as pure - tone average < 25 db hl at the final follow - up . patients with hearing recovery < 15 db were included in the no - recovery group . the overall recovery rate was calculated based on patients in complete , partial , and slight recovery groups . patients received one or more of the following treatments : low - salt diet , short - term steroid injections , vasodilatations , defibrinogenators , plasma expanders , antiviral therapy , anti - inflammatory therapy , the medicines for blood circulation improvement , calcium antagonists , diuretics , and hyperbaric oxygen therapy . whitney u test , fisher 's exact test , and chi - squared test were performed to evaluate clinical characteristics and possible prognostic factors of cssnhl . bonferroni method was used for pair - wise comparisons between multiple samples to adjust the significance level . this retrospective study was performed at department of otolaryngology - head and neck surgery and institute of otolaryngology at chinese pla general hospital in beijing . one hundred and thirty - six hospitalized patients ( 151 ears ) between july 2008 and august 2015 in our department have been included in this study . written informed consents this inclusion criteria of cssnhl patients were : ( 1 ) diagnosed with ssnhl according to the criteria defined in clinical practice guideline : sudden hearing loss ; ( 2 ) aged between 2 and 18 years ; ( 3 ) had a complete audiological and other related inspection report ; and ( 4 ) hospitalized at our department for diagnosis and treatment during the 7-year period from july 2008 to august 2015 . exclusion criteria of cssnhl patients were : ( 1 ) aged under 2 years , because these patients were difficult to distinguish the acquired hearing loss from congenital hearing loss ; ( 2 ) had ear disorders not related to sudden onset hearing loss , including middle ear disease , retrocochlear disorders , auditory neuropathy , and large vestibular aqueduct syndrome ; ( 3 ) had systemic diseases ; ( 4 ) nonorganic hearing loss ; or ( 5 ) genetic or congenital deafness confirmed by genetic screening or neonatal hearing screening . all patients underwent a physical examination of the eardrum , and a neurological examination of the cranial nerves iii , iv , v , vii , and x. audiometry was performed with methods most suitable for the age of the patients . most patients were tested with pure - tone audiometry according to iso 8253 - 1 . moreover , tympanometry , auditory brainstem response ( abr ) , and distortion product otoacoustic emissions ( dpoaes ) were performed . the presence of waves i , iii , and v of abr were used in the analysis . the absolute latencies of abr waves were not analyzed because of the possibility of a latency shift caused by incomplete maturation in children . routine blood tests were performed . viral antibodies against cytomegalovirus ( cmv ) , rubella virus , and herpes simplex virus were detected . the hearing thresholds at 0.50 , 1.00 , 2.00 , and 4.00 khz were measured . the degrees of hearing loss were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hearing loss ( > 80 db hl ) according to the documentation on prevention of blindness and deafness : grades of hearing impairment from the world health organization . five types of audiogram configurations were defined based on the pattern of hearing loss : ascending ( the average threshold of 0.250.50 khz was 20 db higher than the median threshold of 4.008.00 khz ) , descending ( the average threshold of 4.008.00 khz was 20 db higher than the average threshold of 0.250.50 khz ) , flat ( similar threshold observed across the entire frequency range and hearing threshold not exceeding 80 db hl ) , profound ( similar threshold observed across the entire frequency range and hearing threshold over 80 db hl ) , and concave or convex type ( average hearing loss on the mid - tone frequency was 20 db higher than low- and high - frequencies ) . the patients were classified into four groups according to prognosis evaluation : complete recovery , partial recovery , slight recovery , and no - recovery . the complete recovery was defined as pure - tone average < 25 db hl at the final follow - up . patients with hearing recovery < 15 db were included in the no - recovery group . the overall recovery rate was calculated based on patients in complete , partial , and slight recovery groups . patients received one or more of the following treatments : low - salt diet , short - term steroid injections , vasodilatations , defibrinogenators , plasma expanders , antiviral therapy , anti - inflammatory therapy , the medicines for blood circulation improvement , calcium antagonists , diuretics , and hyperbaric oxygen therapy . whitney u test , fisher 's exact test , and chi - squared test were performed to evaluate clinical characteristics and possible prognostic factors of cssnhl . bonferroni method was used for pair - wise comparisons between multiple samples to adjust the significance level . among the 151 ears diagnosed with cssnhl , 53.0% ( 80 ears ) were from males and 47.0% ( 71 ears ) were from females . the 80.1% ( 121 ears ) were suffered by unilateral loss and 19.9% ( 30 ears ) were bilateral loss . the mean age of all of patients was 11.7 years old ( range : 218 years ) . the time intervals between the onset of cssnhl , verification of the diagnosis , and treatment varied from 1 day to approximately 183 days ( 18.5 16.8 days ) . characteristics of all cssnhl based on different hearing loss degrees ( n = 151 ears ) , n ( % ) data were analyzed by non - parametric mann - whitney u test . cssnhl : children sudden sensorineural hearing loss . characteristics of all cssnhl based on different age groups ( n = 151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss . of the 151 ears with ssnhl , the hearing loss was characterized as mild in 11 ears ( 7.3% ) , moderate in 13 ears ( 8.6% ) , severe in 37 ears ( 24.5% ) , and profound in 90 ears ( 59.6% ) . among the five defined types of audiogram curves , 6 ears ( 4.0% ) were classified as ascending , 18 ears ( 11.9% ) as descending , 35 ears ( 23.2% ) as flat , 86 ears ( 57.0% ) as profound , and 6 ears ( 4.0% ) as concave or the convex . dpoaes were performed in 133 ears , among which 22 ears ( 16.5% ) passed , whereas 111 ears ( 83.5% ) failed . for the abr results performed in 112 ears , waves i , iii , and v were evoked and identified in 19 ears ( 17.0% ) and wave v only in 22 ears ( 19.6% ) . tympanometry was performed in 140 ears , and 133 ears ( 95.0% ) showed a type curves and 7 ears ( 5.0% ) showed c type curves . the occurrence of initial degree of hearing loss with respect to side of hearing loss and audiogram curve type differed significantly ( p < 0.05 ) . the distribution of the initial degree of hearing loss with respect to gender , age , ear fullness , tinnitus , and vertigo did not differ significantly ( p > 0.05 ) [ table 1 ] . among the 151 ears with ssnhl , 16 ears ( 10.6% ) represented the age group of 26 years , 66 ears ( 43.7% ) represented the age group of 712 years , and 69 ears ( 45.7% ) represented the age group of 1318 years . the distribution of age with regard to side of hearing loss as well as tinnitus was statistically significant ( p < 0.05 ) . the difference in the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial hearing loss showed no statistical significance ( p > 0.05 ) [ table 2 ] . the 13.9% of patients ( 21 ears ) complained ear fullness , 80.8% of patients ( 122 ears ) reported tinnitus , and 52.3% ( 79 ears ) of patients reported vertigo . among the 151 ears , 95 ears ( 63.0% ) accompanied with no obvious causative factors , 13 ears ( 9.0% ) with epidemic mumps , 29 ears ( 19.0% ) with upper respiratory infections , 6 ears ( 4.0% ) with fatigue , 5 ears ( 3.0% ) with traumatic injury , and 3 ears ( 2.0% ) with others . table 3 shows the number of children with abnormal laboratory tests , including routine blood tests , clinical chemistry examination , virus antibody , immunology examination , and blood coagulation function . laboratory examination of all cssnhl patients in this study cssnhl : children sudden sensorineural hearing loss ; wbc : white blood cell ; plt : platelet ; alp : alkaline phosphatase the percentages of patients in complete recovery , partial recovery , slight recovery , and no improvement were 9.3% ( 14 ears ) , 9.9% ( 15 ears ) , 18.5% ( 28 ears ) , and 62.3% ( 94 ears ) , respectively . multivariate analysis revealed that presence of tinnitus , early treatment , and female had a positive correlation with hearing recovery . bilateral hearing loss and severe to profound hearing loss had a negative relation to recovery ( p < 0.05 ) . in univariate analysis , the unilateral , onset of treatment , initial degree of hearing level , the ascending type audiogram , recorded abr and dpoaes had statistically significant differences between the recovered and no - recovered groups ( p < 0.05 ) . the other factors had no significant difference between the recovered and no - recovered groups ( p > 0.05 ) [ tables 4 and 5 ] . univariate and multivariate analyses of clinical characteristics to hearing recovery in all cssnhl ( n=151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . b : standardized coefficient ; se : standard error ; cssnhl : children sudden sensorineural hearing loss . univariate and multivariate analyses of audiology characteristics to hearing recovery in all cssnhl , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss ; b : standardized coefficient ; se : standard error ; abr : auditory brainstem response ; dpoae : distortion product otoacoustic emission . among the 151 ears diagnosed with cssnhl , 53.0% ( 80 ears ) were from males and 47.0% ( 71 ears ) were from females . the 80.1% ( 121 ears ) were suffered by unilateral loss and 19.9% ( 30 ears ) were bilateral loss . the mean age of all of patients was 11.7 years old ( range : 218 years ) . the time intervals between the onset of cssnhl , verification of the diagnosis , and treatment varied from 1 day to approximately 183 days ( 18.5 16.8 days ) . characteristics of all cssnhl based on different hearing loss degrees ( n = 151 ears ) , n ( % ) data were analyzed by non - parametric mann - whitney u test . cssnhl : children sudden sensorineural hearing loss . characteristics of all cssnhl based on different age groups ( n = 151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss . of the 151 ears with ssnhl , the hearing loss was characterized as mild in 11 ears ( 7.3% ) , moderate in 13 ears ( 8.6% ) , severe in 37 ears ( 24.5% ) , and profound in 90 ears ( 59.6% ) . among the five defined types of audiogram curves , 6 ears ( 4.0% ) were classified as ascending , 18 ears ( 11.9% ) as descending , 35 ears ( 23.2% ) as flat , 86 ears ( 57.0% ) as profound , and 6 ears ( 4.0% ) as concave or the convex . dpoaes were performed in 133 ears , among which 22 ears ( 16.5% ) passed , whereas 111 ears ( 83.5% ) failed . for the abr results performed in 112 ears , waves i , iii , and v were evoked and identified in 19 ears ( 17.0% ) and wave v only in 22 ears ( 19.6% ) . tympanometry was performed in 140 ears , and 133 ears ( 95.0% ) showed a type curves and 7 ears ( 5.0% ) showed c type curves . the occurrence of initial degree of hearing loss with respect to side of hearing loss and audiogram curve type differed significantly ( p < 0.05 ) . the distribution of the initial degree of hearing loss with respect to gender , age , ear fullness , tinnitus , and vertigo did not differ significantly ( p > 0.05 ) [ table 1 ] . among the 151 ears with ssnhl , 16 ears ( 10.6% ) represented the age group of 26 years , 66 ears ( 43.7% ) represented the age group of 712 years , and 69 ears ( 45.7% ) represented the age group of 1318 years . the distribution of age with regard to side of hearing loss as well as tinnitus was statistically significant ( p < 0.05 ) . the difference in the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial hearing loss showed no statistical significance ( p > 0.05 ) [ table 2 ] . the 13.9% of patients ( 21 ears ) complained ear fullness , 80.8% of patients ( 122 ears ) reported tinnitus , and 52.3% ( 79 ears ) of patients reported vertigo . among the 151 ears , 95 ears ( 63.0% ) accompanied with no obvious causative factors , 13 ears ( 9.0% ) with epidemic mumps , 29 ears ( 19.0% ) with upper respiratory infections , 6 ears ( 4.0% ) with fatigue , 5 ears ( 3.0% ) with traumatic injury , and 3 ears ( 2.0% ) with others table 3 shows the number of children with abnormal laboratory tests , including routine blood tests , clinical chemistry examination , virus antibody , immunology examination , and blood coagulation function . laboratory examination of all cssnhl patients in this study cssnhl : children sudden sensorineural hearing loss ; wbc : white blood cell ; plt : platelet ; alp : alkaline phosphatase . the percentages of patients in complete recovery , partial recovery , slight recovery , and no improvement were 9.3% ( 14 ears ) , 9.9% ( 15 ears ) , 18.5% ( 28 ears ) , and 62.3% ( 94 ears ) , respectively . multivariate analysis revealed that presence of tinnitus , early treatment , and female had a positive correlation with hearing recovery . bilateral hearing loss and severe to profound hearing loss in univariate analysis , the unilateral , onset of treatment , initial degree of hearing level , the ascending type audiogram , recorded abr and dpoaes had statistically significant differences between the recovered and no - recovered groups ( p < 0.05 ) . the other factors had no significant difference between the recovered and no - recovered groups ( p > 0.05 ) [ tables 4 and 5 ] . univariate and multivariate analyses of clinical characteristics to hearing recovery in all cssnhl ( n=151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . b : standardized coefficient ; se : standard error ; cssnhl : children sudden sensorineural hearing loss . univariate and multivariate analyses of audiology characteristics to hearing recovery in all cssnhl , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss ; b : standardized coefficient ; se : standard error ; abr : auditory brainstem response ; dpoae : distortion product otoacoustic emission . in this study , 136 patients ( 151 ears ) with cssnhl were identified , accounting for 8.6% of all patients ( 1584 cases ) with ssnhl who were treated in our department between july 2008 and august 2015 . a comparison between ssnhl data and the demographic situation in the referral area showed that the incidence of cssnhl patients was 10- to 20-fold less than the incidence of ssnhl in adults , which was similar to the study of chen et al . among the 79 cases with viral antibody detection , 68 cases ( 86.1% ) had increased cmv igg antibody tests . it can be assumed that viral infection is a major cause of cssnhl . previous studies reported that the main hearing loss caused by cmv infection was severe to profound , with a fluctuating , progressing , and delayed onset . worldwide , cmv infection is the most common environmental cause of cssnhl in very young children , affecting 0.22.5% of live - born neonates . symptomatic infection mostly lead to bilateral hearing loss and asymptomatic infection mainly results in unilateral hearing loss . according to karltorp et al . thus , this test makes it possible for infected children to diagnose congenital cmv infection . it is also important to make a standard protocol to prevent and treat of cmv . as shown in table 1 , cssnhl usually occured unilaterally ( 80.1% ) , which was in line with an earlier study . furthermore , bilateral cssnhl showed a stronger association to profound hearing loss than unilateral cssnhl . regarding to the audiogram patterns , the most common types were flat ( 23.2% ) and profound ( 57.0% ) in conjuncture with severe and profound hearing loss . some studies used 12 years of age as the threshold , some used 15 years , and others used 18 years . in this study , we defined a child as the age below 18 years according to the criteria defined in convention on the rights of the child . as the optimal time for speech and language development in children occurs between 1 and 6 years of age , hearing loss in children can severely affect their speech and cognitive development and increase the burden of society and family . due to the differences associated with age , the patients in this study were grouped into three groups : 26 years , 712 years , and 1318 years . it is not possible to separate cssnhl from congenital or progressive hearing loss with certainty in patients under the age of 2 years and is difficult but possible for the age between 2 and 6 years . since it was important to include young children in the study , especially in studying hearing loss caused by cmv , we included the latter group . as shown in table 2 , more children expressed tinnitus in the older age groups of 712 years and 1318 years . this was possibly related to the ability of older children to describe their symptoms more clearly . more children occurred unilateral in the older age groups of 712 years and 1318 years . hence , children in very young age with a bilateral hearing loss should alert the occurrence of ssnhl . the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial degree of hearing loss had no statistical difference . in our study , twenty - two cases ( 33.8% ) showed increased ige antibodies and six patients ( 9.2% ) showed increased igm antibodies . circulating antibodies cross - react with inner ear antigens or activated t - cells , damaging the inner ear . the 133 patients , 40 patients ( 30.1% ) showed increased platelet ( plt ) levels , indicating increased blood coagulation . promotion of thrombosis may result in a disturbed cochlear microcirculation and an increased risk of cssnhl . the plt results may justify further research into the use of thrombolytic drugs in children with ssnhl . cochlear microcirculation is extremely vulnerable to thrombosis , reduced blood flow , and vascular occlusion . both disturbance of microcirculation and infection by virus in cssnhl may cause thrombosis , damage of vessels , and peripheral blood leukocyte effusion . the present study showed that an increased white blood cell ( wbc ) count had a close relationship with the onset of cssnhl . earlier studies have shown that increased numbers of neutrophils and a changed ratio of neutrophils and lymphocytes are negative prognostic factors of ssnhl . in the present study , 133 patients were subjected to a routine blood test , of which 41 patients ( 30.8% ) showed an increased wbc . , 77 patients were tested for homocysteine levels , of which 17 patients ( 22.1% ) showed increased values . it can damage vascular endothelial cells directly or indirectly , promote vascular smooth muscle cell proliferation , effect the oxidation of low - density lipoprotein , strengthen the function of plt , and promote thrombosis . some patients showed increased levels of cholesterol ( 12.6% ) and triglyceride ( 10.9% ) . blood lipid metabolic disorders can lead to blood vessel wall lesions , increase blood viscosity , and result in cochlear microcirculation disturbance . seventy - three patients ( 65.8% ) showed increased alkaline phosphatase ( alp ) levels . determination of alp is mainly used for the diagnosis and differential diagnosis of bone , liver , and gallbladder diseases . the elevated alp levels monitored in the present study may be due to physiological development , but correlation with cssnhl can not be ruled out . some studies have shown high fibrinogen levels to be a negative prognostic factor of ssnhl . in our study , plasma fibrinogen levels were measured in 96 cases , of which 26 cases ( 27.1% ) exhibited increased levels . unilateral hearing loss and early treatment were determined to be positive prognostic factors of hearing recovery . tinnitus , ascending type audiogram , gender , identifiable abr - waves , and dpoaes were positive prognostic factors regarding hearing recovery in cssnhl . the presence of vertigo is widely used to indicate poor recovery in cssnhl . in the present study , patients with sudden hearing loss accompanied by vertigo accounted for 52.3% of all subjects , with no significant correlation with recovery . some studies suggested that tinnitus is an important positive prognostic factor of cssnhl , while other studies showed independence . in the present study , 80.8% of patients reported tinnitus , with a partial or total recovery rate of 33.1% . the presence of tinnitus was determined to be a positive factor associated with hearing recovery in cssnhl according to the multivariate analysis , but was determined to be unrelated according to univariate analysis . the difference between the results of the multivariate analysis and the univariate analysis with regard to gender , tinnitus , audiogram , abr , and dpoaes may be related to a combined effect . the complex pathogenesis of cssnhl , the poor expression ability of children , and the retrospective nature of this study all limited the significance of this study . in conclusion , cssnhl predominantly occurs unilaterally and is in conjunction with severe hearing loss . in our study , complete recovery initial severe hearing loss and bilateral hearing loss were negative prognostic factors for hearing recovery . tinnitus , gender , ascending type audiogram , identifiable abr - waves , and dpoaes were positive prognostic factors for hearing recovery . we also found that the level of wbc , plt , homocysteine , alp , positive cmv igg antibody , fibrinogen , and some immunologic indicators were closely related to cssnhl . these findings support that several important clinical indicators benefit the diagnosis and treatment of cssnhl . this study was financially supported by grants from the national key basic research program of china ( no . 2014cb943001 ) and the national natural science foundation of china ( no . 81120108009 and no . this study was financially supported by grants from the national key basic research program of china ( no . 2014cb943001 ) and the national natural science foundation of china ( no . 81120108009 and no .

background : the prevalence of sudden sensorineural hearing loss in children ( cssnhl ) is consistently increasing . however , the pathology and prognosis of cssnhl are still poorly understood . this retrospective study evaluated clinical characteristics and possible associated factors of cssnhl.methods:one hundred and thirty - six cssnhl patients treated in department of otolaryngology - head and neck surgery and institute of otolaryngology at chinese pla general hospital between july 2008 and august 2015 were included in this study . these patients were analyzed for clinical characteristics , audiological characteristics , laboratory examinations , and prognostic factors.results:among the 136 patients ( 151 ears ) , 121 patients ( 121 ears , 80.1% ) were diagnosed with unilaterally cssnhl , and 15 patients ( 30 ears , 19.9% ) with bilateral cssnhl . the complete recovery rate of cssnhl was 9.3% , and the overall recovery rate was 37.7% . we found that initial degree of hearing loss , onset of treatment , tinnitus , the ascending type audiogram , gender , side of hearing loss , the recorded auditory brainstem response ( abr ) , and distortion product otoacoustic emissions ( dpoaes ) had prognostic significance . age , ear fullness , and vertigo had no significant correlation with recovery . furthermore , the relevant blood tests showed 30.8% of the children had abnormal white blood cell ( wbc ) counts , 22.1% had elevated homocysteine levels , 65.8% had high alkaline phosphatase ( alp ) , 33.8% had high ige antibody levels , and 86.1% had positive cytomegalovirus ( cmv ) igg antibodies.conclusions:cssnhl commonly occurs unilaterally and results in severe hearing loss . initial severe hearing loss and bilateral hearing loss are negative prognostic factors for hearing recovery , while positive prognostic factors include tinnitus , gender , the ascending type audiogram , early treatment , identifiable abr waves , and dpoaes . age , vertigo , and ear fullness are not correlated with the recovery . some serologic indicators , including the level of wbc , platelet , homocysteine , alp , positive cmv igg antibody , fibrinogen , and some immunologic indicators , are closely related to cssnhl .

I M Study design and patients Treatment Statistical analysis R Clinical characteristics of patients Audiological characteristic Initial hearing loss in children sudden sensorineural hearing loss Age distribution in children sudden sensorineural hearing loss Self-assessed symptoms and possible ethological factors Laboratory examinations of children sudden sensorineural hearing loss Prognostic factors of children sudden sensorineural hearing loss D Financial support and sponsorship Conflicts of interest

sudden sensorineural hearing loss ( ssnhl ) is defined as a hearing loss with a rapid onset in <3 days , and the level of the hearing loss is more than 30 db in at least three contiguous frequencies . however , a series of factors , including viral infections , microcirculatory disorders , autoimmune disorders , and labyrinthine hemorrhage , have been proposed as causative factors . in the present study , we systematically analyzed the clinical and audiological characteristics , laboratory examinations , and prognostic factors of 136 chinese cssnhl patients . this retrospective study was performed at department of otolaryngology - head and neck surgery and institute of otolaryngology at chinese pla general hospital in beijing . one hundred and thirty - six hospitalized patients ( 151 ears ) between july 2008 and august 2015 in our department have been included in this study . written informed consents this inclusion criteria of cssnhl patients were : ( 1 ) diagnosed with ssnhl according to the criteria defined in clinical practice guideline : sudden hearing loss ; ( 2 ) aged between 2 and 18 years ; ( 3 ) had a complete audiological and other related inspection report ; and ( 4 ) hospitalized at our department for diagnosis and treatment during the 7-year period from july 2008 to august 2015 . exclusion criteria of cssnhl patients were : ( 1 ) aged under 2 years , because these patients were difficult to distinguish the acquired hearing loss from congenital hearing loss ; ( 2 ) had ear disorders not related to sudden onset hearing loss , including middle ear disease , retrocochlear disorders , auditory neuropathy , and large vestibular aqueduct syndrome ; ( 3 ) had systemic diseases ; ( 4 ) nonorganic hearing loss ; or ( 5 ) genetic or congenital deafness confirmed by genetic screening or neonatal hearing screening . moreover , tympanometry , auditory brainstem response ( abr ) , and distortion product otoacoustic emissions ( dpoaes ) were performed . the absolute latencies of abr waves were not analyzed because of the possibility of a latency shift caused by incomplete maturation in children . viral antibodies against cytomegalovirus ( cmv ) , rubella virus , and herpes simplex virus were detected . the degrees of hearing loss were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hearing loss ( > 80 db hl ) according to the documentation on prevention of blindness and deafness : grades of hearing impairment from the world health organization . five types of audiogram configurations were defined based on the pattern of hearing loss : ascending ( the average threshold of 0.250.50 khz was 20 db higher than the median threshold of 4.008.00 khz ) , descending ( the average threshold of 4.008.00 khz was 20 db higher than the average threshold of 0.250.50 khz ) , flat ( similar threshold observed across the entire frequency range and hearing threshold not exceeding 80 db hl ) , profound ( similar threshold observed across the entire frequency range and hearing threshold over 80 db hl ) , and concave or convex type ( average hearing loss on the mid - tone frequency was 20 db higher than low- and high - frequencies ) . the patients were classified into four groups according to prognosis evaluation : complete recovery , partial recovery , slight recovery , and no - recovery . patients with hearing recovery < 15 db were included in the no - recovery group . the overall recovery rate was calculated based on patients in complete , partial , and slight recovery groups . patients received one or more of the following treatments : low - salt diet , short - term steroid injections , vasodilatations , defibrinogenators , plasma expanders , antiviral therapy , anti - inflammatory therapy , the medicines for blood circulation improvement , calcium antagonists , diuretics , and hyperbaric oxygen therapy . whitney u test , fisher 's exact test , and chi - squared test were performed to evaluate clinical characteristics and possible prognostic factors of cssnhl . this retrospective study was performed at department of otolaryngology - head and neck surgery and institute of otolaryngology at chinese pla general hospital in beijing . one hundred and thirty - six hospitalized patients ( 151 ears ) between july 2008 and august 2015 in our department have been included in this study . written informed consents this inclusion criteria of cssnhl patients were : ( 1 ) diagnosed with ssnhl according to the criteria defined in clinical practice guideline : sudden hearing loss ; ( 2 ) aged between 2 and 18 years ; ( 3 ) had a complete audiological and other related inspection report ; and ( 4 ) hospitalized at our department for diagnosis and treatment during the 7-year period from july 2008 to august 2015 . exclusion criteria of cssnhl patients were : ( 1 ) aged under 2 years , because these patients were difficult to distinguish the acquired hearing loss from congenital hearing loss ; ( 2 ) had ear disorders not related to sudden onset hearing loss , including middle ear disease , retrocochlear disorders , auditory neuropathy , and large vestibular aqueduct syndrome ; ( 3 ) had systemic diseases ; ( 4 ) nonorganic hearing loss ; or ( 5 ) genetic or congenital deafness confirmed by genetic screening or neonatal hearing screening . moreover , tympanometry , auditory brainstem response ( abr ) , and distortion product otoacoustic emissions ( dpoaes ) were performed . the absolute latencies of abr waves were not analyzed because of the possibility of a latency shift caused by incomplete maturation in children . viral antibodies against cytomegalovirus ( cmv ) , rubella virus , and herpes simplex virus were detected . the degrees of hearing loss were categorized as mild ( 2640 db hl ) , moderate ( 4160 db hl ) , severe ( 6180 db hl ) , and profound hearing loss ( > 80 db hl ) according to the documentation on prevention of blindness and deafness : grades of hearing impairment from the world health organization . five types of audiogram configurations were defined based on the pattern of hearing loss : ascending ( the average threshold of 0.250.50 khz was 20 db higher than the median threshold of 4.008.00 khz ) , descending ( the average threshold of 4.008.00 khz was 20 db higher than the average threshold of 0.250.50 khz ) , flat ( similar threshold observed across the entire frequency range and hearing threshold not exceeding 80 db hl ) , profound ( similar threshold observed across the entire frequency range and hearing threshold over 80 db hl ) , and concave or convex type ( average hearing loss on the mid - tone frequency was 20 db higher than low- and high - frequencies ) . the patients were classified into four groups according to prognosis evaluation : complete recovery , partial recovery , slight recovery , and no - recovery . the overall recovery rate was calculated based on patients in complete , partial , and slight recovery groups . patients received one or more of the following treatments : low - salt diet , short - term steroid injections , vasodilatations , defibrinogenators , plasma expanders , antiviral therapy , anti - inflammatory therapy , the medicines for blood circulation improvement , calcium antagonists , diuretics , and hyperbaric oxygen therapy . whitney u test , fisher 's exact test , and chi - squared test were performed to evaluate clinical characteristics and possible prognostic factors of cssnhl . among the 151 ears diagnosed with cssnhl , 53.0% ( 80 ears ) were from males and 47.0% ( 71 ears ) were from females . the 80.1% ( 121 ears ) were suffered by unilateral loss and 19.9% ( 30 ears ) were bilateral loss . the time intervals between the onset of cssnhl , verification of the diagnosis , and treatment varied from 1 day to approximately 183 days ( 18.5 16.8 days ) . characteristics of all cssnhl based on different hearing loss degrees ( n = 151 ears ) , n ( % ) data were analyzed by non - parametric mann - whitney u test . cssnhl : children sudden sensorineural hearing loss . cssnhl : children sudden sensorineural hearing loss . of the 151 ears with ssnhl , the hearing loss was characterized as mild in 11 ears ( 7.3% ) , moderate in 13 ears ( 8.6% ) , severe in 37 ears ( 24.5% ) , and profound in 90 ears ( 59.6% ) . among the five defined types of audiogram curves , 6 ears ( 4.0% ) were classified as ascending , 18 ears ( 11.9% ) as descending , 35 ears ( 23.2% ) as flat , 86 ears ( 57.0% ) as profound , and 6 ears ( 4.0% ) as concave or the convex . the occurrence of initial degree of hearing loss with respect to side of hearing loss and audiogram curve type differed significantly ( p < 0.05 ) . the distribution of the initial degree of hearing loss with respect to gender , age , ear fullness , tinnitus , and vertigo did not differ significantly ( p > 0.05 ) [ table 1 ] . the distribution of age with regard to side of hearing loss as well as tinnitus was statistically significant ( p < 0.05 ) . the difference in the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial hearing loss showed no statistical significance ( p > 0.05 ) [ table 2 ] . the 13.9% of patients ( 21 ears ) complained ear fullness , 80.8% of patients ( 122 ears ) reported tinnitus , and 52.3% ( 79 ears ) of patients reported vertigo . among the 151 ears , 95 ears ( 63.0% ) accompanied with no obvious causative factors , 13 ears ( 9.0% ) with epidemic mumps , 29 ears ( 19.0% ) with upper respiratory infections , 6 ears ( 4.0% ) with fatigue , 5 ears ( 3.0% ) with traumatic injury , and 3 ears ( 2.0% ) with others . table 3 shows the number of children with abnormal laboratory tests , including routine blood tests , clinical chemistry examination , virus antibody , immunology examination , and blood coagulation function . laboratory examination of all cssnhl patients in this study cssnhl : children sudden sensorineural hearing loss ; wbc : white blood cell ; plt : platelet ; alp : alkaline phosphatase the percentages of patients in complete recovery , partial recovery , slight recovery , and no improvement were 9.3% ( 14 ears ) , 9.9% ( 15 ears ) , 18.5% ( 28 ears ) , and 62.3% ( 94 ears ) , respectively . multivariate analysis revealed that presence of tinnitus , early treatment , and female had a positive correlation with hearing recovery . in univariate analysis , the unilateral , onset of treatment , initial degree of hearing level , the ascending type audiogram , recorded abr and dpoaes had statistically significant differences between the recovered and no - recovered groups ( p < 0.05 ) . univariate and multivariate analyses of clinical characteristics to hearing recovery in all cssnhl ( n=151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss ; b : standardized coefficient ; se : standard error ; abr : auditory brainstem response ; dpoae : distortion product otoacoustic emission . among the 151 ears diagnosed with cssnhl , 53.0% ( 80 ears ) were from males and 47.0% ( 71 ears ) were from females . the 80.1% ( 121 ears ) were suffered by unilateral loss and 19.9% ( 30 ears ) were bilateral loss . the time intervals between the onset of cssnhl , verification of the diagnosis , and treatment varied from 1 day to approximately 183 days ( 18.5 16.8 days ) . characteristics of all cssnhl based on different hearing loss degrees ( n = 151 ears ) , n ( % ) data were analyzed by non - parametric mann - whitney u test . cssnhl : children sudden sensorineural hearing loss . of the 151 ears with ssnhl , the hearing loss was characterized as mild in 11 ears ( 7.3% ) , moderate in 13 ears ( 8.6% ) , severe in 37 ears ( 24.5% ) , and profound in 90 ears ( 59.6% ) . among the five defined types of audiogram curves , 6 ears ( 4.0% ) were classified as ascending , 18 ears ( 11.9% ) as descending , 35 ears ( 23.2% ) as flat , 86 ears ( 57.0% ) as profound , and 6 ears ( 4.0% ) as concave or the convex . the occurrence of initial degree of hearing loss with respect to side of hearing loss and audiogram curve type differed significantly ( p < 0.05 ) . the distribution of the initial degree of hearing loss with respect to gender , age , ear fullness , tinnitus , and vertigo did not differ significantly ( p > 0.05 ) [ table 1 ] . the distribution of age with regard to side of hearing loss as well as tinnitus was statistically significant ( p < 0.05 ) . the difference in the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial hearing loss showed no statistical significance ( p > 0.05 ) [ table 2 ] . the 13.9% of patients ( 21 ears ) complained ear fullness , 80.8% of patients ( 122 ears ) reported tinnitus , and 52.3% ( 79 ears ) of patients reported vertigo . among the 151 ears , 95 ears ( 63.0% ) accompanied with no obvious causative factors , 13 ears ( 9.0% ) with epidemic mumps , 29 ears ( 19.0% ) with upper respiratory infections , 6 ears ( 4.0% ) with fatigue , 5 ears ( 3.0% ) with traumatic injury , and 3 ears ( 2.0% ) with others table 3 shows the number of children with abnormal laboratory tests , including routine blood tests , clinical chemistry examination , virus antibody , immunology examination , and blood coagulation function . laboratory examination of all cssnhl patients in this study cssnhl : children sudden sensorineural hearing loss ; wbc : white blood cell ; plt : platelet ; alp : alkaline phosphatase . the percentages of patients in complete recovery , partial recovery , slight recovery , and no improvement were 9.3% ( 14 ears ) , 9.9% ( 15 ears ) , 18.5% ( 28 ears ) , and 62.3% ( 94 ears ) , respectively . multivariate analysis revealed that presence of tinnitus , early treatment , and female had a positive correlation with hearing recovery . bilateral hearing loss and severe to profound hearing loss in univariate analysis , the unilateral , onset of treatment , initial degree of hearing level , the ascending type audiogram , recorded abr and dpoaes had statistically significant differences between the recovered and no - recovered groups ( p < 0.05 ) . univariate and multivariate analyses of clinical characteristics to hearing recovery in all cssnhl ( n=151 ears ) , n ( % ) * the chi - squared test or fisher 's exact test . cssnhl : children sudden sensorineural hearing loss ; b : standardized coefficient ; se : standard error ; abr : auditory brainstem response ; dpoae : distortion product otoacoustic emission . in this study , 136 patients ( 151 ears ) with cssnhl were identified , accounting for 8.6% of all patients ( 1584 cases ) with ssnhl who were treated in our department between july 2008 and august 2015 . among the 79 cases with viral antibody detection , 68 cases ( 86.1% ) had increased cmv igg antibody tests . symptomatic infection mostly lead to bilateral hearing loss and asymptomatic infection mainly results in unilateral hearing loss . as shown in table 1 , cssnhl usually occured unilaterally ( 80.1% ) , which was in line with an earlier study . furthermore , bilateral cssnhl showed a stronger association to profound hearing loss than unilateral cssnhl . in this study , we defined a child as the age below 18 years according to the criteria defined in convention on the rights of the child . as the optimal time for speech and language development in children occurs between 1 and 6 years of age , hearing loss in children can severely affect their speech and cognitive development and increase the burden of society and family . due to the differences associated with age , the patients in this study were grouped into three groups : 26 years , 712 years , and 1318 years . the distribution of age in relation to gender , ear fullness , vertigo , audiogram curve types , and initial degree of hearing loss had no statistical difference . the 133 patients , 40 patients ( 30.1% ) showed increased platelet ( plt ) levels , indicating increased blood coagulation . the present study showed that an increased white blood cell ( wbc ) count had a close relationship with the onset of cssnhl . earlier studies have shown that increased numbers of neutrophils and a changed ratio of neutrophils and lymphocytes are negative prognostic factors of ssnhl . in the present study , 133 patients were subjected to a routine blood test , of which 41 patients ( 30.8% ) showed an increased wbc . , 77 patients were tested for homocysteine levels , of which 17 patients ( 22.1% ) showed increased values . seventy - three patients ( 65.8% ) showed increased alkaline phosphatase ( alp ) levels . unilateral hearing loss and early treatment were determined to be positive prognostic factors of hearing recovery . tinnitus , ascending type audiogram , gender , identifiable abr - waves , and dpoaes were positive prognostic factors regarding hearing recovery in cssnhl . in the present study , patients with sudden hearing loss accompanied by vertigo accounted for 52.3% of all subjects , with no significant correlation with recovery . some studies suggested that tinnitus is an important positive prognostic factor of cssnhl , while other studies showed independence . in the present study , 80.8% of patients reported tinnitus , with a partial or total recovery rate of 33.1% . the difference between the results of the multivariate analysis and the univariate analysis with regard to gender , tinnitus , audiogram , abr , and dpoaes may be related to a combined effect . the complex pathogenesis of cssnhl , the poor expression ability of children , and the retrospective nature of this study all limited the significance of this study . in conclusion , cssnhl predominantly occurs unilaterally and is in conjunction with severe hearing loss . in our study , complete recovery initial severe hearing loss and bilateral hearing loss were negative prognostic factors for hearing recovery . tinnitus , gender , ascending type audiogram , identifiable abr - waves , and dpoaes were positive prognostic factors for hearing recovery . we also found that the level of wbc , plt , homocysteine , alp , positive cmv igg antibody , fibrinogen , and some immunologic indicators were closely related to cssnhl .

optical coherence tomography ( oct ) provides high - resolution , cross - sectional tomographic images of the human retina and permits direct evaluation of retinal thickness . in recent years the development of spectral - domain oct ( sd - oct ) technology has greatly increased imaging speed and resolution relative to earlier time - domain technology . sd - oct has become invaluable in the management of a variety of retinal diseases including neovascular age - related macular degeneration ( amd ) [ 25 ] and diabetic macular edema [ 6 , 7 ] . this utility is due primarily to the ability to extract estimates of retinal thickness across the macula ( to aid in clinical diagnosis and treatment decisions ) . previous studies on the application of sd - oct to retinal pathology have uncovered multiple sources of error that dramatically decrease the accuracy of these macular thickness measurements [ 8 , 9 ] . perhaps the most obvious source of error is imprecise retinal layer segmentation , which can result from poor signal quality of the sd - oct image or the outright failure in the segmentation algorithm itself in otherwise high - quality images [ 8 , 10 , 11 ] . additional errors inherent to the system can be elucidated by evaluating the reproducibility of sd - oct systems [ 9 , 1217 ] . these reproducibility studies capture all errors inherent to the basic operation of the sd - oct system and represent a baseline level of error that could reasonably be expected even under the best circumstances . however , there are additional sources of inaccuracy that have received considerably less attention and are independent of segmentation and operator errors . rather they pertain to instrument sampling and processing protocols . for example , sadda et al . compared central subfield thickness values from volumes containing 128 b - scans to less densely sampled volumes . as b - scan density is reduced , less retinal area is sampled , leading to less data being included in the retinal thickness calculation . the reduction in data led to differences , or errors , in retinal thickness measurements , the magnitude of which increased as sampling density was decreased . here , we further examined b - scan density as well as factors that are related to assumptions about the patient being imaged , such as errant fixation and variation in axial length among patients . taken together , these variables compromise the accuracy of macular thickness maps . while the degree of inaccuracy depends on the patient , the significance of the inaccuracy depends on the application of the retinal thickness data . one hundred thirteen normal subjects ( 55 male , 58 female ) age 18 years and older were recruited for sd - oct imaging ( mean standard deviation = 27.3 8.3 years ) . normal subjects had normal color vision assessed with the neitz test of color vision and no history of refractive surgery or any vision - limiting ocular pathology . forty - three patients ( 18 male , 25 female ) with various retinal pathologies were also recruited ( mean standard deviation = 40.7 20.1 years ) . pathology included macular dystrophy ( n = 9 ) , blue cone monochromacy ( n = 3 ) , x - linked high myopia ( n = 4 ) , basal laminar drusen ( n = 5 ) , retinitis pigmentosa ( n = 2 ) , amd ( n = 3 ) , plaquenil toxicity ( n = 3 ) , diabetic macular edema ( n = 3 ) , macular telangectasia ( n = 2 ) , central artery occlusion ( n = 2 ) , and one each of oligocone trichromacy , posterior epithelial detachment , oculocutaneous albinism , punctate inner choroidopathy , achromatopsia , cystoid macular edema , and acute zonal occult outer retinopathy . informed consent was obtained from all subjects after explanation of the nature and possible consequences of the study . all research on human subjects followed the tenets of the declaration of helsinki and was approved by the institutional review board at children 's hospital of wisconsin . volumetric sd - oct images of the macula were obtained using the cirrus hd - oct ( carl zeiss meditec , dublin , calif , usa ) . volumes were nominally 6 mm 6 mm and consisted of 128 b - scans ( 512 a - scans / b - scan ) . the internal fixation target of the system was used , which consists of a large green asterisk on a red background , and focus of the lso fundus image was optimized using built - in focus correction . in addition , the polarization setting was optimized using the built - in function for each eye . retinal thickness was calculated using the built - in macular analysis software on the cirrus ( software version 5.0 ) , which is automatically determined by taking the difference between the ilm and rpe boundaries . the positions of the foveal center and retinal thickness data from each volume scan were exported for offline analysis using the zeiss cirrus research browser ( version 5.0 ) . all volumes were manually examined for accuracy of the ilm and rpe segmentation and relative accuracy of the autofovea function . in order to evaluate the acquisition and analysis parameters of interest , we needed to be able to manipulate these macular thickness maps off line . custom matlab ( mathworks , natick , mass , usa ) software was used to generate early treatment diabetic retinopathy study ( etdrs ) thickness maps from the .dat files exported from the zeiss cirrus research browser . as shown in figure 1 , there is good agreement between etdrs segment thicknesses derived from the on - board cirrus software and our offline matlab program , thus demonstrating the fidelity of the data export and validating our use of these matlab - derived etdrs maps for subsequent analysis . to assess the interpolation error in volumetric retinal thickness maps due to decreased b - scan sampling , we created undersampled versions of the retinal thickness volumes exported from the cirrus system . these maps used thickness values from 8 ( every 16th b - scan ) , 16 ( every 8th b - scan ) , 32 ( every 4th b - scan ) , or 64 ( every other b - scan ) of the 128 b - scans initially collected . complete thickness maps were then created by interpolating between these evenly spaced b - scans ( using a matlab spline interpolation function ) . this enabled point - by - point comparison between the native macular thickness map and the undersampled ones , as well as comparison between the corresponding etdrs plots . in all etdrs comparisons , most sd - oct systems assume foveal fixation ; however there is frequently significant discrepancy between the location of the fovea and the preferred retinal locus of fixation . even among individuals with no retinal pathology , there is modest variation in fixation and there is evidence that suggests that the foveal center is not always used for fixation [ 2124 ] . we used the autofovea function of the cirrus hd - oct to identify the location of the foveal pit and generated an etdrs plot centered at this location and a second plot centered at the middle of the volume ( the default setting on most other sd - oct systems ) . manual inspection of each volume confirmed that the fovea was identified by the autofovea function ( though in more severe macular pathology we have seen the algorithm fail ) . comparing these two etdrs plots provides an estimate of the potential error due to improper anchoring of the plot to the scan center . moreover , as we had access to the ( x , y ) coordinate of the fovea within each nominal 6 mm 6 mm volume , we examined error as a function of the displacement of each subject 's fixation from the center of his or her foveal pit . the scan length reported by sd - oct systems ( when reported in mm ) is relative , not absolute . this is because the scanning mirrors are calibrated to a model eye , which assumes a fixed axial length ( typically around 24 mm ) . however there exist significant individual differences in retinal magnification ( primarily caused by differences in axial length ) ; thus the actual scan length will vary from person to person . in fact , using normative axial length data to correct for ocular magnification , we estimate that approximately one - third of individuals would have a scan length that deviates by more than 0.3 mm from the expected length ( with a maximum deviation of nearly 1 mm ) . we obtained axial length measurements using the zeiss iol master ( carl zeiss meditec , dublin , calif , usa ) and subsequently calibrated the lateral scale of each subject 's sd - oct scans in order to generate revised etdrs plots . these plots were then compared to those derived assuming a 24.46 mm axial length ( that of the cirrus model eye ) . despite the macular volume scan nominally subtending a 6 mm 6 mm area , the entire retinal area within that volume is not actually scanned . as shown in figure 2 , even a scan using 512 a - scans / b - scan and 128 b - scans only samples 29% of the retinal area within the volume . using 37 high - resolution b - scans results in less than 10% of the retinal area within the volume actually being scanned . as only retina that gets scanned can actually contribute to plots of retinal thickness measurements , this undersampling can significantly affect the integrity of the resultant macular thickness maps . at first glance , assessment of the effect of b - scan density on macular thickness maps suggests that despite reducing the number of b - scans , the general contour of the map remains qualitatively similar ( figure 3(a ) ) . however in reality , interpolation between b - scans causes overrepresentation and underrepresentation of different features within a given retinal volume ( figure 3(b ) ) . as shown in the normal example , since sampling is being reduced in the vertical direction , the superior and inferior aspects of the fovea show equal magnitude of underrepresentation and overrepresentation of retinal thickness , respectively . this effect is greatly enhanced in a subject with dominant drusen , wherein error is generated not only in the central fovea but also broadly across the retinal volume . we found that in the normal individuals for the central 1 mm subfield , the mean ( standard deviation ) absolute error was 0.19 0.15 m with 64 b - scans , 1.17 0.69 m with 32 b - scans , 7.15 2.35 m with 16 b - scans , and 22.98 7.54 m with 8 b - scans . when expressed as a percentage of subfield thickness we find that the mean percentage error was 0.07 0.06% with 64 b - scans , 0.47 0.29% with 32 b - scans , 2.85 1.08% with 16 b - scans , and 9.19 3.52% with 8 b - scans . for the central 1 mm subfield , the mean ( standard deviation ) absolute error was 0.31 0.36 m with 64 b - scans , 1.36 1.17 m with 32 b - scans , 6.35 3.64 m with 16 b - scans , and 19.56 11.08 m with 8 b - scans . when expressed as a percentage of subfield thickness we find that the mean percentage error was 0.13 0.15% with 64 b - scans , 0.63 0.56% with 32 b - scans , 3.03 2.14% with 16 b - scans , and 9.56 6.92% with 8 b - scans . previous data reveal that the coefficient of repeatability for central subfield measurements on the cirrus is about 4.96 m , indicating that 32 b - scans is sufficient sampling to generate accurate etdrs thickness plots . however , as shown in the difference plots in figure 3 , at neighboring retinal locations where the retinal contour is changing , retinal thickness measurements are in error in opposing directions . thus , reporting retinal thickness for a subregion that averages spatially ( i.e. , etdrs plots ) will not reveal the true extent of the error imparted by undersampling . in order to quantify the effect of b - scan density on the accuracy of retinal thickness at any given point within the 6 mm 6 mm volume , we examined the error per pixel ( a scan ) within the volume . in this case , the retinal thickness measurements utilizing all 128 b - scans were considered to be absolutely accurate for comparison to the undersampled volumes . at 32 b - scans , our analysis revealed that these interpolation errors could be as high as 5.5 m per pixel and 7.5 m per pixel in the normal and pathology groups , respectively . there are 65,536 pixels in each of our thickness maps , native or undersampled . in both groups , on average , the error per pixel increases as the number of b - scans used to construct the retinal thickness map decreases ( figure 4 ) . we compared etdrs thickness plots derived by placing the center of the etdrs grid on the foveal center to those plots centered on the subject 's actual fixation point . we found these plots to differ by over 100 m in some normal individuals ( sum of the error in all nine etdrs segments ) , with the mean error being 14.4 19.3 m ( figure 5(a ) ) . in the 43 pathology cases , the mean error was 30.4 40.9 m , with some individuals exceeding 200 m of total error in their etdrs plots ( figure 5(b ) ) . of course if eccentric fixation is identified by the oct operator , the scan location can be repositioned prior to image acquisition to help reduce this error . for two pathology cases , we acquired one scan at their normal eccentric fixation location and a second after moving the scan to be visually centered on the fovea . at their normal fixation position , these subjects had etdrs plots that deviated by 74.5 m and 101.9 m from an etdrs plot precisely positioned at the foveal center ( using our offline matlab program ) . even after the operator acquired a second scan intentionally centered on the fovea to the best of their ability , etdrs errors persisted of 16.3 m and 17.8 m . regardless , for both normal subjects and subjects with retinal pathology , the greater the distance between the fovea and the center of the sd - oct volume , the less accurate the etdrs thickness map . in just the central subfield thickness , not correcting for scan position results in a mean error of 3.18 6.09 m in the normal subjects ( with a maximum error of 32 m ) and 10.50 19.43 m in the patients with retinal pathology ( with a maximum error of 104 m ) . on average , the central subfield error accounts for 14% and 22% of the total etdrs error in the normal and pathology patients , respectively . axial length varied in our normal subjects from 21.56 to 28.36 mm and in pathology patients from 21.87 to 30.13 mm . using each subject axial length to correct the lateral scale of the nominal 6 mm sd - oct scan , we determined that actual scan sizes range from about 5.29 to 6.96 mm for our normal population and 5.36 to 7.4 mm for our pathological population . we used these corrected scan dimensions to derive corrected etdrs plots , where the rings were actually 1 mm , 3 mm , and 6 mm in diameter . in comparing these plots to the uncorrected ones , we found that the summed error for the nine etdrs segments was as much as 44.9 m , with 37 out of 113 ( 32% ) subjects having more than 20 m of total error . for subjects with retinal pathology the summed error for the nine etdrs segments was as much as 77.3 m , and 13 out of 43 ( 30% ) showed more than 20 m of total error ( figure 6 ) . in just the central subfield alone , the error was as much as 7.86 m ( with an average of 2.56 1.85 m ) for the normals and as much as 12.33 m ( with an average of 2.84 2.46 m ) in the individuals with retinal pathology . in both groups , the error increased with increasing difference in axial length from that of the model eye ( 24.46 mm ) . as illustrated above , not correcting for axial length and not positioning the scan at the center of the fovea introduces significant error in the corresponding etdrs thickness plots . taken together , these artifacts tend to have a cumulative negative effect on the accuracy of the etdrs plots . for example , in considering just the central subfield thickness , not correcting for axial length or scan position results in a mean error of 4.53 5.77 m in the normal subjects ( with a maximum combined error of 33 m ) and 11.29 19.18 m in the patients with retinal pathology ( with a maximum combined error of 105 m ) . this study examined the effects of preventable operational and analytic aspects of the sd - oct on the overall accuracy of etdrs retinal thickness plots . scan density , position of the scan with respect to the foveal center , and magnitude of subject axial length differential all contribute to significant error in computing retinal thickness from sd - oct volumes . an important point to consider is the cumulative nature of the errors reported here ; these parameters should all be accounted for when developing normative databases or analyzing specific retinal features within individual patient data . while the errors were estimated using a single sd - oct device ( cirrus hd - oct ) , they are generic to sd - oct imaging in general . the issue of scan positioning is typically something that can be addressed by the operator by repositioning the etdrs grid ( either manually or using an automatic function like autofovea ) . currently , correcting the lateral scale of oct data / images requires offline correction by the user . in comparing our results to previously published data , we find similarities and differences . in an examination of b - scan density , sadda et al . concluded that 32 b - scans result in only a minimal change in retinal thickness . our data also show that when examining maps of retinal thickness that are based on spatially integrating individual thickness values ( i.e. , etdrs ) , reduced b - scan sampling has minimal impact . however , if interested in deriving absolute measures of retinal thickness at any given point , reduction to 32 b - scans ( a value suggested to provide accurate retinal thickness maps ) , results in an average error of around 3 m per pixel . while this average error is within the system resolution on commercial sd - oct systems , it is worth keeping in mind that the error at any one pixel can be much larger , since not all pixels will contribute equally to the total error ( which is implicit in computing an average error ) . real cost of undersampling , and this would significantly limit the ability to make precise measurements of retinal features ( e.g. , drusen ) . this highlights the importance of considering how the sd - oct data is going to be used when deciding how densely to sample the retina . it is well documented that differences in axial length result in different ocular magnification of retinal images and thus can affect the accuracy of measurements of retinal features . with respect to oct , axial length has been shown to influence measurements of retinal nerve fiber layer ( rnfl ) thickness [ 2831 ] . this of course is based on the fact that rnfl measures are presumed to be taken at a fixed distance from the optic nerve ; thus individual differences in ocular magnificent would result in the rnfl being measured at the wrong location . here we demonstrate that individual differences in ocular magnification also affect the accuracy of macular thickness maps . if the distribution of axial lengths in a normative database does not match that of the subject population being studied , misinterpretation can occur . perhaps more important than retinal thickness maps is the fact that not correcting the nominal scan length for differences in axial length will obviate making reliable measurements in the lateral dimension within a given oct dataset . this could include measuring the area of geographic atrophy , the size of a macular hole , or the size of a druse . despite this , some sd - oct systems still output lateral scale bars on their images that are given in m or provide calipers with which to make lateral measurements in m , despite no correction for axial length having been made . one should avoid using such scale bars to report absolute length measurements , as they are simply not accurate without first taking into account ocular magnification . there have also been previous examinations of the effect of fixation on the accuracy of oct thickness measurements . in glaucoma , it has been shown that if the circular scan is not centered on the onh , the rnfl thickness measurements are inaccurate . examined how intentionally shifting the center of macular volume oct scans ( stratus time - domain ) affected central subfield thickness measurements for 10 normal subjects . they found that scan decentration of 0.50 mm resulted in foveal thickness measurements that were in error by about 45% . for our normal subjects , the average decentration of the sd - oct volume with respect to the foveal center was 0.09 mm and the average error of foveal thickness measurements was about 35% . while this is roughly consistent with the finding of campbell et al . , some discrepancy would be expected given our use of sd - oct ( instead of time domain ) and our ability to precisely determine the exact misalignment between the two scans being compared ( whereas the previous study would have be confounded by errors due to normal fixational instability ) . currently , the cirrus hd - oct will automatically position the etdrs grid over the center of the fovea ( after the scan is taken ) . while this results in a more accurate etdrs map , it may not be valid to compare these maps to a database in which the etdrs maps were not centered on the fovea , though in the case of the cirrus database , good centration of the volume on the fovea was an inclusion criterion . it is generally important to ensure that the scan parameters used to develop the normative database match that of the on - board scan protocol . moreover , the subject composition ( race and gender ) may also need to be considered when comparing a specific patient to a particular normative database . first , in our examination of b - scan sampling , we used 128 b - scans as the truth . this was simply due to a limitation of the specific sd - oct device being used . however , as we showed in figure 2 , 128 b - scans ( at 512 a scans / b - scan ) only sample 29% of the nominal 6 m 6 mm volume . thus these volumes are likely already in error compared to an isotropic volume of 512 b - scans . with the expected availability of even faster oct systems , it will be important to quantify the level of inaccuracy systematically across more densely sampled volumes . in addition , we likely underestimate the real effect of undersampling , as we used simulated thickness maps . if one were to really only acquire 32 b - scans , this could affect the accuracy of segmentation as many oct devices use 3d approaches to make correct assignment of layers . a second limitation is that we corrected for ocular magnification using a linear scaling based on axial length . there are other methods to correct for ocular magnification , and the exact method used for the correction would influence the measured differences in retinal image magnification . finally it seems likely that different retinal pathology would suffer more ( or less ) than others . intuitively , one can conclude that the more uniform the retinal thickness contoured ( as might occur in retinitis pigmentosa , where the retina is uniformly thin ) , the less impact the b - scan sampling , axial length , and scan position would have . likewise , retinal pathology that results in significant peaks and troughs in retinal thickness ( macular holes , amd , diabetic macular edema ) might be more significantly influenced by these parameters . it is important to keep in mind that the relevance of these errors of course ultimately depends on the clinical application . for monitoring patients over time , relative differences in retinal thickness would be generally unaffected by axial length , though comparing populations of patients ( such as in a clinical trial ) where there may be differences in axial length between the groups could result in significant error . if one uses the same sampling density , then the accuracy of these longitudinal measurements of retinal thickness will be on the order of that reported for previous repeatability and reproducibility studies . however , in instances where one is interested in correlating a measure of retinal thickness over a specific retinal area ( e.g. , central subfield thickness ) with some other measure of vision ( such as treatment response ) these errors could reveal correlations that do not exist or hide ones that do exist . moreover , where one is interested in making absolute measurements in the lateral dimension , such as foveal pit morphology [ 12 , 34 ] or drusen volume , it is critical that these sources of error be removed .

sd - oct has become an essential tool for evaluating macular pathology ; however several aspects of data collection and analysis affect the accuracy of retinal thickness measurements . here we evaluated sampling density , scan centering , and axial length compensation as factors affecting the accuracy of macular thickness maps . forty - three patients with various retinal pathologies and 113 normal subjects were imaged using cirrus hd - oct . reduced b - scan density was associated with increased interpolation error in etdrs macular thickness plots . correcting for individual differences in axial length revealed modest errors in retinal thickness maps , while more pronounced errors were observed when the etdrs plot was not positioned at the center of the fovea ( which can occur as a result of errant fixation ) . cumulative error can exceed hundreds of microns , even under ideal observer conditions . this preventable error is particularly relevant when attempting to compare macular thickness maps to normative databases or measuring the area or volume of retinal features .

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

optical coherence tomography ( oct ) provides high - resolution , cross - sectional tomographic images of the human retina and permits direct evaluation of retinal thickness . in recent years the development of spectral - domain oct ( sd - oct ) technology has greatly increased imaging speed and resolution relative to earlier time - domain technology . sd - oct has become invaluable in the management of a variety of retinal diseases including neovascular age - related macular degeneration ( amd ) [ 25 ] and diabetic macular edema [ 6 , 7 ] . this utility is due primarily to the ability to extract estimates of retinal thickness across the macula ( to aid in clinical diagnosis and treatment decisions ) . previous studies on the application of sd - oct to retinal pathology have uncovered multiple sources of error that dramatically decrease the accuracy of these macular thickness measurements [ 8 , 9 ] . perhaps the most obvious source of error is imprecise retinal layer segmentation , which can result from poor signal quality of the sd - oct image or the outright failure in the segmentation algorithm itself in otherwise high - quality images [ 8 , 10 , 11 ] . additional errors inherent to the system can be elucidated by evaluating the reproducibility of sd - oct systems [ 9 , 1217 ] . these reproducibility studies capture all errors inherent to the basic operation of the sd - oct system and represent a baseline level of error that could reasonably be expected even under the best circumstances . compared central subfield thickness values from volumes containing 128 b - scans to less densely sampled volumes . as b - scan density is reduced , less retinal area is sampled , leading to less data being included in the retinal thickness calculation . the reduction in data led to differences , or errors , in retinal thickness measurements , the magnitude of which increased as sampling density was decreased . here , we further examined b - scan density as well as factors that are related to assumptions about the patient being imaged , such as errant fixation and variation in axial length among patients . taken together , these variables compromise the accuracy of macular thickness maps . while the degree of inaccuracy depends on the patient , the significance of the inaccuracy depends on the application of the retinal thickness data . one hundred thirteen normal subjects ( 55 male , 58 female ) age 18 years and older were recruited for sd - oct imaging ( mean standard deviation = 27.3 8.3 years ) . forty - three patients ( 18 male , 25 female ) with various retinal pathologies were also recruited ( mean standard deviation = 40.7 20.1 years ) . volumetric sd - oct images of the macula were obtained using the cirrus hd - oct ( carl zeiss meditec , dublin , calif , usa ) . volumes were nominally 6 mm 6 mm and consisted of 128 b - scans ( 512 a - scans / b - scan ) . the internal fixation target of the system was used , which consists of a large green asterisk on a red background , and focus of the lso fundus image was optimized using built - in focus correction . retinal thickness was calculated using the built - in macular analysis software on the cirrus ( software version 5.0 ) , which is automatically determined by taking the difference between the ilm and rpe boundaries . the positions of the foveal center and retinal thickness data from each volume scan were exported for offline analysis using the zeiss cirrus research browser ( version 5.0 ) . all volumes were manually examined for accuracy of the ilm and rpe segmentation and relative accuracy of the autofovea function . in order to evaluate the acquisition and analysis parameters of interest , we needed to be able to manipulate these macular thickness maps off line . to assess the interpolation error in volumetric retinal thickness maps due to decreased b - scan sampling , we created undersampled versions of the retinal thickness volumes exported from the cirrus system . these maps used thickness values from 8 ( every 16th b - scan ) , 16 ( every 8th b - scan ) , 32 ( every 4th b - scan ) , or 64 ( every other b - scan ) of the 128 b - scans initially collected . complete thickness maps were then created by interpolating between these evenly spaced b - scans ( using a matlab spline interpolation function ) . in all etdrs comparisons , most sd - oct systems assume foveal fixation ; however there is frequently significant discrepancy between the location of the fovea and the preferred retinal locus of fixation . we used the autofovea function of the cirrus hd - oct to identify the location of the foveal pit and generated an etdrs plot centered at this location and a second plot centered at the middle of the volume ( the default setting on most other sd - oct systems ) . manual inspection of each volume confirmed that the fovea was identified by the autofovea function ( though in more severe macular pathology we have seen the algorithm fail ) . comparing these two etdrs plots provides an estimate of the potential error due to improper anchoring of the plot to the scan center . moreover , as we had access to the ( x , y ) coordinate of the fovea within each nominal 6 mm 6 mm volume , we examined error as a function of the displacement of each subject 's fixation from the center of his or her foveal pit . the scan length reported by sd - oct systems ( when reported in mm ) is relative , not absolute . however there exist significant individual differences in retinal magnification ( primarily caused by differences in axial length ) ; thus the actual scan length will vary from person to person . in fact , using normative axial length data to correct for ocular magnification , we estimate that approximately one - third of individuals would have a scan length that deviates by more than 0.3 mm from the expected length ( with a maximum deviation of nearly 1 mm ) . we obtained axial length measurements using the zeiss iol master ( carl zeiss meditec , dublin , calif , usa ) and subsequently calibrated the lateral scale of each subject 's sd - oct scans in order to generate revised etdrs plots . these plots were then compared to those derived assuming a 24.46 mm axial length ( that of the cirrus model eye ) . as shown in figure 2 , even a scan using 512 a - scans / b - scan and 128 b - scans only samples 29% of the retinal area within the volume . using 37 high - resolution b - scans results in less than 10% of the retinal area within the volume actually being scanned . as only retina that gets scanned can actually contribute to plots of retinal thickness measurements , this undersampling can significantly affect the integrity of the resultant macular thickness maps . at first glance , assessment of the effect of b - scan density on macular thickness maps suggests that despite reducing the number of b - scans , the general contour of the map remains qualitatively similar ( figure 3(a ) ) . as shown in the normal example , since sampling is being reduced in the vertical direction , the superior and inferior aspects of the fovea show equal magnitude of underrepresentation and overrepresentation of retinal thickness , respectively . this effect is greatly enhanced in a subject with dominant drusen , wherein error is generated not only in the central fovea but also broadly across the retinal volume . we found that in the normal individuals for the central 1 mm subfield , the mean ( standard deviation ) absolute error was 0.19 0.15 m with 64 b - scans , 1.17 0.69 m with 32 b - scans , 7.15 2.35 m with 16 b - scans , and 22.98 7.54 m with 8 b - scans . when expressed as a percentage of subfield thickness we find that the mean percentage error was 0.07 0.06% with 64 b - scans , 0.47 0.29% with 32 b - scans , 2.85 1.08% with 16 b - scans , and 9.19 3.52% with 8 b - scans . for the central 1 mm subfield , the mean ( standard deviation ) absolute error was 0.31 0.36 m with 64 b - scans , 1.36 1.17 m with 32 b - scans , 6.35 3.64 m with 16 b - scans , and 19.56 11.08 m with 8 b - scans . when expressed as a percentage of subfield thickness we find that the mean percentage error was 0.13 0.15% with 64 b - scans , 0.63 0.56% with 32 b - scans , 3.03 2.14% with 16 b - scans , and 9.56 6.92% with 8 b - scans . previous data reveal that the coefficient of repeatability for central subfield measurements on the cirrus is about 4.96 m , indicating that 32 b - scans is sufficient sampling to generate accurate etdrs thickness plots . however , as shown in the difference plots in figure 3 , at neighboring retinal locations where the retinal contour is changing , retinal thickness measurements are in error in opposing directions . in order to quantify the effect of b - scan density on the accuracy of retinal thickness at any given point within the 6 mm 6 mm volume , we examined the error per pixel ( a scan ) within the volume . in this case , the retinal thickness measurements utilizing all 128 b - scans were considered to be absolutely accurate for comparison to the undersampled volumes . at 32 b - scans , our analysis revealed that these interpolation errors could be as high as 5.5 m per pixel and 7.5 m per pixel in the normal and pathology groups , respectively . there are 65,536 pixels in each of our thickness maps , native or undersampled . in both groups , on average , the error per pixel increases as the number of b - scans used to construct the retinal thickness map decreases ( figure 4 ) . we compared etdrs thickness plots derived by placing the center of the etdrs grid on the foveal center to those plots centered on the subject 's actual fixation point . we found these plots to differ by over 100 m in some normal individuals ( sum of the error in all nine etdrs segments ) , with the mean error being 14.4 19.3 m ( figure 5(a ) ) . at their normal fixation position , these subjects had etdrs plots that deviated by 74.5 m and 101.9 m from an etdrs plot precisely positioned at the foveal center ( using our offline matlab program ) . even after the operator acquired a second scan intentionally centered on the fovea to the best of their ability , etdrs errors persisted of 16.3 m and 17.8 m . regardless , for both normal subjects and subjects with retinal pathology , the greater the distance between the fovea and the center of the sd - oct volume , the less accurate the etdrs thickness map . in just the central subfield thickness , not correcting for scan position results in a mean error of 3.18 6.09 m in the normal subjects ( with a maximum error of 32 m ) and 10.50 19.43 m in the patients with retinal pathology ( with a maximum error of 104 m ) . on average , the central subfield error accounts for 14% and 22% of the total etdrs error in the normal and pathology patients , respectively . axial length varied in our normal subjects from 21.56 to 28.36 mm and in pathology patients from 21.87 to 30.13 mm . using each subject axial length to correct the lateral scale of the nominal 6 mm sd - oct scan , we determined that actual scan sizes range from about 5.29 to 6.96 mm for our normal population and 5.36 to 7.4 mm for our pathological population . for subjects with retinal pathology the summed error for the nine etdrs segments was as much as 77.3 m , and 13 out of 43 ( 30% ) showed more than 20 m of total error ( figure 6 ) . in both groups , the error increased with increasing difference in axial length from that of the model eye ( 24.46 mm ) . as illustrated above , not correcting for axial length and not positioning the scan at the center of the fovea introduces significant error in the corresponding etdrs thickness plots . taken together , these artifacts tend to have a cumulative negative effect on the accuracy of the etdrs plots . for example , in considering just the central subfield thickness , not correcting for axial length or scan position results in a mean error of 4.53 5.77 m in the normal subjects ( with a maximum combined error of 33 m ) and 11.29 19.18 m in the patients with retinal pathology ( with a maximum combined error of 105 m ) . this study examined the effects of preventable operational and analytic aspects of the sd - oct on the overall accuracy of etdrs retinal thickness plots . scan density , position of the scan with respect to the foveal center , and magnitude of subject axial length differential all contribute to significant error in computing retinal thickness from sd - oct volumes . an important point to consider is the cumulative nature of the errors reported here ; these parameters should all be accounted for when developing normative databases or analyzing specific retinal features within individual patient data . while the errors were estimated using a single sd - oct device ( cirrus hd - oct ) , they are generic to sd - oct imaging in general . in an examination of b - scan density , sadda et al . concluded that 32 b - scans result in only a minimal change in retinal thickness . our data also show that when examining maps of retinal thickness that are based on spatially integrating individual thickness values ( i.e. , etdrs ) , reduced b - scan sampling has minimal impact . however , if interested in deriving absolute measures of retinal thickness at any given point , reduction to 32 b - scans ( a value suggested to provide accurate retinal thickness maps ) , results in an average error of around 3 m per pixel . while this average error is within the system resolution on commercial sd - oct systems , it is worth keeping in mind that the error at any one pixel can be much larger , since not all pixels will contribute equally to the total error ( which is implicit in computing an average error ) . real cost of undersampling , and this would significantly limit the ability to make precise measurements of retinal features ( e.g. this highlights the importance of considering how the sd - oct data is going to be used when deciding how densely to sample the retina . it is well documented that differences in axial length result in different ocular magnification of retinal images and thus can affect the accuracy of measurements of retinal features . with respect to oct , axial length has been shown to influence measurements of retinal nerve fiber layer ( rnfl ) thickness [ 2831 ] . this of course is based on the fact that rnfl measures are presumed to be taken at a fixed distance from the optic nerve ; thus individual differences in ocular magnificent would result in the rnfl being measured at the wrong location . here we demonstrate that individual differences in ocular magnification also affect the accuracy of macular thickness maps . if the distribution of axial lengths in a normative database does not match that of the subject population being studied , misinterpretation can occur . perhaps more important than retinal thickness maps is the fact that not correcting the nominal scan length for differences in axial length will obviate making reliable measurements in the lateral dimension within a given oct dataset . this could include measuring the area of geographic atrophy , the size of a macular hole , or the size of a druse . despite this , some sd - oct systems still output lateral scale bars on their images that are given in m or provide calipers with which to make lateral measurements in m , despite no correction for axial length having been made . there have also been previous examinations of the effect of fixation on the accuracy of oct thickness measurements . in glaucoma , it has been shown that if the circular scan is not centered on the onh , the rnfl thickness measurements are inaccurate . examined how intentionally shifting the center of macular volume oct scans ( stratus time - domain ) affected central subfield thickness measurements for 10 normal subjects . for our normal subjects , the average decentration of the sd - oct volume with respect to the foveal center was 0.09 mm and the average error of foveal thickness measurements was about 35% . , some discrepancy would be expected given our use of sd - oct ( instead of time domain ) and our ability to precisely determine the exact misalignment between the two scans being compared ( whereas the previous study would have be confounded by errors due to normal fixational instability ) . currently , the cirrus hd - oct will automatically position the etdrs grid over the center of the fovea ( after the scan is taken ) . while this results in a more accurate etdrs map , it may not be valid to compare these maps to a database in which the etdrs maps were not centered on the fovea , though in the case of the cirrus database , good centration of the volume on the fovea was an inclusion criterion . first , in our examination of b - scan sampling , we used 128 b - scans as the truth . this was simply due to a limitation of the specific sd - oct device being used . however , as we showed in figure 2 , 128 b - scans ( at 512 a scans / b - scan ) only sample 29% of the nominal 6 m 6 mm volume . thus these volumes are likely already in error compared to an isotropic volume of 512 b - scans . if one were to really only acquire 32 b - scans , this could affect the accuracy of segmentation as many oct devices use 3d approaches to make correct assignment of layers . a second limitation is that we corrected for ocular magnification using a linear scaling based on axial length . there are other methods to correct for ocular magnification , and the exact method used for the correction would influence the measured differences in retinal image magnification . intuitively , one can conclude that the more uniform the retinal thickness contoured ( as might occur in retinitis pigmentosa , where the retina is uniformly thin ) , the less impact the b - scan sampling , axial length , and scan position would have . likewise , retinal pathology that results in significant peaks and troughs in retinal thickness ( macular holes , amd , diabetic macular edema ) might be more significantly influenced by these parameters . for monitoring patients over time , relative differences in retinal thickness would be generally unaffected by axial length , though comparing populations of patients ( such as in a clinical trial ) where there may be differences in axial length between the groups could result in significant error . if one uses the same sampling density , then the accuracy of these longitudinal measurements of retinal thickness will be on the order of that reported for previous repeatability and reproducibility studies . however , in instances where one is interested in correlating a measure of retinal thickness over a specific retinal area ( e.g.

despite the expansion of maternal and child health programmes , maternal and neonatal morbidity and mortality remains high in developing countries . while many deaths could be prevented , and complications avoided , if women had access to affordable , good - quality health care , the existence of services does not necessarily lead to their utilisation , and utilisation does not necessarily imply quality ( navaneetham and dharmalingam 2002 , agarwal et al . despite the fact that mumbai is india 's urban economic powerhouse , more than half of its 16 million inhabitants live in informal settlements . neonatal mortality among the urban poor is almost twice that of the urban rich ( national neonatology forum and save the children / us 2004 ) . the estimated crude birth rate in mumbai slums is 17.7 per 1000 and the total fertility rate for women aged 1549 is 1.90 . the neonatal mortality rate is 23.6 per 1000 live births and the infant mortality rate 24.9 ( international institute for population sciences and macro international 2008 ) . health systems in urban areas include a diversity of providers offering a variety of services ( lorenz and garner 1995 , harpham and molyneux 2001 ) . the municipal corporation of greater mumbai ( mcgm ) provides more than one - quarter of the approximately 40,000 hospital beds available across the city ( mcgm 2005 ) . mcgm health - care infrastructure includes four teaching hospitals , five specialised hospitals , 16 peripheral general hospitals and 26 maternity homes . primary care is provided through 168 health posts and 162 dispensaries ( brihan mumbai corporation 2009 ) . public providers compete with a burgeoning private sector , which now accounts for 7080% of outpatient consultations ( bhatia and cleland 2001 , barua 2005 , radwan 2005 ) . the informal sector largely comprises unqualified practitioners with no formal training ( de zoysa et al . reports of malpractice , over - medication , inappropriate prescription practices and treatments , and excessive use of diagnostic tests , are not infrequent ( barua 2005 ) . rates of care - seeking in india are high and the number of consultations with health providers exceeds that in the usa ( das et al . understanding maternal health in such settings requires an examination of how health services are used . while much of the literature focuses on the uptake of routine antenatal and delivery care , we know little about care - seeking for maternal morbidity ( uk all party parliamentary group on population , development and reproductive health 2009 ) . this study considered levels and patterns of care - seeking for a variety of health problems experienced by pregnant women living in mumbai 's slums . our objectives were to document their symptoms and associated care - seeking behaviour , to quantify the choice of sector and health provider for each symptom , to test the hypothesis that chosen care - seeking sites for maternal morbidity mirror those used for antenatal care , to analyse care - seeking delays and to quantify referrals between providers . the study sample consisted of slum areas in six municipal wards ( f / north , g / north , h / east , k / west , m / east and p / north ) . of 92 possible areas , 48 were selected randomly ( eight per ward ) , each comprising 10001500 households . eighteen areas were adjacent to hazards such as polluted bodies of water , railway tracks or garbage dumps ; 62% of families owned their homes ; and 74% lived in pucca ( brick or cement ) houses . infrastructurally , 72% of households had a legal electricity supply , 16% had their own water supply , 62% used a communal tap and 21% purchased their water ; and 94% used public toilets ( fernandez and osrin 2006 ) . a vital registration system was set up to identify births , stillbirths , neonatal deaths and maternal deaths ( shah more et al . data for the study originated from a trial approved by the mcgm , the independent ethics committee for research on human subjects ( mumbai committee , reference iec/06/31 ) and the ethics committee of the institute of child health and great ormond street hospital for children . births were identified by 99 locally resident women , generally two per surveillance area , who covered an average of 600 households each . they received an incentive of 50 rupees ( approximately us$1.10 ) for registering each event , which was confirmed by one of 12 interviewers , each responsible for four areas , who visited mothers at home to arrange and administer interviews approximately 6 weeks after delivery . we used a predominantly closed questionnaire to collect information on demography , socioeconomic factors , illnesses experienced during the index pregnancy and care - seeking behaviour . after an explanation of the data - collection activities , participants were asked for verbal consent to be interviewed and assured of the confidentiality of data . team members who encountered illness in mothers or infants had an ethical responsibility to recommend that they visit a health facility . interviews were subject to a range of systematic and random checks for accuracy and completeness , both in the field and during data entry into a relational database management system in microsoft access ( microsoft corporation ) . the original sample size was based on the trial outcome of neonatal mortality , and this analysis was based on 2 years of data collection . we aimed to accumulate 80100 births per cluster per year . the use of reported symptoms to examine care - seeking for serious and minor illnesses presented some problems . first , apparently minor symptoms such as backache or fatigue could be manifestations of a serious illness , and it was not for us to decide whether care - seeking was inappropriate . second , it was unreasonable to assume that women would know when symptoms were likely to be serious . on the basis of lay knowledge described in a series of women 's group discussions ( shah more et al . 2008 ) , we chose four symptoms that we felt would represent serious illness to both clinicians and women and their families : leaking of waters , vaginal bleeding , a feeling that the baby had stopped moving and convulsions or loss of consciousness . trigger symptoms should unequivocally , we felt , lead to consultation with a health - care provider . to examine potential determinants of choice of health - care provider , we did random effects multivariable logistic regression in stata 10 ( statacorp 2007 ) . site of care was entered as the dependent variable , the clustered nature of the sample was accounted for by grouping on the cluster variable , and a range of factors were entered as independent variables . an asset score for socio - economic status was generated from the first component of a principal components analysis ( filmer and pritchett 2001 , vyas and kumaranayake 2006 ) , including variables describing house ownership and construction , possession of a ration card , source of electricity , type of toilet and possession of a range of consumer durables . we entered continuous variables for maternal age and parity , and binary variables for literacy , hindu religion and previous experience of a miscarriage . we considered entering selected trigger and common symptoms , but decided for the sake of clarity to include dummy variables for each of the morbidities discussed in the paper . as dependent variables , we chose the top four sources of care described by respondents : ( 1 ) single - handed private practitioner ; ( 2 ) private hospital ; ( 3 ) public general hospital ; and ( 4 ) public tertiary hospital . the sponsors had no role in the study design , data collection , analysis , interpretation or writing of the article . study author glyn alcock had full access to all study data and final responsibility for the decision to submit for publication . the study sample consisted of slum areas in six municipal wards ( f / north , g / north , h / east , k / west , m / east and p / north ) . of 92 possible areas , 48 were selected randomly ( eight per ward ) , each comprising 10001500 households . eighteen areas were adjacent to hazards such as polluted bodies of water , railway tracks or garbage dumps ; 62% of families owned their homes ; and 74% lived in pucca ( brick or cement ) houses . infrastructurally , 72% of households had a legal electricity supply , 16% had their own water supply , 62% used a communal tap and 21% purchased their water ; and 94% used public toilets ( fernandez and osrin 2006 ) . a vital registration system was set up to identify births , stillbirths , neonatal deaths and maternal deaths ( shah more et al . data for the study originated from a trial approved by the mcgm , the independent ethics committee for research on human subjects ( mumbai committee , reference iec/06/31 ) and the ethics committee of the institute of child health and great ormond street hospital for children . births were identified by 99 locally resident women , generally two per surveillance area , who covered an average of 600 households each . their role was to keep track of local pregnant women and newborn infants . they received an incentive of 50 rupees ( approximately us$1.10 ) for registering each event , which was confirmed by one of 12 interviewers , each responsible for four areas , who visited mothers at home to arrange and administer interviews approximately 6 weeks after delivery . we used a predominantly closed questionnaire to collect information on demography , socioeconomic factors , illnesses experienced during the index pregnancy and care - seeking behaviour . after an explanation of the data - collection activities , participants were asked for verbal consent to be interviewed and assured of the confidentiality of data . team members who encountered illness in mothers or infants had an ethical responsibility to recommend that they visit a health facility . interviews were subject to a range of systematic and random checks for accuracy and completeness , both in the field and during data entry into a relational database management system in microsoft access ( microsoft corporation ) . the original sample size was based on the trial outcome of neonatal mortality , and this analysis was based on 2 years of data collection . we aimed to accumulate 80100 births per cluster per year . the use of reported symptoms to examine care - seeking for serious and minor illnesses presented some problems . first , apparently minor symptoms such as backache or fatigue could be manifestations of a serious illness , and it was not for us to decide whether care - seeking was inappropriate . second , it was unreasonable to assume that women would know when symptoms were likely to be serious . on the basis of lay knowledge described in a series of women 's group discussions ( shah more et al . 2008 ) , we chose four symptoms that we felt would represent serious illness to both clinicians and women and their families : leaking of waters , vaginal bleeding , a feeling that the baby had stopped moving and convulsions or loss of consciousness . trigger symptoms should unequivocally , we felt , lead to consultation with a health - care provider . to examine potential determinants of choice of health - care provider , we did random effects multivariable logistic regression in stata 10 ( statacorp 2007 ) . site of care was entered as the dependent variable , the clustered nature of the sample was accounted for by grouping on the cluster variable , and a range of factors were entered as independent variables . an asset score for socio - economic status was generated from the first component of a principal components analysis ( filmer and pritchett 2001 , vyas and kumaranayake 2006 ) , including variables describing house ownership and construction , possession of a ration card , source of electricity , type of toilet and possession of a range of consumer durables . we entered continuous variables for maternal age and parity , and binary variables for literacy , hindu religion and previous experience of a miscarriage . we considered entering selected trigger and common symptoms , but decided for the sake of clarity to include dummy variables for each of the morbidities discussed in the paper . as dependent variables , we chose the top four sources of care described by respondents : ( 1 ) single - handed private practitioner ; ( 2 ) private hospital ; ( 3 ) public general hospital ; and ( 4 ) public tertiary hospital . the sponsors had no role in the study design , data collection , analysis , interpretation or writing of the article . study author glyn alcock had full access to all study data and final responsibility for the decision to submit for publication . we recorded 13,467 births between 1 october 2005 and 30 september 2007 , for which detailed information was available on 10,754 ( 80% ) . the main reasons for loss to follow - up were relocation or that women who lived elsewhere had come to the area for delivery . the mean age of respondents was 24 , and 47% were hindu and 46% muslim . data showed that 28% had not attended school , while 57% had completed secondary school and 8% had attended higher education . most lived in a nuclear family ( 54% ) or joint family ( 45% ) ; 17% of women had lived in the community for less than a year , 51% between 1 and 5 years and 32% 6 years or more . only 15% of women had engaged in paid work during the previous 12 months , mostly home - based or as domestic servants . altogether , 6471 ( 60% ) women reported health problems during the index pregnancy . table 1 summarises the four trigger symptoms and the six most commonly reported symptoms : vomiting or diarrhoea ( 40% ) , tiredness or weakness ( 32% ) , headache ( 29% ) , swollen legs ( 25% ) , abdominal pain ( 24% ) and backache ( 16% ) . the table also shows the proportion of women who sought care for each symptom outside the home . institutional care - seeking was high , from 82% ( swollen legs ) to 91% ( abdominal pain ) for common symptoms , and from 88% ( leaking waters ) to 100% ( convulsions or unconsciousness ) for trigger symptoms . exclusive home treatment was minimal ( < 2% ) , and most women who did choose home care also consulted a health provider ; 15% took no curative action . the main reasons for not seeking care for two common and two trigger symptoms are presented in table 2 . the most common reason ( 6483% of responses ) was that the woman had not felt a need to seek medical care for her condition . other important reasons were that she recovered , that family members did not allow her to seek care , and that there was nobody to look after the children . the severity of the condition did not significantly alter the reasons for not seeking care . figure 1 presents graphically the choice of health provider for 4917 women who sought treatment for morbidity during pregnancy at a health - care facility in mumbai . the data using nine of the symptoms identified in table 1 first , while care - seeking sites for three of the trigger symptoms were split evenly between the private and public sectors , there was an overall preference for private health care . this was most marked for the treatment of vaginal bleeding and vomiting or diarrhoea , for which 64% and 62% of clients , respectively , sought treatment in the private sector . almost a third of clients sought treatment for convulsions or unconsciousness at a general hospital . the municipal tertiary hospital and maternity homes were the next most commonly utilised sites , while the use of urban health centres and health posts was low . selected self - reported health problems during pregnancy and institutional care - seeking , for 10,754 women in 48 slum areas , 20052007 . percentages sum to > 100% because most women reported more than one symptom during pregnancy . we examined potential determinants of the chosen site of care in random effects multivariable regression models . there was a clear association between the municipal ward of residence and the source of care . use of single - handed private practitioners was greatest in f / north ward and least in k / west . use of private hospitals was lowest in f / north and greatest in k / west . use of a general hospital was also lowest in f / north and greatest in h / east and use of public - sector maternity homes was lowest in h / east ward and highest in k / west . there was a consistent association of source of care with socio - economic status , although this did not hold for use of single - handed practitioners . increasing wealth was associated with greater use of private hospitals and less use of public general hospitals and maternity homes . for example , the association of greater maternal age with more use of private hospitals and less use of public general hospitals , of parity with more use of public general hospitals , of male infants ( which technically should be unknown during pregnancy ) with less use of private practitioners and of maternal literacy with less use of public maternity homes . there was also some variation between sources of care for common symptoms : diarrhoea or vomiting was associated with more visits to single - handed private practitioners , leg swelling and abdominal pain with fewer such visits and abdominal pain with more visits to public general hospitals . for trigger symptoms , there was a borderline association of leaking waters with fewer visits to single - handed practitioners , and an association of vaginal bleeding with more visits to private hospitals . reasons for not seeking care at a health facility for selected symptoms , for 6471 women in 48 slum areas , 20052007 . figure 2 presents graphically mobility between sectors for women who had both antenatal and curative care in mumbai ( n = 4686 ) . the tendency to seek curative care in the same sector was clear : 46% of women who had received antenatal care in the private sector received treatment in the same sector and 36% received both types of care in the public sector . more clients switched from public to private sector ( 11% ) than from private to public ( 7% ) . further breakdown of the data showed that , of the clients who remained in the private sector , 83% did so at a private hospital and 61% with an individual practitioner . in the public sector , continuation was most common at general hospitals ( 78% ) , the municipal tertiary or government hospitals ( 72% ) and maternity homes ( 69% ) . although continuation was also high at urban health centres ( 61% ) , absolute numbers were low . although we were not able to disaggregate the data into the three delays ( deciding to seek care , reaching a health facility and receiving medical care ; thaddeus and maine 1994 ) , we were able to identify a general pattern across symptoms ( figure 3 ) . most women sought and received treatment within 2 days : 53% who sought care for vaginal bleeding were seen by a health provider within 48 hours and 31% within 24 hours ; for backache , 45% sought care within 48 hours and 17% within 24 hours . less than 2% of clients who sought care for any of the reported symptoms at either public or private providers were referred or transferred to another health facility . choice of health provider for selected symptoms , for 4917 women in 48 slum areas , 20052007 . note : key to public health facilities : tertiary / government hospital large hospital ( > 1000 beds ) and teaching college providing a full range of inpatient and outpatient care and specialised services ; general hospital peripheral hospital ( 50570 beds ) offering a range of inpatient and outpatient care . ten have obstetric wards for antenatal , delivery and postnatal care ; maternity home smaller facility ( 10134 beds ) providing maternal and child health services ; health post community - based facility offering primary health - care services ; and uhc urban health centre offering primary and secondary health - care services . our findings suggest that women and their families made informed choices about care - seeking when health problems arose during pregnancy . symptoms suggesting serious morbidity were reported in fewer than 15% of cases . institutional care - seeking was high across the board ( > 80% ) and slightly higher for trigger symptoms ( > 88% ) . delays were uncommon , and more rapid consultation and treatment was described for trigger symptoms . where comparison is possible , the levels of morbidity concur with national survey data from urban india . these showed that , of women who had given birth in the last 5 years , 5% reported suffering vaginal bleeding , 7% convulsions ( unassociated with fever ) and 28% swelling of the legs , body or face ( international institute for population sciences and macro international 2007 ) . the strongest determinant of site of care was residential location , followed by socio - economic status . the findings for location are consistent with our knowledge of service distribution . in general , use of private hospitals was lowest in f / north ward , the site of a tertiary public hospital with a good reputation , and highest in k / west , the wealthiest of the wards . use of a general hospital was highest in h / east , which has a reputable municipal hospital . public - sector maternity homes were used least in h / east ( where there are none ) , and most in k / west , the site of a large maternity home generally perceived to offer quality care . single - handed private practitioners were consulted across the board , but were perhaps more likely to be chosen for symptoms perceived as less pregnancy - related , such as diarrhoea and vomiting . overall , the wealthier her family , the more likely a pregnant woman was to seek care at a private hospital . random effects multivariable logistic regression models for four choices of health care provider for illness during pregnancy , for 4917 observations in 48 slum areas , 20052007 . note : or , adjusted odds ratio ; ci , confidence interval . choice of health sector and provider for curative care in relation to antenatal care site , for 4686 women in 48 slum areas , 20052007 . note : the size of the outermost shaded box in each quadrant is proportional to the number of clients in the quadrant . within each box , bars indicate the percentage of the quadrant total who sought care at a given type of institution . the strengths of the study were that it was community - based and involved a sample of more than 10,000 women recruited over 2 years . the accuracy of diagnoses could not be verified and our classification of symptoms into common and trigger categories were based on respondents own descriptions . in a study with the same group of women , we reported high uptake of antenatal care ( 93% ) and institutional delivery ( 90% ) ( shah more et al . in contrast to studies from other developing countries , which show a clear tendency for home - based treatment over facility care ( develay et al . 2007 ) , our work in mumbai led us to anticipate high levels of health service use for maternal morbidity . that utilisation rates ranged from 82 to 100% confirmed our hypothesis , as do the findings of the national family health survey ( international institute for population sciences and macro international 2007 ) , and previous community - based research in urban slums ( yesudian 1988 , de zoysa et al . compared to antenatal morbidity , reproductive health problems among women in india 's urban slums are common ( garg et al . 2002 , bhanderi and kannan 2010 ) , as are other conditions such as anaemia ( mayank et al . symptoms that are unrecognised , not thought to be serious or considered normal may lead to underreporting and limited care - seeking . taboos also render some reproductive health problems invisible and women are often expected to endure them ( garg et al . responses to problems during pregnancy may differ because of a perceived risk to the unborn child , and this may help to explain the higher levels of care - seeking . for example , one study showed that women in a delhi slum generally sought care for obstetric morbidity even though their ability to recognise symptoms of serious complications was poor ( mayank et al . several studies affirm the urban preference for private - sector care ( aljunid 1995 , gupta and dasgupta 2000 , bhatia and cleland 2001 ) . among the reasons for this are ease of accessibility , convenient opening hours and a perception that the quality of care is higher ( bennett 1996 , barua 2005 , habtom and ruys 2007 ) . although the use of private facilities is limited by the ability to pay ( shah more et al . 2009a ) , the willingness to meet the costs may be explained by clients expectations that they will receive superior service and more courteous treatment ( the world bank 1996 , de zoysa et al . our previous research showed an association between routine private - sector maternity care and rising socio - economic status ( shah more et al . the least poor chose private hospitals for morbidity care over other types of facility , suggesting that financial constraints do play a part in influencing the choice of provider ( kausar et al . it is estimated that there are well over 1.25 million unqualified practitioners in india ( radwan 2005 ) . we do not know the qualifications of the private practitioners visited by women in our study . however , research across india suggests that most have minimal training , and that individuals trained in non - biomedical disciplines often practice allopathy ( de zoysa et al . importantly , clients may not distinguish between qualified and unqualified practitioners ( de zoysa et al . care - seeking delays for selected symptoms , for 4917 women in 48 slum areas , 20052007 . proportions less than 15% are not indicated . despite the negative publicity that government health facilities have received ( gupta and dasgupta 2000 , barua 2005 , de costa and diwan 2007 ) , our findings show that the public sector is still an important health - care provider . utilisation of municipal hospitals and maternity homes was higher in areas where they were more easily accessible and provided the necessary level of care . similar research in another low - income area of mumbai reported that choice of health - care provider was mediated by accessibility , affordability and quality of services , and that a shortfall in the availability of public facilities left some with no option other than to seek private care ( dilip and duggal 2004 ) . the demand for health - care services for mumbai 's expanding population exceeds the supply of public - health infrastructure a fact acknowledged by the city 's municipal administration ( mcgm 2005 ) and it is plausible that a corresponding increase in the number of peripheral public facilities would result in greater utilisation ( dilip and duggal 2004 ) . despite their easy access , we think that the underutilisation of community - based primary health facilities is largely attributable to their limited services and personnel , poor perceptions of quality and the tendency to seek antenatal and delivery care with the same provider . conversely , the greater use of larger municipal hospitals exacerbates the problems of crowded outpatient departments , queuing , shorter consultation times and the loss of time that results from travelling further from home . these very factors dissuade people from seeking care in a sector that exists to serve them . when faced with a health problem during pregnancy , women did not seem to face major barriers to accessing care . rather than waiting for their next antenatal consultation , few were referred to another provider . while this may be due to a poor referral system ( barua 2005 ) , or women 's reluctance to accept referrals ( de zoysa et al . 1998 ) , we propose that most families care - seeking choices are based on rational decisions about health problems in pregnancy , and that they can usually access the necessary resources to seek care ( matthews et al . furthermore , they are able to navigate a complex health system that comprises multiple sources and types of medical care . our findings suggest that the urban poor recognise symptoms of obstetric complications and understand the need for health care . however , more than 80% of women sought care for non - life - threatening conditions such as tiredness and backache . we are not in a position to judge this , but it seems likely that it reflects a broader picture of care - seeking for all illnesses . we think that the propensity to choose institutional care over self - treatment may reflect acculturation to life in a megacity such as mumbai , a process which might be termed modernisation through migration ( basu 1990 ) . an important question arising from the study is whether the proliferation of private health - care providers in poor urban areas contributes to the medicalisation of pregnancy . that women in mumbai 's urban slums are able to choose from a wide range of health - care providers and that utilisation is high is encouraging . however , access to public facilities is uneven and whether high levels of care - seeking result in better health outcomes are matters for debate ( das et al . seeking care for minor illnesses that could successfully be treated without recourse to a health practitioner places additional financial and social burdens on the poorest and can delay treatment of those with more urgent conditions . conversely , the main reasons for not seeking care suggest that at least some women do not recognise the danger signs of problems during pregnancy , or do not have sufficient mobility or social support to enable them to visit a health provider . in this respect , we are currently conducting a study to quantify women 's agency and its effect on care - seeking behaviour , and we have been working with women 's groups to improve maternal and newborn health practices and encourage appropriate health - care seeking ( shah more et al . the important role that private providers play in the provision of health care for the urban poor needs greater recognition , and ways in which the private and public sectors might collaborate merit investigation . further research is needed on provider activities and behaviour to facilitate effective regulation and improve quality of care . how and why expectant mothers and their families make their care - seeking choices in urban slums , where multiple providers and levels of care coexist , also require more detailed investigation .

this study considers care - seeking patterns for maternal morbidity in mumbai 's slums . our objectives were to document women 's self - reported symptoms and care - seeking , and to quantify their choice of health provider , care - seeking delays and referrals between providers . the hypothesis that care - seeking sites for maternal morbidity mirror those used for antenatal care was also tested . we analysed data for 10,754 births in 48 slum areas and interviewed mothers about their illnesses and care - seeking during pregnancy . institutional care - seeking was high across the board ( > 80% ) , and higher for trigger symptoms suggestive of complications ( > 88% ) . private - sector care was preferred , and increased with socio - economic status , although public providers also played an important role . most women sought treatment at the same site they received their antenatal care , most were treated within 2 days , and less than 2% were referred to other providers . our findings suggest that poor women in mumbai recognise symptoms of obstetric complications and the need for health care . however , that more than 80% also sought care for minor conditions implies that the tendency to seek institutional care for serious conditions reflects a broader picture of care - seeking for all illnesses . the role of private health - care providers needs greater recognition , and further research is required on provider motivations and behaviour .

Introduction Methods Participants Procedures Analysis Role of the funding source Results Discussion

while many deaths could be prevented , and complications avoided , if women had access to affordable , good - quality health care , the existence of services does not necessarily lead to their utilisation , and utilisation does not necessarily imply quality ( navaneetham and dharmalingam 2002 , agarwal et al . despite the fact that mumbai is india 's urban economic powerhouse , more than half of its 16 million inhabitants live in informal settlements . the estimated crude birth rate in mumbai slums is 17.7 per 1000 and the total fertility rate for women aged 1549 is 1.90 . the municipal corporation of greater mumbai ( mcgm ) provides more than one - quarter of the approximately 40,000 hospital beds available across the city ( mcgm 2005 ) . mcgm health - care infrastructure includes four teaching hospitals , five specialised hospitals , 16 peripheral general hospitals and 26 maternity homes . public providers compete with a burgeoning private sector , which now accounts for 7080% of outpatient consultations ( bhatia and cleland 2001 , barua 2005 , radwan 2005 ) . reports of malpractice , over - medication , inappropriate prescription practices and treatments , and excessive use of diagnostic tests , are not infrequent ( barua 2005 ) . rates of care - seeking in india are high and the number of consultations with health providers exceeds that in the usa ( das et al . while much of the literature focuses on the uptake of routine antenatal and delivery care , we know little about care - seeking for maternal morbidity ( uk all party parliamentary group on population , development and reproductive health 2009 ) . this study considered levels and patterns of care - seeking for a variety of health problems experienced by pregnant women living in mumbai 's slums . our objectives were to document their symptoms and associated care - seeking behaviour , to quantify the choice of sector and health provider for each symptom , to test the hypothesis that chosen care - seeking sites for maternal morbidity mirror those used for antenatal care , to analyse care - seeking delays and to quantify referrals between providers . the study sample consisted of slum areas in six municipal wards ( f / north , g / north , h / east , k / west , m / east and p / north ) . of 92 possible areas , 48 were selected randomly ( eight per ward ) , each comprising 10001500 households . data for the study originated from a trial approved by the mcgm , the independent ethics committee for research on human subjects ( mumbai committee , reference iec/06/31 ) and the ethics committee of the institute of child health and great ormond street hospital for children . we used a predominantly closed questionnaire to collect information on demography , socioeconomic factors , illnesses experienced during the index pregnancy and care - seeking behaviour . the original sample size was based on the trial outcome of neonatal mortality , and this analysis was based on 2 years of data collection . the use of reported symptoms to examine care - seeking for serious and minor illnesses presented some problems . first , apparently minor symptoms such as backache or fatigue could be manifestations of a serious illness , and it was not for us to decide whether care - seeking was inappropriate . on the basis of lay knowledge described in a series of women 's group discussions ( shah more et al . 2008 ) , we chose four symptoms that we felt would represent serious illness to both clinicians and women and their families : leaking of waters , vaginal bleeding , a feeling that the baby had stopped moving and convulsions or loss of consciousness . trigger symptoms should unequivocally , we felt , lead to consultation with a health - care provider . to examine potential determinants of choice of health - care provider , we did random effects multivariable logistic regression in stata 10 ( statacorp 2007 ) . site of care was entered as the dependent variable , the clustered nature of the sample was accounted for by grouping on the cluster variable , and a range of factors were entered as independent variables . an asset score for socio - economic status was generated from the first component of a principal components analysis ( filmer and pritchett 2001 , vyas and kumaranayake 2006 ) , including variables describing house ownership and construction , possession of a ration card , source of electricity , type of toilet and possession of a range of consumer durables . we entered continuous variables for maternal age and parity , and binary variables for literacy , hindu religion and previous experience of a miscarriage . as dependent variables , we chose the top four sources of care described by respondents : ( 1 ) single - handed private practitioner ; ( 2 ) private hospital ; ( 3 ) public general hospital ; and ( 4 ) public tertiary hospital . of 92 possible areas , 48 were selected randomly ( eight per ward ) , each comprising 10001500 households . data for the study originated from a trial approved by the mcgm , the independent ethics committee for research on human subjects ( mumbai committee , reference iec/06/31 ) and the ethics committee of the institute of child health and great ormond street hospital for children . we used a predominantly closed questionnaire to collect information on demography , socioeconomic factors , illnesses experienced during the index pregnancy and care - seeking behaviour . the original sample size was based on the trial outcome of neonatal mortality , and this analysis was based on 2 years of data collection . the use of reported symptoms to examine care - seeking for serious and minor illnesses presented some problems . first , apparently minor symptoms such as backache or fatigue could be manifestations of a serious illness , and it was not for us to decide whether care - seeking was inappropriate . 2008 ) , we chose four symptoms that we felt would represent serious illness to both clinicians and women and their families : leaking of waters , vaginal bleeding , a feeling that the baby had stopped moving and convulsions or loss of consciousness . trigger symptoms should unequivocally , we felt , lead to consultation with a health - care provider . to examine potential determinants of choice of health - care provider , we did random effects multivariable logistic regression in stata 10 ( statacorp 2007 ) . site of care was entered as the dependent variable , the clustered nature of the sample was accounted for by grouping on the cluster variable , and a range of factors were entered as independent variables . an asset score for socio - economic status was generated from the first component of a principal components analysis ( filmer and pritchett 2001 , vyas and kumaranayake 2006 ) , including variables describing house ownership and construction , possession of a ration card , source of electricity , type of toilet and possession of a range of consumer durables . we entered continuous variables for maternal age and parity , and binary variables for literacy , hindu religion and previous experience of a miscarriage . as dependent variables , we chose the top four sources of care described by respondents : ( 1 ) single - handed private practitioner ; ( 2 ) private hospital ; ( 3 ) public general hospital ; and ( 4 ) public tertiary hospital . the mean age of respondents was 24 , and 47% were hindu and 46% muslim . most lived in a nuclear family ( 54% ) or joint family ( 45% ) ; 17% of women had lived in the community for less than a year , 51% between 1 and 5 years and 32% 6 years or more . table 1 summarises the four trigger symptoms and the six most commonly reported symptoms : vomiting or diarrhoea ( 40% ) , tiredness or weakness ( 32% ) , headache ( 29% ) , swollen legs ( 25% ) , abdominal pain ( 24% ) and backache ( 16% ) . the table also shows the proportion of women who sought care for each symptom outside the home . institutional care - seeking was high , from 82% ( swollen legs ) to 91% ( abdominal pain ) for common symptoms , and from 88% ( leaking waters ) to 100% ( convulsions or unconsciousness ) for trigger symptoms . exclusive home treatment was minimal ( < 2% ) , and most women who did choose home care also consulted a health provider ; 15% took no curative action . the main reasons for not seeking care for two common and two trigger symptoms are presented in table 2 . the most common reason ( 6483% of responses ) was that the woman had not felt a need to seek medical care for her condition . other important reasons were that she recovered , that family members did not allow her to seek care , and that there was nobody to look after the children . figure 1 presents graphically the choice of health provider for 4917 women who sought treatment for morbidity during pregnancy at a health - care facility in mumbai . the data using nine of the symptoms identified in table 1 first , while care - seeking sites for three of the trigger symptoms were split evenly between the private and public sectors , there was an overall preference for private health care . this was most marked for the treatment of vaginal bleeding and vomiting or diarrhoea , for which 64% and 62% of clients , respectively , sought treatment in the private sector . selected self - reported health problems during pregnancy and institutional care - seeking , for 10,754 women in 48 slum areas , 20052007 . percentages sum to > 100% because most women reported more than one symptom during pregnancy . there was a clear association between the municipal ward of residence and the source of care . use of a general hospital was also lowest in f / north and greatest in h / east and use of public - sector maternity homes was lowest in h / east ward and highest in k / west . there was a consistent association of source of care with socio - economic status , although this did not hold for use of single - handed practitioners . increasing wealth was associated with greater use of private hospitals and less use of public general hospitals and maternity homes . for example , the association of greater maternal age with more use of private hospitals and less use of public general hospitals , of parity with more use of public general hospitals , of male infants ( which technically should be unknown during pregnancy ) with less use of private practitioners and of maternal literacy with less use of public maternity homes . there was also some variation between sources of care for common symptoms : diarrhoea or vomiting was associated with more visits to single - handed private practitioners , leg swelling and abdominal pain with fewer such visits and abdominal pain with more visits to public general hospitals . for trigger symptoms , there was a borderline association of leaking waters with fewer visits to single - handed practitioners , and an association of vaginal bleeding with more visits to private hospitals . reasons for not seeking care at a health facility for selected symptoms , for 6471 women in 48 slum areas , 20052007 . figure 2 presents graphically mobility between sectors for women who had both antenatal and curative care in mumbai ( n = 4686 ) . the tendency to seek curative care in the same sector was clear : 46% of women who had received antenatal care in the private sector received treatment in the same sector and 36% received both types of care in the public sector . in the public sector , continuation was most common at general hospitals ( 78% ) , the municipal tertiary or government hospitals ( 72% ) and maternity homes ( 69% ) . although continuation was also high at urban health centres ( 61% ) , absolute numbers were low . although we were not able to disaggregate the data into the three delays ( deciding to seek care , reaching a health facility and receiving medical care ; thaddeus and maine 1994 ) , we were able to identify a general pattern across symptoms ( figure 3 ) . most women sought and received treatment within 2 days : 53% who sought care for vaginal bleeding were seen by a health provider within 48 hours and 31% within 24 hours ; for backache , 45% sought care within 48 hours and 17% within 24 hours . less than 2% of clients who sought care for any of the reported symptoms at either public or private providers were referred or transferred to another health facility . choice of health provider for selected symptoms , for 4917 women in 48 slum areas , 20052007 . ten have obstetric wards for antenatal , delivery and postnatal care ; maternity home smaller facility ( 10134 beds ) providing maternal and child health services ; health post community - based facility offering primary health - care services ; and uhc urban health centre offering primary and secondary health - care services . our findings suggest that women and their families made informed choices about care - seeking when health problems arose during pregnancy . institutional care - seeking was high across the board ( > 80% ) and slightly higher for trigger symptoms ( > 88% ) . delays were uncommon , and more rapid consultation and treatment was described for trigger symptoms . the strongest determinant of site of care was residential location , followed by socio - economic status . in general , use of private hospitals was lowest in f / north ward , the site of a tertiary public hospital with a good reputation , and highest in k / west , the wealthiest of the wards . public - sector maternity homes were used least in h / east ( where there are none ) , and most in k / west , the site of a large maternity home generally perceived to offer quality care . single - handed private practitioners were consulted across the board , but were perhaps more likely to be chosen for symptoms perceived as less pregnancy - related , such as diarrhoea and vomiting . overall , the wealthier her family , the more likely a pregnant woman was to seek care at a private hospital . random effects multivariable logistic regression models for four choices of health care provider for illness during pregnancy , for 4917 observations in 48 slum areas , 20052007 . choice of health sector and provider for curative care in relation to antenatal care site , for 4686 women in 48 slum areas , 20052007 . within each box , bars indicate the percentage of the quadrant total who sought care at a given type of institution . the strengths of the study were that it was community - based and involved a sample of more than 10,000 women recruited over 2 years . in a study with the same group of women , we reported high uptake of antenatal care ( 93% ) and institutional delivery ( 90% ) ( shah more et al . 2007 ) , our work in mumbai led us to anticipate high levels of health service use for maternal morbidity . that utilisation rates ranged from 82 to 100% confirmed our hypothesis , as do the findings of the national family health survey ( international institute for population sciences and macro international 2007 ) , and previous community - based research in urban slums ( yesudian 1988 , de zoysa et al . compared to antenatal morbidity , reproductive health problems among women in india 's urban slums are common ( garg et al . symptoms that are unrecognised , not thought to be serious or considered normal may lead to underreporting and limited care - seeking . responses to problems during pregnancy may differ because of a perceived risk to the unborn child , and this may help to explain the higher levels of care - seeking . for example , one study showed that women in a delhi slum generally sought care for obstetric morbidity even though their ability to recognise symptoms of serious complications was poor ( mayank et al . several studies affirm the urban preference for private - sector care ( aljunid 1995 , gupta and dasgupta 2000 , bhatia and cleland 2001 ) . among the reasons for this are ease of accessibility , convenient opening hours and a perception that the quality of care is higher ( bennett 1996 , barua 2005 , habtom and ruys 2007 ) . our previous research showed an association between routine private - sector maternity care and rising socio - economic status ( shah more et al . we do not know the qualifications of the private practitioners visited by women in our study . however , research across india suggests that most have minimal training , and that individuals trained in non - biomedical disciplines often practice allopathy ( de zoysa et al . care - seeking delays for selected symptoms , for 4917 women in 48 slum areas , 20052007 . despite the negative publicity that government health facilities have received ( gupta and dasgupta 2000 , barua 2005 , de costa and diwan 2007 ) , our findings show that the public sector is still an important health - care provider . utilisation of municipal hospitals and maternity homes was higher in areas where they were more easily accessible and provided the necessary level of care . similar research in another low - income area of mumbai reported that choice of health - care provider was mediated by accessibility , affordability and quality of services , and that a shortfall in the availability of public facilities left some with no option other than to seek private care ( dilip and duggal 2004 ) . the demand for health - care services for mumbai 's expanding population exceeds the supply of public - health infrastructure a fact acknowledged by the city 's municipal administration ( mcgm 2005 ) and it is plausible that a corresponding increase in the number of peripheral public facilities would result in greater utilisation ( dilip and duggal 2004 ) . despite their easy access , we think that the underutilisation of community - based primary health facilities is largely attributable to their limited services and personnel , poor perceptions of quality and the tendency to seek antenatal and delivery care with the same provider . conversely , the greater use of larger municipal hospitals exacerbates the problems of crowded outpatient departments , queuing , shorter consultation times and the loss of time that results from travelling further from home . rather than waiting for their next antenatal consultation , few were referred to another provider . while this may be due to a poor referral system ( barua 2005 ) , or women 's reluctance to accept referrals ( de zoysa et al . 1998 ) , we propose that most families care - seeking choices are based on rational decisions about health problems in pregnancy , and that they can usually access the necessary resources to seek care ( matthews et al . our findings suggest that the urban poor recognise symptoms of obstetric complications and understand the need for health care . however , more than 80% of women sought care for non - life - threatening conditions such as tiredness and backache . we are not in a position to judge this , but it seems likely that it reflects a broader picture of care - seeking for all illnesses . we think that the propensity to choose institutional care over self - treatment may reflect acculturation to life in a megacity such as mumbai , a process which might be termed modernisation through migration ( basu 1990 ) . an important question arising from the study is whether the proliferation of private health - care providers in poor urban areas contributes to the medicalisation of pregnancy . that women in mumbai 's urban slums are able to choose from a wide range of health - care providers and that utilisation is high is encouraging . however , access to public facilities is uneven and whether high levels of care - seeking result in better health outcomes are matters for debate ( das et al . seeking care for minor illnesses that could successfully be treated without recourse to a health practitioner places additional financial and social burdens on the poorest and can delay treatment of those with more urgent conditions . conversely , the main reasons for not seeking care suggest that at least some women do not recognise the danger signs of problems during pregnancy , or do not have sufficient mobility or social support to enable them to visit a health provider . in this respect , we are currently conducting a study to quantify women 's agency and its effect on care - seeking behaviour , and we have been working with women 's groups to improve maternal and newborn health practices and encourage appropriate health - care seeking ( shah more et al . the important role that private providers play in the provision of health care for the urban poor needs greater recognition , and ways in which the private and public sectors might collaborate merit investigation . further research is needed on provider activities and behaviour to facilitate effective regulation and improve quality of care . how and why expectant mothers and their families make their care - seeking choices in urban slums , where multiple providers and levels of care coexist , also require more detailed investigation .

catalytic swimming devices are small scale , untethered colloids capable of autonomously generating motion in fluidic environments . these devices are attracting significant research interest as they have the potential to enable exciting new functions such as drug delivery , lab - on a chip transport and environmental remediation . these particles get their name from having two distinct sides , or faces ( janus is a two faced roman god ) . one side is catalytically active and able to perform a decomposition reaction , while the other is inert . in the presence of suitable dissolved fuel molecules , the resulting asymmetric chemical reaction creates gradients around the colloids which can produce motion via self - diffusiophoresis / electrophoresis . characterizing the motion for these rapidly moving objects is challenging and many experimental observations to date have been limited to 2d . however , eventual applications are likely to exploit catalytic swimming devices ability to move throughout bulk solutions in 3d . to address this , here we describe a protocol that allows accurate 3d trajectories for swimming devices to be determined . this method is based on interpreting the ring structures produced by out of focus fluorescent colloids observed with a fixed focus objective , and is easy to apply using conventional unmodified microscopes . by clearly describing this method here , other researchers in this field will benefit by being able to access such 3d information . evidence of this potential is given by the recent report of swimming devices being directed by gravity , behavior which can most easily be visualized through the application of 3d tracking . this paper also clearly documents a method to manufacture catalytic janus particle swimming devices , which will be of further benefit to standardize methods across the existing research groups investigating these devices , and additionally guide new researchers interested in making and investigating swimming devices . hydrogen peroxide used in this protocol is harmful , and the evolution of oxygen gas when exposed to platinum poses an explosion risk . use all appropriate safety controls during this protocol including engineering controls while handling peroxide solutions ( fume hood ) and personal protective equipment ( safety glasses , gloves and lab coat ) . prepare glass slide substrate clean an unused standard microscope slide using sequential washing for a few minutes each in deionized ( di ) water , glass decontamination solution and di water . then wash with a 70:30 v : v mixture of ethanol : di water , and finally blow dry in a clean air / nitrogen stream.examine the glass slide under a microscope to verify the surface is clean and free from evidence of particulate contamination . clean an unused standard microscope slide using sequential washing for a few minutes each in deionized ( di ) water , glass decontamination solution and di water . then wash with a 70:30 v : v mixture of ethanol : di water , and finally blow dry in a clean air / nitrogen stream . examine the glass slide under a microscope to verify the surface is clean and free from evidence of particulate contamination . prepare colloidal dispersion for deposition pipette 10 l of aqueous stock fluorescent colloid solution ( 10% wt . ) into 990 l of ethanol . adjust volumes as required depending on stock solution concentration used to arrive at approximately 0.1% wt colloidal suspension.vortex mix for 10 sec . pipette 10 l of aqueous stock fluorescent colloid solution ( 10% wt . ) into 990 l of ethanol . adjust volumes as required depending on stock solution concentration used to arrive at approximately 0.1% wt colloidal suspension . spin coat colloidal dispersion onto glass slide substrate equip a spin coater with the clean glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide.remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide . remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . vacuum evaporate platinum metal onto colloid decorated glass slide insert the colloid coated glass slide into a metal evaporator . ensure the colloid decorated side is facing the evaporation source . note : following metal deposition , samples should be stored under an inert atmosphere . insert the colloid coated glass slide into a metal evaporator . ensure the colloid decorated side is facing the evaporation source . note : following metal deposition , samples should be stored under an inert atmosphere . re - suspend janus colloids into a peroxide containing solution cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate).insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . remove lens tissue.pipette 1 ml of the colloid containing solution into a new container , filled with 1 ml of 30% w / v h2o2 stock solution . gently mix the solutions , and then place in an ultrasonic bath at room temperature for 5 min , followed by another 25 min unstirred incubation period . caution : this solution may evolve oxygen ; do not seal.dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate ) . insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . remove lens tissue . pipette 1 ml of the colloid containing solution into a new container , filled with 1 ml of 30% w / v h2o2 stock solution . gently mix the solutions , and then place in an ultrasonic bath at room temperature for 5 min , followed by another 25 min unstirred incubation period . dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . prepare analysis cuvette add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . note : the prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface.fill a low volume rectangular quartz glass cuvette with the incubated solution . add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . note : the prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface . fill a low volume rectangular quartz glass cuvette with the incubated solution . loosely fit a push - in cap . locate particle of interest load cuvette into a fluorescent microscope equipped with a suitable objective ( e.g. , 20x ) and excite fluorophore emission using a suitable combination of filters ( excitation 450 - 490 nm , emission > 515 nm).manually search for fluorescent colloids within the cuvette . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present . a colloid volume concentration of about 0.003% is a recommended starting point.optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . load cuvette into a fluorescent microscope equipped with a suitable objective ( e.g. , 20x ) and excite fluorophore emission using a suitable combination of filters ( excitation 450 - 490 nm , emission > 515 nm ) . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . record video before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . do not move the focus plane during video capture.record videos of particles of interest . use 30 sec video durations with frame rates in excess of 30 hz to allow detailed trajectory reconstruction . before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . record videos of particles of interest . use 30 sec video durations with frame rates in excess of 30 hz to allow detailed trajectory reconstruction . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy.plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy.plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids . focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane . determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center can then be found . this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy . plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2 . measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . prepare glass slide substrate clean an unused standard microscope slide using sequential washing for a few minutes each in deionized ( di ) water , glass decontamination solution and di water . then wash with a 70:30 v : v mixture of ethanol : di water , and finally blow dry in a clean air / nitrogen stream.examine the glass slide under a microscope to verify the surface is clean and free from evidence of particulate contamination . clean an unused standard microscope slide using sequential washing for a few minutes each in deionized ( di ) water , glass decontamination solution and di water . then wash with a 70:30 v : v mixture of ethanol : di water , and finally blow dry in a clean air / nitrogen stream . examine the glass slide under a microscope to verify the surface is clean and free from evidence of particulate contamination . prepare colloidal dispersion for deposition pipette 10 l of aqueous stock fluorescent colloid solution ( 10% wt . ) into 990 l of ethanol . adjust volumes as required depending on stock solution concentration used to arrive at approximately 0.1% wt colloidal suspension.vortex mix for 10 sec . pipette 10 l of aqueous stock fluorescent colloid solution ( 10% wt . ) into 990 l of ethanol . adjust volumes as required depending on stock solution concentration used to arrive at approximately 0.1% wt colloidal suspension . spin coat colloidal dispersion onto glass slide substrate equip a spin coater with the clean glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide.remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide . remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . vacuum evaporate platinum metal onto colloid decorated glass slide insert the colloid coated glass slide into a metal evaporator . note : following metal deposition , samples should be stored under an inert atmosphere . insert the colloid coated glass slide into a metal evaporator . ensure the colloid decorated side is facing the evaporation source . re - suspend janus colloids into a peroxide containing solution cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate).insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . remove lens tissue.pipette 1 ml of the colloid containing solution into a new container , filled with 1 ml of 30% w / v h2o2 stock solution . gently mix the solutions , and then place in an ultrasonic bath at room temperature for 5 min , followed by another 25 min unstirred incubation period . caution : this solution may evolve oxygen ; do not seal.dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate ) . insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . remove lens tissue . pipette 1 ml of the colloid containing solution into a new container , filled with 1 ml of 30% w / v h2o2 stock solution . gently mix the solutions , and then place in an ultrasonic bath at room temperature for 5 min , followed by another 25 min unstirred incubation period . dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . prepare analysis cuvette add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . note : the prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface.fill a low volume rectangular quartz glass cuvette with the incubated solution . add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . note : the prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface . fill a low volume rectangular quartz glass cuvette with the incubated solution . loosely fit a push - in cap . locate particle of interest load cuvette into a fluorescent microscope equipped with a suitable objective ( e.g. , 20x ) and excite fluorophore emission using a suitable combination of filters ( excitation 450 - 490 nm , emission > 515 nm).manually search for fluorescent colloids within the cuvette . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present . a colloid volume concentration of about 0.003% is a recommended starting point.optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . load cuvette into a fluorescent microscope equipped with a suitable objective ( e.g. , 20x ) and excite fluorophore emission using a suitable combination of filters ( excitation 450 - 490 nm , emission > 515 nm ) . manually search for fluorescent colloids within the cuvette . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . record video before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . do not move the focus plane during video capture.record videos of particles of interest . use 30 sec video durations with frame rates in excess of 30 hz to allow detailed trajectory reconstruction . before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . do not move the focus plane during video capture . use 30 sec video durations with frame rates in excess of 30 hz to allow detailed trajectory reconstruction . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy.plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy.plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids . focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane . determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . when a janus sphere is far from the focal plane its intensity becomes too weak to accurately track it , e.g. , for a 4.8 m diameter colloid a z - range of around 200 m is possible . an alternative non - algorithmic method is to use simple manual measurement of x , y center and radius , however this will reduce accuracy . plot a calibration curve to relate radius to z - position , and fit to an appropriate function ( e.g. , cubic equation ) to allow interpolation . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2 . measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . figure 1 shows a typical dispersion of colloids on a clean glass slide prior to depositing platinum . figure 2 shows a typical back - scattered sem image for a half platinum coated janus swimmer , under this imaging mode the platinum coated region produces bright contrast . figure 3 displays the appearance of a typical fluorescent janus swimmer under optimum illumination conditions fixed in gellan gum . the swimmer appears as a symmetrical ring feature , and it is the radius of the ring that can be used to determine the z - position of the colloid relative to the focus position . figure 4 shows representative cross - sections for the radial brightness intensity distribution that is used in combination with image analysis algorithms to accurately locate the center and apparent radius of the colloid . figure 5 contains a calibration curve obtained using a fixed colloidal sample and a calibrated microscope z - translation stage to relate apparent colloidal size and distance from focus position . this curve is fitted to a cubic function , which is used to convert apparent radius into z - coordinate . finally , figure 6 displays a typical x , y , z trajectory for a fluorescent janus particle swimmer . calibration images of a 4.8 m diameter fluorescent polystyrene sphere fixed in gellan gum recorded using a 20x objective ( 0.4 n.a . ) . the distances below each image indicate the distance of the focal plane of the objective above the sphere . as the image is defocused from 0 m to 200 m the in focus image of a bright disc changes to a bright ring , the radius of which is dependent upon magnification , the sphere size and its distance from the focal plane . a set of self - written algorithms is used to first locate the ( x , y ) center of the bright ring by extracting a series of vertical and horizontal lines and finding the mean mid - point between the bright peaks ( a ) . the ring radius is then calculated from the peak intensity of a spline fitted to the average pixel grey - values radiating out from the ring center ( b ) . z - coordinate calibration chart for janus spheres obtained by measuring the bright ring radius of spheres fixed in gellan gum ( see figures 3 and 4 ) . the calibration chart is used by our algorithms to convert the measured ring radius to a z - coordinate . a sequence of images of the moving swimming device was recorded over a period of 30 sec at a frame rate of 33 hz . the ( x , y , z ) coordinates of the trajectory were obtained by locating the bright ring center ( figure 4 ( a ) ) and comparing the measured ring radius to the calibration chart for each image in the sequence ( figures 4 ( b ) and 5 ) . many variables in the preparation protocol for platinum janus particles will affect the observed trajectories . the parameters as described using 2 m diameter particles will give propulsion velocities in the order of 10 m per second . if smaller particles are used , velocities will increase , while increasing particle size will decrease propulsion velocity . the details of the evaporation protocol will also alter the trajectories observed . in this current protocol , a sparse distribution of colloids is recommended , together with metal evaporation normal to the slide orientation . these conditions result in symmetrical janus structures as shown in figure 2 , which lead to linear trajectories within the limits of brownian rotational diffusion . conversely , if tight packed colloids are subject to glancing angle deposition , then the symmetry of the janus cap can be broken , to induce spinning behavior . the particles produced here display relatively isotropic motion in all three dimensions ; however if thicker platinum coatings , or larger particles are used , an upwards bias or gravitaxis can be imparted . details of the storage of the janus colloids after manufacture may also effect the swimming speeds observed . the high surface energy clean platinum surface emerging from the evaporation stage is susceptible to surface contamination for example from hydrocarbons , and in particular thiols . in addition , the solution properties in which the janus colloids are re - suspended are critical for observing propulsion . low peroxide concentrations will result in slower velocities , as the rate of the decomposition reaction producing motion reduces . in addition , low concentrations of salts will result in a dramatic reduction in propulsion velocity . a key feature of the colloids produced here is their neutral buoyancy , which makes them suitable for 3d tracking . in general the field of swimming devices has paid little attention to 3d effects , partly due to some prominent examples being made from dense metals , causing them to rapidly sediment , but also due to the difficulties and expense associated with making the required measurements . clear drawbacks for some established 3d tracking methods exist for these rapidly moving colloids , for example , confocal scanning laser microscopy can lack the temporal resolution to record sufficient numbers of images to resolve trajectories . in this context , the method we present here has the significant advantage of only requiring a single frame to allow estimation of z - coordinate , which consequently allows high frame rates . also , as z - coordinate reconstruction only relies on the relative contrast of the out - of - focus colloid in single frames , rather than the absolute fluorescence intensity , it is resilient to quenching and blinking effects in the fluorophore . these advantages are possible at the expense of a reduced depth of field over which 3d trajectory reconstruction is possible , and the requirement for well separated non - overlapping colloids . we hope that describing the protocol will allow other research groups with an interest in 3d behavior for their swimming devices to access this information straightforwardly and with a high degree of precision . it is clear that expanding the understanding of these devices to 3d will open up a significant range of interesting future phenomena and applications . readers interested in further details of trajectory analysis are directed towards reference 17 which describes common artifacts in propulsive systems and how to ensure accurate quantification of propulsion velocities .

we report a method to prepare catalytically active janus colloids that " swim " in fluids and describe how to determine their 3d motion using fluorescence microscopy . one commonly deployed method for catalytically active colloids to produce enhanced motion is via an asymmetrical distribution of catalyst . here this is achieved by spin coating a dispersed layer of fluorescent polymeric colloids onto a flat planar substrate , and then using directional platinum vapor deposition to half coat the exposed colloid surface , making a two faced " janus " structure . the janus colloids are then re - suspended from the planar substrate into an aqueous solution containing hydrogen peroxide . hydrogen peroxide serves as a fuel for the platinum catalyst , which is decomposed into water and oxygen , but only on one side of the colloid . the asymmetry results in gradients that produce enhanced motion , or " swimming " . a fluorescence microscope , together with a video camera is used to record the motion of individual colloids . the center of the fluorescent emission is found using image analysis to provide an x and y coordinate for each frame of the video . while keeping the microscope focal position fixed , the fluorescence emission from the colloid produces a characteristic concentric ring pattern which is subject to image analysis to determine the particles relative z position . in this way 3d trajectories for the swimming colloid are obtained , allowing swimming velocity to be accurately measured , and physical phenomena such as gravitaxis , which may bias the colloids motion to be detected .

Introduction Protocol 1. Making Catalytic Janus Particles 2. "Swimming" Janus Particles 3. Microscopic Observation Representative Results Discussion Disclosures

these particles get their name from having two distinct sides , or faces ( janus is a two faced roman god ) . to address this , here we describe a protocol that allows accurate 3d trajectories for swimming devices to be determined . this paper also clearly documents a method to manufacture catalytic janus particle swimming devices , which will be of further benefit to standardize methods across the existing research groups investigating these devices , and additionally guide new researchers interested in making and investigating swimming devices . hydrogen peroxide used in this protocol is harmful , and the evolution of oxygen gas when exposed to platinum poses an explosion risk . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide.remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide . re - suspend janus colloids into a peroxide containing solution cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate).insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . caution : this solution may evolve oxygen ; do not seal.dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate ) . dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . record video before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . by locating the x , y , z position of a janus sphere fixed in gellan gum 30 m from the focal plane , in a time sequence of images , particles can be located with an error of 25 nm along each axis . determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide.remove glass slide from the spin coater , return to the optical microscope and verify that an even dispersion of mainly non - touching separate colloids covers the central region of the glass slide . start spin coater , and when up to speed continuously pipette the prepared solution onto the center of the spinning glass slide . re - suspend janus colloids into a peroxide containing solution cut a 1 x 1 cm square of lens tissue , and dampen the end with 10 l of di water . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate).insert the lens tissue into 1.5 ml of di water and manually shake vigorously for 30 sec in a sealed tube . caution : this solution may evolve oxygen ; do not seal.dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . hold with tweezers , and gently rub along the surface of the platinum coated colloid decorated glass slide ( this step physically removes colloids from the substrate ) . gently mix the solutions , and then place in an ultrasonic bath at room temperature for 5 min , followed by another 25 min unstirred incubation period . dry 100 l of the remaining aqueous colloidal solution onto a scanning electron microscope ( sem ) stub to enable sem verification of the janus colloid structure . prepare analysis cuvette add an additional 1 ml of di water to the incubated solution containing peroxide and platinum coated colloids to arrive at a suitable fuel strength ( 10% ) to allow fast propulsion . note : the prior incubation stages were carried out in higher concentration fuel concentrations to clean the platinum catalyst surface.fill a low volume rectangular quartz glass cuvette with the incubated solution . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . for example , dilution is recommended if the colloid density is high , and numerous oxygen bubbles producing flows are present optimize optical settings for 3d tracking : under suitable illumination conditions , in focus colloids will appear as sharp circular objects . however , as propulsion moves the colloids in and out of the focal plane a distinctive size changing bright ring centered around the sphere will be observed , this is used to determine the z coordinate to enable 3d tracking . record video before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . before starting video capture , focus the microscope so that the particle of interest produces a concentric ring , with the particle " under " the focus position . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . gellan gum solution in water containing a suspension of fluorescent janus particles at 60 c , place in an equivalent cuvette to that used above , and allow to set to form a stiff transparent gelled sample containing fixed static colloids.focus on a single fixed colloid using the same illumination conditions selected above , now record a series of still images as the z - focus is raised by known displacements relative to this plane.determine the radius of the ring at each known focus position . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . note : this is most efficiently performed by using an image analysis algorithm that can be applied as a batch process to all the calibration still frames , and videos . a typical approach involves smoothing the image , thresholding to identify an approximate location of the objects ' center , and then locating the actual x and y coordinates of the ring center by measuring the distance between intensity peaks either side of the ring . the mean radial distance to the intensity peaks from the ring center this allows both the radius of the bright ring and the x - y coordinate to be determined with sub - pixel accuracy . the signal to noise ratio and therefore , the accuracy of the location algorithms is dependent upon the intensity of the detected fluorescence light . calibrate x , y axis record a still frame optical microscope image of a spatial calibration graticule using the same microscope conditions chosen in 3.2.measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . measure the " in pixels " dimension of an object with known real world size from the image of the spatial calibration graticule and use this to establish a pixels to micron conversion factor for the x , y image plane . reconstruct trajectory determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . this procedure can be implemented using an algorithm , or manually.determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . determine the x and y coordinates and radius for each frame of the video sequence as described in 3.3.1.3 , use the function found in 3.3.1.4 to convert radius into z , and the calibration factor found in 3.3.2.2 to convert x and y pixel coordinates into microns . this procedure will result in an accurate x , y , z coordinate for the propulsive particles location as a function of time . determine the derived properties such as average velocity to quantify the extent of observed catalytic swimming . the swimmer appears as a symmetrical ring feature , and it is the radius of the ring that can be used to determine the z - position of the colloid relative to the focus position . figure 4 shows representative cross - sections for the radial brightness intensity distribution that is used in combination with image analysis algorithms to accurately locate the center and apparent radius of the colloid . this curve is fitted to a cubic function , which is used to convert apparent radius into z - coordinate . as the image is defocused from 0 m to 200 m the in focus image of a bright disc changes to a bright ring , the radius of which is dependent upon magnification , the sphere size and its distance from the focal plane . a set of self - written algorithms is used to first locate the ( x , y ) center of the bright ring by extracting a series of vertical and horizontal lines and finding the mean mid - point between the bright peaks ( a ) . in this current protocol , a sparse distribution of colloids is recommended , together with metal evaporation normal to the slide orientation . conversely , if tight packed colloids are subject to glancing angle deposition , then the symmetry of the janus cap can be broken , to induce spinning behavior . the particles produced here display relatively isotropic motion in all three dimensions ; however if thicker platinum coatings , or larger particles are used , an upwards bias or gravitaxis can be imparted . details of the storage of the janus colloids after manufacture may also effect the swimming speeds observed . in addition , the solution properties in which the janus colloids are re - suspended are critical for observing propulsion . a key feature of the colloids produced here is their neutral buoyancy , which makes them suitable for 3d tracking . in this context , the method we present here has the significant advantage of only requiring a single frame to allow estimation of z - coordinate , which consequently allows high frame rates .

preclinical studies have utilized several approaches and different stem cell lines . nevertheless , determination of appropriate stem cell lines as the ideal source for brain - specific restorative neural grafts remains an unsolved problem . second , it is necessary to elucidate cellular mechanisms by which stem cell transplantation leads to functional recovery and structural reorganization . the biological risks of carcinoma stem cells are obvious due to their potential to form donor - derived malignant tumors in the host . on the other hand , it has been previously shown that , when differentiated , carcinoma - derived stem cells lose tumorigenic capacity . furthermore , it has been previously demonstrated that transplantation of neurons derived from a human teratocarcinoma - derived cell line are potentially beneficial in several animal models of neurological disorders , including ischemia - induced injury , spinal cord injury , stroke [ 46 ] , parkinson s disease , huntington s disease and amyotrophic lateral sclerosis . in light of the above , there exists a wide spectrum of human cerebellar ataxias with different pathogenetic etiologies , as well as established animal models of cerebellar disorders [ 1113 ] . despite several therapeutic strategies for spinocerebellar ataxias , none of them is effective enough to stop the degenerative process with substantial recovery of diminished cerebellar function . promising approaches include gene transfer into the cerebellum to prevent the degeneration of purkinje cells and neurotransplantation to provide restorative cells and/or trophic effect of immature grafts ( for review , see ) . the lurcher mutant mouse represents one of the most frequently used natural models of genetically - determined olivocerebellar degeneration . they are heterozygotes ( + /lc ) , carrying a mutation in the glutamate receptor delta2-subunit gene , which is predominantly expressed by cerebellar purkinje cells . lurcher mice suffer from a virtually complete postnatal loss of purkinje cells and a decrease in the number of cerebellar granule , basket and stellate cells and inferior olive neurons . the reduction in the purkinje cell number can be detected from postnatal day 8 ( p8 ) and the degeneration is finalized at the p90 . the death of purkinje cells is a primary effect of the mutation that changes the altered receptor into a leaky membrane channel , leading to chronic depolarization of the cells . the degeneration of the other cerebellar cells and inferior olive neurons is a secondary consequence of the disappearance of target of their axons the purkinje cells and affects 90% of the granule cells and 70% of the inferior olive neurons . homozygous mutants ( lc / lc ) are not viable due to the massive loss of brainstem neurons during prenatal development and they die at birth . homozygous wild type littermates of lurcher mice ( + /+ ) are completely healthy and serve as controls . in contrast , heterozygous lurcher mice suffer from cerebellar ataxia [ 2729 ] , a deterioration of spatial learning or orientation [ 3033 ] , and abnormalities in conditioned eyelid response . for review , we have recently shown that naive carcinoma stem cells and neuroprogenitors derived from these cells are able to survive in a normal mouse cerebellum , although there are some differences in growth character of these 2 types of grafts . accordingly , the aim of the present study was to assess survival of naive and neurally differentiated p19 carcinoma stem cells following transplantation into the cerebellum of lurcher mice and wild type littermates . experimental procedures are identical to those detailed in previous studies and are briefly described below . embryonic carcinoma ( ec ) stem cells of the p19 line isolated from a teratocarcinoma induced in the c3h / he strain of mice were purchased from the european collection of cell culture , wiltshire , uk . they were genetically modified to express the green fluorescent protein ( gfp ) so that they could subsequently be detected after transplantation in histological sections using direct fluorescent microscopy . undifferentiated ec cells were cultured on gelatin - coated tissue culture dishes in dulbecco s modified eagle s medium ( dmem ) containing 10% fetal calf serum , 0.05 mm beta - mercaptoethanol , 100 i.u . /ml penicillin , and 0.1 mg / ml streptomycin ( all gibco brl , chemos cz , prague , czech republic ) . for neurodifferentiation , the ec cells were cultured under serum - free conditions in dmem / f12 ( 1:1 ) media supplemented with a mixture of insulin , transferrin , selenium ( its ) and antibiotics . for the first 2 days , the cells were treated with retinoic acid ( ra , c=510 m ) to induce neurogenesis and then they were cultured for 1 day without ra . at that point , they were used as neuroprogenitors for transplantation . we have previously described the characterization of naive p19 cells and neuroprogenitors in the stage in which they were grafted by immunocytochemical , western blot and quantitative reverse transcriptase real - time pcr analyses . for transplantation of naive p19 cells or neuroprogenitors , the cells were isolated by trypsin , which was neutralized by adding dmem with serum and then centrifuged and resuspended in dmem to give a final cell concentration of 50 000 viable cells/l . transplantations were performed under general anaesthesia ( ketamine 100 mg / kg bw and xylazine 16 mg / kg bw ) . a hole ( 2 mm in diameter ) was drilled in the occipital bone ( bregma 6.57.0 mm , midline ) , and 1 l of cell suspension ( a total amount of 50 000 cells ) was injected with a hamilton syringe at a constant speed of 0.5 l / min . the tip of the needle was inserted 1.6 mm under the surface of the cerebellum in wild type mice and 1.6 or 1.2 mm in lurcher mice . in the lurcher mouse , the cerebellum is flattened , necessitating a lower depth of implantation . during the injection of the cell suspension , upon completion of the administration , the needle remained in situ for 5 minutes to prevent the return of the cells out of the host head . finally , the wound was sutured by 1 layer with chirlac rapid ( chirmax gmbh ) and disinfected . mice were sacrificed 21 days after the transplantation by a lethal dosage of thiopental and were transcardially perfused with phosphate - buffered saline solution ( ph 7.4 ) and 4% phosphate - buffered paraformaldehyde . fixed brainstem and cerebellar blocks were sectioned using a cryostat ( 40 m frontal sections ) . the grafts were detected according to their gfp fluorescence in the native sections under a fluorescent microscope . sections were counter - stained with hematoxylin - eosin or according to the nissl technique to visualize the histological structure of the graft and surrounding host tissue . detection and quantification of purkinje cells and astrocytes was achieved via immunohistochemical staining utilizing a rabbit anti - calbindin - d-28k antiserum ( sigma - aldrich , st . louis , usa ) and a mouse monoclonal glial fibrillary acidic protein ( gfap ) antiserum ( clone g - a-5 , sigma - aldrich , st . sections were incubated in pbs with 10% normal serum , 0.5% tween and 0.1% fbs ( foetal bovine serum ) for 1 hour at room temperature . after washing in pbs , the sections were incubated overnight at 4c with the primary antibody ( dilution of gfap 1:800 and anti - calbindin 1:1000 ) . after a pbs wash , gfap labelling was completed and the sections were mounted in prolong gold antifade reagent with dapi ( invitrogen , eugene , usa ) . sections incubated with anti - calbindin antiserum were washed in pbs and subsequently incubated with the secondary antibody , alexa fluor 568 goat anti rabbit igg ( final dilution 1:400 , invitrogen , eugene , usa ) , for 2 hours at room temperature . after a pbs wash , sections were mounted in prolong gold antifade reagent with dapi and viewed under an epifluorescent microscope ( olympus bx 41 , olympus czech group , prague , czech republic ) . the number and percentage of mice in which the graft survived ( quantified by counting gfp - positive cells ) and the frequency of the occurrence of various features of the grafts ( localization of the graft , the destruction of the tissue , and the characteristic growth of the graft ) were assessed . the differences between the groups of mice were evaluated using fisher s test . in all cases , all animal experiments described in the present study were performed in full compliance with the eu guidelines for scientific experimentation on animals and with the permission of the ethics commission of the faculty of medicine at charles university , pilsen . adult normal wild type and lurcher mutant mice of the b6cba strain were used ; 43 wild type mice were taken from the previous study . the mean age of the animals at the time of transplantation was 261.5 days ( sd=64.3 days , minimum = 156 days , maximum = 385 days ) . the mice were reared under standard conventional conditions with 12:12 hours light : dark cycle ( 6 am 6 pm ) and temperature 2224c . the mice were housed in plastic cages with metal mesh cover ( 1125 cm , 14 cm high for 12 mice or 1825 cm , 14 cm high for 24 mice ) . twenty - five wild type mice ( 12 males and 13 females ) were treated with naive p19 cells and 26 wild type animals ( 13 males and 13 females ) received neuroprogenitor cells . in 25 lurcher mice ( 12 males and 13 females ) naive p19 cells were injected at the depth of 1.6 mm under the surface and in 18 lurcher mice ( 9 males and 9 females ) received neuroprogenitor cells injected at the depth of 1.2 mm . twenty - five lurcher mice ( 13 males and 12 females ) received neuroprogenitor cells injected at the depth of 1.6 mm and 19 lurcher mice ( 9 males and 10 females ) received neuroprogenitor cells injected into the depth of 1.2 mm . embryonic carcinoma ( ec ) stem cells of the p19 line isolated from a teratocarcinoma induced in the c3h / he strain of mice were purchased from the european collection of cell culture , wiltshire , uk . they were genetically modified to express the green fluorescent protein ( gfp ) so that they could subsequently be detected after transplantation in histological sections using direct fluorescent microscopy . undifferentiated ec cells were cultured on gelatin - coated tissue culture dishes in dulbecco s modified eagle s medium ( dmem ) containing 10% fetal calf serum , 0.05 mm beta - mercaptoethanol , 100 i.u . /ml penicillin , and 0.1 mg / ml streptomycin ( all gibco brl , chemos cz , prague , czech republic ) . for neurodifferentiation , the ec cells were cultured under serum - free conditions in dmem / f12 ( 1:1 ) media supplemented with a mixture of insulin , transferrin , selenium ( its ) and antibiotics . for the first 2 days , the cells were treated with retinoic acid ( ra , c=510 m ) to induce neurogenesis and then they were cultured for 1 day without ra . we have previously described the characterization of naive p19 cells and neuroprogenitors in the stage in which they were grafted by immunocytochemical , western blot and quantitative reverse transcriptase real - time pcr analyses . for transplantation of naive p19 cells or neuroprogenitors , the cells were isolated by trypsin , which was neutralized by adding dmem with serum and then centrifuged and resuspended in dmem to give a final cell concentration of 50 000 viable cells/l . transplantations were performed under general anaesthesia ( ketamine 100 mg / kg bw and xylazine 16 mg / kg bw ) . a hole ( 2 mm in diameter ) was drilled in the occipital bone ( bregma 6.57.0 mm , midline ) , and 1 l of cell suspension ( a total amount of 50 000 cells ) was injected with a hamilton syringe at a constant speed of 0.5 l / min . the tip of the needle was inserted 1.6 mm under the surface of the cerebellum in wild type mice and 1.6 or 1.2 mm in lurcher mice . in the lurcher mouse , the cerebellum is flattened , necessitating a lower depth of implantation . during the injection of the cell suspension , upon completion of the administration , the needle remained in situ for 5 minutes to prevent the return of the cells out of the host head . finally , the wound was sutured by 1 layer with chirlac rapid ( chirmax gmbh ) and disinfected . mice were sacrificed 21 days after the transplantation by a lethal dosage of thiopental and were transcardially perfused with phosphate - buffered saline solution ( ph 7.4 ) and 4% phosphate - buffered paraformaldehyde . fixed brainstem and cerebellar blocks were sectioned using a cryostat ( 40 m frontal sections ) . the grafts were detected according to their gfp fluorescence in the native sections under a fluorescent microscope . sections were counter - stained with hematoxylin - eosin or according to the nissl technique to visualize the histological structure of the graft and surrounding host tissue . detection and quantification of purkinje cells and astrocytes was achieved via immunohistochemical staining utilizing a rabbit anti - calbindin - d-28k antiserum ( sigma - aldrich , st . louis , usa ) and a mouse monoclonal glial fibrillary acidic protein ( gfap ) antiserum ( clone g - a-5 , sigma - aldrich , st . , sections were incubated in pbs with 10% normal serum , 0.5% tween and 0.1% fbs ( foetal bovine serum ) for 1 hour at room temperature . after washing in pbs , the sections were incubated overnight at 4c with the primary antibody ( dilution of gfap 1:800 and anti - calbindin 1:1000 ) . after a pbs wash , gfap labelling was completed and the sections were mounted in prolong gold antifade reagent with dapi ( invitrogen , eugene , usa ) . sections incubated with anti - calbindin antiserum were washed in pbs and subsequently incubated with the secondary antibody , alexa fluor 568 goat anti rabbit igg ( final dilution 1:400 , invitrogen , eugene , usa ) , for 2 hours at room temperature . after a pbs wash , sections were mounted in prolong gold antifade reagent with dapi and viewed under an epifluorescent microscope ( olympus bx 41 , olympus czech group , prague , czech republic ) . the number and percentage of mice in which the graft survived ( quantified by counting gfp - positive cells ) and the frequency of the occurrence of various features of the grafts ( localization of the graft , the destruction of the tissue , and the characteristic growth of the graft ) were assessed . the differences between the groups of mice were evaluated using fisher s test . in all cases , all animal experiments described in the present study were performed in full compliance with the eu guidelines for scientific experimentation on animals and with the permission of the ethics commission of the faculty of medicine at charles university , pilsen . adult normal wild type and lurcher mutant mice of the b6cba strain were used ; 43 wild type mice were taken from the previous study . the mean age of the animals at the time of transplantation was 261.5 days ( sd=64.3 days , minimum = 156 days , maximum = 385 days ) . the mice were reared under standard conventional conditions with 12:12 hours light : dark cycle ( 6 am 6 pm ) and temperature 2224c . water and food were available ad libitum . the mice were housed in plastic cages with metal mesh cover ( 1125 cm , 14 cm high for 12 mice or 1825 cm , 14 cm high for 24 mice ) . twenty - five wild type mice ( 12 males and 13 females ) were treated with naive p19 cells and 26 wild type animals ( 13 males and 13 females ) received neuroprogenitor cells . in 25 lurcher mice ( 12 males and 13 females ) naive p19 cells were injected at the depth of 1.6 mm under the surface and in 18 lurcher mice ( 9 males and 9 females ) received neuroprogenitor cells injected at the depth of 1.2 mm . twenty - five lurcher mice ( 13 males and 12 females ) received neuroprogenitor cells injected at the depth of 1.6 mm and 19 lurcher mice ( 9 males and 10 females ) received neuroprogenitor cells injected into the depth of 1.2 mm . the number and percentage of experimental animals with viable grafts are listed in table 1 . notably , the number of viable neuroprogenitor cellular grafts at an injection depth of 1.6 mm was significantly higher in wild type as compared to lurcher mutant mice ( p=0.0078 ) . both p19 cell and neuroprogenitor grafts formed a separate mass containing gfp - positive cells dispersed in non - fluorescent tissue , as previously described for wild type mice . in 11 and 7 wild type mice injected with neuroprogenitor and naive p19 cells , respectively , the graft was typically localized to the vicinity of the injection site in the middle of the cerebellum ( figure 1a ) . in 2 wild type mice injected with neuroprogenitor cells , a viable graft was localized to an area between the cerebellum and the mesencephalon and was strictly delimited against both of these structures . in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum ( figure 1b ) . in 2 lurcher mice injected with neuroprogenitor cells at a depth of 1.6 mm , viable grafts were observed to be in contact with the border between the mesencephalon and the cerebellum , with the mass of the graft strictly delimited against the cerebellum ( figure 1c ) . in a single lurcher mouse injected with naive p19 cells at a depth of 1.6 mm , a large viable graft was observed in the mesencephalon and a small piece of fluorescent graft tissue was found at an angle between the pons and the cerebellum with no signs of direct contact with the cerebellum . finally , in a single lurcher mouse injected with neuroprogenitor cells at a depth of 1.2 mm , gfp - positive cells were observed in the mesencephalon and also on the surface of the medulla oblongata . statistically significant differences were observed in the cerebellar localization of viable grafts in lurcher mutants in comparison to wild type littermates ( for neuroprogenitor graft , depth 1.6 mm p=0.0063 , for naive p19 cells , depth 1.6 mm p=0.0101 ) . immunohistochemical analyses demonstrated that viable grafts contained numerous gfap - positive cells ( figure 2b , d ) , at least some of which were gfp - positive ( figure 2a , c ) . calbindin - positive cells co - expressing gfp fluorescence were not observed in viable grafts . in some of the animals , tissue destruction was observable inside viable grafts , whereas other viable grafts exhibited expansive morphological characteristics . the tabulated number of viable grafts exhibiting these morphological characteristics is depicted in table 3 . in wild type mice , expansive character was more frequent in mice that received naive p19 cells than in those treated with the neuroprogenitors ( p=0.0017 ) . differences in intragraft tissue destruction in viable grafts originating from naive p19 cell and the neuroprogenitor cell injections to wild type mice did not reach statistical significance . due to the low number of surviving grafts in lurcher mice , it was not possible to perform reliable statistical analysis of various morphological features of the grafts . the number and percentage of experimental animals with viable grafts are listed in table 1 . notably , the number of viable neuroprogenitor cellular grafts at an injection depth of 1.6 mm was significantly higher in wild type as compared to lurcher mutant mice ( p=0.0078 ) . both p19 cell and neuroprogenitor grafts formed a separate mass containing gfp - positive cells dispersed in non - fluorescent tissue , as previously described for wild type mice . in 11 and 7 wild type mice injected with neuroprogenitor and naive p19 cells , respectively , the graft was typically localized to the vicinity of the injection site in the middle of the cerebellum ( figure 1a ) . in 2 wild type mice injected with neuroprogenitor cells , a viable graft was localized to an area between the cerebellum and the mesencephalon and was strictly delimited against both of these structures . in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum ( figure 1b ) . in 2 lurcher mice injected with neuroprogenitor cells at a depth of 1.6 mm , viable grafts were observed to be in contact with the border between the mesencephalon and the cerebellum , with the mass of the graft strictly delimited against the cerebellum ( figure 1c ) . in a single lurcher mouse injected with naive p19 cells at a depth of 1.6 mm , a large viable graft was observed in the mesencephalon and a small piece of fluorescent graft tissue was found at an angle between the pons and the cerebellum with no signs of direct contact with the cerebellum . finally , in a single lurcher mouse injected with neuroprogenitor cells at a depth of 1.2 mm , gfp - positive cells were observed in the mesencephalon and also on the surface of the medulla oblongata . statistically significant differences were observed in the cerebellar localization of viable grafts in lurcher mutants in comparison to wild type littermates ( for neuroprogenitor graft , depth 1.6 mm p=0.0063 , for naive p19 cells , depth 1.6 mm p=0.0101 ) . immunohistochemical analyses demonstrated that viable grafts contained numerous gfap - positive cells ( figure 2b , d ) , at least some of which were gfp - positive ( figure 2a , c ) . calbindin - positive cells co - expressing gfp fluorescence were not observed in viable grafts . in some of the animals , tissue destruction was observable inside viable grafts , whereas other viable grafts exhibited expansive morphological characteristics . the tabulated number of viable grafts exhibiting these morphological characteristics is depicted in table 3 . in wild type mice , expansive character was more frequent in mice that received naive p19 cells than in those treated with the neuroprogenitors ( p=0.0017 ) . differences in intragraft tissue destruction in viable grafts originating from naive p19 cell and the neuroprogenitor cell injections to wild type mice did not reach statistical significance . due to the low number of surviving grafts in lurcher mice , it was not possible to perform reliable statistical analysis of various morphological features of the grafts . the present study confirms and elaborates previous preliminary findings that have described cerebellar transplantation of naive p19 carcinoma stem cells and p19-derived neuroprogenitors in wild type mice by including similar analyses in lurcher mutant mice . our major observations indicate that in lurcher mice graft survival is quite poor and survival rate of the neuroprogenitor grafts is significantly lower in comparison to wild type mice when injected at the same tissue depth . the low number of surviving grafts did not allow for a more detailed comparison of morphological characteristics of the 2 types of grafts in lurcher mice . we can conclude , however , that naive p19 cell grafts typically exhibited expansive morphological characteristics without intragraft tissue destruction in both wild type and lurcher mice . in these parameters , the main finding of the present study was the observed regional difference in graft localization between wild type and lurcher mice . it is notable that in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum . this may be attributable to technical limitations due to the flattened cerebellum of the lurcher mutant mouse . in the lurcher mouse cerebellum , there is much less space for viable graft development than in wild type animals and the cerebellum could be easily missed by the tip of the injection needle . moreover , the depth of cell injection suitable for wild type mice could be too deep for the lurcher cerebellum . these anatomical factors could also play a role in the difference in neuroprogenitor graft survival between lurcher mutant and wild type mice . on the other hand , an apparent doubling of survival rate of the neuroprogenitor grafts when injected at the lower depth of 1.2 mm vs. 1.6 mm , together with a lower graft survival in lurcher as compared to wild type mice , suggests that the niche of the lurcher cerebellum is less suitable for the grafted cells than the adjacent mesencephalon . these intrinsic difficulties suggest that neurodegenerative changes of the cerebellar tissue could have a negative impact on the fate of stem cells grafted into the cerebellum of adult lurcher mice . the area of the graft contained numerous astrocytes , at least some of which were donor - derived . additionally , we have no evidence that the grafted cell ( naive embryonic carcinoma cells or neuroprogenitors ) differentiated into the purkinje cells , the main cell type that is missing in lurcher mutant mice . survival of embryonic carcinoma stem cells in lurcher mice is much lower than the survival of embryonic cerebellar tissue . tomey and heckroth , who transplanted a suspension of embryonic cerebellar cells , found surviving graft in 50% of both young and adult lurcher mice 12 months after transplantation . the survival of solid embryonic cerebellar graft 39 weeks after the surgery was 8090% in lurcher mice and it did not differ from wild type mice . however , cells grafted in the form of suspension are in direct and closer contact with the host tissue than cells inside the solid grafts . therefore , the influence of the host tissue on grafted cells development is stronger in cell suspension than in solid grafts . solid grafts are not as suitable as cell suspension for an investigation of the impact of the host environment on the fate of the grafted cells . in addition , the solid embryonic cerebellar graft mediated controversial behavioural effects in lurcher mice . the appearance and low survival rate of the grafts do not show any promise for a sufficient therapeutic effect on cerebellar function that has deteriorated due to degenerative disease in lurcher mice . this is in contrast to the findings of benefit of transplantation of neurons derived from the human teratocarcinoma cell - line in animal models of several other neurological diseases .

summarybackgroundneurotransplantation has great potential for future treatments of various neurodegenerative disorders . preclinically , the lurcher mutant mouse represents an appropriate model of genetically - determined olivocerebellar degeneration . the aim of the present study was to assess survival of nave and neurally differentiated p19 carcinoma stem cells following transplantation into the cerebellum of lurcher mice and wild type littermates.material/methodsadult normal wild type ( n=51 ) and lurcher mutant mice ( n=87 ) of the b6cba strain were used . the mean age of the animals at the time of transplantation was 261.5 days . suspension of naive and neurally differentiated p19 carcinoma stem cells was injected into the cerebellum of the mice . in the lurcher mutants , 2 depths of graft injection were used . three weeks after implantation the brains of experimental animals were examined histologically.resultssurvival of neuroprogenitor grafts at a depth of 1.6 mm was significantly higher in wild type vs. lurcher mutant mice . in wild type mice , the typical graft localization was in the middle of the cerebellum , whereas in lurcher mice the graft was never found inside the degenerated cerebellum and was primarily localized in the mesencephalon.conclusionswe conclude that the appearance and low survival rate of cerebellar p19 carcinoma stem cell grafts in the lurcher mutant mice weigh against the therapeutic value of this cell line in preclinical studies of neurodegeneration .

Background Material and Methods Stem cell culture and neurodifferentiation Graft preparations Transplantations Histological examination Statistical analysis Animals Results Graft survival Graft localization Graft structure Discussion Conclusions

preclinical studies have utilized several approaches and different stem cell lines . second , it is necessary to elucidate cellular mechanisms by which stem cell transplantation leads to functional recovery and structural reorganization . the biological risks of carcinoma stem cells are obvious due to their potential to form donor - derived malignant tumors in the host . in light of the above , there exists a wide spectrum of human cerebellar ataxias with different pathogenetic etiologies , as well as established animal models of cerebellar disorders [ 1113 ] . promising approaches include gene transfer into the cerebellum to prevent the degeneration of purkinje cells and neurotransplantation to provide restorative cells and/or trophic effect of immature grafts ( for review , see ) . the lurcher mutant mouse represents one of the most frequently used natural models of genetically - determined olivocerebellar degeneration . lurcher mice suffer from a virtually complete postnatal loss of purkinje cells and a decrease in the number of cerebellar granule , basket and stellate cells and inferior olive neurons . the reduction in the purkinje cell number can be detected from postnatal day 8 ( p8 ) and the degeneration is finalized at the p90 . the degeneration of the other cerebellar cells and inferior olive neurons is a secondary consequence of the disappearance of target of their axons the purkinje cells and affects 90% of the granule cells and 70% of the inferior olive neurons . homozygous wild type littermates of lurcher mice ( + /+ ) are completely healthy and serve as controls . for review , we have recently shown that naive carcinoma stem cells and neuroprogenitors derived from these cells are able to survive in a normal mouse cerebellum , although there are some differences in growth character of these 2 types of grafts . accordingly , the aim of the present study was to assess survival of naive and neurally differentiated p19 carcinoma stem cells following transplantation into the cerebellum of lurcher mice and wild type littermates . embryonic carcinoma ( ec ) stem cells of the p19 line isolated from a teratocarcinoma induced in the c3h / he strain of mice were purchased from the european collection of cell culture , wiltshire , uk . for neurodifferentiation , the ec cells were cultured under serum - free conditions in dmem / f12 ( 1:1 ) media supplemented with a mixture of insulin , transferrin , selenium ( its ) and antibiotics . for the first 2 days , the cells were treated with retinoic acid ( ra , c=510 m ) to induce neurogenesis and then they were cultured for 1 day without ra . we have previously described the characterization of naive p19 cells and neuroprogenitors in the stage in which they were grafted by immunocytochemical , western blot and quantitative reverse transcriptase real - time pcr analyses . for transplantation of naive p19 cells or neuroprogenitors , the cells were isolated by trypsin , which was neutralized by adding dmem with serum and then centrifuged and resuspended in dmem to give a final cell concentration of 50 000 viable cells/l . a hole ( 2 mm in diameter ) was drilled in the occipital bone ( bregma 6.57.0 mm , midline ) , and 1 l of cell suspension ( a total amount of 50 000 cells ) was injected with a hamilton syringe at a constant speed of 0.5 l / min . the tip of the needle was inserted 1.6 mm under the surface of the cerebellum in wild type mice and 1.6 or 1.2 mm in lurcher mice . in the lurcher mouse , the cerebellum is flattened , necessitating a lower depth of implantation . during the injection of the cell suspension , upon completion of the administration , the needle remained in situ for 5 minutes to prevent the return of the cells out of the host head . finally , the wound was sutured by 1 layer with chirlac rapid ( chirmax gmbh ) and disinfected . the grafts were detected according to their gfp fluorescence in the native sections under a fluorescent microscope . sections were counter - stained with hematoxylin - eosin or according to the nissl technique to visualize the histological structure of the graft and surrounding host tissue . after washing in pbs , the sections were incubated overnight at 4c with the primary antibody ( dilution of gfap 1:800 and anti - calbindin 1:1000 ) . the number and percentage of mice in which the graft survived ( quantified by counting gfp - positive cells ) and the frequency of the occurrence of various features of the grafts ( localization of the graft , the destruction of the tissue , and the characteristic growth of the graft ) were assessed . in all cases , all animal experiments described in the present study were performed in full compliance with the eu guidelines for scientific experimentation on animals and with the permission of the ethics commission of the faculty of medicine at charles university , pilsen . adult normal wild type and lurcher mutant mice of the b6cba strain were used ; 43 wild type mice were taken from the previous study . the mean age of the animals at the time of transplantation was 261.5 days ( sd=64.3 days , minimum = 156 days , maximum = 385 days ) . the mice were reared under standard conventional conditions with 12:12 hours light : dark cycle ( 6 am 6 pm ) and temperature 2224c . twenty - five wild type mice ( 12 males and 13 females ) were treated with naive p19 cells and 26 wild type animals ( 13 males and 13 females ) received neuroprogenitor cells . in 25 lurcher mice ( 12 males and 13 females ) naive p19 cells were injected at the depth of 1.6 mm under the surface and in 18 lurcher mice ( 9 males and 9 females ) received neuroprogenitor cells injected at the depth of 1.2 mm . twenty - five lurcher mice ( 13 males and 12 females ) received neuroprogenitor cells injected at the depth of 1.6 mm and 19 lurcher mice ( 9 males and 10 females ) received neuroprogenitor cells injected into the depth of 1.2 mm . embryonic carcinoma ( ec ) stem cells of the p19 line isolated from a teratocarcinoma induced in the c3h / he strain of mice were purchased from the european collection of cell culture , wiltshire , uk . for neurodifferentiation , the ec cells were cultured under serum - free conditions in dmem / f12 ( 1:1 ) media supplemented with a mixture of insulin , transferrin , selenium ( its ) and antibiotics . we have previously described the characterization of naive p19 cells and neuroprogenitors in the stage in which they were grafted by immunocytochemical , western blot and quantitative reverse transcriptase real - time pcr analyses . for transplantation of naive p19 cells or neuroprogenitors , the cells were isolated by trypsin , which was neutralized by adding dmem with serum and then centrifuged and resuspended in dmem to give a final cell concentration of 50 000 viable cells/l . a hole ( 2 mm in diameter ) was drilled in the occipital bone ( bregma 6.57.0 mm , midline ) , and 1 l of cell suspension ( a total amount of 50 000 cells ) was injected with a hamilton syringe at a constant speed of 0.5 l / min . the tip of the needle was inserted 1.6 mm under the surface of the cerebellum in wild type mice and 1.6 or 1.2 mm in lurcher mice . in the lurcher mouse , the cerebellum is flattened , necessitating a lower depth of implantation . during the injection of the cell suspension , upon completion of the administration , the needle remained in situ for 5 minutes to prevent the return of the cells out of the host head . finally , the wound was sutured by 1 layer with chirlac rapid ( chirmax gmbh ) and disinfected . sections were counter - stained with hematoxylin - eosin or according to the nissl technique to visualize the histological structure of the graft and surrounding host tissue . the number and percentage of mice in which the graft survived ( quantified by counting gfp - positive cells ) and the frequency of the occurrence of various features of the grafts ( localization of the graft , the destruction of the tissue , and the characteristic growth of the graft ) were assessed . in all cases , all animal experiments described in the present study were performed in full compliance with the eu guidelines for scientific experimentation on animals and with the permission of the ethics commission of the faculty of medicine at charles university , pilsen . adult normal wild type and lurcher mutant mice of the b6cba strain were used ; 43 wild type mice were taken from the previous study . the mean age of the animals at the time of transplantation was 261.5 days ( sd=64.3 days , minimum = 156 days , maximum = 385 days ) . the mice were reared under standard conventional conditions with 12:12 hours light : dark cycle ( 6 am 6 pm ) and temperature 2224c . twenty - five wild type mice ( 12 males and 13 females ) were treated with naive p19 cells and 26 wild type animals ( 13 males and 13 females ) received neuroprogenitor cells . in 25 lurcher mice ( 12 males and 13 females ) naive p19 cells were injected at the depth of 1.6 mm under the surface and in 18 lurcher mice ( 9 males and 9 females ) received neuroprogenitor cells injected at the depth of 1.2 mm . twenty - five lurcher mice ( 13 males and 12 females ) received neuroprogenitor cells injected at the depth of 1.6 mm and 19 lurcher mice ( 9 males and 10 females ) received neuroprogenitor cells injected into the depth of 1.2 mm . the number and percentage of experimental animals with viable grafts are listed in table 1 . notably , the number of viable neuroprogenitor cellular grafts at an injection depth of 1.6 mm was significantly higher in wild type as compared to lurcher mutant mice ( p=0.0078 ) . both p19 cell and neuroprogenitor grafts formed a separate mass containing gfp - positive cells dispersed in non - fluorescent tissue , as previously described for wild type mice . in 11 and 7 wild type mice injected with neuroprogenitor and naive p19 cells , respectively , the graft was typically localized to the vicinity of the injection site in the middle of the cerebellum ( figure 1a ) . in 2 wild type mice injected with neuroprogenitor cells , a viable graft was localized to an area between the cerebellum and the mesencephalon and was strictly delimited against both of these structures . in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum ( figure 1b ) . in 2 lurcher mice injected with neuroprogenitor cells at a depth of 1.6 mm , viable grafts were observed to be in contact with the border between the mesencephalon and the cerebellum , with the mass of the graft strictly delimited against the cerebellum ( figure 1c ) . in a single lurcher mouse injected with naive p19 cells at a depth of 1.6 mm , a large viable graft was observed in the mesencephalon and a small piece of fluorescent graft tissue was found at an angle between the pons and the cerebellum with no signs of direct contact with the cerebellum . finally , in a single lurcher mouse injected with neuroprogenitor cells at a depth of 1.2 mm , gfp - positive cells were observed in the mesencephalon and also on the surface of the medulla oblongata . statistically significant differences were observed in the cerebellar localization of viable grafts in lurcher mutants in comparison to wild type littermates ( for neuroprogenitor graft , depth 1.6 mm p=0.0063 , for naive p19 cells , depth 1.6 mm p=0.0101 ) . in some of the animals , tissue destruction was observable inside viable grafts , whereas other viable grafts exhibited expansive morphological characteristics . in wild type mice , expansive character was more frequent in mice that received naive p19 cells than in those treated with the neuroprogenitors ( p=0.0017 ) . differences in intragraft tissue destruction in viable grafts originating from naive p19 cell and the neuroprogenitor cell injections to wild type mice did not reach statistical significance . due to the low number of surviving grafts in lurcher mice , it was not possible to perform reliable statistical analysis of various morphological features of the grafts . the number and percentage of experimental animals with viable grafts are listed in table 1 . notably , the number of viable neuroprogenitor cellular grafts at an injection depth of 1.6 mm was significantly higher in wild type as compared to lurcher mutant mice ( p=0.0078 ) . both p19 cell and neuroprogenitor grafts formed a separate mass containing gfp - positive cells dispersed in non - fluorescent tissue , as previously described for wild type mice . in 11 and 7 wild type mice injected with neuroprogenitor and naive p19 cells , respectively , the graft was typically localized to the vicinity of the injection site in the middle of the cerebellum ( figure 1a ) . in 2 wild type mice injected with neuroprogenitor cells , a viable graft was localized to an area between the cerebellum and the mesencephalon and was strictly delimited against both of these structures . in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum ( figure 1b ) . in 2 lurcher mice injected with neuroprogenitor cells at a depth of 1.6 mm , viable grafts were observed to be in contact with the border between the mesencephalon and the cerebellum , with the mass of the graft strictly delimited against the cerebellum ( figure 1c ) . in a single lurcher mouse injected with naive p19 cells at a depth of 1.6 mm , a large viable graft was observed in the mesencephalon and a small piece of fluorescent graft tissue was found at an angle between the pons and the cerebellum with no signs of direct contact with the cerebellum . finally , in a single lurcher mouse injected with neuroprogenitor cells at a depth of 1.2 mm , gfp - positive cells were observed in the mesencephalon and also on the surface of the medulla oblongata . statistically significant differences were observed in the cerebellar localization of viable grafts in lurcher mutants in comparison to wild type littermates ( for neuroprogenitor graft , depth 1.6 mm p=0.0063 , for naive p19 cells , depth 1.6 mm p=0.0101 ) . in some of the animals , tissue destruction was observable inside viable grafts , whereas other viable grafts exhibited expansive morphological characteristics . in wild type mice , expansive character was more frequent in mice that received naive p19 cells than in those treated with the neuroprogenitors ( p=0.0017 ) . differences in intragraft tissue destruction in viable grafts originating from naive p19 cell and the neuroprogenitor cell injections to wild type mice did not reach statistical significance . due to the low number of surviving grafts in lurcher mice , it was not possible to perform reliable statistical analysis of various morphological features of the grafts . the present study confirms and elaborates previous preliminary findings that have described cerebellar transplantation of naive p19 carcinoma stem cells and p19-derived neuroprogenitors in wild type mice by including similar analyses in lurcher mutant mice . our major observations indicate that in lurcher mice graft survival is quite poor and survival rate of the neuroprogenitor grafts is significantly lower in comparison to wild type mice when injected at the same tissue depth . the low number of surviving grafts did not allow for a more detailed comparison of morphological characteristics of the 2 types of grafts in lurcher mice . we can conclude , however , that naive p19 cell grafts typically exhibited expansive morphological characteristics without intragraft tissue destruction in both wild type and lurcher mice . in these parameters , the main finding of the present study was the observed regional difference in graft localization between wild type and lurcher mice . it is notable that in lurcher mice , viable grafts were localized to the mesencephalon and were never observed to be inside the cerebellum . this may be attributable to technical limitations due to the flattened cerebellum of the lurcher mutant mouse . in the lurcher mouse cerebellum , there is much less space for viable graft development than in wild type animals and the cerebellum could be easily missed by the tip of the injection needle . moreover , the depth of cell injection suitable for wild type mice could be too deep for the lurcher cerebellum . these anatomical factors could also play a role in the difference in neuroprogenitor graft survival between lurcher mutant and wild type mice . on the other hand , an apparent doubling of survival rate of the neuroprogenitor grafts when injected at the lower depth of 1.2 mm vs. 1.6 mm , together with a lower graft survival in lurcher as compared to wild type mice , suggests that the niche of the lurcher cerebellum is less suitable for the grafted cells than the adjacent mesencephalon . these intrinsic difficulties suggest that neurodegenerative changes of the cerebellar tissue could have a negative impact on the fate of stem cells grafted into the cerebellum of adult lurcher mice . the area of the graft contained numerous astrocytes , at least some of which were donor - derived . additionally , we have no evidence that the grafted cell ( naive embryonic carcinoma cells or neuroprogenitors ) differentiated into the purkinje cells , the main cell type that is missing in lurcher mutant mice . survival of embryonic carcinoma stem cells in lurcher mice is much lower than the survival of embryonic cerebellar tissue . tomey and heckroth , who transplanted a suspension of embryonic cerebellar cells , found surviving graft in 50% of both young and adult lurcher mice 12 months after transplantation . the survival of solid embryonic cerebellar graft 39 weeks after the surgery was 8090% in lurcher mice and it did not differ from wild type mice . however , cells grafted in the form of suspension are in direct and closer contact with the host tissue than cells inside the solid grafts . therefore , the influence of the host tissue on grafted cells development is stronger in cell suspension than in solid grafts . in addition , the solid embryonic cerebellar graft mediated controversial behavioural effects in lurcher mice . the appearance and low survival rate of the grafts do not show any promise for a sufficient therapeutic effect on cerebellar function that has deteriorated due to degenerative disease in lurcher mice . this is in contrast to the findings of benefit of transplantation of neurons derived from the human teratocarcinoma cell - line in animal models of several other neurological diseases .

the 2013 american college of cardiology foundation / american heart association ( accf / aha ) guidelines for stelevation myocardial infarction ( stemi ) recommend emergent reperfusion of patients presenting with stemi with a goal first medical contact ( fmc)todevice time of 90 minutes for patients undergoing primary percutaneous coronary intervention ( ppci ) and presenting to a pcicapable hospital.1 for patients presenting to a nonpcicapable hospital , the goal fmc to device time is 120 minutes . the other alternative to ppci is fibrinolysis , when timely transfer from a nonpcicapable to pcicapable hospital is not feasible , with a recommended doortoneedle ( dtn ) time of 30 minutes . these recommendations have evolved from 20042 with goal doortoballoon ( dtb ) time for ppci being 90 minutes and have become a focus of regional and national quality improvement initiatives.3 however , many hospitals in the united states still do not have the capacity to perform ppci or do so during regular hours only , and many of them are located in geographical areas where timely transfer for ppci is not feasible , resulting in failure to meet guidelinerecommended timely reperfusion with ppci.4 a study from the euro heart survey acsiii database examined temporal trends in reperfusion in stemi patients and found an overall decrease in fibrinolytic use with an increase in timely fibrinolysis from 61.7% to 71.1% with a concomitant decrease in dtn time from 20 to 15 minutes between 2006 and 2008.5 it is unclear whether a parallel improvement in the timeliness of reperfusion with fibrinolytic therapy has been observed in the united states and whether it has influenced inhospital mortality . furthermore , compliance with acute myocardial infarction ( ami ) performance measures6 in patients receiving fibrinolysis is not known . therefore , we aimed to examine the frequency and temporal pattern of fibrinolytic use compared to ppci in patients with stemi enrolled in the get with the guidelines coronary artery disease ( gwtgcad ) database . we also evaluated the predictors of fibrinolytic use and the timeliness of reperfusion with either strategy and compared inhospital mortality and performance measures in patients receiving fibrinolysis versus ppci . the gwtgcad database was launched in 2000 and represents a national , prospective , observational registry and quality improvement initiative established by the american heart association ( aha).7 , 8 , 9 , 10 it is a collaborative effort among researchers , professional organizations , and hospitals to provide feedback on performance and strategies to improve the care of patients with cad . the details of the program have been described elsewhere.10 the database includes teaching and nonteaching , rural and urban , large and small hospitals , and community and tertiary referral hospitals from all census regions of the united states . clinical information on patient demographics , medical history , symptoms on arrival , results of laboratory testing , inhospital treatment and events , discharge treatment and counseling , and on patient disposition is submitted using an online , interactive case report form and patient management tool ( patient management tool , outcome sciences inc , cambridge , ma ) . hospitals are encouraged to enroll all eligible patients consecutively with case finding preferentially based on clinical identification of patients.11 all participating institutions were required to comply with local regulatory and privacy guidelines and to submit the gwtgcad protocol for review and approval by their institutional review boards . because data were used primarily at each local site for quality improvement , sites were granted a waiver of informed consent under the common rule . between january 1 , 2003 and december 31 , 2008 , data were available on 238 465 patients enrolled from 415 hospitals participating in the gwtgcad program who were hospitalized with a confirmed clinical diagnosis of cad including patients with acute coronary syndromes , those with stable cad hospitalized for revascularization , and those with documented cad hospitalized for reasons other than heart failure . hospitals with > 25% missing from the medical history panel and patients with unrecorded sex were excluded from the analyses . patients were excluded if they were not diagnosed with stemi , did not receive fibrinolysis or primary pci , had missing discharge status , were transferred to another acute care facility , or left against medical advice ( figure 1 ) . sites with fewer than 5 patients after the prior exclusions were also excluded . following these exclusions , patients who had procedures listed as pci , pci with stent , or percutaneous transluminal angioplasty ( ptca ) and had not received fibrinolytics were classified as patients receiving ppci . we studied the following outcomes : ( 1 ) the overall frequency of fibrinolysis , ( 2 ) the frequency of timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( doortoballoon ( dtb ) 90 minutes ) , and ( 3 ) the association of fibrinolysis with inhospital mortality and compliance with ami performance measures of the gwtgcad program in patients presenting with stemi . we also assessed the temporal trends in fibrinolytic use , timeliness of reperfusion , inhospital mortality , and performance measure compliance during the study period . the performance measures of the gwtgcad program have been previously described8 and included ( 1 ) aspirin therapy within 24 hours of acute myocardial infarction ( ami ) , ( 2 ) aspirin therapy at discharge , ( 3 ) smoking cessation counseling for eligible patients , ( 4 ) angiotensinconverting enzyme inhibitor ( acei ) or angiotensin receptor blocker ( arb ) therapy in patients with left ventricular systolic dysfunction at discharge , ( 5)blocker ( bb ) therapy at discharge , and ( 6 ) lipidlowering therapy for patients with lowdensity lipoprotein ( ldl ) cholesterol levels > 100 mg / dl . a composite performance measure of compliance termed care was also assessed and defined as achievement of all 6 gwtgcad performance measures.12 length of stay was also examined . for the descriptive analyses , patients ' sociodemographic , insurance type and medical history variables , hospital characteristics , clinical performance measures , and inhospital mortality rates were compared between fibrinolytic therapy and ppci . data were reported as meansd values for continuous variables ( or medians and interquartile ranges , 25th75th percentiles ) and as percentages for categorical variables . categorical and continuous variables were compared with the use of the pearson and wilcoxon ranksum tests , respectively . a cochranarmitage test was performed to assess the temporal trend in the timeliness of reperfusion during the study period . multivariable logistic regression analyses were used to examine the predictors of fibrinolytic use and delayed therapy ( dtn > 30 minutes or dtb > 90 minutes ) as well as to evaluate the association of fibrinolysis and timely reperfusion with inhospital outcomes . the generalized estimating equation method with exchangeable working correlation structure was used to account for withinhospital clustering because patients at the same hospital are more likely to have similar treatment relative to patients in other hospitals . transferin patients and sites not capable of pci were also excluded from the analysis of delayed dtb time . the regression models adjusted for the following covariates : patient demographics ( age , sex , race , height , weight , blood pressure , insurance ) , medical history variables ( anemia , atrial fibrillation , atrial flutter , cad , chronic obstructive pulmonary disease [ copd ] or asthma , cardiac resynchronization therapy defibrillator [ crtd ] , crtpacing [ crtp ] , cerebrovascular accident [ cva]/transient ischemic attack [ tia ] , depression , diabetes , dialysis , heart failure , hyperlipidemia , hypertension , icd only , pacemaker , peripheral vascular disease [ pvd ] , coronary artery bypass graft [ cabg ] , prior myocardial infarction [ mi ] , prior pci , renal insufficiency , valvular heart disease , prior cabg , smoking ) , arrival time ( off hours vs regular hours ) , hospital characteristics ( region , hospital type , number of beds , rural vs urban , capability of cardiothoracic surgery ) , and admission year . additional analyses included comparisons of inhospital outcomes and performance measure compliance between patients who received timely therapy and those who did not ( dtn 30 minutes vs dtn > 30 minutes , and dtb 90 minutes vs dtb > 90 minutes ) . most variables had complete data or had a missing rate < 3% , with the exception of insurance , weight , and bmi , which had 7% missing . a p<0.05 was considered statistically significant for all tests , and all tests of statistical significance were 2tailed . all statistical analyses were performed centrally at the duke clinical research institute ( dcri , durham , nc ) with sas software ( version 9.1 , sas institute , cary , nc ) . the gwtgcad database was launched in 2000 and represents a national , prospective , observational registry and quality improvement initiative established by the american heart association ( aha).7 , 8 , 9 , 10 it is a collaborative effort among researchers , professional organizations , and hospitals to provide feedback on performance and strategies to improve the care of patients with cad . the details of the program have been described elsewhere.10 the database includes teaching and nonteaching , rural and urban , large and small hospitals , and community and tertiary referral hospitals from all census regions of the united states . clinical information on patient demographics , medical history , symptoms on arrival , results of laboratory testing , inhospital treatment and events , discharge treatment and counseling , and on patient disposition is submitted using an online , interactive case report form and patient management tool ( patient management tool , outcome sciences inc , cambridge , ma ) . hospitals are encouraged to enroll all eligible patients consecutively with case finding preferentially based on clinical identification of patients.11 all participating institutions were required to comply with local regulatory and privacy guidelines and to submit the gwtgcad protocol for review and approval by their institutional review boards . because data were used primarily at each local site for quality improvement , sites were granted a waiver of informed consent under the common rule . between january 1 , 2003 and december 31 , 2008 , data were available on 238 465 patients enrolled from 415 hospitals participating in the gwtgcad program who were hospitalized with a confirmed clinical diagnosis of cad including patients with acute coronary syndromes , those with stable cad hospitalized for revascularization , and those with documented cad hospitalized for reasons other than heart failure . hospitals with > 25% missing from the medical history panel and patients with unrecorded sex were excluded from the analyses . patients were excluded if they were not diagnosed with stemi , did not receive fibrinolysis or primary pci , had missing discharge status , were transferred to another acute care facility , or left against medical advice ( figure 1 ) . sites with fewer than 5 patients after the prior exclusions were also excluded . following these exclusions , patients who had procedures listed as pci , pci with stent , or percutaneous transluminal angioplasty ( ptca ) and had not received fibrinolytics were classified as patients receiving ppci . we studied the following outcomes : ( 1 ) the overall frequency of fibrinolysis , ( 2 ) the frequency of timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( doortoballoon ( dtb ) 90 minutes ) , and ( 3 ) the association of fibrinolysis with inhospital mortality and compliance with ami performance measures of the gwtgcad program in patients presenting with stemi . we also assessed the temporal trends in fibrinolytic use , timeliness of reperfusion , inhospital mortality , and performance measure compliance during the study period . the performance measures of the gwtgcad program have been previously described8 and included ( 1 ) aspirin therapy within 24 hours of acute myocardial infarction ( ami ) , ( 2 ) aspirin therapy at discharge , ( 3 ) smoking cessation counseling for eligible patients , ( 4 ) angiotensinconverting enzyme inhibitor ( acei ) or angiotensin receptor blocker ( arb ) therapy in patients with left ventricular systolic dysfunction at discharge , ( 5)blocker ( bb ) therapy at discharge , and ( 6 ) lipidlowering therapy for patients with lowdensity lipoprotein ( ldl ) cholesterol levels > 100 mg / dl . a composite performance measure of compliance termed care was also assessed and defined as achievement of all 6 gwtgcad performance measures.12 length of stay was also examined . for the descriptive analyses , patients ' sociodemographic , insurance type and medical history variables , hospital characteristics , clinical performance measures , and inhospital mortality rates were compared between fibrinolytic therapy and ppci . data were reported as meansd values for continuous variables ( or medians and interquartile ranges , 25th75th percentiles ) and as percentages for categorical variables . categorical and continuous variables were compared with the use of the pearson and wilcoxon ranksum tests , respectively . a cochranarmitage test was performed to assess the temporal trend in the timeliness of reperfusion during the study period . multivariable logistic regression analyses were used to examine the predictors of fibrinolytic use and delayed therapy ( dtn > 30 minutes or dtb > 90 minutes ) as well as to evaluate the association of fibrinolysis and timely reperfusion with inhospital outcomes . the generalized estimating equation method with exchangeable working correlation structure was used to account for withinhospital clustering because patients at the same hospital are more likely to have similar treatment relative to patients in other hospitals . transferin patients and sites not capable of pci were also excluded from the analysis of delayed dtb time . the regression models adjusted for the following covariates : patient demographics ( age , sex , race , height , weight , blood pressure , insurance ) , medical history variables ( anemia , atrial fibrillation , atrial flutter , cad , chronic obstructive pulmonary disease [ copd ] or asthma , cardiac resynchronization therapy defibrillator [ crtd ] , crtpacing [ crtp ] , cerebrovascular accident [ cva]/transient ischemic attack [ tia ] , depression , diabetes , dialysis , heart failure , hyperlipidemia , hypertension , icd only , pacemaker , peripheral vascular disease [ pvd ] , coronary artery bypass graft [ cabg ] , prior myocardial infarction [ mi ] , prior pci , renal insufficiency , valvular heart disease , prior cabg , smoking ) , arrival time ( off hours vs regular hours ) , hospital characteristics ( region , hospital type , number of beds , rural vs urban , capability of cardiothoracic surgery ) , and admission year . additional analyses included comparisons of inhospital outcomes and performance measure compliance between patients who received timely therapy and those who did not ( dtn 30 minutes vs dtn > 30 minutes , and dtb 90 minutes vs dtb > 90 minutes ) . most variables had complete data or had a missing rate < 3% , with the exception of insurance , weight , and bmi , which had 7% missing . a p<0.05 was considered statistically significant for all tests , and all tests of statistical significance were 2tailed . all statistical analyses were performed centrally at the duke clinical research institute ( dcri , durham , nc ) with sas software ( version 9.1 , sas institute , cary , nc ) . we evaluated a total of 238 465 patients from 415 hospitals between january 1 , 2003 and december 31 , 2008 from the gwtgcad database . after excluding patients not diagnosed with stemi , those not undergoing reperfusion therapy , patients with missing discharge data , transferout patients , and those leaving against medical advice , the final study cohort included 29 190 stemi patients from 229 hospitals . overall , 38.2% of patients achieved timely fibrinolysis with a median dtn of 37 minutes ( iqr 2165 ) . during hospitalization , 74.4% of these patients underwent coronary angiography , 58.8% underwent pci , and 7.2% underwent cabg surgery . baseline characteristics of patients receiving fibrinolysis compared to ppci are summarized in table 1 . patients receiving fibrinolysis had a lower prevalence of prior cad and dyslipidemia , higher prevalence of diabetes and hypertension , and presented to rural hospitals more frequently . overall , 54.8% of patients undergoing ppci received timely ppci ( dtb 90 minutes ) with a median dtb of 80 ( iqr 51120 ) minutes . baseline characteristics of patients with stemi receiving fibrinolysis vs ppci bmi indicates body mass index ; cabg , coronary artery bypass graft ; cad , coronary artery disease ; copd , chronic obstructive pulmonary disease ; cva , cerebrovascular accident ; pci , percutaneous coronary intervention ; ppci , primary percutaneous coronary intervention ; sd , standard deviation ; stemi , st elevation myocardial infarction ; tia , transient ischemic attack . fibrinolysis decreased from 20.5% in 2003 to 3.7% in 2008 ( p<0.0001 ) ( figure 2a ) . there was an increase in median dtn ( 36 to 60 minutes ; p=0.005 ) , and median dtb decreased ( 94 to 64 minutes ; p<0.0001 ) . as a result , there was a decrease in the proportion of patients with timely fibrinolysis and an increase in the proportion of patients with timely ppci ( figure 2b ) . inhospital mortality with fibrinolysis was higher than that with ppci ( 4.6% vs 3.3% , p=0.001 ) and was consistent during the study period ( figure 2c ) . median length of stay was longer for patients receiving fibrinolysis compared to ppci ( 4 [ iqr 36 ] vs 3 [ iqr 25 ] days , p<0.0001 ) ( table 2 ) . a , annual trends in fibrinolysis and ppci for patients with stemi in the gwtgcad database . the frequency of fibrinolysis decreased from 20.5% in 2003 to 3.7% in 2008 while ppci increased from 79.5% in 2003 to 96.3% in 2008 ( p value from cochranarmitage test < 0.0001 ) . b , annual trends in the proportion of patients receiving timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( dtb 90 minutes ) for patients with stemi in the gwtgcad database . the frequency of timely fibrinolysis peaked at 45.8% in 2006 and decreased to 22.9% in 2008 while timely ppci increased from 37.0% in 2003 to 76.3% in 2008 . ( p value from cochranarmitage test=0.2769 for dtn 30 minutes ; p<0.0001 for dtb 90 minutes . ) inhospital mortality was 4.6% in patients receiving fibrinolysis and 3.3% in patients receiving ppci and did not change significantly over time . ( p value from cochranarmitage test=0.9473 for fibrinolytics patients , p=0.1474 for ppci patients . ) cad indicates coronary artery disease ; dtb , door to balloon ; dtn , door to needle ; gwtg , get with the guidelines ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . inhospital outcomes between patients with stemi receiving fibrinolysis vs ppci dtb indicates door to balloon ; dtn , door to needle ; iqr , interquartile range ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . independent predictors of fibrinolytic use included offhour presentation , midwest and southern regions versus northeast , rural location , smoking , and no hyperlipidemia . independent predictors of delayed fibrinolysis and delayed ppci are summarized in tables s1 through s3 . performance measures and defectfree care were lower among patients receiving fibrinolysis compared to ppci ( defectfree care 81.1% vs 90.1% , p<0.0001 ) ( table 3 ) . however , in patients receiving fibrinolysis , defectfree care increased over the study period ( 74% to 91% , p<0.0001 ) . no difference in compliance with performance measures was noted between patients with timely versus delayed fibrinolysis , although patients with timely ppci were more likely to receive defectfree care than patients with delayed ppci ( 93.3% vs 87.7% , p<0.0001 ) . adherence to cad performance measures between patients with stemi receiving fibrinolysis vs ppci acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; cad , coronary artery disease ; ldl , lowdensity lipoprotein ; lvsd , left ventricular systolic dysfunction ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . after multivariable adjustment , there was no significant association of reperfusion therapy type or timely fibrinolysis ( dtn 30 minutes ) with inhospital mortality . however , an association between timely ppci ( dtb 90 minutes ) and inhospital mortality was seen ( or 0.63 , 95% ci 0.490.79 ; p=0.0001 ) ( table 4 ) . association of outcomes with therapy type and timely reperfusion adjusted for age , sex , race , insurance , body mass index ( bmi ) , height , medical history including anemia , atrial fibrillation , atrial flutter , coronary artery disease ( cad ) , chronic obstructive pulmonary disease ( copd ) or asthma , cardiac resynchronization therapydefibrillator ( crtd ) , cardiac resynchronization therapypacemaker ( crtp ) , cerebrovascular accident ( cva)/transient ischemic attack ( tia ) , diabetes , heart failure , hyperlipidemia , hypertension , implantable cardioverter defibrillator ( icd ) , pacemaker , peripheral vascular disease ( pvd ) , coronary artery bypass graft ( cabg ) , prior myocardial infarction ( mi ) , prior percutaneous coronary intervention ( pci ) , renal insufficiency , valvular heart disease , smoking ; hospital characteristics ( region , hospital type , number of beds , rural vs urban , admission yearquarter ) . acei indicates angiotensin converting enzyme inhibitor ; arb , angiotensin receptor blocker ; cad , coronary artery disease ; ci , confidence interval ; dtb , door to balloon ; dtn , door to needle ; ldl , lowdensity lipoprotein ; lvsd , left ventricular systolic dysfunction ; or , odds ratio ; ppci , primary percutaneous coronary intervention . our data provide an insight into the use of fibrinolytic therapy for acute reperfusion treatment of stemi in a large national registry of hospitals in the united states . we have shown that 8.4% of patients presenting with stemi received fibrinolysis , of whom only 38.2% achieved timely dtn 30 minutes . the use of fibrinolytic therapy decreased from 20.5% in 2003 to 3.7% in 2008 , and its timeliness worsened , with nearly doubling in median dtn times from 36 to 60 minutes ( p=0.005 ) . therefore , it is important to continue to monitor timeliness and outcomes of fibrinolysis in eligible patients as use of fibrinolysis becomes more infrequent . ppci is associated with improved mortality compared to fibrinolytic therapy in patients presenting with stemi and is the preferred reperfusion strategy when feasible in a timely manner.1 , 13 it has been shown that for every 10minute delay in performing pci , the mortality benefit of pci compared to fibrinolysis is decreased by 1% , and both reperfusion strategies become almost equivalent at 62 minutes.14 however , only one third of acute care hospitals in the united states have aroundtheclock pci capability.15 , 16 further , despite investment of effort and resources , limited reductions in interhospital transfer times have occurred.17 the guidelines for management of patients with stemi have evolved with the 1996 and 1999 recommendations18 stating that ppci was an alternative to fibrinolytic therapy for reperfusion and 2004 recommendations2 stating that ppci was the preferred reperfusion strategy with fibrinolysis being used when timely ppci could not be delivered . this likely influenced practice patterns and is consistent with the temporal trend we found in our study with increasing use of ppci and declining fibrinolytic use . the current guidelines for stemi recommend fibrinolysis for eligible patients presenting to nonpcicapable hospitals who would not receive fmctodevice time 120 minutes on transfer . therefore , fibrinolysis plays a key role in the management of eligible stemi patients unable to receive timely ppci . a recent report from the national cardiovascular data registry ( ncdr)4 showed that , among patients presenting to a nonpcicapable hospital and transferred for ppci , only 51.3% of patients achieved dtb 120 minutes . among patients eligible to receive fibrinolysis with estimated drive time over 60 minutes , only 52.7% received fibrinolysis . median dtn was 34 minutes , and only 43.8% achieved dtn 30 minutes , which is consistent with our results . the inhospital mortality of patients receiving fibrinolysis was 3.7% in that report,4 which is also similar to our findings . inhospital mortality of patients undergoing ppci in that report was 3.9% , which is higher than the 3.3% seen in our study and may have been due to delay in transfer with median dtb time of 126 minutes . possible reasons for the worsening in the timeliness of fibrinolysis include the overall decrease in the frequency of fibrinolytic use , resulting in unfamiliarity with its administration protocols , complex care coordination between transferring and receiving centers resulting in delays and cancellation of some transfers , and the perceived hazards of fibrinolytic therapy ( eg , intracranial hemorrhage ) despite delayed transfer resulting in reluctance in its use . another report from the ncdr examined temporal trends in dtb time.19 they found that between 2005 and 2009 , median dtb time had decreased from 83 to 67 minutes while the percentage of patients achieving timely ppci ( dtb 90 minutes ) had increased from 59.7% to 83.1% . despite these significant improvements in dtb times we previously argued that some of the time differential in dtb times observed in clinical studies may be too small to exert a meaningful clinical impact , especially when shortterm outcomes are examined.20 , 21 a prior report from the national registry of myocardial infarction ( nrmi)22 evaluating reperfusion strategies in stemi patients in 1990 through 2006 found a decrease in fibrinolysis from 52.5% to 27.6% with a nearly linear decline in dtn from 59 to 29 minutes and a corresponding decrease in mortality from 7.0% to 6.0% over the study period . the median dtn in our study was lowest in 2006 ( 31 minutes ) , similar to the results from this study . this was followed by an increase to 43.5 minutes in 2007 and 60 minutes in 2008 . overall , the mortality rates in our study were among the lowest reported with both reperfusion strategies . the lack of significant change in inhospital mortality is consistent with other reports and can be attributed to already marked improvement in inhospital mortality rates , making it difficult to achieve further incremental benefit . one may also argue that the total ischemic time from onset of arterial occlusion to reperfusion , rather than medical contact to reperfusion times , may play a more important role in shortterm inhospital outcomes . this may also be related to a low event rate among a small number of patients who received fibrinolysis in our study . further , there has been some renewed interest in fibrinolysis and pharmacoinvasive strategies in which cardiac catheterization is routinely performed within 6 hours of fibrinolytic administration . the trial of routine angioplasty and stenting after fibrinolysis to enhance reperfusion in acute myocardial infarction ( transferami)23 showed that highrisk stemi patients receiving fibrinolysis and routine early pci had improved shortterm outcomes compared to patients receiving standard therapy and delayed pci . a report of 73 belgian hospitals24 found that modern fibrinolytic strategies substantially attenuated the mortality benefit of ppci over fibrinolysis except for patients in the highestrisk subgroup with thrombolysis in myocardial infarction ( timi ) scores of 7 to 14 . the strategic reperfusion early after myocardial infarction ( stream)25 study showed that in patients with stemi unable to undergo ppci within 1 hour , prehospital fibrinolysis and angiography within 6 to 24 hours resulted in effective reperfusion compared to ppci alone . however , fibrinolysis was associated with a slight increase in intracranial bleeding.26 therefore , given the delays in transfer times from nonpcicapable hospitals and the possible lack of mortality benefit of ppci over fibrinolysis in lowrisk patients , the role of timely reperfusion with fibrinolysis followed by routine angiography needs to reconsidered . the acc / aha emphasis on care of patients with stemi has resulted in the public reporting of these performance measures,3 which are also now tied to reimbursement from the centers for medicare and medicaid services27 and therefore have financial implications for institutions . our results , as well as those of others , depict the significant improvements seen in dtb times and timely ppci . however , as our study has shown , this improvement has not occurred in dtn times and timely fibrinolysis . additionally , the improvement noted in defectfree care and adherence to performance measures in patients receiving either reperfusion strategy over the study period indicates the important role played by quality improvement programs . however , defectfree care was found to be significantly lower in patients receiving fibrinolysis and should raise concerns in patients receiving fibrinolysis as use continues to decrease . first , the gwtg program is a quality improvement program , and participation is voluntary . therefore , these hospitals may be highly motivated for quality improvement , and results may not be fully representative of national care patterns and clinical outcomes . second , data on prehospital delay or treatments , 24hour pci capability of hospitals , bleeding outcomes , or postdischarge mortality and morbidity were not available . third , eligibility , type , and time of treatment received were based on documentation in the medical record and were thus dependent on the accuracy of this documentation . fourth , there might be other measured or unmeasured confounding variables that , had they been adjusted for , would have altered outcomes . finally , trends in reperfusion strategies were assessed between 2003 and 2008 , and therefore , contemporary patterns of fibrinolysis may be different . however , our study demonstrates the importance of tracking and improving dtn times in hospitals that do not perform ppci around the clock as well as the need for contemporary data in trends and outcomes of fibrinolytic use for stemi . first , the gwtg program is a quality improvement program , and participation is voluntary . therefore , these hospitals may be highly motivated for quality improvement , and results may not be fully representative of national care patterns and clinical outcomes . second , data on prehospital delay or treatments , 24hour pci capability of hospitals , bleeding outcomes , or postdischarge mortality and morbidity were not available . third , eligibility , type , and time of treatment received were based on documentation in the medical record and were thus dependent on the accuracy of this documentation . fourth , there might be other measured or unmeasured confounding variables that , had they been adjusted for , would have altered outcomes . finally , trends in reperfusion strategies were assessed between 2003 and 2008 , and therefore , contemporary patterns of fibrinolysis may be different . however , our study demonstrates the importance of tracking and improving dtn times in hospitals that do not perform ppci around the clock as well as the need for contemporary data in trends and outcomes of fibrinolytic use for stemi . the use of fibrinolytic therapy for stemi has decreased from 2003 to 2008 , in contradistinction to the use of ppci . despite improvements in timely ppci and defectfree care , a significant delay in timely fibrinolysis inhospital mortality among stemi patients receiving fibrinolysis remained unchanged over the study period and worse than that of their ppci counterparts . our findings highlight important opportunities to improve the use of fibrinolytic therapy and its timeliness among eligible stemi patients . hira was the recipient of a database research seed grant from the american heart association council on clinical cardiology . bhatt discloses the following relationships : advisory board of cardax , elsevier practice update cardiology , medscape cardiology , regado biosciences ; board of directors of boston va research institute , society of cardiovascular patient care ; chair of american heart association quality oversight committee ; data monitoring committee of duke clinical research institute , harvard clinical research institute , mayo clinic , population health research institute ; honoraria from american college of cardiology ( senior associate editor , clinical trials and news , acc.org ) , belvoir publications ( editor in chief , harvard heart letter ) , duke clinical research institute ( clinical trial steering committees ) , harvard clinical research institute ( clinical trial steering committee ) , hmp communications ( editor in chief , journal of invasive cardiology ) , journal of the american college of cardiology ( guest editor ; associate editor ) , population health research institute ( clinical trial steering committee ) , slack publications ( chief medical editor , cardiology today 's intervention ) , society of cardiovascular patient care ( secretary / treasurer ) , webmd ( cme steering committees ) , others including clinical cardiology ( deputy editor ) ; research funding from amarin , amgen , astrazeneca , bristolmyers squibb , eisai , ethicon , forest laboratories , ischemix , medtronic , pfizer , roche , sanofi aventis , the medicines company ; royalties from elsevier ( editor , cardiovascular intervention : a companion to braunwald 's heart disease ) ; site coinvestigator at biotronik and st . jude medical ; trustee of american college of cardiology ; unfunded research for flowco , plx pharma , takeda . virani receives grant / research support ( all significant and paid to the institution , not individual ) from the department of veterans affairs , american diabetes association , and american heart association . eapen discloses the following relationships : novartis ( consultant , advisory board ) , amgen ( consultant , advisory board ) , shl telemedicine ( consultant ) , janssen ( honorarium ) , myokardia ( consultant ) . peterson is the codirector of the gwtg data analytic center at the duke clinical research institute . table s1 . predictors of fibrinolytic use vs ppci table s2 . predictors of delayed fibrinolysis ( dtn > 30 minutes ) versus timely fibrinolysis ( dtn < 30 minutes ) table s3 . predictors of delayed ppci ( dtb > 90 minutes ) vs timely ppci ( dtb < 90 minutes ) click here for additional data file .

backgroundtimely reperfusion after stelevation myocardial infarction ( stemi ) improves survival . guidelines recommend primary percutaneous coronary intervention ( ppci ) within 90 minutes of arrival at a pcicapable hospital . the alternative is fibrinolysis within 30 minutes for those in those for whom timely transfer to a pcicapable hospital is not feasible.methods and resultswe identified stemi patients receiving reperfusion therapy at 229 hospitals participating in the get with the guidelines coronary artery disease ( gwtgcad ) database ( january 1 , 2003 through december 31 , 2008 ) . temporal trends in the use of fibrinolysis and ppci , its timeliness , and inhospital mortality outcomes were assessed . we also assessed predictors of fibrinolysis versus ppci and compliance with performance measures . defectfree care was defined as 100% compliance with all performance measures . we identified 29 190 stemi patients , of whom 2441 ( 8.4% ) received fibrinolysis ; 38.2% of these patients achieved doortoneedle times 30 minutes . median doortoneedle times increased from 36 to 60 minutes ( p=0.005 ) over the study period . among ppci patients , median doortoballoon times decreased from 94 to 64 minutes ( p<0.0001 ) over the same period . inhospital mortality was higher with fibrinolysis than with ppci ( 4.6% vs 3.3% , p=0.001 ) and did not change significantly over time . patients receiving fibrinolysis were less likely to receive defectfree care compared with their ppci counterparts.conclusionsuse of fibrinolysis for stemi has decreased over time with concomitant worsening of doortoneedle times . over the same time period , use of ppci increased with improvement in doortoballoon times . inhospital mortality was higher with fibrinolysis than with ppci . as reperfusion for stemi continues to shift from fibrinolysis to ppci , it will be critical to ensure that doortoneedle times and outcomes do not worsen .

Introduction Methods Study Population Cohort Development and Definitions Study Outcomes and Measures Statistical Analyses Results Discussion Study Limitations Conclusions Sources of Funding Disclosures Supporting information

the 2013 american college of cardiology foundation / american heart association ( accf / aha ) guidelines for stelevation myocardial infarction ( stemi ) recommend emergent reperfusion of patients presenting with stemi with a goal first medical contact ( fmc)todevice time of 90 minutes for patients undergoing primary percutaneous coronary intervention ( ppci ) and presenting to a pcicapable hospital.1 for patients presenting to a nonpcicapable hospital , the goal fmc to device time is 120 minutes . the other alternative to ppci is fibrinolysis , when timely transfer from a nonpcicapable to pcicapable hospital is not feasible , with a recommended doortoneedle ( dtn ) time of 30 minutes . these recommendations have evolved from 20042 with goal doortoballoon ( dtb ) time for ppci being 90 minutes and have become a focus of regional and national quality improvement initiatives.3 however , many hospitals in the united states still do not have the capacity to perform ppci or do so during regular hours only , and many of them are located in geographical areas where timely transfer for ppci is not feasible , resulting in failure to meet guidelinerecommended timely reperfusion with ppci.4 a study from the euro heart survey acsiii database examined temporal trends in reperfusion in stemi patients and found an overall decrease in fibrinolytic use with an increase in timely fibrinolysis from 61.7% to 71.1% with a concomitant decrease in dtn time from 20 to 15 minutes between 2006 and 2008.5 it is unclear whether a parallel improvement in the timeliness of reperfusion with fibrinolytic therapy has been observed in the united states and whether it has influenced inhospital mortality . furthermore , compliance with acute myocardial infarction ( ami ) performance measures6 in patients receiving fibrinolysis is not known . therefore , we aimed to examine the frequency and temporal pattern of fibrinolytic use compared to ppci in patients with stemi enrolled in the get with the guidelines coronary artery disease ( gwtgcad ) database . we also evaluated the predictors of fibrinolytic use and the timeliness of reperfusion with either strategy and compared inhospital mortality and performance measures in patients receiving fibrinolysis versus ppci . between january 1 , 2003 and december 31 , 2008 , data were available on 238 465 patients enrolled from 415 hospitals participating in the gwtgcad program who were hospitalized with a confirmed clinical diagnosis of cad including patients with acute coronary syndromes , those with stable cad hospitalized for revascularization , and those with documented cad hospitalized for reasons other than heart failure . we studied the following outcomes : ( 1 ) the overall frequency of fibrinolysis , ( 2 ) the frequency of timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( doortoballoon ( dtb ) 90 minutes ) , and ( 3 ) the association of fibrinolysis with inhospital mortality and compliance with ami performance measures of the gwtgcad program in patients presenting with stemi . we also assessed the temporal trends in fibrinolytic use , timeliness of reperfusion , inhospital mortality , and performance measure compliance during the study period . the performance measures of the gwtgcad program have been previously described8 and included ( 1 ) aspirin therapy within 24 hours of acute myocardial infarction ( ami ) , ( 2 ) aspirin therapy at discharge , ( 3 ) smoking cessation counseling for eligible patients , ( 4 ) angiotensinconverting enzyme inhibitor ( acei ) or angiotensin receptor blocker ( arb ) therapy in patients with left ventricular systolic dysfunction at discharge , ( 5)blocker ( bb ) therapy at discharge , and ( 6 ) lipidlowering therapy for patients with lowdensity lipoprotein ( ldl ) cholesterol levels > 100 mg / dl . a composite performance measure of compliance termed care was also assessed and defined as achievement of all 6 gwtgcad performance measures.12 length of stay was also examined . for the descriptive analyses , patients ' sociodemographic , insurance type and medical history variables , hospital characteristics , clinical performance measures , and inhospital mortality rates were compared between fibrinolytic therapy and ppci . categorical and continuous variables were compared with the use of the pearson and wilcoxon ranksum tests , respectively . multivariable logistic regression analyses were used to examine the predictors of fibrinolytic use and delayed therapy ( dtn > 30 minutes or dtb > 90 minutes ) as well as to evaluate the association of fibrinolysis and timely reperfusion with inhospital outcomes . additional analyses included comparisons of inhospital outcomes and performance measure compliance between patients who received timely therapy and those who did not ( dtn 30 minutes vs dtn > 30 minutes , and dtb 90 minutes vs dtb > 90 minutes ) . between january 1 , 2003 and december 31 , 2008 , data were available on 238 465 patients enrolled from 415 hospitals participating in the gwtgcad program who were hospitalized with a confirmed clinical diagnosis of cad including patients with acute coronary syndromes , those with stable cad hospitalized for revascularization , and those with documented cad hospitalized for reasons other than heart failure . we studied the following outcomes : ( 1 ) the overall frequency of fibrinolysis , ( 2 ) the frequency of timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( doortoballoon ( dtb ) 90 minutes ) , and ( 3 ) the association of fibrinolysis with inhospital mortality and compliance with ami performance measures of the gwtgcad program in patients presenting with stemi . we also assessed the temporal trends in fibrinolytic use , timeliness of reperfusion , inhospital mortality , and performance measure compliance during the study period . the performance measures of the gwtgcad program have been previously described8 and included ( 1 ) aspirin therapy within 24 hours of acute myocardial infarction ( ami ) , ( 2 ) aspirin therapy at discharge , ( 3 ) smoking cessation counseling for eligible patients , ( 4 ) angiotensinconverting enzyme inhibitor ( acei ) or angiotensin receptor blocker ( arb ) therapy in patients with left ventricular systolic dysfunction at discharge , ( 5)blocker ( bb ) therapy at discharge , and ( 6 ) lipidlowering therapy for patients with lowdensity lipoprotein ( ldl ) cholesterol levels > 100 mg / dl . a composite performance measure of compliance termed care was also assessed and defined as achievement of all 6 gwtgcad performance measures.12 length of stay was also examined . for the descriptive analyses , patients ' sociodemographic , insurance type and medical history variables , hospital characteristics , clinical performance measures , and inhospital mortality rates were compared between fibrinolytic therapy and ppci . categorical and continuous variables were compared with the use of the pearson and wilcoxon ranksum tests , respectively . multivariable logistic regression analyses were used to examine the predictors of fibrinolytic use and delayed therapy ( dtn > 30 minutes or dtb > 90 minutes ) as well as to evaluate the association of fibrinolysis and timely reperfusion with inhospital outcomes . additional analyses included comparisons of inhospital outcomes and performance measure compliance between patients who received timely therapy and those who did not ( dtn 30 minutes vs dtn > 30 minutes , and dtb 90 minutes vs dtb > 90 minutes ) . we evaluated a total of 238 465 patients from 415 hospitals between january 1 , 2003 and december 31 , 2008 from the gwtgcad database . after excluding patients not diagnosed with stemi , those not undergoing reperfusion therapy , patients with missing discharge data , transferout patients , and those leaving against medical advice , the final study cohort included 29 190 stemi patients from 229 hospitals . overall , 38.2% of patients achieved timely fibrinolysis with a median dtn of 37 minutes ( iqr 2165 ) . baseline characteristics of patients with stemi receiving fibrinolysis vs ppci bmi indicates body mass index ; cabg , coronary artery bypass graft ; cad , coronary artery disease ; copd , chronic obstructive pulmonary disease ; cva , cerebrovascular accident ; pci , percutaneous coronary intervention ; ppci , primary percutaneous coronary intervention ; sd , standard deviation ; stemi , st elevation myocardial infarction ; tia , transient ischemic attack . fibrinolysis decreased from 20.5% in 2003 to 3.7% in 2008 ( p<0.0001 ) ( figure 2a ) . there was an increase in median dtn ( 36 to 60 minutes ; p=0.005 ) , and median dtb decreased ( 94 to 64 minutes ; p<0.0001 ) . inhospital mortality with fibrinolysis was higher than that with ppci ( 4.6% vs 3.3% , p=0.001 ) and was consistent during the study period ( figure 2c ) . median length of stay was longer for patients receiving fibrinolysis compared to ppci ( 4 [ iqr 36 ] vs 3 [ iqr 25 ] days , p<0.0001 ) ( table 2 ) . a , annual trends in fibrinolysis and ppci for patients with stemi in the gwtgcad database . the frequency of fibrinolysis decreased from 20.5% in 2003 to 3.7% in 2008 while ppci increased from 79.5% in 2003 to 96.3% in 2008 ( p value from cochranarmitage test < 0.0001 ) . b , annual trends in the proportion of patients receiving timely reperfusion with fibrinolysis ( dtn 30 minutes ) and ppci ( dtb 90 minutes ) for patients with stemi in the gwtgcad database . inhospital mortality was 4.6% in patients receiving fibrinolysis and 3.3% in patients receiving ppci and did not change significantly over time . cad indicates coronary artery disease ; dtb , door to balloon ; dtn , door to needle ; gwtg , get with the guidelines ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . inhospital outcomes between patients with stemi receiving fibrinolysis vs ppci dtb indicates door to balloon ; dtn , door to needle ; iqr , interquartile range ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . performance measures and defectfree care were lower among patients receiving fibrinolysis compared to ppci ( defectfree care 81.1% vs 90.1% , p<0.0001 ) ( table 3 ) . however , in patients receiving fibrinolysis , defectfree care increased over the study period ( 74% to 91% , p<0.0001 ) . no difference in compliance with performance measures was noted between patients with timely versus delayed fibrinolysis , although patients with timely ppci were more likely to receive defectfree care than patients with delayed ppci ( 93.3% vs 87.7% , p<0.0001 ) . adherence to cad performance measures between patients with stemi receiving fibrinolysis vs ppci acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; cad , coronary artery disease ; ldl , lowdensity lipoprotein ; lvsd , left ventricular systolic dysfunction ; ppci , primary percutaneous coronary intervention ; stemi , st elevation myocardial infarction . however , an association between timely ppci ( dtb 90 minutes ) and inhospital mortality was seen ( or 0.63 , 95% ci 0.490.79 ; p=0.0001 ) ( table 4 ) . association of outcomes with therapy type and timely reperfusion adjusted for age , sex , race , insurance , body mass index ( bmi ) , height , medical history including anemia , atrial fibrillation , atrial flutter , coronary artery disease ( cad ) , chronic obstructive pulmonary disease ( copd ) or asthma , cardiac resynchronization therapydefibrillator ( crtd ) , cardiac resynchronization therapypacemaker ( crtp ) , cerebrovascular accident ( cva)/transient ischemic attack ( tia ) , diabetes , heart failure , hyperlipidemia , hypertension , implantable cardioverter defibrillator ( icd ) , pacemaker , peripheral vascular disease ( pvd ) , coronary artery bypass graft ( cabg ) , prior myocardial infarction ( mi ) , prior percutaneous coronary intervention ( pci ) , renal insufficiency , valvular heart disease , smoking ; hospital characteristics ( region , hospital type , number of beds , rural vs urban , admission yearquarter ) . acei indicates angiotensin converting enzyme inhibitor ; arb , angiotensin receptor blocker ; cad , coronary artery disease ; ci , confidence interval ; dtb , door to balloon ; dtn , door to needle ; ldl , lowdensity lipoprotein ; lvsd , left ventricular systolic dysfunction ; or , odds ratio ; ppci , primary percutaneous coronary intervention . we have shown that 8.4% of patients presenting with stemi received fibrinolysis , of whom only 38.2% achieved timely dtn 30 minutes . the use of fibrinolytic therapy decreased from 20.5% in 2003 to 3.7% in 2008 , and its timeliness worsened , with nearly doubling in median dtn times from 36 to 60 minutes ( p=0.005 ) . therefore , it is important to continue to monitor timeliness and outcomes of fibrinolysis in eligible patients as use of fibrinolysis becomes more infrequent . ppci is associated with improved mortality compared to fibrinolytic therapy in patients presenting with stemi and is the preferred reperfusion strategy when feasible in a timely manner.1 , 13 it has been shown that for every 10minute delay in performing pci , the mortality benefit of pci compared to fibrinolysis is decreased by 1% , and both reperfusion strategies become almost equivalent at 62 minutes.14 however , only one third of acute care hospitals in the united states have aroundtheclock pci capability.15 , 16 further , despite investment of effort and resources , limited reductions in interhospital transfer times have occurred.17 the guidelines for management of patients with stemi have evolved with the 1996 and 1999 recommendations18 stating that ppci was an alternative to fibrinolytic therapy for reperfusion and 2004 recommendations2 stating that ppci was the preferred reperfusion strategy with fibrinolysis being used when timely ppci could not be delivered . this likely influenced practice patterns and is consistent with the temporal trend we found in our study with increasing use of ppci and declining fibrinolytic use . a recent report from the national cardiovascular data registry ( ncdr)4 showed that , among patients presenting to a nonpcicapable hospital and transferred for ppci , only 51.3% of patients achieved dtb 120 minutes . possible reasons for the worsening in the timeliness of fibrinolysis include the overall decrease in the frequency of fibrinolytic use , resulting in unfamiliarity with its administration protocols , complex care coordination between transferring and receiving centers resulting in delays and cancellation of some transfers , and the perceived hazards of fibrinolytic therapy ( eg , intracranial hemorrhage ) despite delayed transfer resulting in reluctance in its use . another report from the ncdr examined temporal trends in dtb time.19 they found that between 2005 and 2009 , median dtb time had decreased from 83 to 67 minutes while the percentage of patients achieving timely ppci ( dtb 90 minutes ) had increased from 59.7% to 83.1% . despite these significant improvements in dtb times we previously argued that some of the time differential in dtb times observed in clinical studies may be too small to exert a meaningful clinical impact , especially when shortterm outcomes are examined.20 , 21 a prior report from the national registry of myocardial infarction ( nrmi)22 evaluating reperfusion strategies in stemi patients in 1990 through 2006 found a decrease in fibrinolysis from 52.5% to 27.6% with a nearly linear decline in dtn from 59 to 29 minutes and a corresponding decrease in mortality from 7.0% to 6.0% over the study period . the trial of routine angioplasty and stenting after fibrinolysis to enhance reperfusion in acute myocardial infarction ( transferami)23 showed that highrisk stemi patients receiving fibrinolysis and routine early pci had improved shortterm outcomes compared to patients receiving standard therapy and delayed pci . a report of 73 belgian hospitals24 found that modern fibrinolytic strategies substantially attenuated the mortality benefit of ppci over fibrinolysis except for patients in the highestrisk subgroup with thrombolysis in myocardial infarction ( timi ) scores of 7 to 14 . the strategic reperfusion early after myocardial infarction ( stream)25 study showed that in patients with stemi unable to undergo ppci within 1 hour , prehospital fibrinolysis and angiography within 6 to 24 hours resulted in effective reperfusion compared to ppci alone . additionally , the improvement noted in defectfree care and adherence to performance measures in patients receiving either reperfusion strategy over the study period indicates the important role played by quality improvement programs . however , defectfree care was found to be significantly lower in patients receiving fibrinolysis and should raise concerns in patients receiving fibrinolysis as use continues to decrease . finally , trends in reperfusion strategies were assessed between 2003 and 2008 , and therefore , contemporary patterns of fibrinolysis may be different . finally , trends in reperfusion strategies were assessed between 2003 and 2008 , and therefore , contemporary patterns of fibrinolysis may be different . however , our study demonstrates the importance of tracking and improving dtn times in hospitals that do not perform ppci around the clock as well as the need for contemporary data in trends and outcomes of fibrinolytic use for stemi . the use of fibrinolytic therapy for stemi has decreased from 2003 to 2008 , in contradistinction to the use of ppci . despite improvements in timely ppci and defectfree care , a significant delay in timely fibrinolysis inhospital mortality among stemi patients receiving fibrinolysis remained unchanged over the study period and worse than that of their ppci counterparts . our findings highlight important opportunities to improve the use of fibrinolytic therapy and its timeliness among eligible stemi patients .

protein lysine methyltransferases ( pkmts , also known as histone lysine methyltransferases ( hkmts ) ) catalyze the transfer of the methyl group from the cofactor s - adenosyl - l - methionine ( sam ) to lysine residues of histone and non - histone substrates , leading to lysine mono- , di- , and/or trimethylation . histone lysine methylation has been increasingly recognized as a major epigenetic gene regulation mechanism in eukaryotic cells . therefore , pkmts as a class of potential drug targets have received considerable attention from the medicinal chemistry and chemical biology community . with the exception of dot1l , pkmts contain an evolutionarily conserved set ( su(var ) , e(z ) , and trithorax ) domain . this catalytic domain consists of a substrate binding groove and a cofactor binding site . a number of selective small - molecule inhibitors of pkmts including g9a / glp , ezh2 , smyd2 , dot1l , and setd2 have been discovered . these inhibitors are competitive with either the peptide substrate or the cofactor sam . in addition , a number of selective inhibitors of protein arginine methyltransferases ( prmts ) , another class of protein methyltransferases , have been reported . some of these inhibitors have subsequently been utilized as valuable tools for investigating the role of the corresponding methyltransferases in various human diseases . setd8 ( also known as set8 , pr - set7 , or kmt5a ( lysine methyltransferase 5a ) ) is the sole methyltransferase that catalyzes monomethylation of histone h4 lysine 20 ( h4k20 ) . setd8 and h4k20me ( h4k20 monomethylation ) have been implicated in regulating a diverse set of biological processes including the dna damage response , dna replication , and mitotic condensation . a recent study has shown that ( 1 ) setd8 is physically associated with twist , a master regulator of emt ( epithelial mesenchymal transition ) , ( 2 ) setd8 and twist are functionally interdependent on promoting emt , ( 3 ) setd8 acts on the promoters of the twist target genes such as n - cadherin via exertion of its h4k20 monomethylation activity , and ( 4 ) setd8 expression is positively correlated with metastasis and the expression of twist and n - cadherin in breast cancer cells . in addition to h4k20 , setd8 methylates many non - histone substrates including the tumor suppressor p53 and proliferating cell nuclear antigen ( pcna ) . the monomethylation of p53 at lysine 382 ( p53k382me1 ) catalyzed by setd8 suppresses p53-mediated transcription activation of highly responsive target genes . the monomethylation of pcna at lysine 248 ( pcnak248me1 ) catalyzed by setd8 stabilizes pcna protein , enhances the interaction between pcna and the flap endonuclease fen1 , and promotes the proliferation of cancer cells to date , nahuoic acid a , a marine natural product , is the only known selective inhibitor of setd8 ( figure 1 ) . this inhibitor is competitive with the cofactor sam and noncompetitive with the peptide substrate . here we report the discovery of unc0379 ( 1 ) , the first substrate - competitive inhibitor of setd8 . compound 1 is a synthetic small - molecule inhibitor that displays inhibitory activity in multiple biochemical assays and is selective for setd8 over 15 other methyltransferases . the binding affinity of compound 1 to setd8 was determined using biophysical assays such as itc ( isothermal titration calorimetry ) and spr ( surface plasmon resonance ) and is largely consistent with its potency in biochemical assays . we describe hit identification , analogue synthesis , structure activity relationship ( sar ) findings , and comprehensive characterization of compound 1 in a number of biochemical and biophysical assays including mechanism of action and selectivity studies . we previously reported that 2,4-diaminoquinazolines are selective , substrate - competitive inhibitors of the lysine methyltransferases g9a and glp . to identify a substrate - competitive inhibitor of setd8 , we cross - screened our quinazoline - based inhibitor set , which consists of > 150 compounds , against setd8 . from this study , we discovered compound 1 as an inhibitor of setd8 ( figure 2 ) . interestingly , compound 1 was originally prepared for targeting l3mbtl1 , a methyllysine reader protein , but showed no appreciable activity for l3mbtl1 . on the other hand , compound 1 displayed inhibitory activity with an ic50 of 7.3 1.0 m ( n = 2 ) in a radioactive biochemical assay that measures the transfer of the tritiated methyl group from h - sam to a peptide substrate catalyzed by setd8 ( figure 2 ) . the inhibitory activity of compound 1 was confirmed in an orthogonal biochemical assay , microfluidic capillary electrophoresis ( mce ) assay . this setd8 mce assay was developed analogously to the previously reported g9a mce assay . compound 1 was identified as an inhibitor of setd8 by cross - screening a quinazoline - based inhibitor set . ( a ) structure of compound 1 . ( b ) concentration response curve of compound 1 in the setd8 radioactive methyl transfer assay . to determine sar for this promising hit , we designed and synthesized a number of analogues that contain various 2- and 4-substituents at the quinazoline core . we synthesized compounds 124 from commercially available 2,4-dichloro-6,7-dimethoxyquinazoline and corresponding amines in good yields ( scheme 1 and tables 1 and 2 ) . using the methods developed previously , we displaced the 4-chloro group with the first set of amines at room temperature and the 2-chloro group with the second set of amines under microwave heating conditions to yield the desired 2,4-diamino-6,7-dimethoxyquinazolines . ( a ) r amines , thf , n , n - diisopropylethylamine , room temperature ; ( b ) r amines , n - buoh , dipea , microwave , 160 c . the synthesized compounds were then evaluated in the setd8 radioactive methyl transfer assay . ic50 values of these compounds in this biochemical assay are summarized in tables 1 and 2 . we first explored the 4-amino group of the quinazoline scaffold ( table 1 ) . reducing the length of the alkyl linker between the two amino groups resulted in the decrease of the potency ( compound 1 versus compounds 24 ) . the distal amino group can be changed from the pyrrolidinyl to piperidinyl or dimethylamino without any potency loss ( compound 1 versus compounds 5 and 6 ) . interestingly , the diethylamino analogue ( compound 7 ) was somewhat less potent than compounds 1 , 5 , and 6 . we also attempted to replace the piperidinyl group with a piperazinyl or n - substituted piperazinyl group ( compound 1 versus compounds 812 ) . however , these structural modifications led to a potency loss , suggesting that an additional basic amino group ( compounds 912 ) or a large substituent ( compound 8) is detrimental to inhibiting setd8 . in addition , we found that the basicity of the pyrrolidinyl group in compound 1 was an important contributor to its setd8 inhibitory activity , as the corresponding amide analogue ( compound 13 ) was about 8-fold less potent than compound 1 . we also explored whether the nh group in compound 1 was potentially engaged in a hydrogen bond interaction and found that the n - methyl analogue ( compound 14 ) was drastically less potent than compound 1 , suggesting that the hydrogen of the secondary amine likely serves as a hydrogen bond donor . we next explored the 2-amino group of the quinazoline scaffold ( table 2 ) . the replacement of the pyrrolidinyl group with either the piperidinyl or azepanyl group resulted in a significant loss of the potency ( compound 1 versus compounds 15 and 16 ) , suggesting a large group is disfavored . on the other hand , the dimethylamino group ( compound 17 ) did not lead to any significant potency loss . the introduction of an additional basic amino group ( compound 15 versus the unsubstituted piperazinyl analogue 18 and the n - methylpiperazinyl analogue 19 ) resulted in a complete loss of the potency . we also explored other amino groups such as n - methyl - n - cyclopentylamine ( compound 20 ) and n - methyl - n - cyclohexylamine ( compound 21 ) . compared with compound 1 , compounds interestingly , removing the methyl group from either compound 20 or 21 led to the complete loss of inhibitory activity ( compounds 22 and 23 ) , suggesting that tertiary amino groups are preferred compared with secondary amino groups . taken together , these results suggest that setd8 inhibitory activity is very sensitive to the 2-substituent . compound 1 bound setd8 with a kd of 18.3 3.2 m ( n = 3 ) ( figure 3 ) . in spr studies , compound 1 behaved as a classic reversible inhibitor with a fast on rate ( ka = 2.18 0.36 10 1/ms ) and a fast off rate ( kd = 7.82 0.87 10 1/s ) ( figure 4 ) . the kd of compound 1 was determined to be 36.0 2.3 m ( n = 3 ) . the binding affinity of compound 1 to setd8 determined by itc and spr is largely consistent with its potency in the biochemical assays . compound 1 binds setd8 with a kd of 18.3 3.2 m ( n = 3 ) in itc studies . we next studied the moa ( mechanism of action ) of the setd8 inhibition by compound 1 via varying concentrations of the h4 peptide substrate or the cofactor sam . as illustrated in figure 5a , ic50 values of compound 1 increased linearly with h4 peptide concentrations . on the other hand , ic50 values of compound 1 remained constant in the presence of increasing concentrations of sam ( figure 5b ) . these results indicate that compound 1 is competitive with the peptide substrate and noncompetitive with the cofactor sam . ( a ) compound 1 is competitive with the peptide substrate , as its ic50 values increased linearly with h4 peptide concentrations . ( b ) compound 1 is noncompetitive with the cofactor sam , as its ic50 values remained constant in the presence of increasing concentrations of sam . to confirm the findings from the moa studies , we tested compound 1 and an inactive control ( compound 14 ) in a peptide displacement assay using fluorescence polarization ( fp ) , which measures effects of inhibitors on displacing the h4k20me ( 124 ) peptide with n - terminus labeled by fluorescein isothiocyanate ( fitc ) . as illustrated in figure 6 , compound 1 effectively displaced the fitc - labeled peptide binding to setd8 with an ic50 of 37.7 7.2 m ( n = 2 ) . on the other hand , our negative control ( compound 14 ) did not displace the peptide in this fp assay . compound 1 effectively displaced the fitc - labeled h4k20me ( 114 ) peptide , while an inactive control ( compound 14 ) did not . we next determined the selectivity of inhibitor 1 for setd8 over 15 other methyltransferases ( figure 7 ) . with the exception of prc2 ( polycomb repressive complex 2 ) , the ic50 values of compound 1 for 14 other methyltransferases including g9a and glp were all above 100 m . for prc2 , compound 1 was active only at the two highest concentrations ( 50 and 100 m ) with an estimated ic50 value greater than 50 m . we discovered the first selective , substrate competitive inhibitor of setd8 by cross - screening our quinazoline - based epigenetic library . our sar studies of this series reveal that while the 4-amino moiety can be modified without a significant loss of potency , modifications to the 2-amino moiety are not well tolerated . we characterized compound 1 in a battery of biochemical , biophysical , moa , and selectivity assays and found that the setd8 inhibitory activity of this compound in biochemical assays was largely consistent with its binding affinity to setd8 determined by itc and spr . our moa studies indicate that this inhibitor is competitive with the peptide substrate and noncompetitive with the cofactor sam . this moa was further supported by our finding that compound 1 effectively displaced the fitc - labeled h4 peptide in a fp assay . importantly , this inhibitor is selective for setd8 over 15 other methyltransferases including g9a and glp , the two pkmts that are known for being potently inhibited by quinazolines . these results provide the first evidence that 2,4-diaminoquinazolines can be modified to yield selective , substrate competitive inhibitors of pkmts other than g9a and glp . hplc spectra for all compounds were acquired using an agilent 6110 series system with uv detector set to 254 nm . samples were injected ( 5 l ) onto an agilent eclipse plus 4.6 mm 50 mm , 1.8 m c18 column at room temperature . a linear gradient from 10% to 100% b ( meoh + 0.1% acetic acid ) in 5.0 min was followed by pumping 100% b for another 2 min with a being h2o + 0.1% acetic acid . mass spectra data were acquired in positive ion mode using an agilent 6110 single quadrupole mass spectrometer with an electrospray ionization ( esi ) source . nuclear magnetic resonance ( nmr ) spectra were recorded at varian mercury spectrometer with 400 mhz for proton ( h nmr ) and 100 mhz for carbon ( c nmr ) . preparative hplc was performed on agilent prep 1200 series with uv detector set to 254 nm . samples were injected onto a phenomenex luna 75 mm 30 mm , 5 m c18 column at room temperature . a linear gradient was used with 10% ( or 50% ) of meoh ( a ) in 0.1% tfa in h2o ( b ) to 100% of meoh ( a ) . high - resolution ( positive ion ) mass spectrometry ( hrms ) for compound 1 was performed using a thermo ltqft mass spectrometer under ft control at 100 000 resolution . to a solution of 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 84 mg , 0.17 mmol ) and n - butanol ( 1.5 ml ) were added pyrrolidine ( commercially available , 56 l , 0.68 mmol ) and n , n - diisopropylethylamine ( 89 l , 0.51 mmol ) . the resulting solution was stirred inside a microwave at 160 c for 30 min . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 and washed with brine . the organic layer was dried , concentrated , and purified by hplc to give the title compound as a tfa salt , white solid ( 70 mg , yield 64% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.10 ( s , 1h ) , 3.95 ( s , 3h ) , 3.92 ( s , 3h ) , 3.783.57 ( m , 8h ) , 3.223.16 ( m , 2h ) , 3.103.01 ( m , 2h ) , 2.191.97 ( m , 8h ) , 1.861.73 ( m , 4h ) , 1.551.47 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.40 , 157.17 , 151.52 , 148.80 , 136.95 , 105.04 , 103.62 , 99.62 , 56.93 , 56.79 , 56.11 , 55.08 ( four carbons ) , 42.48 , 29.31 , 26.87 , 25.20 , 23.95 ( four carbons ) . hrms calcd for c23h35n5o2 + h , 414.2869 ; found , 414.2862 [ m + h ] . 2-chloro-6,7-dimethoxy - n-(2-(pyrrolidin-1-yl)ethyl)quinazolin-4-amine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 1-(2-aminoethyl)pyrrolidine ( commercially available ) , n , n - diisopropylethylamine , and thf . to a solution of 2-chloro-6,7-dimethoxy - n-(2-(pyrrolidin-1-yl)ethyl)quinazolin-4-amine ( 72 mg , 0.13 mmol ) and isopropanol ( 0.7 ml ) were added pyrrolidine ( 21 l , 0.26 mmol ) and hcl in dioxane ( 4.0 m , 63 l , 0.26 mmol ) . the resulting solution was stirred inside a microwave at 160 c for 20 min . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 , washed with brine . the organic layer was dried , concentrated , and purified by hplc to give the title compound 2 as a tfa salt , white solid ( 56 mg , yield 73% ) . h nmr ( 400 mhz , meoh - d4 ) 7.50 ( s , 1h ) , 7.12 ( s , 1h ) , 4.06 ( t , j = 6.0 hz , 2h ) , 3.96 ( s , 3h ) , 3.92 ( s , 3h ) , 3.823.71 ( m , 4h ) , 3.683.56 ( m , 4h ) , 3.223.10 ( m , 2h ) , 2.221.99 ( m , 8h ) . 2-chloro-6,7-dimethoxy - n-(3-(pyrrolidin-1-yl)propyl)quinazolin-4-amine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 1-(3-aminopropyl)pyrrolidine ( commercially available ) , n , n - diisopropylethylamine , and thf . compound 3 was prepared according to the procedure for making 2 from 2-chloro-6,7-dimethoxy - n-(3-(pyrrolidin-1-yl)propyl)quinazolin-4-amine ( 66 mg , 0.19 mmol ) , pyrrolidine ( 30 l , 0.37 mmol ) , hcl in dioxane ( 4.0 m , 93 l , 0.37 mmol ) , and isopropanol ( 1.0 ml ) . the title compound 3 was obtained as a tfa salt , brown solid ( 45 mg , yield 40% ) . h nmr ( 400 mhz , meoh - d4 ) 7.53 ( s , 1h ) , 7.10 ( s , 1h ) , 3.95 ( s , 3h ) , 3.91 ( s , 3h ) , 3.833.60 ( m , 8h ) , 3.353.28 ( m , 2h ) , 3.133.02 ( m , 2h ) , 2.252.00 ( m , 10h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.68 , 157.34 , 151.49 , 148.83 , 137.08 , 105.03 , 103.57 , 99.57 , 56.91 , 56.82 , 55.11 ( four carbons ) , 53.95 , 39.75 , 26.51 , 23.96 ( four carbons ) . 2-chloro-6,7-dimethoxy - n-(4-(pyrrolidin-1-yl)butyl)quinazolin-4-amine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 1-pyrrolidinebutanamine ( commercially available ) , n , n - diisopropylethylamine , and thf . compound 4 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(4-(pyrrolidin-1-yl)butyl)quinazolin-4-amine ( 109 mg , 0.30 mmol ) , pyrrolidine ( 99 l , 1.2 mmol ) , n , n - diisopropylethylamine ( 105 l , 0.60 mmol ) , and n - butanol ( 1.0 ml ) . the title compound 4 was obtained as a tfa salt , white solid ( 144 mg , yield 77% ) . h nmr ( 400 mhz , meoh - d4 ) 7.54 ( s , 1h ) , 7.09 ( s , 1h ) , 3.94 ( s , 3h ) , 3.91 ( s , 3h ) , 3.793.54 ( m , 8h ) , 3.283.19 ( m , 2h ) , 3.123.01 ( m , 2h ) , 2.191.97 ( m , 8h ) , 1.891.79 ( m , 4h ) . hplc purity : > 95% ; tr = 3.02 min . ms ( esi ) : 400 [ m + h ] . 2-chloro-6,7-dimethoxy - n-(5-(piperidin-1-yl)pentyl)quinazolin-4-amine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 1-piperidinepentanamine ( commercially available ) , n , n - diisopropylethylamine , and thf . compound 5 was prepared according to the procedure for making 2 from 2-chloro-6,7-dimethoxy - n-(5-(piperidin-1-yl)pentyl)quinazolin-4-amine ( 79 mg , 0.13 mmol ) , pyrrolidine ( 21 l , 0.26 mmol ) , hcl in dioxane ( 4.0 m , 63 l , 0.26 mmol ) , and isopropanol ( 0.7 ml ) . the title compound 5 was obtained as a tfa salt , brown solid ( 58 mg , yield 68% ) . h nmr ( 400 mhz , meoh - d4 ) 7.55 ( s , 1h ) , 7.09 ( s , 1h ) , 3.95 ( s , 3h ) , 3.91 ( s , 3h ) , 3.793.57 ( m , 6h ) , 3.563.47 ( m , 2h ) , 3.123.05 ( m , 2h ) , 2.90 ( td , j = 12.5 , 2.7 hz , 2h ) , 2.222.01 ( m , 4h ) , 1.971.89 ( m , 2h ) , 1.881.70 ( m , 7h ) , 1.581.43 ( m , 3h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.40 , 157.17 , 151.53 , 148.81 , 136.96 , 105.05 , 103.62 , 99.62 , 58.12 , 56.93 , 56.79 , 54.27 ( four carbons ) , 42.50 , 29.32 , 25.29 , 24.92 , 24.24 ( four carbons ) , 22.72 . n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - dimethylpentane-1,5-diamine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 5-(dimethylamino)amylamine ( commercially available ) , n , compound 6 was prepared according to the procedure for making 2 from n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - dimethylpentane-1,5-diamine ( 70 mg , 0.15 mmol ) , pyrrolidine ( 21 l , 0.30 mmol ) , hcl in dioxane ( 4.0 m , 75 l , 0.30 mmol ) , and isopropanol ( 1.0 ml ) . the title compound 6 was obtained as a tfa salt , white solid ( 57 mg , yield 62% ) . h nmr ( 400 mhz , meoh - d4 ) 7.55 ( s , 1h ) , 7.09 ( s , 1h ) , 3.95 ( s , 3h ) , 3.91 ( s , 3h ) , 3.783.54 ( m , 6h ) , 3.163.09 ( m , 2h ) , 2.87 ( s , 6h ) , 2.221.98 ( m , 4h ) , 1.891.72 ( m , 4h ) , 1.581.39 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.41 , 157.19 , 151.53 , 148.82 , 136.96 , 105.05 , 103.62 , 99.62 , 58.87 , 56.93 , 56.79 , 43.37 ( four carbons ) , 42.48 , 29.32 ( two carbons ) , 25.47 ( two carbons ) , 25.06 . n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - diethylpentane-1,5-diamine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 5-(diethylamino)pentylamine ( commercially available ) , n , n - diisopropylethylamine , and thf . compound 7 was prepared according to the procedure for making 1 from n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - diethylpentane-1,5-diamine ( 62 mg , 0.16 mmol ) , pyrrolidine ( 54 l , 0.64 mmol ) , n , n - diisopropylethylamine ( 113 l , 0.64 mmol ) , and n - butanol ( 0.2 ml ) . the title compound 7 was obtained as a tfa salt , amber solid ( 79 mg , yield 76% ) . h nmr ( 400 mhz , meoh - d4 ) 7.53 ( s , 1h ) , 7.07 ( s , 1h ) , 3.93 ( s , 3h ) , 3.90 ( s , 3h ) , 3.773.54 ( m , 6h ) , 3.22 ( q , j = 7.4 hz , 4h ) , 3.163.09 ( m , 2h ) , 2.212.01 ( m , 4h ) , 1.871.72 ( m , 4h ) , 1.571.45 ( m , 2h ) , 1.30 ( t , j = 7.3 hz , 6h ) . tert - butyl 4-(5-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)pentyl)piperazine-1-carboxylate was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , tert - butyl 4-(5-aminopentyl)piperazine-1-carboxylate ( commercially available ) , n , n - diisopropylethylamine , and thf . compound 8 was prepared according to the procedure for making 1 from tert - butyl 4-(5-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)pentyl)piperazine-1-carboxylate ( 200 mg , 0.40 mmol ) , pyrrolidine ( 135 l , 1.6 mmol ) , n , n - diisopropylethylamine ( 280 l , 1.6 mmol ) , and n - butanol ( 0.5 ml ) . the title compound 8 was obtained as a tfa salt , yellow solid ( 180 mg , yield 60% ) . h nmr ( 400 mhz , meoh - d4 ) 7.54 ( s , 1h ) , 7.09 ( s , 1h ) , 4.334.08 ( m , 2h ) , 3.95 ( s , 3h ) , 3.91 ( s , 3h ) , 3.773.50 ( m , 8h ) , 3.293.12 ( m , 4h ) , 3.102.90 ( m , 2h ) , 2.212.01 ( m , 4h ) , 1.871.77 ( m , 4h ) , 1.551.49 ( m , 2h ) , 1.47 ( s , 9h ) . to the solution of tert - butyl 4-(5-((6,7-dimethoxy-2-(pyrrolidin-1-yl)quinazolin-4-yl)amino)pentyl)piperazine-1-carboxylate ( 8) in meoh and thf was added tfa at room temperature and stirred overnight at 50 c . after removal of the solvent by rotary evaporation , the residue was purified by hplc to give 14 mg of the title compound 9 as a tfa salt , white solid ( 14 mg , yield 80% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.11 ( s , 1h ) , 3.96 ( s , 3h ) , 3.92 ( s , 3h ) , 3.823.49 ( m , 14h ) , 3.233.17 ( m , 2h ) , 2.212.02 ( m , 4h ) , 1.871.78 ( m , 4h ) , 1.561.47 ( m , 2h ) . ms ( esi ) : 429 [ m + h ] . to the solution of 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) in meoh ( 1.0 ml ) were added formaldehyde ( commercially available , 39 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) , and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) at 0 c . after removal of the solvent by rotary evaporation , the residue was purified by hplc to give the title compound 10 as a tfa salt , gray solid ( 25 mg , yield 31% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.10 ( s , 1h ) , 3.96 ( s , 3h ) , 3.92 ( s , 3h ) , 3.813.53 ( m , 14h ) , 3.263.19 ( m , 2h ) , 2.97 ( s , 3h ) , 2.222.02 ( m , 4h ) , 1.881.77 ( m , 4h ) , 1.561.46 ( m , 2h ) . compound 11 was prepared according to the procedure for making 10 from 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) , acetaldehyde ( commercially available , 56 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) and meoh ( 1.0 ml ) . the title compound 11 was obtained as a tfa salt , gray solid ( 74 mg , yield 90% ) . h nmr ( 400 mhz , meoh - d4 ) 7.55 ( s , 1h ) , 7.10 ( s , 1h ) , 3.95 ( s , 3h ) , 3.92 ( s , 3h ) , 3.813.55 ( m , 14h ) , 3.353.26 ( m , 2h ) , 3.263.20 ( m , 2h ) , 2.182.02 ( m , 4h ) , 1.881.78 ( m , 4h ) , 1.561.48 ( m , 2h ) , 1.37 ( t , j = 7.3 hz , 3h ) . compound 12 was prepared according to the procedure for making 10 from 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) , acetone ( commercially available , 103 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) and meoh ( 1.0 ml ) . the title compound 12 was obtained as a tfa salt , brown solid ( 61 mg , yield 73% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.10 ( s , 1h ) , 3.96 ( s , 3h ) , 3.92 ( s , 3h ) , 3.823.48 ( m , 15h ) , 3.243.18 ( m , 2h ) , 2.202.03 ( m , 4h ) , 1.881.78 ( m , 4h ) , 1.561.47 ( m , 2h ) , 1.40 ( s , 3h ) , 1.38 ( s , 3h ) . 5-((tert - butoxycarbonyl)amino)pentanoic acid ( commercially available , 0.93 g , 4.3 mmol ) and pyrrolidine ( 238 l , 2.9 mmol ) were dissolved in 23 ml of dmf . to this solution were added n , n - diisopropylethylamine ( 2.5 ml , 14.5 mmol ) and hatu ( 2.18 g , 5.8 mmol ) . the resulting solution was stirred at 50 c for 3 h. after the mixture was cooled , tlc indicated the completion of the reaction . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 , washed with brine . the organic layer was dried , concentrated , and purified by isco to give tert - butyl ( 5-oxo-5-(pyrrolidin-1-yl)pentyl)carbamate . to the solution of tert - butyl ( 5-oxo-5-(pyrrolidin-1-yl)pentyl)carbamate in meoh and thf after removal of the solvent by rotary evaporation , the product 5-amino-1-(pyrrolidin-1-yl)pentan-1-one obtained was used for the next step without further purification . 5-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)-1-(pyrrolidin-1-yl)pentan-1-one was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 5-amino-1-(pyrrolidin-1-yl)pentan-1-one , n , n - diisopropylethylamine , and thf . compound 13 was prepared according to the procedure for making 1 from 5-((2-chloro-6,7-dimethoxyquinazolin-4-yl)amino)-1-(pyrrolidin-1-yl)pentan-1-one ( 87 mg , 0.22 mmol ) , pyrrolidine ( 74 l , 0.88 mmol ) , n , n - diisopropylethylamine ( 154 l , 0.88 mmol ) , and n - butanol ( 0.25 ml ) . the title compound 13 was obtained as a tfa salt , brown solid ( 16 mg , yield 13% ) . h nmr ( 400 mhz , meoh - d4 ) 7.57 ( s , 1h ) , 7.09 ( s , 1h ) , 3.96 ( s , 3h ) , 3.93 ( s , 3h ) , 3.803.59 ( m , 6h ) , 3.48 ( t , j = 6.8 hz , 2h ) , 3.40 ( t , j = 6.9 hz , 2h ) , 2.41 ( t , j = 7.0 hz , 2h ) , 2.202.03 ( m , 4h ) , 2.011.93 ( m , 2h ) , 1.911.84 ( m , 2h ) , 1.831.71 ( m , 4h ) . 2-chloro-6,7-dimethoxy - n - methyl - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , n - methyl-5-(pyrrolidin-1-yl)pentan-1-amine ( synthesized according to the precedures preported previously),n , n - diisopropylethylamine , and thf . compound 14 was prepared according to the procedure for making 2 from 2-chloro-6,7-dimethoxy - n - methyl - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 50 mg , 0.13 mmol ) , pyrrolidine ( 21 l , 0.26 mmol ) , hcl in dioxane ( 4.0 m , 65 l , 0.26 mmol ) , and isopropanol ( 0.7 ml ) . the title compound 14 was obtained as a tfa salt , white solid ( 25 mg , yield 30% ) . h nmr ( 400 mhz , meoh - d4 ) 7.47 ( s , 1h ) , 7.17 ( s , 1h ) , 3.97 ( s , 3h ) , 3.91 ( s , 3h ) , 3.903.85 ( m , 2h ) , 3.753.60 ( m , 6h ) , 3.57 ( s , 3h ) , 3.243.17 ( m , 2h ) , 3.113.01 ( m , 2h ) , 2.222.06 ( m , 6h ) , 2.051.97 ( m , 2h ) , 1.961.87 ( m , 2h ) , 1.861.77 ( m , 2h ) , 1.551.45 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 162.30 , 157.06 , 150.22 , 147.38 , 139.69 , 109.50 , 104.02 , 99.83 , 56.97 , 56.79 , 56.04 ( two carbons ) , 55.09 ( two carbons ) , 54.45 ( two carbons ) , 41.10 , 27.43 , 26.91 ( two carbons ) , 25.08 ( two carbons ) , 23.97 ( two carbons ) . compound 15 was prepared according to the procedure for making 2 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 70 mg , 0.19 mmol ) , piperidine ( commercially available , 37 l , 0.38 mmol ) , hcl in dioxane ( 4.0 m , 95 l , 0.38 mmol ) , and isopropanol ( 1.0 ml ) . the title compound 15 was obtained as a tfa salt , light yellow solid ( 101 mg , yield 83% ) . h nmr ( 400 mhz , meoh - d4 ) 7.54 ( s , 1h ) , 7.09 ( s , 1h ) , 3.94 ( s , 3h ) , 3.91 ( s , 3h ) , 3.883.82 ( m , 4h ) , 3.693.61 ( m , 4h ) , 3.223.16 ( m , 2h ) , 3.103.01 ( m , 2h ) , 2.202.08 ( m , 2h ) , 2.071.96 ( m , 2h ) , 1.851.69 ( m , 10h ) , 1.551.46 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.55 , 157.21 , 152.43 , 149.01 , 137.00 , 104.87 , 103.58 , 99.79 , 56.90 , 56.81 , 56.08 , 55.06 ( two carbons ) , 47.16 ( two carbons ) , 42.56 , 29.29 , 26.87 , 26.69 ( two carbons ) , 25.19 , 25.13 , 23.95 ( two carbons ) . compound 16 was prepared according to the procedure for making 2 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 70 mg , 0.19 mmol ) , hexamethyleneimine ( commercially available , 42 l , 0.38 mmol ) , hcl in dioxane ( 4.0 m , 95 l , 0.38 mmol ) , and isopropanol ( 1.0 ml ) . the title compound 16 was obtained as a tfa salt , light yellow solid ( 35 mg , yield 28% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.18 ( s , 1h ) , 3.95 ( s , 3h ) , 3.92 ( s , 3h ) , 3.903.70 ( m , 4h ) , 3.703.61 ( m , 4h ) , 3.223.16 ( m , 2h ) , 3.103.01 ( m , 2h ) , 2.192.08 ( m , 2h ) , 2.071.97 ( m , 2h ) , 1.951.86 ( m , 4h ) , 1.851.75 ( m , 4h ) , 1.691.61 ( m , 4h ) , 1.561.47 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.40 , 157.23 , 152.91 , 148.99 , 137.10 , 104.89 , 103.68 , 99.85 , 56.91 , 56.80 , 56.10 , 55.09 ( two carbons ) , 49.16 ( two carbons ) , 42.59 , 29.47 , 28.58 ( three carbons ) , 27.85 , 26.91 , 25.23 , 23.95 ( two carbons ) . ms ( esi ) : 442 [ m + h ] . to a solution of pd(oac)2 ( 2 mg , 0.01 mmol ) , ( + ) -binap ( 6 mg , 0.01 mmol ) , and thf ( 0.5 ml ) were added 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 46 mg , 0.10 mmol ) , dimethylamine hydrochloride ( commercially available , 10 mg , 0.12 mmol ) , and cs2co3 ( 73 mg , 0.24 mmol ) . the resulting solution was stirred inside a microwave at 140 c for 30 min . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 , washed with brine . the organic layer was dried , concentrated , and purified by hplc to give 7.0 mg of the title compound 17 as a tfa salt , white solid ( yield 13% ) . h nmr ( 400 mhz , meoh - d4 ) 7.57 ( s , 1h ) , 7.15 ( s , 1h ) , 3.97 ( s , 3h ) , 3.92 ( s , 3h ) , 3.71 ( t , j = 7.1 hz , 2h ) , 3.683.60 ( m , 2h ) , 3.31 ( s , 6h ) , 3.233.16 ( m , 2h ) , 3.103.01 ( m , 2h ) , 2.212.09 ( m , 2h ) , 2.071.96 ( m , 2h ) , 1.871.75 ( m , 4h ) , 1.561.48 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.39 , 157.35 , 153.70 , 149.10 , 137.07 , 104.92 , 103.55 , 99.79 , 56.93 , 56.82 , 56.13 , 55.14 ( two carbons ) , 42.57 ( two carbons ) , 37.96 , 29.36 , 26.92 , 25.23 , 23.96 ( two carbons ) . boc - protected compound 18 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , 1-boc - piperazine ( commercially available , 82 mg , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . to the resulting mixture after removal of the solvent by rotary evaporation , the residue was purified by hplc to give 40 mg of the title compound 18 as a tfa salt , white solid ( yield 55% , two steps ) . h nmr ( 400 mhz , meoh - d4 ) 7.62 ( s , 1h ) , 7.16 ( s , 1h ) , 4.224.14 ( m , 4h ) , 3.96 ( s , 3h ) , 3.93 ( s , 3h ) , 3.71 ( t , j = 7.0 hz , 2h ) , 3.693.61 ( m , 2h ) , 3.483.38 ( m , 4h ) , 3.233.16 ( m , 2h ) , 3.113.01 ( m , 2h ) , 2.202.09 ( m , 2h ) , 2.071.96 ( m , 2h ) , 1.861.73 ( m , 4h ) , 1.571.48 ( m , 2h ) . compound 19 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , 1-methylpiperazine ( commercially available , 49 l , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . the title compound 19 was obtained as a tfa salt , gray solid ( 54 mg , yield 73% ) . h nmr ( 400 mhz , meoh - d4 ) 7.62 ( s , 1h ) , 7.16 ( s , 1h ) , 3.96 ( s , 3h ) , 3.93 ( s , 3h ) , 3.903.24 ( m , 12h ) , 3.223.16 ( m , 2h ) , 3.103.02 ( m , 2h ) , 2.99 ( s , 3h ) , 2.192.10 ( m , 2h ) , 2.071.96 ( m , 2h ) , 1.861.75 ( m , 4h ) , 1.561.47 ( m , 2h ) . compound 20 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , n - methylcyclopentanamine ( commercially available , 44 mg , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . the title compound 20 was obtained as a tfa salt , yellow solid ( 40 mg , yield 54% ) . h nmr ( 400 mhz , meoh - d4 ) 7.57 ( s , 1h ) , 7.18 ( s , 1h ) , 5.155.01 ( m , 1h ) , 3.96 ( s , 3h ) , 3.92 ( s , 3h ) , 3.723.61 ( m , 4h ) , 3.233.16 ( m , 2h ) , 3.14 ( s , 3h ) , 3.103.01 ( m , 2h ) , 2.192.10 ( m , 2h ) , 2.051.93 ( m , 4h ) , 1.891.68 ( m , 10h ) , 1.561.47 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.35 , 157.29 , 153.54 , 149.07 , 137.09 , 104.90 , 103.75 , 99.90 , 58.92 , 56.92 , 56.80 , 56.10 , 55.10 ( three carbons ) , 42.62 , 30.41 , 29.65 ( two carbons ) , 29.39 , 26.93 , 25.28 , 25.25 , 23.95 ( two carbons ) . compound 21 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , n - methylcyclohexylamine ( commercially available , 44 mg , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . the title compound 21 was obtained as a tfa salt , brown solid ( 47 mg , yield 62% ) . h nmr ( 400 mhz , meoh - d4 ) 7.56 ( s , 1h ) , 7.18 ( s , 1h ) , 4.654.49 ( m , 1h ) , 3.95 ( s , 3h ) , 3.92 ( s , 3h ) , 3.713.61 ( m , 4h ) , 3.233.16 ( m , 2h ) , 3.14 ( s , 3h ) , 3.103.01 ( m , 2h ) , 2.212.08 ( m , 2h ) , 2.071.98 ( m , 2h ) , 1.971.90 ( m , 2h ) , 1.861.75 ( m , 6h ) , 1.711.60 ( m , 2h ) , 1.561.42 ( m , 4h ) , 1.401.17 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 160.35 , 157.24 , 153.14 , 149.04 , 137.14 , 104.89 , 103.79 , 99.93 , 57.59 , 56.91 , 56.80 , 56.08 , 55.09 ( three carbons ) , 42.68 , 30.85 ( two carbons ) , 30.09 , 29.40 , 26.93 ( two carbons ) , 26.38 , 25.34 , 23.95 ( two carbons ) . compound 22 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , cyclopentylamine ( commercially available , 44 l , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . the title compound 22 was obtained as a tfa salt , light yellow solid ( 41 mg , yield 57% ) . h nmr ( 400 mhz , meoh - d4 ) 7.52 ( s , 1h ) , 6.95 ( s , 1h ) , 4.414.28 ( m , 1h ) , 3.95 ( s , 3h ) , 3.90 ( s , 3h ) , 3.733.60 ( m , 4h ) , 3.233.16 ( m , 2h ) , 3.102.99 ( m , 2h ) , 2.201.98 ( m , 6h ) , 1.881.75 ( m , 6h ) , 1.731.60 ( m , 4h ) , 1.561.46 ( m , 2h ) . compound 23 was prepared according to the procedure for making 1 from 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 56 mg , 0.11 mmol ) , n - cyclohexylamine ( commercially available , 50 l , 0.44 mmol ) , n , n - diisopropylethylamine ( 57 l , 0.33 mmol ) , and n - butanol ( 0.7 ml ) . the title compound 23 was obtained as a tfa salt , brown semisolid ( 45 mg , yield 61% ) . h nmr ( 400 mhz , meoh - d4 ) 7.50 ( s , 1h ) , 6.91 ( s , 1h ) , 4.003.85 ( m , 1h ) , 3.94 ( s , 3h ) , 3.89 ( s , 3h ) , 3.703.61 ( m , 4h ) , 3.233.15 ( m , 2h ) , 3.103.01 ( m , 2h ) , 2.192.10 ( m , 2h ) , 2.061.99 ( m , 3h ) , 1.881.75 ( m , 7h ) , 1.541.28 ( m , 8h ) . to the solution of 2,4-dichloro-6,7-dimethoxyquinazoline ( commercially available , 1.00 g , 3.86 mmol ) in thf ( 9.5 ml ) was added 5-(pyrrolidin-1-yl)pentan-1-amine ( commercially available , 1.33 g , 8.49 mmol ) , followed by the addition of n , n - diisopropylethylamine ( 612 l , 3.51 mmol ) . and the resulting mixture was stirred at room temperature for 6 h until tlc showed that the starting material had disappeared . water was added to the reaction mixture , and the resulting solution was extracted with ethyl acetate . the organic layer was washed with brine , dried , and concentrated to give the crude product , which was purified on isco using 24 g silica gel column , eluting with hexane ethyl acetate to give 754 mg of the title compound 24 as a yellow semisolid ( yield 52% ) . h nmr ( 400 mhz , meoh - d4 ) 7.61 ( s , 1h ) , 6.99 ( s , 1h ) , 3.98 ( s , 3h ) , 3.96 ( s , 3h ) , 3.73 ( t , j = 7.2 hz , 2h ) , 3.693.61 ( m , 2h ) , 3.243.16 ( m , 2h ) , 3.113.01 ( m , 2h ) , 2.202.09 ( m , 2h ) , 2.071.96 ( m , 2h ) , 1.871.76 ( m , 4h ) , 1.571.47 ( m , 2h ) ; c nmr ( 100 mhz , meoh - d4 ) 161.38 , 157.90 , 152.68 , 151.65 , 141.15 , 107.16 , 103.64 , 102.16 , 57.02 , 56.99 , 56.08 , 55.12 ( two carbons ) , 42.73 , 29.27 , 26.64 , 24.82 , 23.95 ( two carbons ) . radioactive assay was developed to screen our chemical library for small molecule inhibitors . methylation ( 10 l ) reactions were carried out in a buffer containing 50 mm tris - hcl ( ph 8.0 ) , 10 mm gsh , 0.1%triton x-100 , at room temperature using 50 nm setd8 , 1.5 m tritium labeled sam ( catalog no . net155v250uc , perkinelmer ) , and 5 m biotinylated h4 ( 124 ) peptide substrate ( sgrgkggkglgkggakrhrkvlrdk - biotin ) in 384-well plates in the presence of 50 m compounds . the reactions were then quenched by addition of equal volume of 7.5 m guanidine hydrochloride after a 1 h incubation . then 40 l of buffer ( 20 mm tris - hcl , ph 8.0 ) was added into the quenched samples , and all samples were then transferred into a streptavidin / scintillant - coated microplate ( catalog no . the amount of methylated peptide was quantified by tracing the radioactivity ( counts per minute ) as measured after 1 h using a topcount plate reader ( perkinelmer ) . for ic50 values determination , the compounds were serially diluted 2-fold in dmso for a total of 11 concentrations , beginning at 0.25 mm and tested in the same condition . inhibition of setd8 methyltransferase activity was analyzed by monitoring decrease in methylation of the fluorescein labeled peptide , tw21 ( nh2-lgkggakrhrkvlrdniqgitk(5fam)-oh ) , essentially as described previously . the test compounds were solubilized in 100% dmso to 10 mm and then plated in greiner 384-well polypropylene plates using 3-fold dilution scheme over 10 points spanning the concentration range from 3 mm to 0.15 m using tecan genesis liquid handler . then 1 l of the serial dilution was transferred into compound dilution plate using nanoscreen multimek liquid handling robot . prior to performing the assay the compounds were diluted 10-fold in 1 assay buffer ( 20 mm tris , ph 8.0 , 25 mm nacl , 2 mm dtt , and 0.05% tween-20 ) , and an amount of 2.5 l of the resulting dilution was transferred into assay plate by nanoscreen multimek liquid handling robot . to this plate 20 l of 50 nm setd8 and 2 m tw21 peptide cocktail in 1 assay buffer were added using multidrop liquid dispenser . following a 10 min incubation of the compounds with the enzyme / peptide mix , the reaction was initiated by adding 2.5 l of 150 m sam in 1 buffer . for 100% inhibition controls 1 the reaction was allowed to proceed at room temperature for 120 min , and then the reaction was terminated by adding 35 l of 0.08 ng/l endo - lysc protease solution . following an additional 1 h incubation the plate was read on a caliper life sciences ez reader ii using upstream voltage 500 v , downstream voltage 1800 v , and pressure of 1.5 psi . all itc measurements were performed at 25 c using an autoitc200 microcalorimeter ( microcal / ge healthcare ) . the calorimeter cell ( volume 200 l ) was loaded with setd8 protein in the full salt dialysis buffer ( 150 mm nacl ) at a concentration of 100 m . the syringe was loaded with compound 1 ( dissolved in the same buffer ) at a concentration of 1 mm . a typical injection protocol included a single 0.2 l first injection followed by 26 1.5 l injections of the compound into the calorimeter cell . the spacing between injections was kept at 180 s and the reference power at 8 cal / s . a control experiment was performed by titrating compound 1 into buffer under identical settings to determine the heat signals that arose from compound dilution ; these were subtracted from the heat signals of protein the data were analyzed using origin for itc , version 7.0 , software supplied by the manufacturer and fitted well to a one - site binding model . the interaction between compound 1 and protein setd8 was further explored using a proteon xpr36 biosensor ( bio - rad laboratories , inc . ) at 25 c . pbs buffer ( phosphate buffered saline , ph 7.4 ) supplemented with 0.005% tween-20 was used as the running buffer . a glh ( catalog no . sensor chip was first activated by flowing a mixture of 20 mm sulfo - nhs and 20 mm edc over the chip surface for 5 min at a flow rate of 30 l / min . setd8 was then diluted to 20 and 10 g / ml in 5 mm naoac , ph 5.0 , and immobilized onto two ligand channels ( 30 l / min for 2 min ) . the surface was then deactivated by flowing 1 m ethanolamine for 5 min at a flow rate 60 l / min . a blank injection of the running buffer was made , after which four concentrations of the compound in the running buffer ( 100 , 33.3 , 11.1 , and 3.7 m ) and a running buffer were injected simultaneously at a flow rate of 50 l / min for 60 s. the sensorgrams obtained at the four concentrations of the compound were fit simultaneously after subtracting a ligand reference ( inner spot ) and a double compound reference ( buffer ) using a langmuir model to obtain on ( ka ) and off ( kd ) rates . the kinetic kd was calculated based on the on and off rates for three replicates . ic50 values were determined for compound 1 at varying concentrations of sam , 50 nm setd8 , 200 m peptide h4 ( 124 ) or at varying concentrations of substrate h4 ( 124 ) peptide , 50 nm of setd8 , 250 m sam . the reaction mixtures were incubated 15 min at 23 c . to stop the enzymatic reactions , an equal volume of 7.5 m guanidine hydrochloride then 10 l of mixture was spotted onto a square of sam biotin capture membrane ( catalog no . v2861 , promega ) , allowed absorption for 5 min at room temperature , washed with 2 m of nacl solution several times followed by two deionized water wash , and dried , and scintillation liquid was added and cpm ( counts per minute ) were read using the scintillation counter . ( sgrgkggkglgkggakrhrkme1vlrd ) with n - terminus labelled by fitc was ordered from tufts university core services ( boston , ma ) . the sample was prepared in a buffer containing 50 mm tris - hcl ( ph 8.0 ) , 10 mm gsh , 0.01% triton x-100 . 50 nm peptide was incubated with 20 m setd8 protein , 1 mm s - adenosyl - l - homocysteine ( sah ) , and different concentrations of compounds . the fp measurement was performed in black , 384-well pcr plate ( catalog no . 47744 - 828 , vwr ) , and signal was read with a synergy h4 microplate reader ( biotek ) . the polarization values in millipolarization units were measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm . selectivity of compound 1 against a panel of methyltransferases was assessed as previously reported . in brief , the effect of compound 1 on activity of g9a , setdb1 , glp , suv39h2 , setd7 , prmt3 , prmt5-mep50 complex , prmt1 , suv420h1 , suv420h2 , smyd2 , dnmt1 , prc2 complex , mll1 complex , and dot1l was assessed by monitoring the incorporation of tritium - labeled methyl group to lysine or arginine residues of peptide substrates using radioactive assay . assays were performed in a 20 l reaction mixture containing h - sam ( catalog no . net155v250uc , perkin elmer ) at substrate concentrations close to km values for each enzyme . compound concentrations from 100 nm to 100 m were used in all selectivity assays . to stop the enzymatic reactions , 7.5 m guanidine hydrochloride was added , followed by 180 l of buffer ( 20 mm tris , ph 8.0 ) , mixed , and then transferred to a 96-well flashplate ( catalog no . , the reaction mixtures in flashplates were incubated for 1 h and the cpm were measured using topcount plate reader ( perkinelmer ) . the cpm in the absence of compound for each data set were defined as 100% activity . in the absence of the enzyme , for dnmt1 the dsdna substrate was prepared by annealing two complementary strands ( biotintlated forward strand b - gagcccgtaagcccgttcaggtcg and reverse strand cgacctgaacgggcttacgggctc ) , synthesized by eurofins mwg operon . for dot1l , a filter - based assay was used . in this assay , 20 l of reaction mixtures was incubated at room temperature for 1 h , 100 l of 10% trichloroacetic acid ( tca ) was added , mixed , and transferred to filter plates ( catalog no . plates were centrifuged at 2000 rpm ( allegra x-15r ; beckman coulter , inc . ) for 2 min followed by two additional 10% tca washes and one ethanol wash ( 180 l ) followed by centrifugation . plates were dried , and 100 l of microscint - o ( catalog no . 6013611 , perkinelmer ) was added to each well , centrifuged , and removed . then 70 l of microscint - o was added again , and cpm were measured using topcount plate reader .

the lysine methyltransferase setd8 is the only known methyltransferase that catalyzes monomethylation of histone h4 lysine 20 ( h4k20 ) . monomethylation of h4k20 has been implicated in regulating diverse biological processes including the dna damage response . in addition to h4k20 , setd8 monomethylates non - histone substrates including proliferating cell nuclear antigen ( pcna ) and promotes carcinogenesis by deregulating pcna expression . however , selective inhibitors of setd8 are scarce . the only known selective inhibitor of setd8 to date is nahuoic acid a , a marine natural product , which is competitive with the cofactor . here , we report the discovery of the first substrate - competitive inhibitor of setd8 , unc0379 ( 1 ) . this small - molecule inhibitor is active in multiple biochemical assays . its affinity to setd8 was confirmed by itc ( isothermal titration calorimetry ) and spr ( surface plasmon resonance ) studies . importantly , compound 1 is selective for setd8 over 15 other methyltransferases . we also describe structure activity relationships ( sar ) of this series .

Introduction Results and Discussion Conclusions Experimental Section

protein lysine methyltransferases ( pkmts , also known as histone lysine methyltransferases ( hkmts ) ) catalyze the transfer of the methyl group from the cofactor s - adenosyl - l - methionine ( sam ) to lysine residues of histone and non - histone substrates , leading to lysine mono- , di- , and/or trimethylation . with the exception of dot1l , pkmts contain an evolutionarily conserved set ( su(var ) , e(z ) , and trithorax ) domain . a number of selective small - molecule inhibitors of pkmts including g9a / glp , ezh2 , smyd2 , dot1l , and setd2 have been discovered . these inhibitors are competitive with either the peptide substrate or the cofactor sam . in addition , a number of selective inhibitors of protein arginine methyltransferases ( prmts ) , another class of protein methyltransferases , have been reported . some of these inhibitors have subsequently been utilized as valuable tools for investigating the role of the corresponding methyltransferases in various human diseases . setd8 ( also known as set8 , pr - set7 , or kmt5a ( lysine methyltransferase 5a ) ) is the sole methyltransferase that catalyzes monomethylation of histone h4 lysine 20 ( h4k20 ) . setd8 and h4k20me ( h4k20 monomethylation ) have been implicated in regulating a diverse set of biological processes including the dna damage response , dna replication , and mitotic condensation . a recent study has shown that ( 1 ) setd8 is physically associated with twist , a master regulator of emt ( epithelial mesenchymal transition ) , ( 2 ) setd8 and twist are functionally interdependent on promoting emt , ( 3 ) setd8 acts on the promoters of the twist target genes such as n - cadherin via exertion of its h4k20 monomethylation activity , and ( 4 ) setd8 expression is positively correlated with metastasis and the expression of twist and n - cadherin in breast cancer cells . in addition to h4k20 , setd8 methylates many non - histone substrates including the tumor suppressor p53 and proliferating cell nuclear antigen ( pcna ) . the monomethylation of p53 at lysine 382 ( p53k382me1 ) catalyzed by setd8 suppresses p53-mediated transcription activation of highly responsive target genes . the monomethylation of pcna at lysine 248 ( pcnak248me1 ) catalyzed by setd8 stabilizes pcna protein , enhances the interaction between pcna and the flap endonuclease fen1 , and promotes the proliferation of cancer cells to date , nahuoic acid a , a marine natural product , is the only known selective inhibitor of setd8 ( figure 1 ) . this inhibitor is competitive with the cofactor sam and noncompetitive with the peptide substrate . here we report the discovery of unc0379 ( 1 ) , the first substrate - competitive inhibitor of setd8 . compound 1 is a synthetic small - molecule inhibitor that displays inhibitory activity in multiple biochemical assays and is selective for setd8 over 15 other methyltransferases . the binding affinity of compound 1 to setd8 was determined using biophysical assays such as itc ( isothermal titration calorimetry ) and spr ( surface plasmon resonance ) and is largely consistent with its potency in biochemical assays . we describe hit identification , analogue synthesis , structure activity relationship ( sar ) findings , and comprehensive characterization of compound 1 in a number of biochemical and biophysical assays including mechanism of action and selectivity studies . we previously reported that 2,4-diaminoquinazolines are selective , substrate - competitive inhibitors of the lysine methyltransferases g9a and glp . to identify a substrate - competitive inhibitor of setd8 , we cross - screened our quinazoline - based inhibitor set , which consists of > 150 compounds , against setd8 . from this study , we discovered compound 1 as an inhibitor of setd8 ( figure 2 ) . interestingly , compound 1 was originally prepared for targeting l3mbtl1 , a methyllysine reader protein , but showed no appreciable activity for l3mbtl1 . on the other hand , compound 1 displayed inhibitory activity with an ic50 of 7.3 1.0 m ( n = 2 ) in a radioactive biochemical assay that measures the transfer of the tritiated methyl group from h - sam to a peptide substrate catalyzed by setd8 ( figure 2 ) . the inhibitory activity of compound 1 was confirmed in an orthogonal biochemical assay , microfluidic capillary electrophoresis ( mce ) assay . compound 1 was identified as an inhibitor of setd8 by cross - screening a quinazoline - based inhibitor set . to determine sar for this promising hit , we designed and synthesized a number of analogues that contain various 2- and 4-substituents at the quinazoline core . using the methods developed previously , we displaced the 4-chloro group with the first set of amines at room temperature and the 2-chloro group with the second set of amines under microwave heating conditions to yield the desired 2,4-diamino-6,7-dimethoxyquinazolines . we first explored the 4-amino group of the quinazoline scaffold ( table 1 ) . reducing the length of the alkyl linker between the two amino groups resulted in the decrease of the potency ( compound 1 versus compounds 24 ) . we also attempted to replace the piperidinyl group with a piperazinyl or n - substituted piperazinyl group ( compound 1 versus compounds 812 ) . in addition , we found that the basicity of the pyrrolidinyl group in compound 1 was an important contributor to its setd8 inhibitory activity , as the corresponding amide analogue ( compound 13 ) was about 8-fold less potent than compound 1 . we also explored whether the nh group in compound 1 was potentially engaged in a hydrogen bond interaction and found that the n - methyl analogue ( compound 14 ) was drastically less potent than compound 1 , suggesting that the hydrogen of the secondary amine likely serves as a hydrogen bond donor . the replacement of the pyrrolidinyl group with either the piperidinyl or azepanyl group resulted in a significant loss of the potency ( compound 1 versus compounds 15 and 16 ) , suggesting a large group is disfavored . the introduction of an additional basic amino group ( compound 15 versus the unsubstituted piperazinyl analogue 18 and the n - methylpiperazinyl analogue 19 ) resulted in a complete loss of the potency . we also explored other amino groups such as n - methyl - n - cyclopentylamine ( compound 20 ) and n - methyl - n - cyclohexylamine ( compound 21 ) . compound 1 bound setd8 with a kd of 18.3 3.2 m ( n = 3 ) ( figure 3 ) . in spr studies , compound 1 behaved as a classic reversible inhibitor with a fast on rate ( ka = 2.18 0.36 10 1/ms ) and a fast off rate ( kd = 7.82 0.87 10 1/s ) ( figure 4 ) . the binding affinity of compound 1 to setd8 determined by itc and spr is largely consistent with its potency in the biochemical assays . we next studied the moa ( mechanism of action ) of the setd8 inhibition by compound 1 via varying concentrations of the h4 peptide substrate or the cofactor sam . as illustrated in figure 5a , ic50 values of compound 1 increased linearly with h4 peptide concentrations . on the other hand , ic50 values of compound 1 remained constant in the presence of increasing concentrations of sam ( figure 5b ) . these results indicate that compound 1 is competitive with the peptide substrate and noncompetitive with the cofactor sam . ( a ) compound 1 is competitive with the peptide substrate , as its ic50 values increased linearly with h4 peptide concentrations . ( b ) compound 1 is noncompetitive with the cofactor sam , as its ic50 values remained constant in the presence of increasing concentrations of sam . to confirm the findings from the moa studies , we tested compound 1 and an inactive control ( compound 14 ) in a peptide displacement assay using fluorescence polarization ( fp ) , which measures effects of inhibitors on displacing the h4k20me ( 124 ) peptide with n - terminus labeled by fluorescein isothiocyanate ( fitc ) . as illustrated in figure 6 , compound 1 effectively displaced the fitc - labeled peptide binding to setd8 with an ic50 of 37.7 7.2 m ( n = 2 ) . we next determined the selectivity of inhibitor 1 for setd8 over 15 other methyltransferases ( figure 7 ) . with the exception of prc2 ( polycomb repressive complex 2 ) , the ic50 values of compound 1 for 14 other methyltransferases including g9a and glp were all above 100 m . for prc2 , compound 1 was active only at the two highest concentrations ( 50 and 100 m ) with an estimated ic50 value greater than 50 m . we discovered the first selective , substrate competitive inhibitor of setd8 by cross - screening our quinazoline - based epigenetic library . our sar studies of this series reveal that while the 4-amino moiety can be modified without a significant loss of potency , modifications to the 2-amino moiety are not well tolerated . we characterized compound 1 in a battery of biochemical , biophysical , moa , and selectivity assays and found that the setd8 inhibitory activity of this compound in biochemical assays was largely consistent with its binding affinity to setd8 determined by itc and spr . our moa studies indicate that this inhibitor is competitive with the peptide substrate and noncompetitive with the cofactor sam . importantly , this inhibitor is selective for setd8 over 15 other methyltransferases including g9a and glp , the two pkmts that are known for being potently inhibited by quinazolines . these results provide the first evidence that 2,4-diaminoquinazolines can be modified to yield selective , substrate competitive inhibitors of pkmts other than g9a and glp . a linear gradient was used with 10% ( or 50% ) of meoh ( a ) in 0.1% tfa in h2o ( b ) to 100% of meoh ( a ) . to a solution of 2-chloro-6,7-dimethoxy - n-(5-(pyrrolidin-1-yl)pentyl)quinazolin-4-amine ( 24 , 84 mg , 0.17 mmol ) and n - butanol ( 1.5 ml ) were added pyrrolidine ( commercially available , 56 l , 0.68 mmol ) and n , n - diisopropylethylamine ( 89 l , 0.51 mmol ) . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 and washed with brine . to a solution of 2-chloro-6,7-dimethoxy - n-(2-(pyrrolidin-1-yl)ethyl)quinazolin-4-amine ( 72 mg , 0.13 mmol ) and isopropanol ( 0.7 ml ) were added pyrrolidine ( 21 l , 0.26 mmol ) and hcl in dioxane ( 4.0 m , 63 l , 0.26 mmol ) . n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - dimethylpentane-1,5-diamine was prepared according to the procedure for making 24 from 2,4-dichloro-6,7-dimethoxylquinazoline , 5-(dimethylamino)amylamine ( commercially available ) , n , compound 6 was prepared according to the procedure for making 2 from n-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-n , n - dimethylpentane-1,5-diamine ( 70 mg , 0.15 mmol ) , pyrrolidine ( 21 l , 0.30 mmol ) , hcl in dioxane ( 4.0 m , 75 l , 0.30 mmol ) , and isopropanol ( 1.0 ml ) . after removal of the solvent by rotary evaporation , the residue was purified by hplc to give 14 mg of the title compound 9 as a tfa salt , white solid ( 14 mg , yield 80% ) . to the solution of 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) in meoh ( 1.0 ml ) were added formaldehyde ( commercially available , 39 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) , and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) at 0 c . compound 11 was prepared according to the procedure for making 10 from 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) , acetaldehyde ( commercially available , 56 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) and meoh ( 1.0 ml ) . compound 12 was prepared according to the procedure for making 10 from 6,7-dimethoxy - n-(5-(piperazin-1-yl)pentyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine ( 9 , 80 mg , 0.12 mmol ) , acetone ( commercially available , 103 l , 1.4 mmol ) , acetic acid ( 80 l , 1.4 mmol ) and sodium cyanoborohydride ( 44 mg , 0.7 mmol ) and meoh ( 1.0 ml ) . to the solution of tert - butyl ( 5-oxo-5-(pyrrolidin-1-yl)pentyl)carbamate in meoh and thf after removal of the solvent by rotary evaporation , the product 5-amino-1-(pyrrolidin-1-yl)pentan-1-one obtained was used for the next step without further purification . after removal of the solvent by rotary evaporation , the residue was redissolved in ch2cl2 , washed with brine . the organic layer was washed with brine , dried , and concentrated to give the crude product , which was purified on isco using 24 g silica gel column , eluting with hexane ethyl acetate to give 754 mg of the title compound 24 as a yellow semisolid ( yield 52% ) . inhibition of setd8 methyltransferase activity was analyzed by monitoring decrease in methylation of the fluorescein labeled peptide , tw21 ( nh2-lgkggakrhrkvlrdniqgitk(5fam)-oh ) , essentially as described previously . then 1 l of the serial dilution was transferred into compound dilution plate using nanoscreen multimek liquid handling robot . following a 10 min incubation of the compounds with the enzyme / peptide mix , the reaction was initiated by adding 2.5 l of 150 m sam in 1 buffer . the interaction between compound 1 and protein setd8 was further explored using a proteon xpr36 biosensor ( bio - rad laboratories , inc . ) a blank injection of the running buffer was made , after which four concentrations of the compound in the running buffer ( 100 , 33.3 , 11.1 , and 3.7 m ) and a running buffer were injected simultaneously at a flow rate of 50 l / min for 60 s. the sensorgrams obtained at the four concentrations of the compound were fit simultaneously after subtracting a ligand reference ( inner spot ) and a double compound reference ( buffer ) using a langmuir model to obtain on ( ka ) and off ( kd ) rates . ic50 values were determined for compound 1 at varying concentrations of sam , 50 nm setd8 , 200 m peptide h4 ( 124 ) or at varying concentrations of substrate h4 ( 124 ) peptide , 50 nm of setd8 , 250 m sam . in brief , the effect of compound 1 on activity of g9a , setdb1 , glp , suv39h2 , setd7 , prmt3 , prmt5-mep50 complex , prmt1 , suv420h1 , suv420h2 , smyd2 , dnmt1 , prc2 complex , mll1 complex , and dot1l was assessed by monitoring the incorporation of tritium - labeled methyl group to lysine or arginine residues of peptide substrates using radioactive assay .

based on a definition from the psychologist and philosopher williams james , attention is taking possession of the mind in clear and vivid form , of one out of what may seem several simultaneously possible objects or trains of thoughts . it implies withdrawal from some things in order to deal effectively with others " ( 1).along these lines , visual attention as a cognitive property is known to provide a critical base for learning ( 2 ) , working memory ( 3 ) , self - regulation ( 4 ) and word learning ( 5 ) . according to multiple models one of the most important types of attention with a significant role in learning and enhancing school achievement is selective or focused attention ( 7 ) . selective attention is defined as a preferential allocation of limited processing resources to events that have become behaviorally relevant ( 8) and depends on working memory capacity ( 9 , 10 ) . overall , sustained selective attention has an important role in academic performance ( 11 , 12 ) such as reading efficiency ( 13 - 15 ) and mathematical skills ( 16 ) . according to posner and rothbart , the stimulation of brain networks which involve in attention mechanism could enhance this skill in early childhood ( 17 ) . therefore , it is worthwhile to investigate the possible effect of interventions in this critical period . results underscore the need for research on the course of attention problems and the necessity to test interventions on children s early attention problems and their effects on subsequent academic achievement ( 18 ) . different tools areavailable to assess various types of attention . some currently validated neuropsychological tests for attention in children include the dtest ( 19 ) , test of everyday attention for children ( tea - ch ) ( 20 ) , stroop ( 21 ) and trail making test(part b ) ( 22 ) which allow measuring the visual selective attention , and there is alsothe persian version of the sustained auditory attention capacity test ( 23)which is designed to assess auditory sustain attention . since recently , there exist some computerized instruments to test cognitive skills such as attention which provide abundant proof . these instruments include test of variables of attention ( tova ) ( 24 , 25 ) , the integrated visual and auditory continuous performance test ( iva plus ) ( 26 ) and the connors ' continuous performance test(cpt ) ( 27)which assesses sustained attention . such computer - based assessments have two major benefits : first , they can be used to score thetests promptly , theyare ableto keep proper record of reaction time and accurate responses and can also generate an interpretive profile based on the normative data and provide concurrent stimuli ( 28 ) ; andsecond , they seem to be quite interesting for children and canincrease their motivation to have more cooperation and participation during the evaluation ( 29 ) . although a variety of computer - based assessments for auditory selective / sustained attention , such as paced auditory serial addition test ( pasat ) ( 30)and test of sustained selective attention ( tossa ) ( 31),are available , it seems there is a limitation in computer - based assessmentofvisual selective attention . stroop interference testwasoriginally developed as a paper - based tool to measure selective attention and cognitive flexibility ( 32 ) , ability to set shifting ( 33 ) , inhibition ( 34)and extinction ( 35 ) . the original stroop has been translated into numerous languages such as chinese , czechoslovakian , german , hebrew , swedish , japanese ( 35 ) , spanish ( 36 ) and persian ( 37 ) . there are a variation of stroop tasks i.e. , word - color and number - color . some limitations of this test are that the tool may not be administered to illiterate subjects such as preschoolers and is not specific for selective attention as it assesses set shifting and executive function as well . while attention difficulties are widespread among preschool children ( 7 ) , development of a tool to evaluatethe selective visual attention which fits into children s conditions and requirements seems crucial . toachievethis goal and upon designing the selective attention test complying with children s requirements , four important factors were considered asessential and they are as follows : 1)when children are illiterate , using the pictorial stimuli is the preferred method as elementary school children can grasp visualized concepts more easily and they can reply to picture - based questions as instantly as written concepts ( 38);2)tasks were preferred to be designed in agame format because games are considered the most significant means of communication in elementary school children ( 29 ) and may notably make them motivated once experimenters try to evaluate their cognitive skills . games can also allow children to have enhanced attentional control as well as greater cognitive flexibility . this would be served as a new route to better address developmental disorders ( 39 ) ; 3 ) simple design and use of familiar stimuli should be preferred . attention tests rarely resemble daily life activities and most such tests require quite a lot of focus , probably much more than daily life activities;4)the investigation of attention to both visual fields is required . it is well known that right hemisphere distributes attention in both hemispheres and visual fields . the activation of the attentional network appears to occur primarily in the right hemisphere ( 6 ) . the objectives of the current study were to design and develop a computer - based visual selective attention test ( sevat ) with stimuli requiring the attention level in daily life , and secondly , to evaluate the psychometric properties including content , face and convergent validity and test - retest and internal consistency reliability of the developed test . the test was designed as a game in order to engage children in a play and prevent them from feeling bored . the study involved the assessment of original and parallel forms of the computer - based selective visual attention test and was carried out in two phases : - phase 1 : the development of computer - based selective visual attention test ( sevat):the parallel form of the computer - based sevatwas designed toimprove the test reliability and eliminate the carry over and learned effect . the reliability for the parallel test was measured by comparing two different tests with similar but not fully equal content . this wasbasically done by creating a bank of questions or stimuli which measure the same quality after which stimuli are randomly divided into two separate tests . absent or insufficient consistency in each test ( for instance difference in question items or the content ) or measurement error diminishes the test reliability ( 40 ) . to create the preliminary design of this test , all variables involved in the computer - based selective attention test were taken into account . this was based on the available evidence and inputs provided from our experts panelwhich has been described in our previous study ( 41 ) . the first phase of this study ( the development of sevat ) is outlined below . stimuli selection : based on our previous record , 20 experts from various related disciplines including cognitive science , rehabilitation and computer - based game designer ( 9 occupational therapists , twocognitive neuroscientists , twopsychologists , sixcomputer game designers and one pediatric psychologist ) validated the selected stimuli(n = 200 ) for both the original and parallel versions . the stimuli were selected from a picture bank consisting of 600 pictures in different categories including cloths , familiar cartoons characters ( which are popular in media ) , fruits , foods , animals , toys , geometric shapes and signs , letters and numbers ( 42 - 44 ) . based on the consensus reached bythe experts panel , pictures from cloths , familiar cartoons characters , fruits , foods , animals and toys categories were selected . these pictures were shown to children while they were being asked to decidewhether the pictures were attractive , familiar and simple to recognize . results demonstrated that the clothing subgroup was the least attractive ( 97%agreements ) while the food and fruit subgroups ( 100% agreements ) were the most attractive items . eventually , the test contained 5 subgroups which were attractive to children and acceptable based onthe view points of the experts . task : the test comprised of20 trials ( 10 right-10 left visual fields in order to assess the visual field effect on attention allocation ) . in each trial , fivepictures of each subtest were framed . they were shown in the column of theright or left side of the screen , and one of them was taken as the target . in each trial , first , the target picture was largely displayed at the middle of the screen and was then paused for 1500 milliseconds based on theexperts ' opinion ( 41 ) . next , the target picture shrank to small size and moved above a column with fivecells in theright ( or left ) side of the screen and stayed there till the end of trial presentation ( in order to decrease the memory effect ) . each cell of thecolumns was differently colored . then , five pictures moved horizontally from the left side of the screen towards this column . after the first 10 replications , the position of thecolumn and direction of thepictures were changed in order to compare the asymmetric effect of the attention network . software : the software codes were defined in action script 3 and the test was executed in adobe flash professional cs 5 , adobe air 3 , adobe photoshop cs 5.5 , sublime text 2 and notepad 9.8 . the attribute of this test were reaction time in millisecond , accuracy and false reaction . - phase 2 : the psychometric properties of computer - based sevat content validity : ten experts ( occupational therapists ) who did not take part in the first stage endorsed the appropriateness of thequality and quantity of each trial of the test . the inclusion criteria were as follows:1)being the author of at least onerelevant article and 2)acquiring 10-years academic work experience as a faculty member . the content validity ratio ( cvr ) and content validity index ( cvi ) were calculated based on thelawshe method ( 45 ) . responses from experts were pooled and the number indicated the " essentiality " for each item . face validity : after validatingeach stimulus , tenchildren(7 years old ) who did not take part in the first stageassessed the degree of attractiveness , the presentation time and degree of simplicity of each trial . besides , they identified how much each task was interesting using the visual analog scale ( vas ) where 0 indicated boring and 10 indicated very interesting . in addition , once the test was over , all participants ( 60 children ) answered the open question of " do you want to play it again ? convergent validity : sixty first - grade students were enrolled to measure the test - retest reliability and convergent validity of the test . the participantswere all selected through convenience sampling method from two elementary schools located in the1district of tehran during april - may 2013 . participants were 30 girls with the mean age of 80.83 months and standard deviation of 3.26 and 33 boys with the mean age of 81.50 months and standard deviation of 2.58 . the inclusion criteria were age 78 - 84 months years old , normal visual acuity and hearing , no color blindness confirmed by the ishihara test , normal visual field documented by visual confrontation test performed by an occupational therapist , normal intelligence score iq90 based on raven intelligence questionnaire for children and fluency in reading color names ability . the participants were excluded from the study if they revealed any history of a neurological disorder , loss of consciousness due to head injury , any medical condition that might affect cerebral functioning and epilepsy based on their medical record and interview with their parent(s ) . lack of adhd signs was confirmed using the teacher version of conners rating scales for children in elementary schools ( 46 ) which was confirmed by their teachers . three boys were excluded from thestudy due to fever and lack of proper cooperation . in order to evaluate the convergent validity , the correlation between theaverage of reaction time and accuracy of computer - based sevatand thepersian version of computerized stroop color - word test was determined . the persian version of computerized stroop color - word test ( ravansinainc , iran)includes two stages . in the first stage , the training phase , theparticipant should choose the color of the circle which is shown on the monitor in four possible colors of blue , red , yellow and green and press the keys which are covered with colorful labels ( v ( blue ) , b ( red ) , n ( yellow ) , m ( green)]on the keyboard . the main part of the test consists of 96 colorful words - 48 colorful congruent words ( the meaning of the word complies with the ink color in which the word is written ) and 48 colorful incongruent words ( the meaning of the word does not comply with the ink color in which the word is written ) - which wasdisplayed inpseudo - randomly order on the middle of the monitor for 2000 milliseconds ( ms ) with 800 ms inter - stimulus interval ( isi ) . the participantswere asked to identify the color of the words regardless of their meaning ( 47 ) . reliability : to examine the test - retest reliability , the parallel version was designed and all the 60 children were administered the original and parallel versions in a single session . procedure : all children participating in this study completed the two versions ( original and parallel ) of thecomputer - based sevatand the persian version of computerized stroop color - word test in a random order . all children had a snack before the experiment with 5 - 10 minutes resting time between the tests . participants were seated comfortably on a chair in aquiet room at their schools in the morning during 8 - 12 am . the participantswere initially briefed about the overall procedure ( by training in practice block ) and clicked on the correct picture using the mouse . the ethical protocol of this study was based on the approval from the ethic committeeof iran university of medical sciences ( iums ) and wassigned by all theparticipants and one of their parents . result of the kolomogorov - smirnov test determined the non - normal distribution of the stroop test data and normal distribution of theoriginal and parallel versions of sevat . as such , the spearman correlation coefficients , intra - class correlation coefficient ( icc ) and cronbach 's alpha coefficients were used to examine the convergent validity , test - retest reliability and internal consistency , respectively . the reliability correlation coefficients less than 0.4 , between 0.4 and 0.7 and more than 0.7 were considered as weak , tolerable to fine and great reliability , respectively ( 49 ) . result of the kolomogorov - smirnov test determined the non - normal distribution of the stroop test data and normal distribution of theoriginal and parallel versions of sevat . as such , the spearman correlation coefficients , intra - class correlation coefficient ( icc ) and cronbach 's alpha coefficients were used to examine the convergent validity , test - retest reliability and internal consistency , respectively . the reliability correlation coefficients less than 0.4 , between 0.4 and 0.7 and more than 0.7 were considered as weak , tolerable to fine and great reliability , respectively ( 49 ) . content validity : the content validity ratio ( cvr ) of the sevat s 20 trials was determined by 10 occupational therapists . while the experts marked 19 trials as " essential " , the cvr for all trials was1 except forone trial ( animals ' pictures cvr=0.8 ) . the content validity index ( cvi ) face validity : based on the inputs collected from10 first - grade participants , the test was perceived to be attractive ( n=10 , 100% ) , the time duration for each trial was sufficient ( n=8 , 80% ) and the task was simple ( n=8 , 80% ) . besides , they acknowledgedthat the task was interesting ( mean vas= 9.5).the answers of all theparticipants to the question " do you want to play it again ? " were yes . test - retest reliability : with regards to the test - retest reliability using the icc , there was a correlation between the original and parallel version of sevat(p<0.001 , r=0.778 ) ( table 1 ) . sem : standard error of measurement , sd : standard deviation , m : mean , ul : upper , limit , ll : lower limit , cn : number of correct answers internal consistency : the internal consistencies ( cronbach 's alpha ) of scores in the original and parallel tests were 0.857 and 0.831 , respectively . cronbach s alpha of each subgroups revealed that no item needed to be deleted , since all were less than the total score ( table 2 ) . * : all subgroups are significantly correlated at p<0.05 the correlation between the subgroups and the total scores in the both versions aredemonstratedin table 2 . there wasa significantrelationship between the subgroups and the total scores in the original ( 0.759 < r < 0.818 ) as well as the parallel ( 0.714 < r < 0.802 ) versions of thesevat . the correlation between the subgroups and the total score for theoriginal version of sevat was shown to be tolerable to fine ( 0.434 < r < 0.601),and it was weak to tolerable ( 0.394 < r < 0.575)for theparallel version of sevat . convergent validity : the spearman s correlation coefficient demonstrated asignificant positive relationship between the number of correct answers and reaction time to correct answers in the original / parallel versions of sevat with the congruent / incongruent stimuli of the stroop test ( table 3 ) . cn : number of correct answers , rt : reaction time to corrected answers , * * : p<0.001 is significant , * : p<0.05 is significant , r : spearman s rho correlation , ms : millisecond repeated - measure anova demonstrated neither a significant main / interaction effects of the right and left visual field in the original ( f ( 1 , 53 ] = 0.003 , p= 0.956 ) and parallel ( f ( 1 , 57 ] = 0.379 , p= 0.541 ) tests on the reaction time to corrected answers nor on the number of correct answer of these two versions of the computer - based original ( f ( 1 , 57 ] = 0.970 , p= 0.329 ) and parallel ( f ( 1 , 59 ] = 0.506 , p= 0.480 ) sevat . the aim of this study was to develop and validate an instrument , which is interesting to children , to test their selective attention . to do so , a computer - based sevat was designed and developed . following theadministration of the test to first - grade children , the validity ( content , face and convergent ) and reliability ( test - retest and internal consistency ) of the test was assessed . one limitation of applying the attentional models onyounger children was thegreater overlap with other developing skills ; for instance , executive function , language , visuospatial skills ( 51 ) . hence , the design of the current test was based on the perceptual matching tasks method in which childrenwatched thetarget picturesin the monitor and chose the picture which was the same as the target picture among the5 other pictures . in other words , the participantswere asked to track the target pictureamong the other stimuli which were moving from theright to theleft or vice versa ( based on the visual field ) with the task being somehow similar to visual tracking . basically , these two methods are similar to other tasks for selective attention testing as described by mahone and schneiderin 2012 perceptual matching tasks , central - incidental learning tasks and visual search tasks as common methods to measure the selective attention ( 7 ) . hayakawa and colleagues recorded the responses of 111 childrento colorful pictures to be more rapid than the gray scale regardless of the content as compared to the adults ( 52 ) . task designers believed that illiterate childrencould grasp concepts in pictures faster than letters , numbers and words as confirmed by evidence ( 53)as well , and they could understand concepts in familiar pictures more quickly than theunfamiliar ones . the target stimuli in thesevat was shown at the middle part of the monitor and in a larger size than other stimuli and then became equal to others in thesevat based on the experts opinion which was recorded in our previous study ( 41 ) . this might have rooted in the fact that concentration and fixation in visual field relies on the center and larger objects are processed in shorter period of time which is in agreement with the results foundby yao and colleagues ( 2011 ) . they declared that categorization of the complex natural images may occur within a limited area of the visual field , referred to as " field of attention " ( fa).the fa is limited to20 x 24 of visual field within almost 0.1 second without eye movement . as such , the target stimuli in the stroop test arein the middle of the visual field ( 48 ) . in thesevat , each of the5 stimuli in thetrial moves horizontally in a straight line from the right to the left and vice versa on the monitor . thenchildrenshould track the stimulus which is found as same as the target.chosing this design for sevat was done based on the agreements of the experts panel ( 41).firstly , thevisual tracking is considered as an essential skill for reading ( 55 ) . ( 56)and maunsell et al . ( 57 ) studies , moving objects and orientation changes may attract more attention capacity . based on the lieberman et al . study , moving objects are more interesting for childrenand cause more excitement ( 58 ) . the hierarchical visual perception processing which begins with eye movement is followed by visual attentionand is completed with visual memory ( 59 ) . as stated by burnham et al.(2014 ) , parallel with load theory , the visual and spatial working memory may influence selective attention ( 9 ) . consequently , test designers are suggested to reduce the impact of working memory by selecting 5 stimuli as distracters since normal working memory capacity for adult is 72 , or by letting the target stimulus remain displayed on the screen until the end of thereplication . studies.they showed that the responses of children in the multiple - object tracking task ( parts tracking and recognizing the moving position)were affectedbytheir age ( 60 ) . thus,6-year - old children can track the positions of more than four moving itemsbecause of their working memory capacity ( 61 , 62 ) . no significant difference was found between the two visual fieldswith regards to the reaction time and the number of correct answerswhen the two versions were compared . they found a significant difference in responses from left vs. right visual fields and confirmed the existence of hemispheric asymmetry in selective attention and concluded that stimuli similarity may play a critical role in this asymmetry ( 63 ) . although such result should be validated by examining the test in adhd or patients with unilateral neglect , some possible reasons for this disparity may be small sample size , unrestricted head movement during the test , lack of sensitivity of the test ( for which some electrophysiology modalities such as erp or eye tracking are preferred ) and presenting 10 trials for each right or left visual field orderly instead of randomly;tojustify this , conductingfurther research is suggested . validity : one of the most important factors in developing tests is their validity . regarding the content validity stages of the computer - based sevat , the value of both versions of the test was approved by the experts panel . according to the experts analysis , the content validity of thewhole test , stimuli , psychophysics properties and the homogeneity of computer - basedsevat subgroups werequite favorable forassessing selective attention . with regards all children found this test to be interesting and they wanted to play it again . however , they indicated that the stroop test was boring for them and madethem feel asanxious as at the time of theexamination . with respect to the convergent validity , there was a positive and significant correlation between raw scores in the original and parallel versions of the sevat test and stroop test . therefore , the sevatmay be a proper tool to measure selective attention in 7 year - old children . this means that there were very strong relationships between different subgroups of the sevat with the total scores . in addition , the different subgroups of the computer - based sevat were strongly interrelated , indicating a good internal consistency for the test . meanwhile , exclusion of each subgroup decreased the internal consistency of the scale which confirmed the sevat measuring a specific area . concerning the test - retest reliability of the computer - based sevat , findings onthe repeatability of thescores , usingtheparallel version to examine test retest in asingle session , demonstrated agreat relationship . this suggested that the original and parallel versions of the computer - based sevat may be conducted interchangeably . further studies should be conductedto assess the differentiated validity of thesevat in other age groups of preschool childrenand in other disorders such as adhd , learning disorders and pdd . however , the main limitation of this study was the lack of other computer - based visual selective attention tests to measure convergent validity in children , and expectedly lack of relevant evidence to compare the results . moreover , lack of any alternative test rather than thestrooptestmadeus to wait till the end of the first grade in whichkids acquire an acceptable literacy level . the computer - based selective visual attention test is an easily administered instrument to assess selective attention in children who were not literate . considering the good validity and reliability of thesevat , itcanbe used as a test besides other children s cognitive assessment toolbox . children were found to perceive it as agame they like to play and theydemonstrated very good cooperation during the test . the applicability of such a test alsoneeds to be examined in other age groups ( 3 - 7 year - old kids ) and in different subgroups with neuro - developmental predicaments .

background : visual attention is known as a critical base for learning . the purpose of the present study was to design , develop and evaluate the test - retest and internal consistency reliability as well as face , content and convergent validity of the computer- based selective visual attention test ( sevat ) for healthy first - grade school children . methods : in the first phase of this study , the computer - based sevat was developed in two versions of original and parallel . ten experts in occupational therapy helped to measure the content validity using the cvr and cvi methods . face validity was measured through opinions collected from 10 first - grade children . the convergent validity of the test was examined using the spearman correlation between the sevat and stroop test . in addition , test - retest reliability was determined by measuring the intra - class correlation ( icc ) between the original and parallel versions of the sevat in a single session . the internal consistency was calculated by cronbach 's alpha coefficients . sixty first grade children ( 30 girls/30boys ) participated in this study . results : the developed test was found to have good content and face validity . the sevat showed an excellent test - retest reliability ( icc= 0.778 , p<0.001 ) and internal consistency ( cronbach 's alpha of original and parallel tests were 0.857 and 0.831 , respectively ) . sevat and stroop test demonstrated a positive correlation upon the convergent validity testing . conclusion : our results suggested an acceptable reliability and validity for the computer - based sevat in the assessment of selective attention in children . further research may warrant the differential validity of such a test in other age groups and neuro - cognitively disordered populations .

Introduction Methods Statistical Analyses Results Discussion Conclusion

based on a definition from the psychologist and philosopher williams james , attention is taking possession of the mind in clear and vivid form , of one out of what may seem several simultaneously possible objects or trains of thoughts . it implies withdrawal from some things in order to deal effectively with others " ( 1).along these lines , visual attention as a cognitive property is known to provide a critical base for learning ( 2 ) , working memory ( 3 ) , self - regulation ( 4 ) and word learning ( 5 ) . selective attention is defined as a preferential allocation of limited processing resources to events that have become behaviorally relevant ( 8) and depends on working memory capacity ( 9 , 10 ) . overall , sustained selective attention has an important role in academic performance ( 11 , 12 ) such as reading efficiency ( 13 - 15 ) and mathematical skills ( 16 ) . therefore , it is worthwhile to investigate the possible effect of interventions in this critical period . some currently validated neuropsychological tests for attention in children include the dtest ( 19 ) , test of everyday attention for children ( tea - ch ) ( 20 ) , stroop ( 21 ) and trail making test(part b ) ( 22 ) which allow measuring the visual selective attention , and there is alsothe persian version of the sustained auditory attention capacity test ( 23)which is designed to assess auditory sustain attention . these instruments include test of variables of attention ( tova ) ( 24 , 25 ) , the integrated visual and auditory continuous performance test ( iva plus ) ( 26 ) and the connors ' continuous performance test(cpt ) ( 27)which assesses sustained attention . such computer - based assessments have two major benefits : first , they can be used to score thetests promptly , theyare ableto keep proper record of reaction time and accurate responses and can also generate an interpretive profile based on the normative data and provide concurrent stimuli ( 28 ) ; andsecond , they seem to be quite interesting for children and canincrease their motivation to have more cooperation and participation during the evaluation ( 29 ) . although a variety of computer - based assessments for auditory selective / sustained attention , such as paced auditory serial addition test ( pasat ) ( 30)and test of sustained selective attention ( tossa ) ( 31),are available , it seems there is a limitation in computer - based assessmentofvisual selective attention . stroop interference testwasoriginally developed as a paper - based tool to measure selective attention and cognitive flexibility ( 32 ) , ability to set shifting ( 33 ) , inhibition ( 34)and extinction ( 35 ) . the original stroop has been translated into numerous languages such as chinese , czechoslovakian , german , hebrew , swedish , japanese ( 35 ) , spanish ( 36 ) and persian ( 37 ) . some limitations of this test are that the tool may not be administered to illiterate subjects such as preschoolers and is not specific for selective attention as it assesses set shifting and executive function as well . while attention difficulties are widespread among preschool children ( 7 ) , development of a tool to evaluatethe selective visual attention which fits into children s conditions and requirements seems crucial . toachievethis goal and upon designing the selective attention test complying with children s requirements , four important factors were considered asessential and they are as follows : 1)when children are illiterate , using the pictorial stimuli is the preferred method as elementary school children can grasp visualized concepts more easily and they can reply to picture - based questions as instantly as written concepts ( 38);2)tasks were preferred to be designed in agame format because games are considered the most significant means of communication in elementary school children ( 29 ) and may notably make them motivated once experimenters try to evaluate their cognitive skills . games can also allow children to have enhanced attentional control as well as greater cognitive flexibility . the activation of the attentional network appears to occur primarily in the right hemisphere ( 6 ) . the objectives of the current study were to design and develop a computer - based visual selective attention test ( sevat ) with stimuli requiring the attention level in daily life , and secondly , to evaluate the psychometric properties including content , face and convergent validity and test - retest and internal consistency reliability of the developed test . the test was designed as a game in order to engage children in a play and prevent them from feeling bored . the study involved the assessment of original and parallel forms of the computer - based selective visual attention test and was carried out in two phases : - phase 1 : the development of computer - based selective visual attention test ( sevat):the parallel form of the computer - based sevatwas designed toimprove the test reliability and eliminate the carry over and learned effect . the reliability for the parallel test was measured by comparing two different tests with similar but not fully equal content . absent or insufficient consistency in each test ( for instance difference in question items or the content ) or measurement error diminishes the test reliability ( 40 ) . to create the preliminary design of this test , all variables involved in the computer - based selective attention test were taken into account . the first phase of this study ( the development of sevat ) is outlined below . stimuli selection : based on our previous record , 20 experts from various related disciplines including cognitive science , rehabilitation and computer - based game designer ( 9 occupational therapists , twocognitive neuroscientists , twopsychologists , sixcomputer game designers and one pediatric psychologist ) validated the selected stimuli(n = 200 ) for both the original and parallel versions . eventually , the test contained 5 subgroups which were attractive to children and acceptable based onthe view points of the experts . task : the test comprised of20 trials ( 10 right-10 left visual fields in order to assess the visual field effect on attention allocation ) . they were shown in the column of theright or left side of the screen , and one of them was taken as the target . in each trial , first , the target picture was largely displayed at the middle of the screen and was then paused for 1500 milliseconds based on theexperts ' opinion ( 41 ) . next , the target picture shrank to small size and moved above a column with fivecells in theright ( or left ) side of the screen and stayed there till the end of trial presentation ( in order to decrease the memory effect ) . then , five pictures moved horizontally from the left side of the screen towards this column . after the first 10 replications , the position of thecolumn and direction of thepictures were changed in order to compare the asymmetric effect of the attention network . software : the software codes were defined in action script 3 and the test was executed in adobe flash professional cs 5 , adobe air 3 , adobe photoshop cs 5.5 , sublime text 2 and notepad 9.8 . - phase 2 : the psychometric properties of computer - based sevat content validity : ten experts ( occupational therapists ) who did not take part in the first stage endorsed the appropriateness of thequality and quantity of each trial of the test . the content validity ratio ( cvr ) and content validity index ( cvi ) were calculated based on thelawshe method ( 45 ) . face validity : after validatingeach stimulus , tenchildren(7 years old ) who did not take part in the first stageassessed the degree of attractiveness , the presentation time and degree of simplicity of each trial . in addition , once the test was over , all participants ( 60 children ) answered the open question of " do you want to play it again ? convergent validity : sixty first - grade students were enrolled to measure the test - retest reliability and convergent validity of the test . in order to evaluate the convergent validity , the correlation between theaverage of reaction time and accuracy of computer - based sevatand thepersian version of computerized stroop color - word test was determined . in the first stage , the training phase , theparticipant should choose the color of the circle which is shown on the monitor in four possible colors of blue , red , yellow and green and press the keys which are covered with colorful labels ( v ( blue ) , b ( red ) , n ( yellow ) , m ( green)]on the keyboard . the main part of the test consists of 96 colorful words - 48 colorful congruent words ( the meaning of the word complies with the ink color in which the word is written ) and 48 colorful incongruent words ( the meaning of the word does not comply with the ink color in which the word is written ) - which wasdisplayed inpseudo - randomly order on the middle of the monitor for 2000 milliseconds ( ms ) with 800 ms inter - stimulus interval ( isi ) . reliability : to examine the test - retest reliability , the parallel version was designed and all the 60 children were administered the original and parallel versions in a single session . procedure : all children participating in this study completed the two versions ( original and parallel ) of thecomputer - based sevatand the persian version of computerized stroop color - word test in a random order . all children had a snack before the experiment with 5 - 10 minutes resting time between the tests . participants were seated comfortably on a chair in aquiet room at their schools in the morning during 8 - 12 am . the participantswere initially briefed about the overall procedure ( by training in practice block ) and clicked on the correct picture using the mouse . the ethical protocol of this study was based on the approval from the ethic committeeof iran university of medical sciences ( iums ) and wassigned by all theparticipants and one of their parents . result of the kolomogorov - smirnov test determined the non - normal distribution of the stroop test data and normal distribution of theoriginal and parallel versions of sevat . as such , the spearman correlation coefficients , intra - class correlation coefficient ( icc ) and cronbach 's alpha coefficients were used to examine the convergent validity , test - retest reliability and internal consistency , respectively . the reliability correlation coefficients less than 0.4 , between 0.4 and 0.7 and more than 0.7 were considered as weak , tolerable to fine and great reliability , respectively ( 49 ) . result of the kolomogorov - smirnov test determined the non - normal distribution of the stroop test data and normal distribution of theoriginal and parallel versions of sevat . as such , the spearman correlation coefficients , intra - class correlation coefficient ( icc ) and cronbach 's alpha coefficients were used to examine the convergent validity , test - retest reliability and internal consistency , respectively . the reliability correlation coefficients less than 0.4 , between 0.4 and 0.7 and more than 0.7 were considered as weak , tolerable to fine and great reliability , respectively ( 49 ) . content validity : the content validity ratio ( cvr ) of the sevat s 20 trials was determined by 10 occupational therapists . while the experts marked 19 trials as " essential " , the cvr for all trials was1 except forone trial ( animals ' pictures cvr=0.8 ) . the content validity index ( cvi ) face validity : based on the inputs collected from10 first - grade participants , the test was perceived to be attractive ( n=10 , 100% ) , the time duration for each trial was sufficient ( n=8 , 80% ) and the task was simple ( n=8 , 80% ) . test - retest reliability : with regards to the test - retest reliability using the icc , there was a correlation between the original and parallel version of sevat(p<0.001 , r=0.778 ) ( table 1 ) . sem : standard error of measurement , sd : standard deviation , m : mean , ul : upper , limit , ll : lower limit , cn : number of correct answers internal consistency : the internal consistencies ( cronbach 's alpha ) of scores in the original and parallel tests were 0.857 and 0.831 , respectively . cronbach s alpha of each subgroups revealed that no item needed to be deleted , since all were less than the total score ( table 2 ) . * : all subgroups are significantly correlated at p<0.05 the correlation between the subgroups and the total scores in the both versions aredemonstratedin table 2 . there wasa significantrelationship between the subgroups and the total scores in the original ( 0.759 < r < 0.818 ) as well as the parallel ( 0.714 < r < 0.802 ) versions of thesevat . the correlation between the subgroups and the total score for theoriginal version of sevat was shown to be tolerable to fine ( 0.434 < r < 0.601),and it was weak to tolerable ( 0.394 < r < 0.575)for theparallel version of sevat . convergent validity : the spearman s correlation coefficient demonstrated asignificant positive relationship between the number of correct answers and reaction time to correct answers in the original / parallel versions of sevat with the congruent / incongruent stimuli of the stroop test ( table 3 ) . cn : number of correct answers , rt : reaction time to corrected answers , * * : p<0.001 is significant , * : p<0.05 is significant , r : spearman s rho correlation , ms : millisecond repeated - measure anova demonstrated neither a significant main / interaction effects of the right and left visual field in the original ( f ( 1 , 53 ] = 0.003 , p= 0.956 ) and parallel ( f ( 1 , 57 ] = 0.379 , p= 0.541 ) tests on the reaction time to corrected answers nor on the number of correct answer of these two versions of the computer - based original ( f ( 1 , 57 ] = 0.970 , p= 0.329 ) and parallel ( f ( 1 , 59 ] = 0.506 , p= 0.480 ) sevat . the aim of this study was to develop and validate an instrument , which is interesting to children , to test their selective attention . to do so , a computer - based sevat was designed and developed . following theadministration of the test to first - grade children , the validity ( content , face and convergent ) and reliability ( test - retest and internal consistency ) of the test was assessed . hence , the design of the current test was based on the perceptual matching tasks method in which childrenwatched thetarget picturesin the monitor and chose the picture which was the same as the target picture among the5 other pictures . in other words , the participantswere asked to track the target pictureamong the other stimuli which were moving from theright to theleft or vice versa ( based on the visual field ) with the task being somehow similar to visual tracking . basically , these two methods are similar to other tasks for selective attention testing as described by mahone and schneiderin 2012 perceptual matching tasks , central - incidental learning tasks and visual search tasks as common methods to measure the selective attention ( 7 ) . hayakawa and colleagues recorded the responses of 111 childrento colorful pictures to be more rapid than the gray scale regardless of the content as compared to the adults ( 52 ) . the target stimuli in thesevat was shown at the middle part of the monitor and in a larger size than other stimuli and then became equal to others in thesevat based on the experts opinion which was recorded in our previous study ( 41 ) . they declared that categorization of the complex natural images may occur within a limited area of the visual field , referred to as " field of attention " ( fa).the fa is limited to20 x 24 of visual field within almost 0.1 second without eye movement . as such , the target stimuli in the stroop test arein the middle of the visual field ( 48 ) . thenchildrenshould track the stimulus which is found as same as the target.chosing this design for sevat was done based on the agreements of the experts panel ( 41).firstly , thevisual tracking is considered as an essential skill for reading ( 55 ) . (2014 ) , parallel with load theory , the visual and spatial working memory may influence selective attention ( 9 ) . no significant difference was found between the two visual fieldswith regards to the reaction time and the number of correct answerswhen the two versions were compared . they found a significant difference in responses from left vs. right visual fields and confirmed the existence of hemispheric asymmetry in selective attention and concluded that stimuli similarity may play a critical role in this asymmetry ( 63 ) . although such result should be validated by examining the test in adhd or patients with unilateral neglect , some possible reasons for this disparity may be small sample size , unrestricted head movement during the test , lack of sensitivity of the test ( for which some electrophysiology modalities such as erp or eye tracking are preferred ) and presenting 10 trials for each right or left visual field orderly instead of randomly;tojustify this , conductingfurther research is suggested . regarding the content validity stages of the computer - based sevat , the value of both versions of the test was approved by the experts panel . according to the experts analysis , the content validity of thewhole test , stimuli , psychophysics properties and the homogeneity of computer - basedsevat subgroups werequite favorable forassessing selective attention . however , they indicated that the stroop test was boring for them and madethem feel asanxious as at the time of theexamination . with respect to the convergent validity , there was a positive and significant correlation between raw scores in the original and parallel versions of the sevat test and stroop test . therefore , the sevatmay be a proper tool to measure selective attention in 7 year - old children . this means that there were very strong relationships between different subgroups of the sevat with the total scores . in addition , the different subgroups of the computer - based sevat were strongly interrelated , indicating a good internal consistency for the test . meanwhile , exclusion of each subgroup decreased the internal consistency of the scale which confirmed the sevat measuring a specific area . concerning the test - retest reliability of the computer - based sevat , findings onthe repeatability of thescores , usingtheparallel version to examine test retest in asingle session , demonstrated agreat relationship . this suggested that the original and parallel versions of the computer - based sevat may be conducted interchangeably . further studies should be conductedto assess the differentiated validity of thesevat in other age groups of preschool childrenand in other disorders such as adhd , learning disorders and pdd . however , the main limitation of this study was the lack of other computer - based visual selective attention tests to measure convergent validity in children , and expectedly lack of relevant evidence to compare the results . moreover , lack of any alternative test rather than thestrooptestmadeus to wait till the end of the first grade in whichkids acquire an acceptable literacy level . the computer - based selective visual attention test is an easily administered instrument to assess selective attention in children who were not literate . considering the good validity and reliability of thesevat , itcanbe used as a test besides other children s cognitive assessment toolbox . children were found to perceive it as agame they like to play and theydemonstrated very good cooperation during the test . the applicability of such a test alsoneeds to be examined in other age groups ( 3 - 7 year - old kids ) and in different subgroups with neuro - developmental predicaments .

the sibling bond is the marathon runner of human relationships ; it is the longest lasting relationship , enduring from the birth of the youngest sibling to the death of the first to go . it is therefore reasonable to assume the importance of this relationship across the lifespan . it has been established that the younger siblings of individuals with autism spectrum disorder ( asd ) are at heightened risk of developmental problems in comparison to the general population . some of these developmental difficulties include : social and cognitive deficiencies , and neurocognitive and behavioural delays , specifically executive function and repetitive behaviours . the above examples of sibling research are commonly used to provide evidence for the genetic basis of asd with elevated levels of autistic traits in siblings representing potential markers of a broader autism phenotype ( bap ; ) . the nurture side of the debate how are these siblings and families impacted by the disorder in regard to their quality of life ( qol ) and psychosocial outcomes ? the american psychiatric association describes the essential features of autistic disorder as the presence of markedly abnormal or impaired development in social interactions and communication and a markedly restricted repertoire of activity and interests this is commonly referred to as the triad of autism diagnosis , representing delays or deficits in three essential categories : social interaction , communication , and restricted interests . characteristic symptoms may include self - stimulation behaviours ( e.g. , hand flapping and rocking ) , self - injury behaviours ( e.g. , head banging , skin picking , hair pulling ) , lack of emotional and social reciprocity , and inflexible adherence to routines or rituals . in addition to common comorbidity with other medical , psychiatric , and developmental disorders , these asd characteristics can be difficult for families to manage . epilepsy , allergies , sleep disorders , conduct disorder , attention deficit / hyperactivity disorder ( adhd ) , gastro - intestinal disorders , anxiety disorders , intellectual disability and language disorders often cooccur with a diagnosis of asd . furthermore , asd requires substantial financial investment in therapy , medical treatment , and early intervention education ; increased vigilance of the individual with autism ; micromanagement of the family environment to ensure minimal sensory discomfort and maintenance of a highly predictable routine ; often limited communication with the family member with autism ; and many other family disturbances . ensuring that an individual with asd lives in an environment that caters for their complex needs requires a large commitment from the family . it is empirically intuitive to assume that quality of life ( qol ) is impacted in these families . this has yet to be supported by research , with a lack of studies focusing on qol outcomes in asd families . a review of the sibling literature reveals both positive and negative effects of growing up with an individual with a disability . siblings of children with autism have been shown to do more poorly on a number of outcome measures in comparison to siblings of other developmental disabilities and those with only typically developing siblings . this distinct population of siblings has been shown to exhibit higher levels of internalising and externalising disorders [ 5 , 6 ] , social and behavioural adjustment problems , hassles with sibling behaviour , and distressing emotions such as guilt . in contrast , other researchers have found no difference in levels of adjustment between siblings of individuals with asd and those with only typically developing siblings or those with siblings with other developmental diagnoses . even more promising , the positive impacts of growing up with a sibling with asd are becoming known . siblings of individuals with autism have been reported to have less conflict in the sibling relationship , family resilience , and increased self - perceived competence [ 6 , 13 , 14 ] . they have also been shown to have a more positive opinion of the sibling relationship , positive psychosocial and emotional development conditional upon limited demographic risk factors , feelings of empathy for their sibling , and increased maturity . the autism sibling literature is yet to reach consensus ; however , macks and reeve [ 14 , page 1065 ] succinctly conclude that having a sibling with autism may not be a risk factor in and of itself , and children with autism may even have a positive influence on the life of the nondisabled sibling . however , when multiple demographic risk factors are present , it becomes more difficult for the nondisabled sibling to deal with the child with autism , both emotionally and psychologically . macks and reeve consider the contribution this potentially makes to the inconsistency currently plaguing sibling research . if those studies reporting positive influences included samples of demographically stable families , while those samples that exhibited negative impacts consisted of participants experiencing a high number of demographic risk factors , the true effects of growing up with a sibling with autism would be masked , justifying continued exploration of sibling outcome . in order to investigate this masking effect at surface level , the author conducted closer inspection of the studies listed above that showed no difference between siblings of individuals with asd and comparison siblings . study were part of high - income families ( 25 out of 27 families ) . a large proportion of the sample in the kaminsky and dewey study reported receiving high levels of social support . the majority of bayat 's resilient families used in his 2007 study were intact families with average incomes of 81,000 usd per year . although the study included families with low income ( 23% ) , over 60 percent were in the high - income category . had these samples included more demographically disadvantaged siblings , their outcome may have been poorer than comparison siblings . the low level of demographic risk may have contributed to the healthy levels of adjustment reported in these studies , a consideration that further supports macks and reeve 's proposal . recent literature reviews report mixed results and emphasise the large gaps in our current knowledge of sibling outcome , due in large part to inconsistency in methodology and a lack of rigorous sampling procedures [ 18 , 19 ] . the purpose of this literature review was to summarise studies of the psychosocial impacts of growing up with a sibling with asd , identify gaps in the literature , and propose future directions for sibling research in the autism spectrum field . although it is recognised that genetic factors play an integral part in any research regarding asd , the current paper focuses on psychosocial rather than biological components of sibling outcome . in the context of the multitude of challenging behaviours exhibited by an individual with autism , it is intuitively appealing to predict that the well - being of siblings in these homes may be compromised ; this literature review attempted to summarise this impact . electronic databases including pubmed , psycinfo , google scholar , psycarticles , medline , scopus , wiley online library , and proquest psychology journals were reviewed for studies published in peer - reviewed journals within the last decade . a large number of the identified studies were published in 2003 , signalling an increase in focus on siblings around this time and a suitable start point for this historical investigation . fourteen articles were accessed using key words to identify research within the sibling well - being field and further filtered in order to critically appraise the most relevant studies . due to the small number of robust and empirically sound studies focussing specifically on siblings of children with autism , a number of studies focussing on the functioning of the whole family in addition to studies targeting a range of developmental disabilities were considered . relevant studies were also identified if they were cited within the articles accessed through the above database search . within the sibling field a number of pertinent concepts were identified ; the current article will discuss the outcome of siblings of children with autism according to psychological health , overall quality of life , emotional intelligence , family coping , and child and family factors . the original criteria used to filter relevant articles allowed for inclusion of databased articles that were asd - specific and included a control comparison group . however , these criteria needed to be relaxed as only 4 studies met this stringent category . siblings of individuals with autism appear to be socially and emotional well adjusted according to a study that compared them to siblings of individuals with developmental language disorders ( specific learning disability ) and intellectual disability . despite the absence of a typically developing sibling control comparison , these findings are still promising due to the low number of the siblings of individuals with asd that fell into the clinical diagnostic range ( 13.4% ) on a number of behavioural adjustment measures . kaminsky and dewey . with the advantage of including comparison groups of siblings of individuals with another disability ( down syndrome ) and those with only typically developing siblings , kaminsky and dewey reported low levels of loneliness in siblings of individuals with asd . they also reported that siblings of individuals with asd were no more likely to have adjustment problems than comparison siblings . despite all of the sibling groups falling into the normal range for adjustment , kaminsky and dewey concluded that it is more likely for siblings of individuals with asd who live in larger families to be well - adjusted . the addition of other typically developing children in the family appeared to buffer the negative impacts of growing up with autism in the home . many siblings reported high levels of social support , a promising finding that may have buffered against the adjustment problems reported in other studies . children and adolescent 's self - concepts appear to benefit from them having a sibling with asd , when demographic risk factors are limited . the risk factors that are important for psychological well - being were not detailed in the study ; however , birth order , family size , gender , and socioeconomic status ( ses ) were considered in their analyses . it is not clear which gender or birth order constitutes a risk for psychosocial and emotional maladjustment or whether a small family constituted a risk as reported previously ; however , other studies reported below have investigated these variables . the important finding to note is that demographic factors may have a cumulative effect in producing poorer outcome . the social , behavioural , academic , and psychological adjustment of siblings was investigated in families with and without a child with asd . there was an interaction effect , however , between maternal well - being and sibling adjustment . both teacher and parent reports suggested that higher levels of daily stress and depression in the mother correlated with higher ratings of problem behaviours in siblings . ross and cuskelly . in a study that categorised 40 percent of siblings of individuals with autism in the borderline or clinical range for behavioural and emotional dysfunction , ross and cuskelly concluded that the risk of developing internalising behaviour problems is heightened in siblings of individuals with asd . an interaction between the number of stressful life events experienced by siblings of individuals with asd and their level of subthreshold asd characteristics was used as evidence to partially support a diathesis - stress model of sibling adjustment . genetic vulnerability to asd ( broad autism phenotype ) was found to increase depressive and anxious traits only in the presence of high levels of stress , such as in maternal depression and challenging behaviours in the individual with asd . the adolescent sisters in the sample reported more depression and anxiety symptoms than brothers but were comparable to community samples . it was reported that maternal depression was related to increased risk of depression in the adolescent siblings of individuals with asd . behaviour problems and social competence in siblings of individuals with high - functioning asd were found to be comparable to a control group of siblings of typically developing individuals . siblings appear to be capable of adapting to the environmental demands of high - functioning autism . siblings of individuals with asd were rated higher on measures of behavioural dysfunction and exhibiting fewer prosocial behaviours . maternal stress and behaviour problems in the child with autism were not predictive of sibling adjustment ; however , the low sample size reduces predictive power , a common difficulty in sibling research . the siblings with asd were all recruited from one school , reducing demographic variability and lowering ability to generalise these results . the study also failed to control for bap ( as does the majority of the sibling studies ) , so it is unclear whether these behaviour problems have genetic or environmental basis , particularly when the outcome measure selected is based on two important components of the triad of autism diagnosis ( challenging behaviours and social dysfunction ) . hastings provides only a brief report and recognises the limitations of his data ; however , his contribution is important , particularly as his analyses took into account important variables such as age , gender , gender matches , birth order , and living arrangements of siblings . a case study methodology revealed a number of stressful life conditions that siblings of individuals face regularly . a number of emotional reactions were identified within the seven domains of the sibling experience : empathy , sympathy , fear , anxiety ; and social isolation . the siblings revealed that : ( 1 ) they felt obligated under a sense of precocious responsibility for protecting the individual with asd and helping their parents ; ( 2 ) they felt sorry for their sibling with asd ; ( 3 ) they were exposed to frightening and abnormal behaviour ; ( 4 ) they held empathetic feelings for their sibling with asd ; ( 5 ) they hoped and wished that their family , and the individual with asd may have some relief with the individual in a group home ; ( 6 ) impulsive and uncontrolled physical violence made the siblings feel anxious and unsafe ; and ( 7 ) their relationships with friends were negatively affected . despite some positive emotions , the overwhelming feel of the sibling experience was negative . moyson and roeyers . in a recent qualitative investigation into the quality of life of siblings of children with asd ( sibqol ) , moyson and roeyers identified a number of integral domains within this concept . an important theme contributing to sibqol appeared to be the apparent invisibility of asd ( page 46 ) . a heterogeneous disorder , asd does not always present uniformly and there are no physical features distinguishing individuals with asd from their typically developing peers . it may also ensure that others do not stare at the individual with asd or express their negative opinions . it does , however , lessen the likelihood that the outside world can accurately assess the impact of a child with asd on their sibling / s , possibly lessening the likelihood that the sibling 's feelings will be validated and that they will receive appropriate services . the siblings felt it was important that the individual with asd could communicate with them ; they were disappointed that their sibling did not always want to talk , and they consistently felt misunderstood . moyson and roeyers suggested that the characteristic behaviour of an individual with asd , reported by their siblings as being sometimes bizarre , aggressive , or annoying , siblings do appear to eventually develop awareness that the individual with asd is often unable to control their behaviour , resulting in some forbearance on the part of the sibling . the qualitative findings of the study suggested that the siblings developed patience and tolerance to these challenging behaviours . longitudinal studies would provide important information about the common trajectories across the lifespan for siblings . quality of life in families of children with a disability was significantly accounted for by a number of family variables . these variables included : parental perceptions of the disability ; experiences of family - centred professional support ; perceived intensity of child behavioural problems ; and support from extended family members . this has important implications for providing parents with the skills and opportunities to apply positive meaning to disability and model this to their family . it has been reported that parents use both positive and negative appraisals of childhood developmental disability . these findings also suggest that disability services should focus on family approaches to intervention . siblings of individuals with asd have been reported to conceptualise the self in a positively enhanced manner in comparison to the general population . siblings of individuals with asd were more likely to have developed positive perspectives towards their behaviour , intelligence , academic ability , and levels of anxiety . they also held a more positive view of their overall personal characteristics than siblings of typically developing individuals . furthermore , qualitative reports revealed empathetic feelings towards siblings with autism , showing an emotional maturity also reported by gray . a diagnosis of asd often brings with it a period of family loss : feelings of loss for the individual with the disorder , parental ideas and plans for their family 's future , and the loss of the normal sibling relationship for the typically developing child . once the period of grieving is over , however , families often show great resilience in the face of adversity . bayat identified a number of coping strategies used by families facing asd that fit nicely with the foundational concepts of the resilience theoretical framework [ 25 , 26 ] . using qualitative and quantitative investigations , bayat reported evidence of family unity , mobilisation of resources , greater life appreciation , and spiritual and personal growth . qualitative data was used to identify adolescent perceptions and coping responses towards a sibling with a disability . adolescent siblings resisted expressing feelings about the individual with the disability ; however , they did express emotions related to guilt . unfortunately this study was not specific to asd , but it gives justification that qualitative studies should be pursued in the asd field as the findings may have important implications for what components of their experience siblings feel comfortable in expressing . quintero and mcintyre . maternal well - being correlated negatively with higher problem behaviours in siblings of individuals with asd ; however , there was no significant difference between siblings of individuals with asd and those with typically developing siblings on measures of adjustment ( behavioural outcome and socialisation skills ) . ses ; past attendance at a sibling group ; parent stress ; family time and routines ; family problem - solving and communication ; and family hardiness were found to be important predictors of sibling adjustment in families of children with a disability . the study consisted of children with intellectual , sensory , physical , or developmental disabilities , with 30.6 percent of participating families including a child with asd . more positive effect has been reported within sibling relationships in which the member with asd had fewer problem behaviours . the present study aimed to determine the extent of negative and positive impacts of growing up with a sibling with asd . the scarce research available on psychosocial outcome points to some promising outcomes ; the review revealed positive impacts of growing up with a sibling with asd contingent upon limited risk factors . only four studies met the more rigorous criteria of : quantitative study , focused exclusively on siblings of individuals with asd with comparisons to controls with only typically developing siblings , and targeting behavioural and emotional psychosocial adjustment rather than the quality of the sibling relationship [ 10 , 13 , 14 , 20 ] . all of these studies suggested that the sample siblings were well adjusted under optimal environmental conditions ( limited demographic risk factors , large family , no compromised maternal well - being , sibling has high - functioning asd ) . this suggests that the focal mechanism for poor outcome may not be the autism spectrum disorder but a number of other child and family variables . these studies , however , still involved a mixture of children and adolescents , or in the case of quintero and mcintyre , primary school - aged children only ; this creates a challenge in attempting to make conclusive remarks regarding adolescent sibling outcome and a complete lack of ability to comment on the adult experience . furthermore , none of the databased studies explicitly measured qol , and they all involved less than one hundred participants . the majority of studies involved children and adolescent samples , leading to confounding results and an inability to draw accurate insights and conclusions about the distinct life stages of childhood and adolescence . this is concerning due to the large changes that occur during transition through these life periods . siblings play a vital role in each other 's lives within the family system . during childhood , children rely heavily on siblings for a multitude of functions : as role models ; in the development of theory of mind ; in seeking personal identity and their niche in the family and then the world ; and in developing socialisation skills . during this period siblings may fail to understand a complex disability like asd and may not understand why their brother or sister will not play with them , why their brother or sister gets different rules , and why their parents spend more time with their brother or sister . during adolescence , teenagers begin to spend less time with siblings and family and more time with friends ; they may begin to understand the diagnosis of asd more and feel guilt for any of their previous behaviours toward their sibling . they have usually formed strong social awareness and may become embarrassed by disability in the family , potentially resulting in a conflict between loyalty to their sibling and a desire to fit in with their peers . with such different concerns , child and adolescent siblings need to be studied separately . out of the two studies that focussed exclusively on an adolescent sample , one was not autism - specific and included multiple severe disabilities . limited family interaction and uncomfortable emotions were some concerning results that arose from this qualitative study . the other study concluded with a strong genetic environment interaction as a basis for the depressive and anxious traits found in the siblings of individuals with asd , whereas the current literature review was more concerned with the psychosocial components of sibling outcome . when qol was investigated using qualitative measures , communication within the sibling relationship and the invisibility of autism emerged as important themes . the severity of communication deficits in the individual with asd may be a mediating factor in qol outcome studies and should be investigated in future research . in regard to behavioural and emotional adjustment , internalising difficulties appear to be a concern for siblings of individuals with autism but perhaps mostly in sisters and in the presence of high levels of stress or maternal depression [ 20 , 21 ] . on a more positive note , bayat reported evidence of family unity , mobilisation of resources , greater life appreciation , and spiritual and personal growth . the families of individuals with asd also appeared to attach positive meaning to the disability in many instances . opperman and alant did find , however , that siblings of individuals with a disability felt that they could not express their feelings regarding the disability . this needs to be investigated in asd - specific samples , with implications for service providers to give siblings the opportunity to express emotions such as guilt . investigations into how we can foster resilience in our most at - risk families seem important . asd may be conceptualised as a developmental disorder that is particularly disruptive to the family unit . this is supported by reports that siblings of individuals with intellectual disability without asd were emotionally and behaviourally better adjusted than those siblings of individuals with intellectual disability and comorbid asd . this supports the notion that autism contributes to environmental stress that a sibling needs to adapt to , that it produces a unique set of circumstances for the sibling to navigate , and that autism specific studies , in the midst of such mixed results , need to become more prevalent in addition to disability studies . despite reports that the large majority of siblings of individuals with asd may be well adjusted , a significant proportion of the research indicates some vulnerability for behavioural and emotional dysfunction . this suggests that siblings need to be considered when planning interventions for asd , as important units within whole family functioning . the current review helps to identify potentially at - risk siblings , such as those in small families and those with little external support . furthermore , while sisters appear to internalise the stress of growing up with a sibling with asd , brothers and younger siblings appear to be at heightened risk for externalising disorders and antisocial behaviours . in summarising current knowledge of sibling outcome in asd research and identifying the gaps in this knowledge , the current literature review provides a theoretical and empirical basis from which to develop a conceptual framework to test the important research questions that have yet to be answered . what impact does having a sibling with asd have on children , adolescents , and adults as distinct life stages ? what transitions are important in the life of the typically developing sibling and the individual with asd ? most importantly , how can we lessen the impact of autism on siblings and families ? with findings that suggest the true impacts of growing up with a sibling with a disability may be masked by samples containing too many confounding factors such as age and gender [ 14 , 21 , 30 ] , further studies are vital in presenting conclusive findings . studies using systematic sampling procedures to identify participants within a narrow age range and controlling for confounding variables such as gender and birth order need to become more common in the sibling literature . according to hodapp et al . , research into the siblings of individuals with disabilities needs to meet certain vital criteria before the sibling community can reach a consensus . they identified six themes that appeared to be creating challenges in the sibling literature , providing a framework for guiding future research in this field . methodological challenges , measurement inconsistencies , developmental and life - course perspectives , factors and predictors , cultural issues , and balanced views were revealed as potential challenges in presenting conclusive data . ( 1 ) methodological challenges included the need to consider genetic and environmental influences , the use of systematic sampling procedures that take important characteristics into account ( age , gender , and birth order ) , and the need for matched control groups . these difficulties present significant potential for confounding yet are common in the sibling literature to date . ( 3 ) a developmental perspective is yet to be arrived at due to the use of both children and adolescents within sibling samples . ( 4 ) explicit research on mediators and moderators of sibling outcome specifically for asd is scarce and has been identified by hodapp et al . as an important consideration for future researchers . ( 5 ) furthermore , there is a complete paucity of studies examining cultural factors in sibling research . ( 6 ) lastly , hodapp et al . valued the presentation of both positive and negative aspects of sibling adjustment . there is a need within the literature to further define well - being , a term that currently appears to encompass a large range of sibling outcome measures , as being distinct from adjustment and qol , two potentially separate components of the well - being construct . it has been shown that psychosocial outcome and qol are related , but distinct constructs and that psychosocial outcome may be a necessary but insufficient component of overall qol . a clearer distinction is needed of the terms used to represent the outcome measures used in future research . despite normal levels of psychosocial adjustment in adolescents following childhood traumatic brain injury ( tbi ) , this suggests that qol can still be affected in the presence of normal levels of psychosocial health . this justifies further investigation into qol in disability studies , even if sibling adjustment appears normal , which is still debatable at this point . as it is yet to be explored empirically , it is only theoretically pertinent to propose at this point that siblings of individuals with asd may be behaving well and reporting no ill effects of their home environment due to empathy for their sibling and a desire to assist their parents , despite implicitly suffering compromised satisfaction with life . the studies that reported that siblings of individuals with asd were well adjusted included mostly demographically stable samples ; hence , future research using demographically disadvantaged siblings is warranted . many family and child factors have been linked to poorer outcome in siblings of individuals with a disability . these factors need to be considered in future studies so we can begin to make conclusive remarks on sibling outcome . these mediating variables do suggest , however , that there is the potential for siblings of individuals with asd to become well adjusted and happy adults under the right conditions . these conditions need to be rigorously studied so we can optimise sibling well - being . it appears that quantitative examples of the sibling experience , in which researchers categorise siblings into clinical and nonclinical or rate their symptom level in the desired emotional and behavioural domains , show that siblings may be well adjusted if not for a small proportion of vulnerable children . qualitative data , however , reveals some disturbing emotional and cognitive challenges for siblings of individuals with asd , justifying further investigation into subjective well - being outcomes .

objective . the purpose of this review of the literature was to summarise studies regarding the psychosocial impact of growing up with a sibling with autism and to identify gaps in the related literature . methods . electronic databases were reviewed in order to critically appraise the 14 articles relevant to the topic . the search included a combination of the following key words : autism , quality of life , well - being , sibling , asd , asd sibling , family , adjust , psychological functioning . results . the majority of studies involved mixed children and adolescent samples , leading to confounding results and an inability to draw accurate conclusions about these distinct life stages . autism appears to contribute to unique environmental stressors for the typically developing sibling . when experienced in the context of additional demographic risk factors , these stressors can result in difficulties adjusting to the demands of a special - needs child . despite some vulnerability to behavioural and emotional dysfunction in at - risk children , siblings have the potential to not only adjust but to thrive in the face of disability adversity . conclusion . growing up with a sibling with autism appears to manifest in both positive and negative outcomes for siblings , depending upon important demographical , family , and individual variables .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusions

the sibling bond is the marathon runner of human relationships ; it is the longest lasting relationship , enduring from the birth of the youngest sibling to the death of the first to go . it is therefore reasonable to assume the importance of this relationship across the lifespan . it has been established that the younger siblings of individuals with autism spectrum disorder ( asd ) are at heightened risk of developmental problems in comparison to the general population . some of these developmental difficulties include : social and cognitive deficiencies , and neurocognitive and behavioural delays , specifically executive function and repetitive behaviours . the above examples of sibling research are commonly used to provide evidence for the genetic basis of asd with elevated levels of autistic traits in siblings representing potential markers of a broader autism phenotype ( bap ; ) . the nurture side of the debate how are these siblings and families impacted by the disorder in regard to their quality of life ( qol ) and psychosocial outcomes ? , head banging , skin picking , hair pulling ) , lack of emotional and social reciprocity , and inflexible adherence to routines or rituals . in addition to common comorbidity with other medical , psychiatric , and developmental disorders , these asd characteristics can be difficult for families to manage . epilepsy , allergies , sleep disorders , conduct disorder , attention deficit / hyperactivity disorder ( adhd ) , gastro - intestinal disorders , anxiety disorders , intellectual disability and language disorders often cooccur with a diagnosis of asd . furthermore , asd requires substantial financial investment in therapy , medical treatment , and early intervention education ; increased vigilance of the individual with autism ; micromanagement of the family environment to ensure minimal sensory discomfort and maintenance of a highly predictable routine ; often limited communication with the family member with autism ; and many other family disturbances . it is empirically intuitive to assume that quality of life ( qol ) is impacted in these families . this has yet to be supported by research , with a lack of studies focusing on qol outcomes in asd families . a review of the sibling literature reveals both positive and negative effects of growing up with an individual with a disability . siblings of children with autism have been shown to do more poorly on a number of outcome measures in comparison to siblings of other developmental disabilities and those with only typically developing siblings . in contrast , other researchers have found no difference in levels of adjustment between siblings of individuals with asd and those with only typically developing siblings or those with siblings with other developmental diagnoses . even more promising , the positive impacts of growing up with a sibling with asd are becoming known . siblings of individuals with autism have been reported to have less conflict in the sibling relationship , family resilience , and increased self - perceived competence [ 6 , 13 , 14 ] . they have also been shown to have a more positive opinion of the sibling relationship , positive psychosocial and emotional development conditional upon limited demographic risk factors , feelings of empathy for their sibling , and increased maturity . the autism sibling literature is yet to reach consensus ; however , macks and reeve [ 14 , page 1065 ] succinctly conclude that having a sibling with autism may not be a risk factor in and of itself , and children with autism may even have a positive influence on the life of the nondisabled sibling . however , when multiple demographic risk factors are present , it becomes more difficult for the nondisabled sibling to deal with the child with autism , both emotionally and psychologically . macks and reeve consider the contribution this potentially makes to the inconsistency currently plaguing sibling research . if those studies reporting positive influences included samples of demographically stable families , while those samples that exhibited negative impacts consisted of participants experiencing a high number of demographic risk factors , the true effects of growing up with a sibling with autism would be masked , justifying continued exploration of sibling outcome . in order to investigate this masking effect at surface level , the author conducted closer inspection of the studies listed above that showed no difference between siblings of individuals with asd and comparison siblings . a large proportion of the sample in the kaminsky and dewey study reported receiving high levels of social support . the majority of bayat 's resilient families used in his 2007 study were intact families with average incomes of 81,000 usd per year . although the study included families with low income ( 23% ) , over 60 percent were in the high - income category . had these samples included more demographically disadvantaged siblings , their outcome may have been poorer than comparison siblings . the low level of demographic risk may have contributed to the healthy levels of adjustment reported in these studies , a consideration that further supports macks and reeve 's proposal . recent literature reviews report mixed results and emphasise the large gaps in our current knowledge of sibling outcome , due in large part to inconsistency in methodology and a lack of rigorous sampling procedures [ 18 , 19 ] . the purpose of this literature review was to summarise studies of the psychosocial impacts of growing up with a sibling with asd , identify gaps in the literature , and propose future directions for sibling research in the autism spectrum field . although it is recognised that genetic factors play an integral part in any research regarding asd , the current paper focuses on psychosocial rather than biological components of sibling outcome . in the context of the multitude of challenging behaviours exhibited by an individual with autism , it is intuitively appealing to predict that the well - being of siblings in these homes may be compromised ; this literature review attempted to summarise this impact . electronic databases including pubmed , psycinfo , google scholar , psycarticles , medline , scopus , wiley online library , and proquest psychology journals were reviewed for studies published in peer - reviewed journals within the last decade . fourteen articles were accessed using key words to identify research within the sibling well - being field and further filtered in order to critically appraise the most relevant studies . due to the small number of robust and empirically sound studies focussing specifically on siblings of children with autism , a number of studies focussing on the functioning of the whole family in addition to studies targeting a range of developmental disabilities were considered . within the sibling field a number of pertinent concepts were identified ; the current article will discuss the outcome of siblings of children with autism according to psychological health , overall quality of life , emotional intelligence , family coping , and child and family factors . the original criteria used to filter relevant articles allowed for inclusion of databased articles that were asd - specific and included a control comparison group . however , these criteria needed to be relaxed as only 4 studies met this stringent category . siblings of individuals with autism appear to be socially and emotional well adjusted according to a study that compared them to siblings of individuals with developmental language disorders ( specific learning disability ) and intellectual disability . despite the absence of a typically developing sibling control comparison , these findings are still promising due to the low number of the siblings of individuals with asd that fell into the clinical diagnostic range ( 13.4% ) on a number of behavioural adjustment measures . with the advantage of including comparison groups of siblings of individuals with another disability ( down syndrome ) and those with only typically developing siblings , kaminsky and dewey reported low levels of loneliness in siblings of individuals with asd . despite all of the sibling groups falling into the normal range for adjustment , kaminsky and dewey concluded that it is more likely for siblings of individuals with asd who live in larger families to be well - adjusted . the addition of other typically developing children in the family appeared to buffer the negative impacts of growing up with autism in the home . children and adolescent 's self - concepts appear to benefit from them having a sibling with asd , when demographic risk factors are limited . the risk factors that are important for psychological well - being were not detailed in the study ; however , birth order , family size , gender , and socioeconomic status ( ses ) were considered in their analyses . it is not clear which gender or birth order constitutes a risk for psychosocial and emotional maladjustment or whether a small family constituted a risk as reported previously ; however , other studies reported below have investigated these variables . there was an interaction effect , however , between maternal well - being and sibling adjustment . both teacher and parent reports suggested that higher levels of daily stress and depression in the mother correlated with higher ratings of problem behaviours in siblings . in a study that categorised 40 percent of siblings of individuals with autism in the borderline or clinical range for behavioural and emotional dysfunction , ross and cuskelly concluded that the risk of developing internalising behaviour problems is heightened in siblings of individuals with asd . genetic vulnerability to asd ( broad autism phenotype ) was found to increase depressive and anxious traits only in the presence of high levels of stress , such as in maternal depression and challenging behaviours in the individual with asd . the adolescent sisters in the sample reported more depression and anxiety symptoms than brothers but were comparable to community samples . it was reported that maternal depression was related to increased risk of depression in the adolescent siblings of individuals with asd . siblings appear to be capable of adapting to the environmental demands of high - functioning autism . maternal stress and behaviour problems in the child with autism were not predictive of sibling adjustment ; however , the low sample size reduces predictive power , a common difficulty in sibling research . the study also failed to control for bap ( as does the majority of the sibling studies ) , so it is unclear whether these behaviour problems have genetic or environmental basis , particularly when the outcome measure selected is based on two important components of the triad of autism diagnosis ( challenging behaviours and social dysfunction ) . hastings provides only a brief report and recognises the limitations of his data ; however , his contribution is important , particularly as his analyses took into account important variables such as age , gender , gender matches , birth order , and living arrangements of siblings . a number of emotional reactions were identified within the seven domains of the sibling experience : empathy , sympathy , fear , anxiety ; and social isolation . the siblings revealed that : ( 1 ) they felt obligated under a sense of precocious responsibility for protecting the individual with asd and helping their parents ; ( 2 ) they felt sorry for their sibling with asd ; ( 3 ) they were exposed to frightening and abnormal behaviour ; ( 4 ) they held empathetic feelings for their sibling with asd ; ( 5 ) they hoped and wished that their family , and the individual with asd may have some relief with the individual in a group home ; ( 6 ) impulsive and uncontrolled physical violence made the siblings feel anxious and unsafe ; and ( 7 ) their relationships with friends were negatively affected . despite some positive emotions , the overwhelming feel of the sibling experience was negative . in a recent qualitative investigation into the quality of life of siblings of children with asd ( sibqol ) , moyson and roeyers identified a number of integral domains within this concept . a heterogeneous disorder , asd does not always present uniformly and there are no physical features distinguishing individuals with asd from their typically developing peers . it does , however , lessen the likelihood that the outside world can accurately assess the impact of a child with asd on their sibling / s , possibly lessening the likelihood that the sibling 's feelings will be validated and that they will receive appropriate services . the siblings felt it was important that the individual with asd could communicate with them ; they were disappointed that their sibling did not always want to talk , and they consistently felt misunderstood . moyson and roeyers suggested that the characteristic behaviour of an individual with asd , reported by their siblings as being sometimes bizarre , aggressive , or annoying , siblings do appear to eventually develop awareness that the individual with asd is often unable to control their behaviour , resulting in some forbearance on the part of the sibling . quality of life in families of children with a disability was significantly accounted for by a number of family variables . these variables included : parental perceptions of the disability ; experiences of family - centred professional support ; perceived intensity of child behavioural problems ; and support from extended family members . it has been reported that parents use both positive and negative appraisals of childhood developmental disability . siblings of individuals with asd have been reported to conceptualise the self in a positively enhanced manner in comparison to the general population . siblings of individuals with asd were more likely to have developed positive perspectives towards their behaviour , intelligence , academic ability , and levels of anxiety . furthermore , qualitative reports revealed empathetic feelings towards siblings with autism , showing an emotional maturity also reported by gray . a diagnosis of asd often brings with it a period of family loss : feelings of loss for the individual with the disorder , parental ideas and plans for their family 's future , and the loss of the normal sibling relationship for the typically developing child . once the period of grieving is over , however , families often show great resilience in the face of adversity . bayat identified a number of coping strategies used by families facing asd that fit nicely with the foundational concepts of the resilience theoretical framework [ 25 , 26 ] . using qualitative and quantitative investigations , bayat reported evidence of family unity , mobilisation of resources , greater life appreciation , and spiritual and personal growth . qualitative data was used to identify adolescent perceptions and coping responses towards a sibling with a disability . unfortunately this study was not specific to asd , but it gives justification that qualitative studies should be pursued in the asd field as the findings may have important implications for what components of their experience siblings feel comfortable in expressing . maternal well - being correlated negatively with higher problem behaviours in siblings of individuals with asd ; however , there was no significant difference between siblings of individuals with asd and those with typically developing siblings on measures of adjustment ( behavioural outcome and socialisation skills ) . ses ; past attendance at a sibling group ; parent stress ; family time and routines ; family problem - solving and communication ; and family hardiness were found to be important predictors of sibling adjustment in families of children with a disability . the present study aimed to determine the extent of negative and positive impacts of growing up with a sibling with asd . the scarce research available on psychosocial outcome points to some promising outcomes ; the review revealed positive impacts of growing up with a sibling with asd contingent upon limited risk factors . only four studies met the more rigorous criteria of : quantitative study , focused exclusively on siblings of individuals with asd with comparisons to controls with only typically developing siblings , and targeting behavioural and emotional psychosocial adjustment rather than the quality of the sibling relationship [ 10 , 13 , 14 , 20 ] . all of these studies suggested that the sample siblings were well adjusted under optimal environmental conditions ( limited demographic risk factors , large family , no compromised maternal well - being , sibling has high - functioning asd ) . these studies , however , still involved a mixture of children and adolescents , or in the case of quintero and mcintyre , primary school - aged children only ; this creates a challenge in attempting to make conclusive remarks regarding adolescent sibling outcome and a complete lack of ability to comment on the adult experience . furthermore , none of the databased studies explicitly measured qol , and they all involved less than one hundred participants . the majority of studies involved children and adolescent samples , leading to confounding results and an inability to draw accurate insights and conclusions about the distinct life stages of childhood and adolescence . this is concerning due to the large changes that occur during transition through these life periods . during childhood , children rely heavily on siblings for a multitude of functions : as role models ; in the development of theory of mind ; in seeking personal identity and their niche in the family and then the world ; and in developing socialisation skills . they have usually formed strong social awareness and may become embarrassed by disability in the family , potentially resulting in a conflict between loyalty to their sibling and a desire to fit in with their peers . out of the two studies that focussed exclusively on an adolescent sample , one was not autism - specific and included multiple severe disabilities . the other study concluded with a strong genetic environment interaction as a basis for the depressive and anxious traits found in the siblings of individuals with asd , whereas the current literature review was more concerned with the psychosocial components of sibling outcome . in regard to behavioural and emotional adjustment , internalising difficulties appear to be a concern for siblings of individuals with autism but perhaps mostly in sisters and in the presence of high levels of stress or maternal depression [ 20 , 21 ] . on a more positive note , bayat reported evidence of family unity , mobilisation of resources , greater life appreciation , and spiritual and personal growth . the families of individuals with asd also appeared to attach positive meaning to the disability in many instances . opperman and alant did find , however , that siblings of individuals with a disability felt that they could not express their feelings regarding the disability . this needs to be investigated in asd - specific samples , with implications for service providers to give siblings the opportunity to express emotions such as guilt . investigations into how we can foster resilience in our most at - risk families seem important . asd may be conceptualised as a developmental disorder that is particularly disruptive to the family unit . this supports the notion that autism contributes to environmental stress that a sibling needs to adapt to , that it produces a unique set of circumstances for the sibling to navigate , and that autism specific studies , in the midst of such mixed results , need to become more prevalent in addition to disability studies . despite reports that the large majority of siblings of individuals with asd may be well adjusted , a significant proportion of the research indicates some vulnerability for behavioural and emotional dysfunction . the current review helps to identify potentially at - risk siblings , such as those in small families and those with little external support . furthermore , while sisters appear to internalise the stress of growing up with a sibling with asd , brothers and younger siblings appear to be at heightened risk for externalising disorders and antisocial behaviours . what impact does having a sibling with asd have on children , adolescents , and adults as distinct life stages ? what transitions are important in the life of the typically developing sibling and the individual with asd ? with findings that suggest the true impacts of growing up with a sibling with a disability may be masked by samples containing too many confounding factors such as age and gender [ 14 , 21 , 30 ] , further studies are vital in presenting conclusive findings . studies using systematic sampling procedures to identify participants within a narrow age range and controlling for confounding variables such as gender and birth order need to become more common in the sibling literature . methodological challenges , measurement inconsistencies , developmental and life - course perspectives , factors and predictors , cultural issues , and balanced views were revealed as potential challenges in presenting conclusive data . these difficulties present significant potential for confounding yet are common in the sibling literature to date . ( 3 ) a developmental perspective is yet to be arrived at due to the use of both children and adolescents within sibling samples . valued the presentation of both positive and negative aspects of sibling adjustment . there is a need within the literature to further define well - being , a term that currently appears to encompass a large range of sibling outcome measures , as being distinct from adjustment and qol , two potentially separate components of the well - being construct . a clearer distinction is needed of the terms used to represent the outcome measures used in future research . despite normal levels of psychosocial adjustment in adolescents following childhood traumatic brain injury ( tbi ) , this suggests that qol can still be affected in the presence of normal levels of psychosocial health . these mediating variables do suggest , however , that there is the potential for siblings of individuals with asd to become well adjusted and happy adults under the right conditions . these conditions need to be rigorously studied so we can optimise sibling well - being . it appears that quantitative examples of the sibling experience , in which researchers categorise siblings into clinical and nonclinical or rate their symptom level in the desired emotional and behavioural domains , show that siblings may be well adjusted if not for a small proportion of vulnerable children . qualitative data , however , reveals some disturbing emotional and cognitive challenges for siblings of individuals with asd , justifying further investigation into subjective well - being outcomes .

obesity has been a major world - wide public health challenge . during the past 10 years , there has been a dramatic increase in the prevalence of obesity in both developed and developing countries . it is imperative to identify effective and simple life - style intervention strategies to reduce the risk of obesity . adipose tissue as an endocrine organ producing adipokines , are thought to be key regulators of inflammation . low grade chronic inflammation is a characteristic of obese state and has an important role in pathogenesis and progression of chronic disease such as metabolic syndrome , diabetes and cardiovascular disorders . it is becoming increasingly evident that over nutrition , physical inactivity and aging would result in hypersecretion inflammatory mediators and eventually lead to obesity - induced disorders . . for centuries rice has been traditional staple food in asian countries and more than 70% of rice consumed is in the form of white rice ( wr ) . several cohort studies indicate that higher wr intake is associated with elevated risk of diabetes . in a prospective study conducted in japan recent studies shown that intake of whole grain as a component of healthy eating pattern has been associated with lower risk of chronic disease . brown rice ( br ) is the unrefined whole grain contains various and important nutrient in bran layer that is located between white center and outer bran . removing bran layer results in losing potential health benefits of rice . due to the milling process and different degree of processing , compared with wr , br has a greater amount of dietary fiber , magnesium , vitamin b , -aminobutyric acid ( gaba ) , -oryzanol ( -orz ) , phytoestrogens , lignin and essential fatty acid . major previous studies had focused on the effects of br on diabetic risk factors which show br is associated whit a lower risk of diabetes . in br , gaba that is known as a neurotransmitter has the ability to regulate the blood pressure and increase insulin secretion . furthermore , -orz as a potent anti - oxidant by antihypercholestrolemic effects and vitamin e may prevent the progression of cardiovascular complication and may confer protection against metabolic disorders . few studies that had considered the influence of substituting wr with br on metabolic risk factors show no significant result . previously conflicting results regarding the effect of br on some inflammation mediators such as plasminogen activator inhibitor type-1 ( pai-1 ) and tumor necrosis factor - alpha ( tnf- ) in rats were reported . in the 14 week clinical trial using cross - over design , we aim to determine the effects of br consumption on high - sensitivity c - reactive protein ( hs - crp ) as an inflammatory marker and some cardiovascular risk factors among non - menopausal overweight or obese female adults . participants were eligible if they have a body mass index ( bmi ) more than 25 kg / m and non - menopausal , non - pregnant , non - lactating and non - smoking women in the range of 18 - 50 years old were included in this study . individual with a history of chronic disease , consuming medicine and flowing special regimen were excluded . the sample size calculation was determined using the serum hs - crp level as a key dependent variable in the standard formula suggested for cross - over trials : n = [ ( z 1 /2 + z 1 ) . s]/2 ( 1 2 ) ; where z 1 /2 was 1.96 , z 1 was 0.84 , s was 0.01 , 1 2 was 0 and was 0.04 . in the present study , a total of 40 eligible volunteers enrolled into the present study and allocated to group 1 ( treatment from br to wr ; n = 20 ) and group 2 ( treatment from wr to br ; n = 20 ) . all of them were instructed to use br in one period of trial and wr in another period . each patient received two diets . in group 2 , 2 subjects in the first intervention period and 3 subjects in the second intervention period were eliminated from this study , due to : busy schedule : n = 3 , intervention unrelated surgery problem : n = 1 and pregnancy : n = 1 . finally this study was conducted with 15 subjects in group 2 and 20 subjects in grup1 . signing the written consent the study protocol was approved by food security research center isfahan university of medical science and nutrition department . patients flow diagram in the present study in the run in period subjects were allowed to continue performing daily living and normal activities . after 2 week run in , participants were randomly allocated to two experimental periods of wr or br , each one for 6 weeks in a crossover design . first intervention period was 0 - 6 week , second intervention period was 8 - 14 week and 2 week was set for washout phase from 6 to 8 of the study [ figure 2 ] . in the present work participant were not blinded and all of them were on a weight reducing diet in duration of the study . two kinds of rice used in this study were from the same iranian rice variety ( tarom ) . raw rice packages were given to each subject and all of them were instructed to eat 150 g of cooked rice daily . participants were allowed to continue their usual physical activity and prepared their own meal according to prescribed diet . study cross - over design to observe the effect of white rice and brown rice on inflammatory marker and cardiovascular risk factors among overweight or obese female adults two diets were prescribed for each patient : ( 1 ) diet with wr ( 2 ) diet with br . the macronutrient composition of both diet were : 50 - 60% carbohydrate , 15 - 20% protein and < 30% fat respectively . for assessing subject adherence , dietary intake based on 3-day food records , were obtained and analysis at baseline and end of each intervention period . as derived from diet prescription and food records , no significant differences were obtained between consumed amount and prescribed amount from each of the five food group . in the both of intervention periods , we provided weight reducing diet with 200 - 500 kcal / day deficit for each female based on the bmi range . in br period 10 spoon ( 150 g ) br was replaced instead of 10 spoons ( 150 g ) wr . individual diet was prescribed for each participant and provided an exchange list and educated them how to record their food intake and method of using exchange list . composition of white rice and brown rice after an overnight fasting , blood samples were collected from patients between 9 and 11 am . after centrifuging at 4c and 500 g for 10 min , tubes were frozen at 80c until analyzing . serum glucose , total cholesterol ( tc ) , triacylglycerol ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) were measured by photometric enzyme assay ( pars azmoon , iran ) . hs - crp was determined by latex - immunoturbidemetric assay ( bionic , iran ) . both intra and inter assay coefficients of variation for all of measurements were < 5% . body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm respectively and bmi was calculated . waist circumference was obtained at the midpoint between the lowest rib and the iliac crest . when participants were in comfortable seat position , after 10 min rest , blood pressure was measured on the right arm using electronic blood monitor . except height , other physical characteristics and blood pressure were measured 4 times , in study week 0 , 6 , 8 , 14 respectively . different value between first and last intervention in the each period was calculated and then difference between them was determined as mean difference for both group 1 and group 2 . we used paired t - test for comparing means difference to detect any statistical significant level as main effect of br and wr diets . we also check the period and carry - over effects using independent t - test . data were analyzed using spss ( version 15 ) and a 2-sided p < 0.05 was considered to be significant . participants were eligible if they have a body mass index ( bmi ) more than 25 kg / m and non - menopausal , non - pregnant , non - lactating and non - smoking women in the range of 18 - 50 years old were included in this study . individual with a history of chronic disease , consuming medicine and flowing special regimen were excluded . the sample size calculation was determined using the serum hs - crp level as a key dependent variable in the standard formula suggested for cross - over trials : n = [ ( z 1 /2 + z 1 ) . s]/2 ( 1 2 ) ; where z 1 /2 was 1.96 , z 1 was 0.84 , s was 0.01 , 1 2 was 0 and was 0.04 . in the present study , a total of 40 eligible volunteers enrolled into the present study and allocated to group 1 ( treatment from br to wr ; n = 20 ) and group 2 ( treatment from wr to br ; n = 20 ) . all of them were instructed to use br in one period of trial and wr in another period . each patient received two diets . in group 2 , 2 subjects in the first intervention period and 3 subjects in the second intervention period were eliminated from this study , due to : busy schedule : n = 3 , intervention unrelated surgery problem : n = 1 and pregnancy : n = 1 . finally this study was conducted with 15 subjects in group 2 and 20 subjects in grup1 . signing the written consent the study protocol was approved by food security research center isfahan university of medical science and nutrition department . in the run in period subjects were allowed to continue performing daily living and normal activities . after 2 week run in , participants were randomly allocated to two experimental periods of wr or br , each one for 6 weeks in a crossover design . first intervention period was 0 - 6 week , second intervention period was 8 - 14 week and 2 week was set for washout phase from 6 to 8 of the study [ figure 2 ] . in the present work participant were not blinded and all of them were on a weight reducing diet in duration of the study . two kinds of rice used in this study were from the same iranian rice variety ( tarom ) . raw rice packages were given to each subject and all of them were instructed to eat 150 g of cooked rice daily . participants were allowed to continue their usual physical activity and prepared their own meal according to prescribed diet . study cross - over design to observe the effect of white rice and brown rice on inflammatory marker and cardiovascular risk factors among overweight or obese female adults two diets were prescribed for each patient : ( 1 ) diet with wr ( 2 ) diet with br . the macronutrient composition of both diet were : 50 - 60% carbohydrate , 15 - 20% protein and < 30% fat respectively . for assessing subject adherence , dietary intake based on 3-day food records , were obtained and analysis at baseline and end of each intervention period . as derived from diet prescription and food records , no significant differences were obtained between consumed amount and prescribed amount from each of the five food group . in the both of intervention periods , we provided weight reducing diet with 200 - 500 kcal / day deficit for each female based on the bmi range . in br period 10 spoon ( 150 g ) br individual diet was prescribed for each participant and provided an exchange list and educated them how to record their food intake and method of using exchange list . after an overnight fasting , blood samples were collected from patients between 9 and 11 am . after centrifuging at 4c and 500 g for 10 min , tubes were frozen at 80c until analyzing . serum glucose , total cholesterol ( tc ) , triacylglycerol ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) were measured by photometric enzyme assay ( pars azmoon , iran ) . hs - crp was determined by latex - immunoturbidemetric assay ( bionic , iran ) . both intra and inter assay coefficients of variation for all of measurements were < 5% . body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm respectively and bmi was calculated . waist circumference was obtained at the midpoint between the lowest rib and the iliac crest . when participants were in comfortable seat position , after 10 min rest , blood pressure was measured on the right arm using electronic blood monitor . except height , other physical characteristics and blood pressure were measured 4 times , in study week 0 , 6 , 8 , 14 respectively . different value between first and last intervention in the each period was calculated and then difference between them was determined as mean difference for both group 1 and group 2 . we used paired t - test for comparing means difference to detect any statistical significant level as main effect of br and wr diets . we also check the period and carry - over effects using independent t - test . data were analyzed using spss ( version 15 ) and a 2-sided p < 0.05 was considered to be significant . after randomization , the two groups were balanced but during the study , 5 person withdrew duo to busy schedule ( n = 3 ) , intervention unrelated surgery problem ( n = 1 ) and pregnancy ( n = 1 ) . thus , a total of 40 participants , 35 non - menopausal over weight and obese female completed the study . the period and carry - over effects were checked and none of them were significant in this study . the mean age of the patient was 32.6 6 years and the mean of bmi was 29.38 4 . except dietary magnesium content which was significantly ( p < 0.001 ) higher during br consumption , no significant differences were found between wr and br diets regarding each of the five food groups , polyunsaturated fatty acid ( pufa ) , monounsaturated fatty acid ( mufa ) , saturated fatty acids and majority of micronutrients as derived from the food record . energy and nutrient intake in the first and second intervention period was shown in table 2 . they were instructed to avoid change in exercise habits , hence their physical activity was similar and it was checked by researchers . energy and nutrient intakes in the first and second intervention periods in groups 1 and 2 br diet in comparison with wr could decrease physical characteristics such as : weight , waist and hip circumference and bmi significantly . mean difference changes were 1.2 kg ( 95% confidence interval [ ci ] = 0.60 - 1.96 , p = 0.001 ) , 2.38 cm ( 95% ci = 0.51 - 4.25 , p = 0.014 ) , 2.98 cm ( 95% ci = 1.55 - 4.42 , p 0.001 ) and 0.52 kg / m2 ( 95% ci = 0.26 - 0.78 , p 0.001 ) respectively . in the br diet , inflammatory marker ( hs - crp ) level decreased significantly in comparison with wr diet . mean difference changes was 0.98 mg / l ( 95% ci = 0.23 - 1.73 , p = 0.012 ) . no significant changes were appeared regarding serum lipids concentration such as tg , tc , hdl , ldl and fasting blood glucose ( fbg ) . diastole blood pressure level was significantly ( p = 0.032 ) decreased after consuming br diet compared with wr diet . physical characteristics and biochemical parameters in first and second intervention phase in group 1 and 2 , as shown in table 3 and table 4 . physical characteristics in the first and second intervention period in groups 1 and 2 biochemical parameters in the first and second intervention periods in groups 1 and 2 this is due to the high concentration of this nutrient in bran layer of br , which has potential benefit for the cardiovascular system . the present results show that br diet in comparison with wr diet could significantly reduce weight , waist and hip circumference , bmi and hs - crp . br as unrefined whole grain contain protective factors for diabetics and cardiovascular disease , such as vitamins , magnesium and other minerals , fiber and essential fatty acids . by milling and polishing br and convert it to wr , 50% fiber 84% magnesium and 69% of total mufa and pufa have been eliminated respectively . despite these benefits , previous studies indicated no significant changes on body weight following br consumption but the size of adipocytes was reduced by feeding with pre germinated br in diabetic rats . in this study diet with br could reduce waist circumference , a strong risk factor for cardiovascular disease and enhanced weight loss . there are many reports which show the ability of fiber to slow down the process of absorption of nutrients by modifying the metabolic response to intake , its ability to lowering fasting insulin level , increase the excretion of biliary acid and the metabolic effects of the short - chain fatty acids ( scfa ) produced in the bacterial fermentation of fiber . recent human studies reported relationships between the composition of the intestinal microbiota and obesity . in a recent study , br could increase large bowel scfa and have a beneficially effect on the gut microbial composition in humans . hence anti - obesity effects of br may be related to the profile and activity of the intestinal microbiota . leptin is released from adipocytes and controls food intake and energy expenditure by brain mechanisms . gaba , an important component of rice bran , has the ability to prevent obesity by linking to leptin as the co - transmitter . finding from an animal study indicated that br has the anti - obesity effect through down regulation of lipogenic gene . the organelle responsible for protein synthesis , folding and trafficking is endoplasmic reticulum ( er ) . creating a toxic condition due to accumulation of newly synthesized unfolded proteins defined as er stress , which induces apoptotic cell death . hypothalamic er stress is linked to leptin resistance , which leads to dysregulation of appetite and energy expenditure in obese mice . -orz ( ferulate ester of triterpen alcohols ) in bran layer acts as a chemical chaperone ; so it can decrease hypothalamic er stress , ameliorated hypothalamic leptin resistance and attenuate the preference for dietary fat . overall this evidence highlights that br may have potential for the enhanced weight loss in humans . there are conflicting results regarding the effect of br on serum lipid profiles and fbg . in a recent study substituting wr with br for 16 weeks was not associated with improvement in fasting serum glucose , insulin , serum lipids among people with diabetes . two recent short - term interventions indicated no significant benefits of substituting whole grains for refined grains on fasting glucose , insulin and blood lipids . but in the study with cross - over design diabetic patients were instructed to consume 180 g of rice 3 times a day for 6 week and blood glucose management , lipid related markers were improved on br diet . these results were similar to the results of another study in which blood glucose and insulin reaction were lower for br when compared with wr . higher content of dietary fiber in br ( 2 g/100 g ) than in wr ( 0.4 g/100 g ) might be responsible for this effect . fiber in bran layer raps -amylase , the enzyme to digest starch and substrates ( saccharides ) in the intestine , ultimately suppresses increasing in postprandial blood glucose levels . gaba that is richer in br than wr ( contents of gaba in raw br and wr were 7 mg/100 and 3 mg/100 respectively ) , stimulate the pancreatic beta cell and insulin secretion from pancreas and injection gaba agonist to streptozotocin - induced diabetic rats ameliorated the elevation of blood glucose concentration and increased their insulin levels . br contains -orz that are effective for improving hypercholesterolemia by suppressing cholesterol absorption from intestine , in both human and animal 's models . br diet can also suppress hypercholesterolemia via enhancing fecal bile acids secretion without affecting on the cholesterol synthesis of host liver . comparing with wr , br contains more gaba with hypocholestrolemic effect , although the exact mechanisms are not yet clear . however in our study br diet in comparison with wr diet , did not have any significant effects on serum lipid profiles and fbg . this might be related to the low amount of br intake in the current study . in the present study , we observed that comparing with wr diet ; br diet could reduce diastole blood pressure . this result was similar to the findings of the study which showed the br intervention had a beneficial effect on diastole pressure among diabetic person . furthermore in another study consumption of diet with br / whole wheat for 5 week could reduce blood pressure significantly in hypercholestrolemic patients . several mechanisms have been proposed to be responsible for the reduction in blood pressure , which occurs with increased fiber intake including increased water and electrolyte excretion with insoluble fiber intake . past studies have shown that gaba is not only a neurotransmitter but also it can decrease blood pressure . the major bioactive peptide of the renin - angiotensin system , angiotensin ii , is an important growth factor causing hypertrophy and hyperplasia of vascular smooth muscle cells that is believed to be pivotal for progression of hypertension . extract from the bran layer of br has a protective effect toward hypertension by interfering with signal transduction induced by ang ii . few studies are available about the effects of whole grain or br on inflammatory risk markers . hs - crp is a strong inflammatory marker for predicting future cardiovascular events . compared with wr , br ameliorated the increase in both plasma tnf- and pai-1 and decrease in adiponectin in diabetic rats this result was similar to another study which showed pai-1 significantly decreased in diabetic rats by taking pgbr for 7 week . two previous studies did not find significant benefits of substituting whole grains for refined grains on interleukin-6 and hs - crp . in a study consumption of 3 portions / d whole - grain foods for 12 week this result was consistent with results from a 14-week pilot trial using a crossover design in 30 participants . in our short - term trial br diet in comparison with wr diet , significantly reduced level of hs - crp . gene expression of adhesion molecules in vascular endothelium including intercellular adhesion molecule , vascular cell adhesion molecule and e - selection regulate by nf-b . a recent study showed that -orz as well as it 's component does - dependently reduced adhesion - molecules expression through nf-b inhibition in vascular endothelium . -orz probably can have meditative role on crp expression , at least , partly through the modulation of nf-b dependent path way , but the exact mechanisms are not known yet . however , a few human studies are available about the effect of br on inflammatory and cardiovascular risk factors . furthermore , we assessed the effect of br in cross - over design as the strong method for conducting this trial . one important difference between our study and the other research was the amount of br intake . the subjects were instructed to consume only 150 g br accompany with weigh loss diet and some significantly effects were seen for mentioned variables . in the present study we just recommended food item but br and wr were provided for participants . we analyzed patient food records and their diets controlled exactly . in non - menopausal female , since lipid profiles variation may be affected by change in blood estrogen level , patient biochemical measurement were assessed based on menstrual date and all of them were in the same hormone phase . this study was conducted on healthy adults without any specific disorder ; hence , our trial should have good external validity . br replacement in the diet as a staple food can be useful to improve weight loss , waist and hip circumference , bmi , diastole pressure and hs - crp level significantly , among over weight and obese female adults . our findings show that in overweight or obese female adults br diet in comparison with wr diet resulted in decreased diastole blood pressure and inflammatory marker ( hs - crp ) . furthermore , br replacement in the diet can be useful to improve weight loss and visceral obesity significantly . replacement of white rice by brown rice did not have any significant effects on serum lipid profiles and fbg .

background : brown rice ( br ) is unpolished rice with various beneficial compounds such as vitamins , magnesium and other minerals , dietary fiber , essential fatty acids , -oryzanol and -aminobutyric acid . in the present study , we compared the effects of white rice ( wr ) and br on inflammatory marker high - sensitivity c - reactive protein ( hs - crp ) and cardiovascular risk factors among non - menopausal overweight or obese female.methods:in a randomized cross - over clinical trial , 40 overweight or obese ( body mass index ( bmi ) > 25 ) women were randomly allocated to group 1 ( n = 20 ) : treatment with br diet and group 2 ( n = 20 ) : treatment with wr diet for 6 weeks ( first intervention period ) . two participants in group 2 dropped out during this period . after a 2-week washout period , individuals were switched to the alternate diet for an additional 6 weeks ( second intervention period ) and three subjects in group 2 did not follow this period and eliminated , finally this study was completed with 35 subjects ( group 1 = 20 and group 2 = 15 ) . each one was instructed to consume 150 g cooked wr or br daily in each intervention period . cardiovascular risk factors including bmi , waist and hip circumference , blood pressure , serum lipid profiles , fasting blood glucose ( fbg ) and hs - crp as an inflammatory marker , were measured 4 times ( in study week 0 , 6 , 8 , 14).results : br diet in comparison with wr diet could significantly reduce weight , waist and hip circumference , bmi , diastole blood pressure and hs - crp . no significant differences between the two diets were found regarding lipid profiles and fbg.conclusions:the present results suggest that br replacement in the diet may be useful to decrease inflammatory marker level and several cardiovascular risk factors among non - menopausal overweight or obese female .

INTRODUCTION METHODS Participants Study design Diets Variables assessment Statistical analysis RESULTS DISCUSSION CONCLUSIONS

obesity has been a major world - wide public health challenge . during the past 10 years , there has been a dramatic increase in the prevalence of obesity in both developed and developing countries . adipose tissue as an endocrine organ producing adipokines , are thought to be key regulators of inflammation . low grade chronic inflammation is a characteristic of obese state and has an important role in pathogenesis and progression of chronic disease such as metabolic syndrome , diabetes and cardiovascular disorders . for centuries rice has been traditional staple food in asian countries and more than 70% of rice consumed is in the form of white rice ( wr ) . brown rice ( br ) is the unrefined whole grain contains various and important nutrient in bran layer that is located between white center and outer bran . due to the milling process and different degree of processing , compared with wr , br has a greater amount of dietary fiber , magnesium , vitamin b , -aminobutyric acid ( gaba ) , -oryzanol ( -orz ) , phytoestrogens , lignin and essential fatty acid . major previous studies had focused on the effects of br on diabetic risk factors which show br is associated whit a lower risk of diabetes . in br , gaba that is known as a neurotransmitter has the ability to regulate the blood pressure and increase insulin secretion . furthermore , -orz as a potent anti - oxidant by antihypercholestrolemic effects and vitamin e may prevent the progression of cardiovascular complication and may confer protection against metabolic disorders . few studies that had considered the influence of substituting wr with br on metabolic risk factors show no significant result . previously conflicting results regarding the effect of br on some inflammation mediators such as plasminogen activator inhibitor type-1 ( pai-1 ) and tumor necrosis factor - alpha ( tnf- ) in rats were reported . in the 14 week clinical trial using cross - over design , we aim to determine the effects of br consumption on high - sensitivity c - reactive protein ( hs - crp ) as an inflammatory marker and some cardiovascular risk factors among non - menopausal overweight or obese female adults . participants were eligible if they have a body mass index ( bmi ) more than 25 kg / m and non - menopausal , non - pregnant , non - lactating and non - smoking women in the range of 18 - 50 years old were included in this study . the sample size calculation was determined using the serum hs - crp level as a key dependent variable in the standard formula suggested for cross - over trials : n = [ ( z 1 /2 + z 1 ) . s]/2 ( 1 2 ) ; where z 1 /2 was 1.96 , z 1 was 0.84 , s was 0.01 , 1 2 was 0 and was 0.04 . in the present study , a total of 40 eligible volunteers enrolled into the present study and allocated to group 1 ( treatment from br to wr ; n = 20 ) and group 2 ( treatment from wr to br ; n = 20 ) . all of them were instructed to use br in one period of trial and wr in another period . each patient received two diets . in group 2 , 2 subjects in the first intervention period and 3 subjects in the second intervention period were eliminated from this study , due to : busy schedule : n = 3 , intervention unrelated surgery problem : n = 1 and pregnancy : n = 1 . finally this study was conducted with 15 subjects in group 2 and 20 subjects in grup1 . patients flow diagram in the present study in the run in period subjects were allowed to continue performing daily living and normal activities . after 2 week run in , participants were randomly allocated to two experimental periods of wr or br , each one for 6 weeks in a crossover design . first intervention period was 0 - 6 week , second intervention period was 8 - 14 week and 2 week was set for washout phase from 6 to 8 of the study [ figure 2 ] . in the present work participant were not blinded and all of them were on a weight reducing diet in duration of the study . two kinds of rice used in this study were from the same iranian rice variety ( tarom ) . raw rice packages were given to each subject and all of them were instructed to eat 150 g of cooked rice daily . study cross - over design to observe the effect of white rice and brown rice on inflammatory marker and cardiovascular risk factors among overweight or obese female adults two diets were prescribed for each patient : ( 1 ) diet with wr ( 2 ) diet with br . for assessing subject adherence , dietary intake based on 3-day food records , were obtained and analysis at baseline and end of each intervention period . as derived from diet prescription and food records , no significant differences were obtained between consumed amount and prescribed amount from each of the five food group . in the both of intervention periods , we provided weight reducing diet with 200 - 500 kcal / day deficit for each female based on the bmi range . in br period 10 spoon ( 150 g ) br was replaced instead of 10 spoons ( 150 g ) wr . composition of white rice and brown rice after an overnight fasting , blood samples were collected from patients between 9 and 11 am . after centrifuging at 4c and 500 g for 10 min , tubes were frozen at 80c until analyzing . serum glucose , total cholesterol ( tc ) , triacylglycerol ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) were measured by photometric enzyme assay ( pars azmoon , iran ) . hs - crp was determined by latex - immunoturbidemetric assay ( bionic , iran ) . body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm respectively and bmi was calculated . waist circumference was obtained at the midpoint between the lowest rib and the iliac crest . when participants were in comfortable seat position , after 10 min rest , blood pressure was measured on the right arm using electronic blood monitor . except height , other physical characteristics and blood pressure were measured 4 times , in study week 0 , 6 , 8 , 14 respectively . different value between first and last intervention in the each period was calculated and then difference between them was determined as mean difference for both group 1 and group 2 . we also check the period and carry - over effects using independent t - test . data were analyzed using spss ( version 15 ) and a 2-sided p < 0.05 was considered to be significant . participants were eligible if they have a body mass index ( bmi ) more than 25 kg / m and non - menopausal , non - pregnant , non - lactating and non - smoking women in the range of 18 - 50 years old were included in this study . individual with a history of chronic disease , consuming medicine and flowing special regimen were excluded . the sample size calculation was determined using the serum hs - crp level as a key dependent variable in the standard formula suggested for cross - over trials : n = [ ( z 1 /2 + z 1 ) . in the present study , a total of 40 eligible volunteers enrolled into the present study and allocated to group 1 ( treatment from br to wr ; n = 20 ) and group 2 ( treatment from wr to br ; n = 20 ) . all of them were instructed to use br in one period of trial and wr in another period . each patient received two diets . in group 2 , 2 subjects in the first intervention period and 3 subjects in the second intervention period were eliminated from this study , due to : busy schedule : n = 3 , intervention unrelated surgery problem : n = 1 and pregnancy : n = 1 . finally this study was conducted with 15 subjects in group 2 and 20 subjects in grup1 . signing the written consent the study protocol was approved by food security research center isfahan university of medical science and nutrition department . in the run in period subjects were allowed to continue performing daily living and normal activities . after 2 week run in , participants were randomly allocated to two experimental periods of wr or br , each one for 6 weeks in a crossover design . first intervention period was 0 - 6 week , second intervention period was 8 - 14 week and 2 week was set for washout phase from 6 to 8 of the study [ figure 2 ] . in the present work participant were not blinded and all of them were on a weight reducing diet in duration of the study . two kinds of rice used in this study were from the same iranian rice variety ( tarom ) . raw rice packages were given to each subject and all of them were instructed to eat 150 g of cooked rice daily . study cross - over design to observe the effect of white rice and brown rice on inflammatory marker and cardiovascular risk factors among overweight or obese female adults two diets were prescribed for each patient : ( 1 ) diet with wr ( 2 ) diet with br . for assessing subject adherence , dietary intake based on 3-day food records , were obtained and analysis at baseline and end of each intervention period . as derived from diet prescription and food records , no significant differences were obtained between consumed amount and prescribed amount from each of the five food group . in the both of intervention periods , we provided weight reducing diet with 200 - 500 kcal / day deficit for each female based on the bmi range . in br period 10 spoon ( 150 g ) br individual diet was prescribed for each participant and provided an exchange list and educated them how to record their food intake and method of using exchange list . after an overnight fasting , blood samples were collected from patients between 9 and 11 am . serum glucose , total cholesterol ( tc ) , triacylglycerol ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) were measured by photometric enzyme assay ( pars azmoon , iran ) . hs - crp was determined by latex - immunoturbidemetric assay ( bionic , iran ) . body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm respectively and bmi was calculated . waist circumference was obtained at the midpoint between the lowest rib and the iliac crest . when participants were in comfortable seat position , after 10 min rest , blood pressure was measured on the right arm using electronic blood monitor . except height , other physical characteristics and blood pressure were measured 4 times , in study week 0 , 6 , 8 , 14 respectively . different value between first and last intervention in the each period was calculated and then difference between them was determined as mean difference for both group 1 and group 2 . we also check the period and carry - over effects using independent t - test . data were analyzed using spss ( version 15 ) and a 2-sided p < 0.05 was considered to be significant . after randomization , the two groups were balanced but during the study , 5 person withdrew duo to busy schedule ( n = 3 ) , intervention unrelated surgery problem ( n = 1 ) and pregnancy ( n = 1 ) . thus , a total of 40 participants , 35 non - menopausal over weight and obese female completed the study . the period and carry - over effects were checked and none of them were significant in this study . except dietary magnesium content which was significantly ( p < 0.001 ) higher during br consumption , no significant differences were found between wr and br diets regarding each of the five food groups , polyunsaturated fatty acid ( pufa ) , monounsaturated fatty acid ( mufa ) , saturated fatty acids and majority of micronutrients as derived from the food record . energy and nutrient intake in the first and second intervention period was shown in table 2 . they were instructed to avoid change in exercise habits , hence their physical activity was similar and it was checked by researchers . energy and nutrient intakes in the first and second intervention periods in groups 1 and 2 br diet in comparison with wr could decrease physical characteristics such as : weight , waist and hip circumference and bmi significantly . mean difference changes were 1.2 kg ( 95% confidence interval [ ci ] = 0.60 - 1.96 , p = 0.001 ) , 2.38 cm ( 95% ci = 0.51 - 4.25 , p = 0.014 ) , 2.98 cm ( 95% ci = 1.55 - 4.42 , p 0.001 ) and 0.52 kg / m2 ( 95% ci = 0.26 - 0.78 , p 0.001 ) respectively . in the br diet , inflammatory marker ( hs - crp ) level decreased significantly in comparison with wr diet . no significant changes were appeared regarding serum lipids concentration such as tg , tc , hdl , ldl and fasting blood glucose ( fbg ) . diastole blood pressure level was significantly ( p = 0.032 ) decreased after consuming br diet compared with wr diet . physical characteristics and biochemical parameters in first and second intervention phase in group 1 and 2 , as shown in table 3 and table 4 . physical characteristics in the first and second intervention period in groups 1 and 2 biochemical parameters in the first and second intervention periods in groups 1 and 2 this is due to the high concentration of this nutrient in bran layer of br , which has potential benefit for the cardiovascular system . the present results show that br diet in comparison with wr diet could significantly reduce weight , waist and hip circumference , bmi and hs - crp . br as unrefined whole grain contain protective factors for diabetics and cardiovascular disease , such as vitamins , magnesium and other minerals , fiber and essential fatty acids . by milling and polishing br and convert it to wr , 50% fiber 84% magnesium and 69% of total mufa and pufa have been eliminated respectively . despite these benefits , previous studies indicated no significant changes on body weight following br consumption but the size of adipocytes was reduced by feeding with pre germinated br in diabetic rats . in this study diet with br could reduce waist circumference , a strong risk factor for cardiovascular disease and enhanced weight loss . there are many reports which show the ability of fiber to slow down the process of absorption of nutrients by modifying the metabolic response to intake , its ability to lowering fasting insulin level , increase the excretion of biliary acid and the metabolic effects of the short - chain fatty acids ( scfa ) produced in the bacterial fermentation of fiber . recent human studies reported relationships between the composition of the intestinal microbiota and obesity . in a recent study , br could increase large bowel scfa and have a beneficially effect on the gut microbial composition in humans . hence anti - obesity effects of br may be related to the profile and activity of the intestinal microbiota . finding from an animal study indicated that br has the anti - obesity effect through down regulation of lipogenic gene . hypothalamic er stress is linked to leptin resistance , which leads to dysregulation of appetite and energy expenditure in obese mice . overall this evidence highlights that br may have potential for the enhanced weight loss in humans . there are conflicting results regarding the effect of br on serum lipid profiles and fbg . in a recent study substituting wr with br for 16 weeks was not associated with improvement in fasting serum glucose , insulin , serum lipids among people with diabetes . two recent short - term interventions indicated no significant benefits of substituting whole grains for refined grains on fasting glucose , insulin and blood lipids . but in the study with cross - over design diabetic patients were instructed to consume 180 g of rice 3 times a day for 6 week and blood glucose management , lipid related markers were improved on br diet . these results were similar to the results of another study in which blood glucose and insulin reaction were lower for br when compared with wr . higher content of dietary fiber in br ( 2 g/100 g ) than in wr ( 0.4 g/100 g ) might be responsible for this effect . fiber in bran layer raps -amylase , the enzyme to digest starch and substrates ( saccharides ) in the intestine , ultimately suppresses increasing in postprandial blood glucose levels . gaba that is richer in br than wr ( contents of gaba in raw br and wr were 7 mg/100 and 3 mg/100 respectively ) , stimulate the pancreatic beta cell and insulin secretion from pancreas and injection gaba agonist to streptozotocin - induced diabetic rats ameliorated the elevation of blood glucose concentration and increased their insulin levels . br diet can also suppress hypercholesterolemia via enhancing fecal bile acids secretion without affecting on the cholesterol synthesis of host liver . comparing with wr , br contains more gaba with hypocholestrolemic effect , although the exact mechanisms are not yet clear . however in our study br diet in comparison with wr diet , did not have any significant effects on serum lipid profiles and fbg . this might be related to the low amount of br intake in the current study . in the present study , we observed that comparing with wr diet ; br diet could reduce diastole blood pressure . this result was similar to the findings of the study which showed the br intervention had a beneficial effect on diastole pressure among diabetic person . furthermore in another study consumption of diet with br / whole wheat for 5 week could reduce blood pressure significantly in hypercholestrolemic patients . several mechanisms have been proposed to be responsible for the reduction in blood pressure , which occurs with increased fiber intake including increased water and electrolyte excretion with insoluble fiber intake . past studies have shown that gaba is not only a neurotransmitter but also it can decrease blood pressure . few studies are available about the effects of whole grain or br on inflammatory risk markers . hs - crp is a strong inflammatory marker for predicting future cardiovascular events . compared with wr , br ameliorated the increase in both plasma tnf- and pai-1 and decrease in adiponectin in diabetic rats this result was similar to another study which showed pai-1 significantly decreased in diabetic rats by taking pgbr for 7 week . two previous studies did not find significant benefits of substituting whole grains for refined grains on interleukin-6 and hs - crp . in our short - term trial br diet in comparison with wr diet , significantly reduced level of hs - crp . however , a few human studies are available about the effect of br on inflammatory and cardiovascular risk factors . furthermore , we assessed the effect of br in cross - over design as the strong method for conducting this trial . the subjects were instructed to consume only 150 g br accompany with weigh loss diet and some significantly effects were seen for mentioned variables . in the present study we just recommended food item but br and wr were provided for participants . we analyzed patient food records and their diets controlled exactly . in non - menopausal female , since lipid profiles variation may be affected by change in blood estrogen level , patient biochemical measurement were assessed based on menstrual date and all of them were in the same hormone phase . this study was conducted on healthy adults without any specific disorder ; hence , our trial should have good external validity . br replacement in the diet as a staple food can be useful to improve weight loss , waist and hip circumference , bmi , diastole pressure and hs - crp level significantly , among over weight and obese female adults . our findings show that in overweight or obese female adults br diet in comparison with wr diet resulted in decreased diastole blood pressure and inflammatory marker ( hs - crp ) . furthermore , br replacement in the diet can be useful to improve weight loss and visceral obesity significantly . replacement of white rice by brown rice did not have any significant effects on serum lipid profiles and fbg .

glucose homeostasis is controlled by an intricate network composed of organs , glands , and molecular messengers , whose primary aim is to maintain an optimal balance between energy stores and immediately available fuel for cellular processes . hence , it is not surprising that the brain is endowed with mechanisms for sensing glucose levels . in addition , cerebral areas such as the basal hypothalamus and the brainstem contain neuronal populations which act as controllers of physiological and behavioral reactions ( i.e. , regulation of feeding behavior ) in response to oscillations in glucose levels and bodily energy demands . strikingly , glucose sensing in the brain appears to be also involved in the modulation of brain cell functions having no direct relationships with metabolism . indeed , glucose - related signaling has a strong impact on neuronal activity . in particular , we will here attempt to review the increasing body of evidence indicating that messengers essential to glucose homeostasis also affect at multiple levels the activity of the hippocampus , which is a brain area critically involved in cognitive functions . we will focus on a few key molecules : insulin and insulin - response substrates ( irss ) , insulin - like growth factor - i ( igf - i ) , glucagon - like peptide-1 ( glp-1 ) , and ghrelin . after briefly summarizing nonmetabolic glucose - related signaling in the brain , we will try to convey the message that these molecules exert multiple actions on hippocampal physiology by affecting structural and functional neuroplasticity . this is , in turn , correlated to modifications in hippocampal - dependent learning and memory processes ( figure 1 ) . finally , we will give an overview of the relevance of these phenomena for pathology , since the involvement of metabolic dysregulation in neuronal function impairment is an emerging topic with promising translational implications . in response to physiological stimuli and environmental conditions , the central nervous system undergoes structural and functional changes , both during development and throughout adulthood . this process of plasticity involves neurogenesis , that is , proliferation and differentiation of neural stem cells ( nscs ) , as well as changes in the morphology and activity of differentiated neurons . these adjustments are instrumental to the brain orchestration of various peripheral organs functions , in order to adapt energy expenditure to nutrient availability . in this regard , the hypothalamus - pituitary axis integrates humoral signals and coordinates behavioral and metabolic responses of the whole body . however , the hypothalamus is only one of the brain areas sensitive to hormones and metabolic signals . indeed , food seeking during fasting is a complex activity which involves information processing to identify or remember the location of resources necessary for survival . in keeping with this , brain areas not involved in feeding control synthesize receptors for insulin , insulin - like peptides such as insulin - like growth factor - i ( igf - i ) , glucagon - like peptide-1 ( glp-1 ) , and ghrelin [ 4 , 5 ] . accordingly , the activity of neural circuits in the hippocampus is influenced by metabolic stimuli and energy supply . moreover , neurons are high energy - consuming cells and their function is markedly affected by the energy status . in the brain , most energy is consumed to generate action potentials and postsynaptic potentials [ 7 , 8 ] . additionally , glucose metabolism provides energy for the biosynthesis of neurotransmitters in differentiated neurons and for nsc fate determination . importantly , the astrocytic energy sources glycogen and lactate seem to be directly relevant for learning and memory , although the underlying mechanisms have not yet been elucidated . finally , the transporters glut1 and glut3 mediate glucose uptake from extracellular fluid into glial and neuronal cells , respectively . glut1 and glut3 are insulin - independent transporters , suggesting that the impact of insulin and related signals on brain plasticity should be independent of glucose uptake . in addition to a direct effect of glucose levels on neuronal metabolism , hormones involved in glucose homeostasis activate different signal transduction cascades in the brain . this process results in effects which go well beyond the regulation of neuronal energy demand and metabolism ( see below ) . indeed , insulin and igf - i activate the phosphatidylinositol trisphosphate kinase ( pi3k)/akt and ras / mapk - erk pathways , thus affecting gene expression , with huge consequences for nsc proliferation and neuronal activity [ 14 , 15 ] . glp-1 is secreted by the gut in response to satiation and participates in glucose homeostasis . indeed , it was first characterized for its ability to enhance insulin release from pancreatic -cells , thus leading to increased glucose sensitivity . subsequent research in rodents has shown that glp-1 receptors are also present on neurons , with intense expression in the ca hippocampal region [ 17 , 18 ] , and that their activation stimulates the activity of the map kinase pathway . on the other hand , the stomach secretes ghrelin , which stimulates feeding behavior , and exerts a global counterregulatory action in comparison to insulin . in the brain , ghrelin binds the growth hormone secretagogue receptor 1a ( ghs - r1a ) and controls the g protein - mediated activation of the pi3k / akt , ras / mapk - erk , and pka / creb pathways [ 2224 ] . in addition , it is interesting to notice the close similarity between the intracellular signaling pathways activated by glucose metabolism regulators and those controlled by neurotrophins . this convergence emphasizes the importance of metabolic mediators for proper functioning of neural circuits . in particular , the camp - responsive element binding ( creb ) transcription factor has been largely studied as mediator of neurotrophin - triggered neuronal differentiation , survival , and plasticity in the brain , and it has been characterized as metabolic sensor modulated by nutrient depletion and fasting hormones [ 26 , 27 ] . calorie restriction also induces the expression of the nad - dependent histone deacetylase sirtuin 1 ( sirt1 ) , which has been recently identified as a partner in creb - dependent gene expression , thus highlighting a novel mechanism linking metabolic homeostasis and brain health . in a mouse model of brain insulin resistance , intracerebral injection of streptozotocin reduces the activity of sirt1 and causes cognitive impairment , an alteration prevented by administration of the sirt1 activator resveratrol . indeed , sirt1 promotes the creb - dependent expression of brain - derived neurotrophic factor ( bdnf ) and other neuroprotective genes . moreover , creb - dependent transactivation of genes regulating neuronal survival , metabolism , and plasticity ( like pgc1 and nnos ) in calorie - restricted mice requires sirt1 . in keeping with these data , electrophysiological and cognitive brain responses to calorie restriction are similarly impaired in mice harboring brain - specific inactivation of sirt1 or creb . finally , in the mouse hippocampus , sirt1 transcription is induced by creb during calorie restriction and may , in turn , increase creb expression ( and function ) through a mirna - mediated mechanism . consistently , recent evidence obtained in pc12 cell cultures indicates that the induction of creb expression by igf - i is mediated by downregulation of the microrna mir-181a . taken together , the above evidence suggests that the complex interplay between sirt1 and creb , while affecting nutrient sensing and glucose homeostasis in peripheral tissues , may also play a pivotal role in the metabolic regulation of neuronal plasticity and of high - order brain functions . hippocampal ca1 neurons display increased expression of the glucose transporter glut1 during the execution of a behavioral test . this is an expected homeostatic reaction , aimed at fulfilling the increased metabolic demand of neurons challenged by a cognitive task . on the other hand , the finding that the molecular network outlined above ( see section 2 ) can actually modulate performance in behavioral tests involving learning and memory is less trivial . indeed , zucker rats display impaired insulin sensitivity , which correlates with poor performance in the morris water maze ( mwm ) . in agreement with this finding , heterozygous knockout mice for insulin receptor show lower performance in the novel object recognition test . interestingly , lower values for glycosylated hemoglobin ( hba1c ) and glycemia indicate improved glucose homeostasis and are associated with better performance on memory tasks in human subjects . moreover , db / db transgenic mice , which are a knockout for the gene encoding the leptin receptor , are also characterized by insulin - resistant diabetes . improving glucose homeostasis of db / db mice by means of physical exercise or calorie restriction it is noteworthy that this improvement is accompanied by increased expression of the bdnf gene , possibly as a result of restored insulin signaling . analogous results come from studies on the ucd - t2d mouse model of type 2 diabetes , which displays reduced hippocampal insulin signaling and reduced activation of the bdnf receptor , trkb . moreover , knockout mice for the insulin receptor substrate p53 ( irsp53 ) display impaired learning and acquisition of a navigation task ( mwm ) and poor recognition memory ( novel object recognition test , nor ) . on the other hand , insulin receptor substrate 2 ( irs2 ) forebrain - specific knockout mice exhibit improved memory retention in the mwm test , whereas the learning curve is unaffected , indicating that different effectors of insulin can exert opposite actions on behavior . glucose intolerance can also result from exposure to a high - fat diet ( hfd ) during early postnatal life and is associated with impaired learning of an operant conditioning task ( pressing a lever to obtain reward ) and in the radial arm maze task . strikingly , this hippocampal - dependent behavioural task is unaffected if mice are subjected to hfd starting from adulthood . it is tempting to speculate about the existence of a specific critical period(s ) for developmental programming of proper sensitivity of hippocampal circuits to the various components of glucose signaling , in close analogy to what has been demonstrated for programming by environmental stimuli of the set point for hypothalamic leptin sensitivity and the development of cortical sensory systems in response to early experience . similar data have been obtained from liver - specific , igf - i knockout mice . they exhibit deficits in both the learning and memory retention phases of the mwm , which can be detected as early as two months of age and still persist at 18 months of age . in addition , treatment of young rats with an igf - i antiserum impairs learning of a passive avoidance task . these findings globally indicate that loss of insulin signaling results in decreased cognitive performance ; conversely , administration of ghrelin and glp-1 has been shown to improve learning and memory of new tasks . indeed , administration of ghrelin after training in a t - maze foot - shock avoidance test improves memory retention and , conversely , ghrelin knockout mice show impaired performance in the nor test . consistently , bilateral intrahippocampal infusion of ghrelin for 4 days , prior to training in the mwm , enhances acquisition and memory retention of the task . lastly , glp-1 receptor knockout mice display decreased memory retention in both the nor and the mwm tests . conversely , intrahippocampal administration of glp-1 to wild - type mice enhances spatial learning in both the passive avoidance and the mwm tests . moreover , administration of exendin-4 , a glp-1r agonist , for two weeks prior to training in a radial arm maze task is associated with improved spatial reference memory . taken together , these findings lend support to the view that molecules involved in glucose signaling play a key role in modulating learning and memory , with the intriguing implication that they can be exploited to potentiate cognitive function and to ameliorate pathological deficits . the hippocampus is one of the few areas where neurogenesis persists throughout adulthood , thus supporting learning and memory , in addition to potentially contributing to brain repair . in the adult mammalian brain , the subventricular and subgranular zones represent the two hippocampal neurogenic niches , populated by nscs that proliferate and differentiate to generate new neurons . a proper balance between the proliferative expansion of these populations and their maturation underlies the maintenance of both the hippocampus stemness reservoir and cognitive function [ 56 , 57 ] . although the regenerative potential of stem cell niches in the brain is still debated , a growing body of evidence indicates that , in the hippocampus , newborn neurons integrate into existing circuits to play a pivotal role in learning , memory , and neurological disorders . studies carried out both in vitro and in vivo suggest that insulin and igf - i promote neurogenesis by affecting nsc proliferation , differentiation , and survival [ 5961 ] . moreover , insulin is a crucial trophic factor for nervous system development and maintenance of neurogenic niches . indeed , activation of the insulin / igf - i pathway is required for neuroblasts to exit quiescence [ 62 , 63 ] . however , a chronic hyperstimulation of insulin / igf - i effectors can lead to premature impoverishment of the nsc pool . therefore , insulin may exert either trophic or harmful effects on nscs depending on the timing and the duration of stimulation . on the other hand , animals undergoing calorie restriction exhibit lower plasma levels of glucose and insulin , in parallel with increased neurogenesis in the dentate gyrus and slowdown of the age - related stemness decline . induction of the expression of the bdnf gene may at least partly explain the trophic action of nutrient deprivation on the nsc compartment . in addition , nutrient depletion may directly preserve the nsc capacity to self - renew and differentiate . in this regard , sirt1 works as an epigenetic repressor and it modulates the neurogenic potential of neural precursors in the adult mouse brain niches . according to an interesting scenario emerging from various experimental models , under metabolic and oxidative stress sirt1 represses nsc self - renewal and promotes their differentiation . in summary , sirt1 might serve as a metabolic sensor regulating the balance between nsc self - renewal and differentiation and controlling the preservation of the stem cell niche . conversely , knocking out the genes encoding the nutrient- and insulin - regulated foxo transcription factors causes sustained activation of nutrient replenishment signaling , thus leading to unbalanced proliferation and rapid exhaustion of neural progenitors both in vivo and in vitro . hence , absence of foxos mimics insulin hyperstimulation and promotes a premature senescence of the stem cell niche . similarly , stimulation of the nutrient - dependent mtor pathway causes reduced self - renewal and earlier nsc differentiation , resulting in altered brain development . accordingly , glp-1 receptor agonist exendin-4 stimulates neurogenesis in the dentate gyrus , evaluated by bromodeoxyuridine incorporation assay , as well as by the expression of the newborn neuron marker doublecortin . moreover , the antidiabetic drug sitagliptin , in concomitance with the amelioration of peripheral glucose homeostasis , improves hippocampal neurogenesis and recognition memory through the upregulation of hippocampal glp-1 receptor , in addition to modifying the expression of key genes involved in cognitive decline . together , this evidence supports the idea that nutrient - related signals control nsc fate , actively participating in neural plasticity processes , under both physiological and pathological ( i.e. , overnutrition , diabetes and see section 6 ) conditions . modifications in the activity of synapses , that is , potentiation or depression , or in their function and number , for example , generation of new dendritic spines , represent the functional and structural substrates underlying the integration of neurons into networks . the interaction between these different phenomena is , in turn , instrumental to acquire and consolidate behavioral modifications ( see section 3 ) . treatment of primary cultures of rat hippocampal neurons results in higher frequency of miniature excitatory postsynaptic currents ( mepscs ) , suggesting an increased basal neurotransmitter release from presynaptic terminals . this functional effect is paralleled by increased density of dendritic spines , involving activation of the akt pathway and of the rho gtpase rac1 , an important mediator of cytoskeleton rearrangement . first , zucker rats display loss of insulin sensitivity and a concurrent reduction in long - term potentiation ( ltp ) at ca3ca1 synapses , whereas long - term depression ( ltd ) is unaffected . in addition , heterozygous knockout mice for insulin receptor have normal basal synaptic transmission and induction of ltp that , however , fails to be consolidated owing to reduced activation of the akt pathway . on the other hand , in physiological conditions insulin van der heide and colleagues have shown that insulin application results in a leftward shift in the input - output relationship of excitatory postsynaptic potentials ( epsps ) response as a function of stimulation frequency . indeed , under control conditions , ltd or ltp can be achieved using stimulation frequencies of 1 hz and 50100 hz , respectively . however , in the presence of insulin ltd or ltp is obtained in response to stimulation frequencies of 0.033 hz or 10 hz ( which would yield no effect under control conditions ) , respectively . plasticity of plasticity phenomenon that results in a lower stimulation frequency threshold , called inducer of synaptic plasticity . in keeping with the findings shown in section 2 , this effect requires activation of the pi3k pathway and results in increased exocytosis of n - methyl - d - aspartate receptors ( nmdars ) . moreover , nmdar function is also transiently enhanced by phosphorylation of the nr2a and nr2b subunits , which correlates with the potentiation of nmdar - mediated currents . it is worth noting that nr2a and nr2b subunits are responsible for different nmda current kinetics . in addition , nr2b confers a higher time constant to nmda responses , predominates during early cortical and hippocampal development , and is downregulated in adulthood , when nr2a becomes more expressed . insulin treatment of hippocampal cultures also increases phosphorylation of the glur1 subunit of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampars ) , indicating that multiple sites of action are responsible for the effect of insulin on synaptic plasticity . finally , in thalamocortical organotypic slices this hormone stimulates maturation of silent synapses , that is , those mainly containing nmdars that represent a substrate for circuit potentiation through ampars insertion . notably , these data were obtained following acute insulin applications , whereas chronic elevation of cerebral insulin levels by means of intracerebroventricular infusion greatly reduces ltp in the ca1 area in response to high - frequency stimulation . however , the mechanisms responsible for this time - dependent change in the polarity of insulin effect on synaptic plasticity still need to be better addressed . insulin receptor substrates are essential to the actuation of the above described effects . for instance , the synapse - specific irsp53 interacts with the activated rho gtpase rac and with the postsynaptic protein psd-95 . accordingly , overexpression of irsp53 stimulates dendritic spine formation . however , transgenic mice lacking the gene encoding this protein show enhanced ltp of the schaffer collateral pathway , although they do not display any obvious change in dendritic spine density and morphology . the higher propensity of irsp53 knockout mice for ltp correlates with increased nmdar - dependent synaptic transmission , although no obvious changes in the expression of nmdar and ampar subunits could be detected . it is noteworthy that another group independently generated an irsp53 transgenic mouse line and found a small ( 17% ) , but significant , reduction in postsynaptic density area , in addition to the upregulation in the expression of nr2a and nr2b proteins in both juvenile and adult animals . taken together , these data point to a role of irsp53 in promoting generation of dendritic spines which , on the other hand , are less sensitive to display ltp , although the underlying mechanisms are still unclear . interestingly , also irs2 deletion affects structural and functional plasticity of the hippocampus , but with different outcomes in comparison to irsp53 . indeed , irs2 knockout mice have higher density of ca1 dendritic spines , in addition to showing decreased ltp at the schaffer collateral pathway , as a result of impaired akt activation and lower phosphorylation of nr2b subunits . it is important to notice that the morphology of dendritic spines ( e.g. , unstable filopodia versus stable mushroom spines ) was not assessed in these studies , and elucidation of this issue would contribute to understanding the seemingly contrasting effects of manipulating the expression of different irss on structural and functional plasticity . considering the convergence on the same molecular effectors as insulin , it is not surprising that igf-1 treatment stimulates structural plasticity in cortical cultures , as assessed by increased immunoreactivity for synaptic markers such as synapsin-1 and psd-95 . in addition , igf - i knockout mice have reduced dendritic complexity and number of dendritic spines of cortical layer ii - iii neurons . this can represent one of the substrates for the role of igf-1 in promoting plasticity . a similar action is likely exerted in the adult hippocampus , since administration of igf - i antiserum partially blocks the increase in spine density of ca1 basal dendrites in response to physical exercise . moreover , liver - specific igf - i knockout mice exhibit impaired ltp at perforant path - dentate gyrus synapses . this deficit is partially rescued if the inhibitory tone is decreased by bath perfusion with the gabaa receptor antagonist bicuculline . consistently , igf - i knockout mice have reduced density of glutamatergic synapses , which leads to a lower excitation / inhibition ratio . during development , brain - specific overexpression of human igf - i results in boosting of postnatal synaptogenesis in the molecular layer of the dentate gyrus peaking at 35 days of age . this phenomenon is likely the result of accelerated maturation , since the final number of synapses is not different from that of wild - type controls . further investigation is required to understand whether igf - i overexpression has any consequence on the developmental curve of hippocampal neurons at functional , structural , and behavioral levels . besides , the therapeutic potential of igf-1 in diseases characterized by impaired hippocampal function needs to be better investigated , especially considering evidence pointing to brain insulin and igf-1 resistance in alzheimer 's disease patients . analogous effects are exerted by ghrelin , which crosses the blood - brain barrier to bind its hippocampal receptors . indeed , peripheral administration of ghrelin results in higher density of dendritic spines in the ca1 area and in augmentation of ltp of the schaffer collateral pathway . moreover , addition of ghrelin to rat hippocampal slices increases the density of phalloidin - positive puncta , which indicates higher abundance of polymerized f - actin , thus representing an indirect measurement of dendritic spine reorganization . although more accurate measurements need to be performed , for instance , with the use of time - lapse imaging on slices from gfp - expressing transgenic mice , this finding is suggestive of increased dendritic spine dynamics . recent data indicate that ghs - r1as are expressed in the vicinity of hippocampal excitatory synapses and , indeed , their pharmacological activation triggers surface exposure of glua1 subunits of the ampa glutamate receptor . this structural change is paralleled by facilitation of nmda receptor - dependent ltp via pi3k / akt activation . in addition , ghrelin also stimulates phosphorylation of the nr1 subunit of nmdars , which can further facilitate activity - dependent synaptic plasticity . moreover , experiments on rat brain slices containing substantia nigra pars compacta have shown that ghrelin enhances excitability also by inhibiting kv7 channels . it would be interesting to study whether a similar mechanism is also present in the hippocampus . finally , glp-1 appears to affect mainly basal inhibitory synaptic transmission , as it has been shown to increase both frequency and amplitude of gabaergic spontaneous inhibitory postsynaptic currents ( sipscs ) recorded from ca3 neurons . accordingly , glp-1 reduces excitability of hippocampal cultures by acting on glutamate- and depolarization - induced ca influx ; this effect has been hypothesized to have the purpose of protecting neurons from glutamate excitotoxicity , as it occurs , for instance , in epilepsy . indeed , glp-1 receptor knockout mice have lower threshold for and higher severity of kainic acid - induced seizures . however , it should be taken into account that neuronal response to glp-1 can vary according to the time - scale considered . as it has been shown for ca1 neurons by oka and colleagues using in vivo electrophysiological recordings in anesthetized rats , an initial increase in single - unit activity is followed by a decrease . moreover , the effect of glp-1 on synaptic plasticity appears to be radically different from that on basal transmission . indeed , administration of glp-1 receptor agonists such as liraglutide increases ltp , whereas glp-1 receptor knockout mice show impairment in this form of synaptic plasticity . hence , the data summarized above indicate that structural and functional aspects of hippocampal plasticity are strongly sensitive to key mediators of glucose homeostasis . moreover , they suggest the existence of multiple interactions and synergies between the different molecular players , and understanding the details of this network appears to be one of the main goals of future research . the data reviewed in the previous paragraphs support the view that glucose homeostasis imbalances can alter signaling pathways involved in adult neurogenesis and synaptic plasticity , thereby leading to reduced mindspan ( the maintenance of mental abilities throughout life ) and increased risk of neurodegenerative disorders . moreover , it is widely known that energy restriction promotes neuronal survival and improves cognitive function . conversely , the excess of nutrients impinges on brain health and impairs synaptic transmission and plasticity leading to accelerated cognitive decline ( cd ) [ 102 , 103 ] . in line with these concepts , humans in the western world are thought to be unnaturally overfed and sedentary , a state of chronic positive energy balance that results in suboptimal health . in addition , the incidence of metabolic disorders , including type 2 diabetes ( t2d ) , is increasing at alarming rates worldwide , largely due to poor lifestyle habits . in parallel epidemiological / clinical observations have accumulated showing that diabetic patients are significantly more likely to develop cognitive impairment and exhibit increased susceptibility to dementia . importantly , impaired metabolic parameters , such as hyperglycemia and hyperinsulinemia , positively correlate with cd . elevated blood glucose levels increase the risk of dementia in both diabetic and nondiabetic individuals by 40% and 18% , respectively , and are associated with cd and reduced hippocampal volume . these findings indicate that fluctuations in blood glucose levels negatively impact on brain function , even in the absence of overt t2d or impaired glucose tolerance . chronic hyperglycemia and hyperinsulinemia primarily stimulate the formation of advanced glucose end products , which leads to an overproduction of reactive oxygen species and alteration of intracellular second messenger pathways . whereas insulin is clearly neurotrophic at moderate concentrations , too much insulin in the brain may be associated with increased amyloid- deposition due to competition for their common and main clearance mechanism , the insulin - degrading enzyme . in this regard , it has even been proposed that alzheimer 's disease may be considered a form of type 3 diabetes , based on the evidence for insulin resistance and impaired insulin - response pathways in the alzheimer's - affected brain . however , glucose and insulin levels changes are not the only metabolic factors involved in hippocampal plasticity alterations produced by glucose dyshomeostasis . interestingly , in insulin - deficient rats and insulin - resistant mice , diabetes impairs hippocampus - dependent memory , impinging on both synaptic plasticity and adult neurogenesis , and the glucocorticoid system contributes to these adverse effects . in this regard , nsc proliferation and adult neurogenesis are impaired in t2d and prediabetes animal models . moreover , as mentioned above , several gut hormones are able to impact on hippocampal function . an additional aspect to be taken into account is that microbiota dysbiosis could affect the gut - brain axis , thus promoting insulin resistance and cognitive impairment . in addition , germ - free mice show a significant alteration of serotonergic system metabolites concentration and serotonergic neurotransmission in the hippocampus , which can have a negative impact on synaptic plasticity . hence , a key topic in current research is understanding which metabolic factors are most harmful to brain plasticity and which drugs suitable for metabolic disorders can also have an effect on cognitive functions . in particular , a challenge for the upcoming years will be investigating whether there are common molecular mechanisms underlying metabolic and neurodegenerative diseases and whether the glycemic memory of particular brain areas ( e.g. , the hippocampus ) may be a risk factor for early cd . molecules involved in metabolic homeostasis are now recognized to exert a deep influence on hippocampal plasticity and alteration of their equilibrium has a strong impact at the functional and behavioral levels . it is worth noting that , for instance , experimental paradigms such as physical exercise or environmental enrichment , that is , coupling of motor activity to sensory stimulation , social interaction , and enhancement of exploratory behavior , dramatically affect neural plasticity of several brain districts , including the hippocampus , during development and in adulthood , as well as in aging [ 115117 ] . therefore , metabolically active molecules can act as a bridge between a healthy body and a healthy brain by communicating a status of optimal metabolic homeostasis , which can represent a modulatory ( or permissive ) factor for the activation of neuroplasticity , that is , a highly energy - demanding process . in this regard , understanding how brain sensitivity to insulin and other metabolic players can be controlled may be an effective way to impact on pathologies characterized by impaired neural plasticity , especially alzheimer 's disease . indeed , expression of the insulin receptor mrna is maximal during development and decreases with aging , and this , in addition to representing another similarity between insulin sensitivities inside and outside the brain , could represent a key contributor to the decline in neural plasticity of the elderly . thus , acting on insulin signaling might be a promising strategy for overcoming age - associated plasticity deficits . finally , a crucial point is also represented by understanding interactions between glucose and lipid homeostasis , since soluble factors involved in this latter process , such as leptin , have also been demonstrated to modulate synaptic plasticity and to have a role in age - associated cd [ 45 , 119 , 120 ] . in summary , drawing a comprehensive picture of the interactions between metabolism , hippocampal circuits , and cognitive performance , in addition to elucidating the underlying molecular mechanisms , can represent an important step forward , from a conceptual and translational point of view , towards a deeper understanding of the mechanisms regulating neural plasticity in health and neurodegeneration .

hormones and peptides involved in glucose homeostasis are emerging as important modulators of neural plasticity . in this regard , increasing evidence shows that molecules such as insulin , insulin - like growth factor - i , glucagon - like peptide-1 , and ghrelin impact on the function of the hippocampus , which is a key area for learning and memory . indeed , all these factors affect fundamental hippocampal properties including synaptic plasticity ( i.e. , synapse potentiation and depression ) , structural plasticity ( i.e. , dynamics of dendritic spines ) , and adult neurogenesis , thus leading to modifications in cognitive performance . here , we review the main mechanisms underlying the effects of glucose metabolism on hippocampal physiology . in particular , we discuss the role of these signals in the modulation of cognitive functions and their potential implications in dysmetabolism - related cognitive decline .

1. Introduction 2. Outlines of Glucose Homeostasis-Related Signals Acting on the Hippocampus 3. Behavioral Outcomes and Effects on Learning and Memory 4. Impact on Hippocampal Neurogenesis 5. Effects on Synaptogenesis and Synaptic Plasticity 6. Effects of Glucose Homeostasis Dysregulation on Hippocampal Plasticity 7. Concluding Remarks and Future Perspectives

glucose homeostasis is controlled by an intricate network composed of organs , glands , and molecular messengers , whose primary aim is to maintain an optimal balance between energy stores and immediately available fuel for cellular processes . in addition , cerebral areas such as the basal hypothalamus and the brainstem contain neuronal populations which act as controllers of physiological and behavioral reactions ( i.e. strikingly , glucose sensing in the brain appears to be also involved in the modulation of brain cell functions having no direct relationships with metabolism . indeed , glucose - related signaling has a strong impact on neuronal activity . in particular , we will here attempt to review the increasing body of evidence indicating that messengers essential to glucose homeostasis also affect at multiple levels the activity of the hippocampus , which is a brain area critically involved in cognitive functions . we will focus on a few key molecules : insulin and insulin - response substrates ( irss ) , insulin - like growth factor - i ( igf - i ) , glucagon - like peptide-1 ( glp-1 ) , and ghrelin . after briefly summarizing nonmetabolic glucose - related signaling in the brain , we will try to convey the message that these molecules exert multiple actions on hippocampal physiology by affecting structural and functional neuroplasticity . this is , in turn , correlated to modifications in hippocampal - dependent learning and memory processes ( figure 1 ) . finally , we will give an overview of the relevance of these phenomena for pathology , since the involvement of metabolic dysregulation in neuronal function impairment is an emerging topic with promising translational implications . this process of plasticity involves neurogenesis , that is , proliferation and differentiation of neural stem cells ( nscs ) , as well as changes in the morphology and activity of differentiated neurons . in this regard , the hypothalamus - pituitary axis integrates humoral signals and coordinates behavioral and metabolic responses of the whole body . however , the hypothalamus is only one of the brain areas sensitive to hormones and metabolic signals . indeed , food seeking during fasting is a complex activity which involves information processing to identify or remember the location of resources necessary for survival . in keeping with this , brain areas not involved in feeding control synthesize receptors for insulin , insulin - like peptides such as insulin - like growth factor - i ( igf - i ) , glucagon - like peptide-1 ( glp-1 ) , and ghrelin [ 4 , 5 ] . accordingly , the activity of neural circuits in the hippocampus is influenced by metabolic stimuli and energy supply . importantly , the astrocytic energy sources glycogen and lactate seem to be directly relevant for learning and memory , although the underlying mechanisms have not yet been elucidated . glut1 and glut3 are insulin - independent transporters , suggesting that the impact of insulin and related signals on brain plasticity should be independent of glucose uptake . in addition to a direct effect of glucose levels on neuronal metabolism , hormones involved in glucose homeostasis activate different signal transduction cascades in the brain . indeed , insulin and igf - i activate the phosphatidylinositol trisphosphate kinase ( pi3k)/akt and ras / mapk - erk pathways , thus affecting gene expression , with huge consequences for nsc proliferation and neuronal activity [ 14 , 15 ] . glp-1 is secreted by the gut in response to satiation and participates in glucose homeostasis . indeed , it was first characterized for its ability to enhance insulin release from pancreatic -cells , thus leading to increased glucose sensitivity . subsequent research in rodents has shown that glp-1 receptors are also present on neurons , with intense expression in the ca hippocampal region [ 17 , 18 ] , and that their activation stimulates the activity of the map kinase pathway . on the other hand , the stomach secretes ghrelin , which stimulates feeding behavior , and exerts a global counterregulatory action in comparison to insulin . in the brain , ghrelin binds the growth hormone secretagogue receptor 1a ( ghs - r1a ) and controls the g protein - mediated activation of the pi3k / akt , ras / mapk - erk , and pka / creb pathways [ 2224 ] . in particular , the camp - responsive element binding ( creb ) transcription factor has been largely studied as mediator of neurotrophin - triggered neuronal differentiation , survival , and plasticity in the brain , and it has been characterized as metabolic sensor modulated by nutrient depletion and fasting hormones [ 26 , 27 ] . calorie restriction also induces the expression of the nad - dependent histone deacetylase sirtuin 1 ( sirt1 ) , which has been recently identified as a partner in creb - dependent gene expression , thus highlighting a novel mechanism linking metabolic homeostasis and brain health . moreover , creb - dependent transactivation of genes regulating neuronal survival , metabolism , and plasticity ( like pgc1 and nnos ) in calorie - restricted mice requires sirt1 . finally , in the mouse hippocampus , sirt1 transcription is induced by creb during calorie restriction and may , in turn , increase creb expression ( and function ) through a mirna - mediated mechanism . taken together , the above evidence suggests that the complex interplay between sirt1 and creb , while affecting nutrient sensing and glucose homeostasis in peripheral tissues , may also play a pivotal role in the metabolic regulation of neuronal plasticity and of high - order brain functions . on the other hand , the finding that the molecular network outlined above ( see section 2 ) can actually modulate performance in behavioral tests involving learning and memory is less trivial . indeed , zucker rats display impaired insulin sensitivity , which correlates with poor performance in the morris water maze ( mwm ) . moreover , db / db transgenic mice , which are a knockout for the gene encoding the leptin receptor , are also characterized by insulin - resistant diabetes . improving glucose homeostasis of db / db mice by means of physical exercise or calorie restriction it is noteworthy that this improvement is accompanied by increased expression of the bdnf gene , possibly as a result of restored insulin signaling . analogous results come from studies on the ucd - t2d mouse model of type 2 diabetes , which displays reduced hippocampal insulin signaling and reduced activation of the bdnf receptor , trkb . on the other hand , insulin receptor substrate 2 ( irs2 ) forebrain - specific knockout mice exhibit improved memory retention in the mwm test , whereas the learning curve is unaffected , indicating that different effectors of insulin can exert opposite actions on behavior . it is tempting to speculate about the existence of a specific critical period(s ) for developmental programming of proper sensitivity of hippocampal circuits to the various components of glucose signaling , in close analogy to what has been demonstrated for programming by environmental stimuli of the set point for hypothalamic leptin sensitivity and the development of cortical sensory systems in response to early experience . they exhibit deficits in both the learning and memory retention phases of the mwm , which can be detected as early as two months of age and still persist at 18 months of age . these findings globally indicate that loss of insulin signaling results in decreased cognitive performance ; conversely , administration of ghrelin and glp-1 has been shown to improve learning and memory of new tasks . indeed , administration of ghrelin after training in a t - maze foot - shock avoidance test improves memory retention and , conversely , ghrelin knockout mice show impaired performance in the nor test . consistently , bilateral intrahippocampal infusion of ghrelin for 4 days , prior to training in the mwm , enhances acquisition and memory retention of the task . taken together , these findings lend support to the view that molecules involved in glucose signaling play a key role in modulating learning and memory , with the intriguing implication that they can be exploited to potentiate cognitive function and to ameliorate pathological deficits . the hippocampus is one of the few areas where neurogenesis persists throughout adulthood , thus supporting learning and memory , in addition to potentially contributing to brain repair . a proper balance between the proliferative expansion of these populations and their maturation underlies the maintenance of both the hippocampus stemness reservoir and cognitive function [ 56 , 57 ] . although the regenerative potential of stem cell niches in the brain is still debated , a growing body of evidence indicates that , in the hippocampus , newborn neurons integrate into existing circuits to play a pivotal role in learning , memory , and neurological disorders . studies carried out both in vitro and in vivo suggest that insulin and igf - i promote neurogenesis by affecting nsc proliferation , differentiation , and survival [ 5961 ] . moreover , insulin is a crucial trophic factor for nervous system development and maintenance of neurogenic niches . indeed , activation of the insulin / igf - i pathway is required for neuroblasts to exit quiescence [ 62 , 63 ] . therefore , insulin may exert either trophic or harmful effects on nscs depending on the timing and the duration of stimulation . on the other hand , animals undergoing calorie restriction exhibit lower plasma levels of glucose and insulin , in parallel with increased neurogenesis in the dentate gyrus and slowdown of the age - related stemness decline . induction of the expression of the bdnf gene may at least partly explain the trophic action of nutrient deprivation on the nsc compartment . in this regard , sirt1 works as an epigenetic repressor and it modulates the neurogenic potential of neural precursors in the adult mouse brain niches . conversely , knocking out the genes encoding the nutrient- and insulin - regulated foxo transcription factors causes sustained activation of nutrient replenishment signaling , thus leading to unbalanced proliferation and rapid exhaustion of neural progenitors both in vivo and in vitro . accordingly , glp-1 receptor agonist exendin-4 stimulates neurogenesis in the dentate gyrus , evaluated by bromodeoxyuridine incorporation assay , as well as by the expression of the newborn neuron marker doublecortin . moreover , the antidiabetic drug sitagliptin , in concomitance with the amelioration of peripheral glucose homeostasis , improves hippocampal neurogenesis and recognition memory through the upregulation of hippocampal glp-1 receptor , in addition to modifying the expression of key genes involved in cognitive decline . together , this evidence supports the idea that nutrient - related signals control nsc fate , actively participating in neural plasticity processes , under both physiological and pathological ( i.e. modifications in the activity of synapses , that is , potentiation or depression , or in their function and number , for example , generation of new dendritic spines , represent the functional and structural substrates underlying the integration of neurons into networks . this functional effect is paralleled by increased density of dendritic spines , involving activation of the akt pathway and of the rho gtpase rac1 , an important mediator of cytoskeleton rearrangement . on the other hand , in physiological conditions insulin van der heide and colleagues have shown that insulin application results in a leftward shift in the input - output relationship of excitatory postsynaptic potentials ( epsps ) response as a function of stimulation frequency . however , in the presence of insulin ltd or ltp is obtained in response to stimulation frequencies of 0.033 hz or 10 hz ( which would yield no effect under control conditions ) , respectively . moreover , nmdar function is also transiently enhanced by phosphorylation of the nr2a and nr2b subunits , which correlates with the potentiation of nmdar - mediated currents . insulin treatment of hippocampal cultures also increases phosphorylation of the glur1 subunit of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampars ) , indicating that multiple sites of action are responsible for the effect of insulin on synaptic plasticity . however , the mechanisms responsible for this time - dependent change in the polarity of insulin effect on synaptic plasticity still need to be better addressed . it is noteworthy that another group independently generated an irsp53 transgenic mouse line and found a small ( 17% ) , but significant , reduction in postsynaptic density area , in addition to the upregulation in the expression of nr2a and nr2b proteins in both juvenile and adult animals . taken together , these data point to a role of irsp53 in promoting generation of dendritic spines which , on the other hand , are less sensitive to display ltp , although the underlying mechanisms are still unclear . interestingly , also irs2 deletion affects structural and functional plasticity of the hippocampus , but with different outcomes in comparison to irsp53 . indeed , irs2 knockout mice have higher density of ca1 dendritic spines , in addition to showing decreased ltp at the schaffer collateral pathway , as a result of impaired akt activation and lower phosphorylation of nr2b subunits . it is important to notice that the morphology of dendritic spines ( e.g. , unstable filopodia versus stable mushroom spines ) was not assessed in these studies , and elucidation of this issue would contribute to understanding the seemingly contrasting effects of manipulating the expression of different irss on structural and functional plasticity . considering the convergence on the same molecular effectors as insulin , it is not surprising that igf-1 treatment stimulates structural plasticity in cortical cultures , as assessed by increased immunoreactivity for synaptic markers such as synapsin-1 and psd-95 . in addition , igf - i knockout mice have reduced dendritic complexity and number of dendritic spines of cortical layer ii - iii neurons . this can represent one of the substrates for the role of igf-1 in promoting plasticity . a similar action is likely exerted in the adult hippocampus , since administration of igf - i antiserum partially blocks the increase in spine density of ca1 basal dendrites in response to physical exercise . consistently , igf - i knockout mice have reduced density of glutamatergic synapses , which leads to a lower excitation / inhibition ratio . during development , brain - specific overexpression of human igf - i results in boosting of postnatal synaptogenesis in the molecular layer of the dentate gyrus peaking at 35 days of age . further investigation is required to understand whether igf - i overexpression has any consequence on the developmental curve of hippocampal neurons at functional , structural , and behavioral levels . indeed , peripheral administration of ghrelin results in higher density of dendritic spines in the ca1 area and in augmentation of ltp of the schaffer collateral pathway . moreover , addition of ghrelin to rat hippocampal slices increases the density of phalloidin - positive puncta , which indicates higher abundance of polymerized f - actin , thus representing an indirect measurement of dendritic spine reorganization . recent data indicate that ghs - r1as are expressed in the vicinity of hippocampal excitatory synapses and , indeed , their pharmacological activation triggers surface exposure of glua1 subunits of the ampa glutamate receptor . in addition , ghrelin also stimulates phosphorylation of the nr1 subunit of nmdars , which can further facilitate activity - dependent synaptic plasticity . it would be interesting to study whether a similar mechanism is also present in the hippocampus . indeed , administration of glp-1 receptor agonists such as liraglutide increases ltp , whereas glp-1 receptor knockout mice show impairment in this form of synaptic plasticity . hence , the data summarized above indicate that structural and functional aspects of hippocampal plasticity are strongly sensitive to key mediators of glucose homeostasis . moreover , they suggest the existence of multiple interactions and synergies between the different molecular players , and understanding the details of this network appears to be one of the main goals of future research . the data reviewed in the previous paragraphs support the view that glucose homeostasis imbalances can alter signaling pathways involved in adult neurogenesis and synaptic plasticity , thereby leading to reduced mindspan ( the maintenance of mental abilities throughout life ) and increased risk of neurodegenerative disorders . conversely , the excess of nutrients impinges on brain health and impairs synaptic transmission and plasticity leading to accelerated cognitive decline ( cd ) [ 102 , 103 ] . these findings indicate that fluctuations in blood glucose levels negatively impact on brain function , even in the absence of overt t2d or impaired glucose tolerance . whereas insulin is clearly neurotrophic at moderate concentrations , too much insulin in the brain may be associated with increased amyloid- deposition due to competition for their common and main clearance mechanism , the insulin - degrading enzyme . in this regard , it has even been proposed that alzheimer 's disease may be considered a form of type 3 diabetes , based on the evidence for insulin resistance and impaired insulin - response pathways in the alzheimer's - affected brain . interestingly , in insulin - deficient rats and insulin - resistant mice , diabetes impairs hippocampus - dependent memory , impinging on both synaptic plasticity and adult neurogenesis , and the glucocorticoid system contributes to these adverse effects . in this regard , nsc proliferation and adult neurogenesis are impaired in t2d and prediabetes animal models . moreover , as mentioned above , several gut hormones are able to impact on hippocampal function . in addition , germ - free mice show a significant alteration of serotonergic system metabolites concentration and serotonergic neurotransmission in the hippocampus , which can have a negative impact on synaptic plasticity . hence , a key topic in current research is understanding which metabolic factors are most harmful to brain plasticity and which drugs suitable for metabolic disorders can also have an effect on cognitive functions . in particular , a challenge for the upcoming years will be investigating whether there are common molecular mechanisms underlying metabolic and neurodegenerative diseases and whether the glycemic memory of particular brain areas ( e.g. molecules involved in metabolic homeostasis are now recognized to exert a deep influence on hippocampal plasticity and alteration of their equilibrium has a strong impact at the functional and behavioral levels . it is worth noting that , for instance , experimental paradigms such as physical exercise or environmental enrichment , that is , coupling of motor activity to sensory stimulation , social interaction , and enhancement of exploratory behavior , dramatically affect neural plasticity of several brain districts , including the hippocampus , during development and in adulthood , as well as in aging [ 115117 ] . in this regard , understanding how brain sensitivity to insulin and other metabolic players can be controlled may be an effective way to impact on pathologies characterized by impaired neural plasticity , especially alzheimer 's disease . indeed , expression of the insulin receptor mrna is maximal during development and decreases with aging , and this , in addition to representing another similarity between insulin sensitivities inside and outside the brain , could represent a key contributor to the decline in neural plasticity of the elderly . finally , a crucial point is also represented by understanding interactions between glucose and lipid homeostasis , since soluble factors involved in this latter process , such as leptin , have also been demonstrated to modulate synaptic plasticity and to have a role in age - associated cd [ 45 , 119 , 120 ] . in summary , drawing a comprehensive picture of the interactions between metabolism , hippocampal circuits , and cognitive performance , in addition to elucidating the underlying molecular mechanisms , can represent an important step forward , from a conceptual and translational point of view , towards a deeper understanding of the mechanisms regulating neural plasticity in health and neurodegeneration .

obesity and overweight have a significant influence on reproductive functions in females as the extra body fat may cause problems related to ovulatory dysfunction and infertility . this abnormality is frequently associated with the polycystic ovary syndrome ( pcos ) . around 47% of reproductive - age women about 3075% of the patients with pcos are obese and previous data have reported that 5060% of patients with pcos have a central adiposity irrespective of their body mass index ( bmi ) . complex interaction between obesity , insulin and leptin resistance , and the endocrine abnormalities in pcos . leptin is a product of the ob gene that acts as a sensor to the hypothalamus about the adipose tissue stores as well as a regulator of food intake and the energy balance . several studies suggested that leptin may be involved in the reproductive axis function at both central and peripheral levels [ 9 , 10 ] . high leptin may interfere with the development of the mature oocyte and may directly activate ovarian 17- hydroxylase enzyme that is involved in ovarian and adrenal steroidogenesis . leptin in the circulation is present in two main forms : a protein bound and a free form that is the biological active form . the soluble leptin receptor ( sob - r ) circulates in human plasma and is accomplished by binding to leptin and symbolizes the significant leptin binding activity in humans . in lean subjects , leptin circulates principally in the bound form while in obesity the leptin circulates mainly as a free form due to small sob - r concentrations . the fraction of the total leptin concentrations to the sob - r designates the free leptin index ( fli ) . the role of sob - r in the reproductive system especially in pcos is still uncertain . few studies addressed the potential role of sob - r in pcos that showed inconsistent results [ 1618 ] . in addition , conflicting results have been described on circulating leptin levels among women with different bmi , lean , overweight , and obese women with pcos . world health organization ( who ) survey in year 2010 indicated alarming levels of obesity and overweight in qatar and it was reported that the prevalence of obesity was 38.1% while overweight was 70.2% in females . in addition , no data is available about the leptin / sob - r level among young females at the reproductive age whether they are lean or overweight / obese subjects and had clinical and biochemical features suggestive of pcos in qatar . the objective of this study is to explore the circulating total leptin hormone and the sob - r levels in pcos subjects and to assess its relationship with the metabolic and hormonal parameters in these subjects . a cross - sectional study was carried out from october 2011 to january 2013 at the health sciences department , qatar university . questionnaire about the family history of pcos , diabetes , infertility , and medical history such as the regularity of the menses , abnormal hair growth , or distribution was performed before the blood collection . the diagnosis of pcos in this study is based on nih criteria . for diagnosis of pcos , the adult female subject should present the two following criteria : menstrual irregularity such as oligomenorrhea and amenorrhea ( cycle length > 35 days ) and hyperandrogenism ( clinical by assessment of the hair growth based on the ferriman - gallwey score and/or biochemical such as elevated total testosterone > 2.0 nmol / l and free androgen index [ fai ] > 3.8 ) . the subjects were divided into two main groups : pcos subjects and control non - pcos , and each group is subdivided based upon the cutoff value of bmi of 25 kg / m into overweight / obese subgroup ( 25 kg / m ) and lean subgroup ( < 25 kg / m ) . the exclusion criteria of the study included females who have thyroid disorders , cushing syndrome , congenital adrenal hyperplasia , acromegaly , hypothalamic disorders , hyperprolactinemia and systemic inflammatory diseases and on hormonal treatment in last three months . the study received an ethical approval from qatar university ( qu - irb 58-e/11 ) , and an informed consent was obtained from each participant after explanation of the procedures in full detail . the informed consent and ethical guidelines were followed , based on the deceleration of helsinki for year 2000 . diagnosis of pcos in this study is based on nih criteria ( 3 ) . ( a ) anthropometric measurements , questionnaire , and ferriman - gallwey assessment . the weight and height were measured in kg and cm down to one decimal point , and body mass index ( bmi ) was calculated by dividing the weight ( kg ) by the square meter of height ( m ) . all participants filled out a questionnaire about their general demographic information , anthropometric measurements , and medical history ( pcos , diabetes , hypertension , and dyslipidemia and family history ) . in the second part , the subjects were asked about their menstrual history , diet , and lifestyle , including pharmaceutical history . the modified ferriman - gallwey was self - scored after explanation and demonstration in full detail to all participants . a score above 17 was considered hyperandrogenism [ ha ] as previously published in this geographical area . women were asked about the hair growth in a male pattern in 19 different body parts based on the modified ferriman - gallwey score in 2001 . ultrasound imaging was advised to those subjects who had irregular periods and/or ha to examine the ovaries for the stigma of pcos . blood samples were collected in the follicular phase of menstrual cycles from students with regular cycles and collected independently from the day of menses from students with menstrual irregularity . overnight fasting blood samples were drawn , followed by centrifugation , and serum was collected into aliquots and stored at 80c for further hormonal assay . blood glucose was measured by an enzymatic colorimetric assay in the clinical chemistry laboratory at qatar university . the test principle of the hormonal assay was chemiluminescent microparticle immunoassay ( cmia ) for the qualitative determination of the following hormones : thyroid - stimulating hormone ( tsh ) , free thyroxine ( ft4 ) prolactin , insulin , estradiol , progesterone , testosterone , dehydroepiandrostedione sulfate ( dhea - s ) , and sex hormone binding globulin ( shbg ) . the hormonal assay was measured at the endocrine chemistry lab at hmc , qatar , by architect . leptin and sob - r were measured by elisa techniques based on the manufacturer ( cat . the interassay cv% of the measured hormones was shbg 10% , estradiol 7% , progesterone for low control 10% and for medium and high controls 7% , free t4 8% , tsh 10% , dheas 10% , and testosterone 10% , prolactin 6% , and insulin 10% . the interassay cv% and intra - assay cv% were 5.5% and 8.2% for leptin and 8.7% and 7.10% for sob - r , respectively . free androgen index ( fai ) was calculated as the total testosterone level in nmol / l over the sex hormone binding globulin ( shbg ) level in nmol / l and then multiplied by 100 . the free testosterone ( ft ) was calculated from total testosterone levels , albumin , and shbg , using a specified formula that has been used as a diagnostic tool for hyperandrogenism . the cutoff value for homa - ir was 2.6 , and it is calculated as ( fasting serum insulin ( u / ml ) fasting plasma glucose ( mmol l)/22.5 ) . data were explored for outliers , skewness , and normality and transformed when necessary if the normality assumption was violated . continuous data are expressed as mean sd for normally distributed variables , median and interquartile range [ 25%75% ] for nondistributed continuous data , and number and percentage for categorical data . two student 's t - tests and nonparametric mann - whitney test were used to evaluate the differences between the continuous variables , accordingly for the analysis . anova was used to evaluate the difference between three groups if the data were normally distributed and/or wilcoxon / kruskal - wallis tests ( rank sums ) test if the data were not - normally distributed were used accordingly for the analysis . spearman correlation was used to evaluate the linear correlations between the studied variables . partial correlation analysis with adjustment for bmi was used to assess the correlations of leptin , sob - r with the different variables of the study . odds ratios ( ors ) , 95% confidence interval ( ci ) , and corresponding p values were analyzed by the logistic regression analysis to examine the relationship between pcos and cofounders . the two - tailed p < 0.05 was considered as the cutoff value for significance . spss program ( version 22 , ibm , usa ) for mac was used for all of the statistical analysis . the graphic presentation was performed by the use of the graphpad prism version 6.00 for mac ( graphpad software , la jolla , ca , usa , http://www.graphpad.com/ ) . table 1 shows the mean and sd for age and bmi and the median values and iq values for mfg , glucose , homa - ir , fai , and hormone concentrations of estradiol , progesterone , dheas , shbg , testosterone [ total ( t ) and free ( f ) ] , insulin , and tsh of the 4 studied groups . the mean age did not differ significantly between the studied groups ( p = 0.997 ) . bmi was significantly higher in overweight / obese [ ow / ob ] subjects than in the lean subjects whether they were pcos or non - pcos groups , respectively ( p < 0.0001 ) . there was a significant difference among the studied groups ( intragroup ) for bmi , mfg score , testosterone ( t and f ) , dheas , and fai , as shown in table 1 . overweight / obese pcos subjects had significantly higher levels of the mfg score , total testosterone , dheas , fai , homa ( ir ) , and free testosterone than non - pcos overweight / obese subjects as shown in table 1 . in addition , overweight / obese pcos subjects had significantly higher median values of the mfg score , testosterone , dheas , fai , homa ( ir ) , and free testosterone than other lean pcos subjects . shbg is significantly lower in ow / ob , ow / ob pcos , and lean pcos than in lean , healthy control subjects as shown in table 1 . other variables such as prolactin , tsh , free t4 , insulin , and female sex hormones are not significantly different between the various groups . the calculated homa - ir is significantly higher in ow / ob pcos than in other groups without pcos ( lean and ow / ob ) and lean pcos subjects , respectively . furthermore , we classified the study groups in subjects with and without the insulin resistance . in addition , the frequency of ir was evident in 24/78 ( 30.77% ) in all study subjects . ir was insignificantly higher ( 9/22 or 40.91% ) in ow / ob subjects than in lean subjects ( 15/56 or 26.79% ) , p = 0.278 . in addition , ir was more prevalent in ow / ob pcos ( 5/8 or 62.5% ) than in lean pcos ( 3/10 or 30.0% ) with p value = 0.133 . pcos subjects had significantly higher median leptin levels in ng / ml ( 44.29 ) than in control non - pcos ( 15.16 ) with p value = 0.013 and a significant increase of fli ( 2.81 ) versus 0.86 with p = 0.001 . in addition , a significant decrease of sob - r ( ng / ml ) was detected in pcos than in the control subjects with p value ( 0.002 ) as shown in table 2 . furthermore , to detect the effect of overweight / obesity in pcos subjects , ow / ob pcos subjects had significantly higher leptin concentrations and fli than lean pcos subjects ( p = 0.013 ; p = 0.000 ) , respectively , and a significant reduction for sob - r was detected between the two groups ( p = 0.042 ) , respectively . furthermore , to evaluate effect of pcos on leptin system in bmi - matched subjects , the results showed that pcos in lean subjects had significantly higher fli and reduced leptin receptor than in the results of lean subjects without pcos ( p = 0.002 and p = 0.041 ) , respectively , while no significant difference was detected for leptin levels in both groups ( p = 0.282 ) . further analysis of ow / ob subjects with and without pcos indicated that only leptin level was significantly increased in pcos subjects ( p = 0.029 ) without significant changes in sob - r and fli between both groups ( p = 0.786 and p = 0.172 ) , respectively . these data are presented in table 2 and figure 1 . to identify the outcome of insulin resistance ( ir ) in pcos , the results showed that fli was not significantly different between both groups of pcos with and without insulin resistance ( p = 0.613 ) , as illustrated in figure 1 . bmi and ir are well - known factors that affect leptin level and sob - r as shown in previous studies . we performed a multiple regression analysis to examine the effects of bmi and ir on leptin level . as shown in table 3 , only bmi is the significant predictor of leptin and sob - r in pcos subjects with beta unstandardized coefficients of 2.252 and 1.649 and p values of 0.008 and 0.000 , respectively . the results demonstrated that insulin resistance ( homa - ir ) is not a significant predictor of leptin and sob - r in pcos subjects with beta unstandardized coefficients of 1.716 and 1.267 and p values of 0.287 and 0.084 , respectively . next , we evaluated the relationship between the pcos , plasma leptin , and sob - r with the other parameters including the endocrine parameters and the other variables in the pcos subjects , by partial pearson 's correlation coefficient adjusted for bmi ( table 4 ) . regression analysis showed a significant positive correlation between leptin and bmi ( r = 0.588 , p = 0.011 ) and a significant negative correlation between sob - r and bmi ( r = 0.443 , p = 0.048 ) in pcos subjects ( data are not shown , in omitted figures ) . therefore , bmi was included in the partial correlation analysis since it significantly associated with leptin and its soluble receptor . the partial coefficient correlation analysis revealed that leptin level had a significant positive correlation with pcos and negative correlation with sob - r . pcos is directly significantly correlated with free testosterone , dheas , fai , irregular menstrual cycles , and leptin and inversely with sob - r , as shown in table 4 . further we calculated the odds ratio and 95% confidence intervals ( ci ) ( figure 2 ) between pcos as a dependent variable and the following independent variables bmi and homa - ir in addition to the following variables : free testosterone , leptin , dheas , and menstrual cycle , which showed significant associations with pcos and leptin . the results showed that the following independent variables have a significant impact ( p value < 0.05 ) on pcos : the menstrual cycle irregularities ( 69.2 ) times , free testosterone ( 7.9 ) times , and leptin ( 1.33 ) times as shown in figure 2 . leptin is an adipokine produced by the adipose tissue , and it is proposed to be one of the associates between obesity , insulin resistance , and pcos [ 9 , 10 ] . there is no single coherent model for the pathogenesis of pcos that could explain all observed cases . the aim of this study was to evaluate the level of the circulating leptin hormone and its sob - r in association with the clinical and metabolic parameters of pcos subjects among the female students in qatar university . this correlation can explore the role of leptin and its soluble receptor in women with pcos . the current findings of this study showed that leptin hormone and the free leptin index are significantly higher , and sob - r is significantly lower in all pcos subjects compared to the healthy control of age - matched subjects . moreover , age - matched overweight / obese pcos females showed that leptin and free leptin index are significantly higher than in lean pcos subjects while they showed opposite results for sob - r . in addition , pcos significantly increases the free leptin index and decreases the leptin receptor in lean subjects . moreover , in age- and bmi - matched ow / ob subjects , only the leptin increases in ow / ob pcos subjects without significant effects on sob - r and fli . the present data also demonstrated that leptin and soluble leptin receptor are significantly associated with pcos independently of ir and it is dependent on bmi . overweight / obese females had higher levels of leptin and biochemical parameters of hyperandrogenism including testosterone and dheas and reduced sob - r . leptin , sob - r , testosterone , and menstrual irregularities are the significant independent factors for pcos among the study subjects . several studies reported increased circulating leptin concentrations in pcos and suggested that leptin has a role in its pathogenesis [ 31 , 32 ] , which are similar to the findings of the current study . ( 1998 ) reported an increased leptin level in obese pcos subjects compared to lean pcos subjects . ( 2003 ) demonstrated an increase in leptin level in overweight women with pcos and insulin resistance . a recent study by olszanecka - glinianowicz et al . ( 2013 ) demonstrated higher leptin levels in pcos subjects compared with age- and bmi - matched control group . furthermore , they observed more elevated leptin in obese subjects than in lean pcos subjects . other studies showed elevated leptin level in obese pcos patients [ 34 , 35 ] , which is consistent with the data of the current study as shown in figure 2 . on the contrary , several studies showed no significant difference of leptin between subjects with and without pcos [ 36 , 37 ] . the difference in such studies with the current data could be due to the sample size , type of sample collection , and the heterogeneity nature of the pcos syndrome . previous studies evaluated the role of the soluble leptin receptor in pcos subjects [ 16 , 30 ] . hahn et al . ( 2006 ) demonstrated a significant stepwise increase in leptin , with a significant stepwise reduction in sob - r from lean to overweight to obese subjects , respectively . the investigator also reported that lean pcos patients had lower sob - r levels and higher free leptin indices , compared with bmi - matched lean controls , respectively . these data are consistent with the findings of the current study where ( 1 ) sob - r levels were reduced in all pcos subjects compared to bmi - matched healthy controls . in addition , in overweight and obesity pcos subjects , the sob - r level decreases and fli increases significantly compared with lean pcos subjects . moreover , in lean pcos subjects , the sob - r level decreases significantly compared to lean control ( pcos ) subjects while fli showed opposite effects . similar findings were demonstrated in pcos patients from different countries [ 27 , 36 ] . ( 2006 ) demonstrated no significant change in leptin , sob - r , and fli between pcos women with insulin resistance and bmi - matched controls , which is not consistent with the current findings . the difference in such study with the present data could be due to the sample size , type of sample collection , and the heterogeneity nature of the pcos syndrome . we also assessed the possible association between the leptin system ( hormone and its soluble receptor ) and the hormonal profile , bmi , and the menstrual irregularities . only a significant association was detected between bmi , leptin , and leptin receptor as shown in previous studies [ 30 , 38 ] . moreover , the current data demonstrated that soluble leptin receptor is negatively correlated with leptin , as reported in previous studies [ 30 , 39 ] . the correlation between total leptin and leptin receptor in pcos cohort with the female gonadal , thyroid , and androgen hormones failed to detect any significant correlations after adjusting bmi . such findings are in agreement with previous studies [ 30 , 36 ] though previous studies reported a significant correlation between dheas levels and the free leptin index . conflicting results regarding the correlations between androgen and estradiol with the leptin were observed in other studies [ 16 , 40 ] . the controversy could be due to differences in the subject populations and the sample size and the heterogenic nature of the pcos syndrome . hyperinsulinemia and insulin resistance play a critical role in the development of the polycystic ovary syndrome ( pcos ) . insulin upregulates leptin mrna in adipocytes , signifying its potential role in leptin secretion . on the contrary insulin resistance and hyperinsulinemia are present in up to 65% of obese women with pcos and in up to 20% of lean women with pcos . the high leptin in hyperinsulinemia of pcos women may be a secondary consequence of insulin - stimulated leptin synthesis . several studies showed that leptin is correlated with insulin level and insulin resistance in pcos subjects [ 34 , 44 ] , though other studies demonstrated no correlation [ 16 , 32 ] . the data of the current study did not illustrate any noteworthy correlation between insulin and leptin and homa with leptin and sob - r . the controversy could be attributed to the differences in the sample size , the study populations , and the heterogenic nature of the pcos syndrome . in obesity , leptin resistance or inadequate leptin action is indicated by high leptin level , and several mechanisms were postulated to explain the defect of leptin action in obese subjects . hahn et al . ( 2006 ) hypothesized that the reduction of sob - r is a tentative mechanism to overcome the leptin resistance or the defective leptin action . in the current study , pcos and the obesity status increases leptin level and decresaes the soluble leptin receptor . to gain insight into these findings , we evaluated the impact of both factors , the body mass index as a representative of the obesity and the insulin resistance calculated by homa - ir in the current study by regression analysis on leptin and sob - r levels . the current data indicated that hyperleptinemia and decreased sob - r are independent of insulin resistance and only related to the bmi and adiposity of the pcos subjects . these findings are supported by earlier studies [ 17 , 30 ] , which showed correlations of leptin with adiposity independent of insulin resistance . thus , the correlation between the bmi and leptin and its sob - r observed in this study and in the previous studies may indicate that adiposity ( bmi ) may represent the primary determinant of the leptin system in pcos , as well as being known in the general populations . thus , it could be concluded that the higher incidence of obesity , rather than hyperinsulinemia , could be a confounding factor in hyperleptinemia observed in ow / ob pcos patients . furthermore , the current data showed that even with bmi - matched subjects , in lean and in ow / ob subjects with and without pcos leptin levels , sob - r and fli were significantly different . such findings could also highlight a direct role of leptin in the pathogenesis of pcos independent of obesity / bmi and insulin resistance and other hormonal factors such as androgens in the current study . the reduction of sob - r in pcos could be a tentative mechanism to overcome the defect of leptin action in these patients . the difference in androgen levels in the studied subgroups could explain the current results for leptin and sob - r and fli in bmi - matched subjects as shown in previous studies [ 28 , 47 ] . the genetic background variability and the heterogenicity of pcos could be another factor for the variation of leptin and its soluble receptor in the bmi - matched pcos subjects . our data may require further studies to evaluate the role of leptin and the soluble receptors in a well - characterized large cohort . it seems that heterogenicity of pcos could explain different mechanisms for leptin 's action on the reproductive system in these patients . the current data showed that the leptin and sob - r with testosterone and menstrual irregularities are the significant factors contributing to the pcos in the present study . the current finding highlights that leptin and sob - r could be independent factors in the pathogenesis of the pcos , independent of bmi . several studies suggested a different mechanism through which leptin could affect the reproductive functions [ 9 , 10 ] . low leptin level in amenorrheic athletes with low body fat and the leptin application in females with hypothalamic amenorrhea restore the reproduction . in addition , hyperleptinemia may inhibit the development of the mature oocyte development directly and affect ovarian and adrenal steroidogenesis . defective leptin action that is observed in pcos subjects and its consequences may be explained by the impaired central action of leptin in the hypothalamus with more weight gain and adiposity and/or peripherally via the impaired action of leptin on mature ovum production . the soluble leptin receptor ( sob - r ) that circulates in human plasma is capable of binding to leptin that could protect against obesity . the role of sob - r in the reproductive system especially in pcos could be explained to compensate for the defective leptin action observed in pcos subjects whether lean or obese as suggested by another study . the present study has some limitations , such as the small size of the studied samples , the narrow range for the age of the studied subjects , and the cross - sectional nature of the study . further studies are needed with large sample size , including a wide variety of ages and occupations and of longitudinal nature . this could help to understand the detailed associations and actions of insulin and androgen on leptin , sob - r , and fli in pcos . in conclusion , pcos is associated with hyperleptinemia , decreased sob - r , and increased free leptin index . decreased sob - r could be a compensatory mechanism for the defective action of leptin . elevated serum leptin in pcos women could be due to the positive correlation between leptin and bmi and is independent of insulin resistance . leptin and its soluble receptor are implicated in the pathophysiology of pcos that needs further investigations .

background and aim . leptin has two forms in the circulation : free and bound forms . the soluble leptin receptor ( sob - r ) circulates in the blood and can bind to leptin . the aim of this study is to assess the concentrations of the leptin and the sob - r in pcos and its relation to adiposity , insulin resistance , and androgens . methods . a cross - sectional study included 78 female students aged 1725 years . fasting serum leptin and sob - r concentrations were measured . the anthropometric variables and the hormonal profile such as insulin , female and male sex hormones , and prolactin were assessed . results . in pcos , leptin level ( ng / ml ) and free leptin index ( fli ) increased significantly while sob - r ( ng / ml ) significantly decreased compared to control subjects . in age - matched subjects , obese pcos had increased leptin level in ng / ml ( median level with interquartile levels ) of 45.67 ( 41.9848.04 ) and decreased sob - r in ng / ml 11.47 ( 7.5916.44 ) compared to lean pcos 16.97 ( 10.6045.55 ) for leptin and 16.62 ( 11.6117.96 ) for sob - r with p values 0.013 and 0.042 , respectively . however , body mass index ( bmi ) is significantly correlated with leptin and s - obr , while no significant correlations with parameters of insulin resistance were detected . conclusion . pcos is associated with hyperleptinemia and increased free leptin index . decreased sob - r could be a compensatory mechanism for the defective action of leptin .

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

around 47% of reproductive - age women about 3075% of the patients with pcos are obese and previous data have reported that 5060% of patients with pcos have a central adiposity irrespective of their body mass index ( bmi ) . complex interaction between obesity , insulin and leptin resistance , and the endocrine abnormalities in pcos . leptin in the circulation is present in two main forms : a protein bound and a free form that is the biological active form . the soluble leptin receptor ( sob - r ) circulates in human plasma and is accomplished by binding to leptin and symbolizes the significant leptin binding activity in humans . in lean subjects , leptin circulates principally in the bound form while in obesity the leptin circulates mainly as a free form due to small sob - r concentrations . the fraction of the total leptin concentrations to the sob - r designates the free leptin index ( fli ) . the role of sob - r in the reproductive system especially in pcos is still uncertain . few studies addressed the potential role of sob - r in pcos that showed inconsistent results [ 1618 ] . in addition , no data is available about the leptin / sob - r level among young females at the reproductive age whether they are lean or overweight / obese subjects and had clinical and biochemical features suggestive of pcos in qatar . the objective of this study is to explore the circulating total leptin hormone and the sob - r levels in pcos subjects and to assess its relationship with the metabolic and hormonal parameters in these subjects . a cross - sectional study was carried out from october 2011 to january 2013 at the health sciences department , qatar university . questionnaire about the family history of pcos , diabetes , infertility , and medical history such as the regularity of the menses , abnormal hair growth , or distribution was performed before the blood collection . the diagnosis of pcos in this study is based on nih criteria . for diagnosis of pcos , the adult female subject should present the two following criteria : menstrual irregularity such as oligomenorrhea and amenorrhea ( cycle length > 35 days ) and hyperandrogenism ( clinical by assessment of the hair growth based on the ferriman - gallwey score and/or biochemical such as elevated total testosterone > 2.0 nmol / l and free androgen index [ fai ] > 3.8 ) . the subjects were divided into two main groups : pcos subjects and control non - pcos , and each group is subdivided based upon the cutoff value of bmi of 25 kg / m into overweight / obese subgroup ( 25 kg / m ) and lean subgroup ( < 25 kg / m ) . the study received an ethical approval from qatar university ( qu - irb 58-e/11 ) , and an informed consent was obtained from each participant after explanation of the procedures in full detail . diagnosis of pcos in this study is based on nih criteria ( 3 ) . the weight and height were measured in kg and cm down to one decimal point , and body mass index ( bmi ) was calculated by dividing the weight ( kg ) by the square meter of height ( m ) . all participants filled out a questionnaire about their general demographic information , anthropometric measurements , and medical history ( pcos , diabetes , hypertension , and dyslipidemia and family history ) . the test principle of the hormonal assay was chemiluminescent microparticle immunoassay ( cmia ) for the qualitative determination of the following hormones : thyroid - stimulating hormone ( tsh ) , free thyroxine ( ft4 ) prolactin , insulin , estradiol , progesterone , testosterone , dehydroepiandrostedione sulfate ( dhea - s ) , and sex hormone binding globulin ( shbg ) . leptin and sob - r were measured by elisa techniques based on the manufacturer ( cat . the interassay cv% of the measured hormones was shbg 10% , estradiol 7% , progesterone for low control 10% and for medium and high controls 7% , free t4 8% , tsh 10% , dheas 10% , and testosterone 10% , prolactin 6% , and insulin 10% . the interassay cv% and intra - assay cv% were 5.5% and 8.2% for leptin and 8.7% and 7.10% for sob - r , respectively . free androgen index ( fai ) was calculated as the total testosterone level in nmol / l over the sex hormone binding globulin ( shbg ) level in nmol / l and then multiplied by 100 . the cutoff value for homa - ir was 2.6 , and it is calculated as ( fasting serum insulin ( u / ml ) fasting plasma glucose ( mmol l)/22.5 ) . partial correlation analysis with adjustment for bmi was used to assess the correlations of leptin , sob - r with the different variables of the study . odds ratios ( ors ) , 95% confidence interval ( ci ) , and corresponding p values were analyzed by the logistic regression analysis to examine the relationship between pcos and cofounders . table 1 shows the mean and sd for age and bmi and the median values and iq values for mfg , glucose , homa - ir , fai , and hormone concentrations of estradiol , progesterone , dheas , shbg , testosterone [ total ( t ) and free ( f ) ] , insulin , and tsh of the 4 studied groups . bmi was significantly higher in overweight / obese [ ow / ob ] subjects than in the lean subjects whether they were pcos or non - pcos groups , respectively ( p < 0.0001 ) . there was a significant difference among the studied groups ( intragroup ) for bmi , mfg score , testosterone ( t and f ) , dheas , and fai , as shown in table 1 . overweight / obese pcos subjects had significantly higher levels of the mfg score , total testosterone , dheas , fai , homa ( ir ) , and free testosterone than non - pcos overweight / obese subjects as shown in table 1 . in addition , overweight / obese pcos subjects had significantly higher median values of the mfg score , testosterone , dheas , fai , homa ( ir ) , and free testosterone than other lean pcos subjects . shbg is significantly lower in ow / ob , ow / ob pcos , and lean pcos than in lean , healthy control subjects as shown in table 1 . other variables such as prolactin , tsh , free t4 , insulin , and female sex hormones are not significantly different between the various groups . the calculated homa - ir is significantly higher in ow / ob pcos than in other groups without pcos ( lean and ow / ob ) and lean pcos subjects , respectively . in addition , ir was more prevalent in ow / ob pcos ( 5/8 or 62.5% ) than in lean pcos ( 3/10 or 30.0% ) with p value = 0.133 . pcos subjects had significantly higher median leptin levels in ng / ml ( 44.29 ) than in control non - pcos ( 15.16 ) with p value = 0.013 and a significant increase of fli ( 2.81 ) versus 0.86 with p = 0.001 . in addition , a significant decrease of sob - r ( ng / ml ) was detected in pcos than in the control subjects with p value ( 0.002 ) as shown in table 2 . furthermore , to detect the effect of overweight / obesity in pcos subjects , ow / ob pcos subjects had significantly higher leptin concentrations and fli than lean pcos subjects ( p = 0.013 ; p = 0.000 ) , respectively , and a significant reduction for sob - r was detected between the two groups ( p = 0.042 ) , respectively . furthermore , to evaluate effect of pcos on leptin system in bmi - matched subjects , the results showed that pcos in lean subjects had significantly higher fli and reduced leptin receptor than in the results of lean subjects without pcos ( p = 0.002 and p = 0.041 ) , respectively , while no significant difference was detected for leptin levels in both groups ( p = 0.282 ) . further analysis of ow / ob subjects with and without pcos indicated that only leptin level was significantly increased in pcos subjects ( p = 0.029 ) without significant changes in sob - r and fli between both groups ( p = 0.786 and p = 0.172 ) , respectively . to identify the outcome of insulin resistance ( ir ) in pcos , the results showed that fli was not significantly different between both groups of pcos with and without insulin resistance ( p = 0.613 ) , as illustrated in figure 1 . bmi and ir are well - known factors that affect leptin level and sob - r as shown in previous studies . as shown in table 3 , only bmi is the significant predictor of leptin and sob - r in pcos subjects with beta unstandardized coefficients of 2.252 and 1.649 and p values of 0.008 and 0.000 , respectively . the results demonstrated that insulin resistance ( homa - ir ) is not a significant predictor of leptin and sob - r in pcos subjects with beta unstandardized coefficients of 1.716 and 1.267 and p values of 0.287 and 0.084 , respectively . next , we evaluated the relationship between the pcos , plasma leptin , and sob - r with the other parameters including the endocrine parameters and the other variables in the pcos subjects , by partial pearson 's correlation coefficient adjusted for bmi ( table 4 ) . regression analysis showed a significant positive correlation between leptin and bmi ( r = 0.588 , p = 0.011 ) and a significant negative correlation between sob - r and bmi ( r = 0.443 , p = 0.048 ) in pcos subjects ( data are not shown , in omitted figures ) . therefore , bmi was included in the partial correlation analysis since it significantly associated with leptin and its soluble receptor . the partial coefficient correlation analysis revealed that leptin level had a significant positive correlation with pcos and negative correlation with sob - r . pcos is directly significantly correlated with free testosterone , dheas , fai , irregular menstrual cycles , and leptin and inversely with sob - r , as shown in table 4 . further we calculated the odds ratio and 95% confidence intervals ( ci ) ( figure 2 ) between pcos as a dependent variable and the following independent variables bmi and homa - ir in addition to the following variables : free testosterone , leptin , dheas , and menstrual cycle , which showed significant associations with pcos and leptin . leptin is an adipokine produced by the adipose tissue , and it is proposed to be one of the associates between obesity , insulin resistance , and pcos [ 9 , 10 ] . the aim of this study was to evaluate the level of the circulating leptin hormone and its sob - r in association with the clinical and metabolic parameters of pcos subjects among the female students in qatar university . this correlation can explore the role of leptin and its soluble receptor in women with pcos . the current findings of this study showed that leptin hormone and the free leptin index are significantly higher , and sob - r is significantly lower in all pcos subjects compared to the healthy control of age - matched subjects . moreover , age - matched overweight / obese pcos females showed that leptin and free leptin index are significantly higher than in lean pcos subjects while they showed opposite results for sob - r . in addition , pcos significantly increases the free leptin index and decreases the leptin receptor in lean subjects . moreover , in age- and bmi - matched ow / ob subjects , only the leptin increases in ow / ob pcos subjects without significant effects on sob - r and fli . the present data also demonstrated that leptin and soluble leptin receptor are significantly associated with pcos independently of ir and it is dependent on bmi . overweight / obese females had higher levels of leptin and biochemical parameters of hyperandrogenism including testosterone and dheas and reduced sob - r . leptin , sob - r , testosterone , and menstrual irregularities are the significant independent factors for pcos among the study subjects . several studies reported increased circulating leptin concentrations in pcos and suggested that leptin has a role in its pathogenesis [ 31 , 32 ] , which are similar to the findings of the current study . ( 1998 ) reported an increased leptin level in obese pcos subjects compared to lean pcos subjects . ( 2003 ) demonstrated an increase in leptin level in overweight women with pcos and insulin resistance . other studies showed elevated leptin level in obese pcos patients [ 34 , 35 ] , which is consistent with the data of the current study as shown in figure 2 . the difference in such studies with the current data could be due to the sample size , type of sample collection , and the heterogeneity nature of the pcos syndrome . previous studies evaluated the role of the soluble leptin receptor in pcos subjects [ 16 , 30 ] . ( 2006 ) demonstrated a significant stepwise increase in leptin , with a significant stepwise reduction in sob - r from lean to overweight to obese subjects , respectively . the investigator also reported that lean pcos patients had lower sob - r levels and higher free leptin indices , compared with bmi - matched lean controls , respectively . these data are consistent with the findings of the current study where ( 1 ) sob - r levels were reduced in all pcos subjects compared to bmi - matched healthy controls . in addition , in overweight and obesity pcos subjects , the sob - r level decreases and fli increases significantly compared with lean pcos subjects . moreover , in lean pcos subjects , the sob - r level decreases significantly compared to lean control ( pcos ) subjects while fli showed opposite effects . ( 2006 ) demonstrated no significant change in leptin , sob - r , and fli between pcos women with insulin resistance and bmi - matched controls , which is not consistent with the current findings . the difference in such study with the present data could be due to the sample size , type of sample collection , and the heterogeneity nature of the pcos syndrome . we also assessed the possible association between the leptin system ( hormone and its soluble receptor ) and the hormonal profile , bmi , and the menstrual irregularities . only a significant association was detected between bmi , leptin , and leptin receptor as shown in previous studies [ 30 , 38 ] . moreover , the current data demonstrated that soluble leptin receptor is negatively correlated with leptin , as reported in previous studies [ 30 , 39 ] . the correlation between total leptin and leptin receptor in pcos cohort with the female gonadal , thyroid , and androgen hormones failed to detect any significant correlations after adjusting bmi . such findings are in agreement with previous studies [ 30 , 36 ] though previous studies reported a significant correlation between dheas levels and the free leptin index . the controversy could be due to differences in the subject populations and the sample size and the heterogenic nature of the pcos syndrome . hyperinsulinemia and insulin resistance play a critical role in the development of the polycystic ovary syndrome ( pcos ) . several studies showed that leptin is correlated with insulin level and insulin resistance in pcos subjects [ 34 , 44 ] , though other studies demonstrated no correlation [ 16 , 32 ] . the data of the current study did not illustrate any noteworthy correlation between insulin and leptin and homa with leptin and sob - r . the controversy could be attributed to the differences in the sample size , the study populations , and the heterogenic nature of the pcos syndrome . in obesity , leptin resistance or inadequate leptin action is indicated by high leptin level , and several mechanisms were postulated to explain the defect of leptin action in obese subjects . ( 2006 ) hypothesized that the reduction of sob - r is a tentative mechanism to overcome the leptin resistance or the defective leptin action . in the current study , pcos and the obesity status increases leptin level and decresaes the soluble leptin receptor . to gain insight into these findings , we evaluated the impact of both factors , the body mass index as a representative of the obesity and the insulin resistance calculated by homa - ir in the current study by regression analysis on leptin and sob - r levels . the current data indicated that hyperleptinemia and decreased sob - r are independent of insulin resistance and only related to the bmi and adiposity of the pcos subjects . these findings are supported by earlier studies [ 17 , 30 ] , which showed correlations of leptin with adiposity independent of insulin resistance . thus , the correlation between the bmi and leptin and its sob - r observed in this study and in the previous studies may indicate that adiposity ( bmi ) may represent the primary determinant of the leptin system in pcos , as well as being known in the general populations . furthermore , the current data showed that even with bmi - matched subjects , in lean and in ow / ob subjects with and without pcos leptin levels , sob - r and fli were significantly different . such findings could also highlight a direct role of leptin in the pathogenesis of pcos independent of obesity / bmi and insulin resistance and other hormonal factors such as androgens in the current study . the reduction of sob - r in pcos could be a tentative mechanism to overcome the defect of leptin action in these patients . the difference in androgen levels in the studied subgroups could explain the current results for leptin and sob - r and fli in bmi - matched subjects as shown in previous studies [ 28 , 47 ] . the genetic background variability and the heterogenicity of pcos could be another factor for the variation of leptin and its soluble receptor in the bmi - matched pcos subjects . our data may require further studies to evaluate the role of leptin and the soluble receptors in a well - characterized large cohort . the current data showed that the leptin and sob - r with testosterone and menstrual irregularities are the significant factors contributing to the pcos in the present study . the current finding highlights that leptin and sob - r could be independent factors in the pathogenesis of the pcos , independent of bmi . low leptin level in amenorrheic athletes with low body fat and the leptin application in females with hypothalamic amenorrhea restore the reproduction . defective leptin action that is observed in pcos subjects and its consequences may be explained by the impaired central action of leptin in the hypothalamus with more weight gain and adiposity and/or peripherally via the impaired action of leptin on mature ovum production . the soluble leptin receptor ( sob - r ) that circulates in human plasma is capable of binding to leptin that could protect against obesity . the role of sob - r in the reproductive system especially in pcos could be explained to compensate for the defective leptin action observed in pcos subjects whether lean or obese as suggested by another study . the present study has some limitations , such as the small size of the studied samples , the narrow range for the age of the studied subjects , and the cross - sectional nature of the study . this could help to understand the detailed associations and actions of insulin and androgen on leptin , sob - r , and fli in pcos . in conclusion , pcos is associated with hyperleptinemia , decreased sob - r , and increased free leptin index . decreased sob - r could be a compensatory mechanism for the defective action of leptin . elevated serum leptin in pcos women could be due to the positive correlation between leptin and bmi and is independent of insulin resistance . leptin and its soluble receptor are implicated in the pathophysiology of pcos that needs further investigations .

the thalassemias together with sickle cell anemia ( sca ) and its variants are the world 's most common form of inherited anemia . the beta thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult beta - globin gene . beta thalassemia is characterized by reduced synthesis of the hemoglobin subunit beta globin protein that results in microcytic hypochromic anemia , an abnormal peripheral blood smear with nucleated red blood cells , and reduced amounts of hemoglobin a on hemoglobin analysis . for instance , beta thalassemia major ( btm ) is a severe transfusion - dependent anemia . sickle cell disease ( scd ) is an autosomal recessive disorder in the gene encoding the -chain of hemoglobin causing a weakening systemic syndrome characterized by chronic anemia , acute painful episodes , tissue ischemia , and chronic organ damage in addition to a significant reduction in life expectancy . sca is the homozygoid form of scd , which includes a group of genetic disorders characterized by production of an abnormal hemoglobin s , hemolysis , and vasoocclusive phenomena with recurrent painful episodes that can lead to life - long disabilities and death . the patients with transfusion - dependent iron overload conditions such as btm and sca require frequent blood transfusions . the conventional approach to treatment of btm and sca is based on the correction of hemoglobin status through regular blood transfusions and iron chelation therapy ( ict ) for iron overload . on the other hand , iron overload causes iron deposition and accumulation in the liver , heart , skin , and other tissues resulting in serious tissue damages . thus , there is a need to remove excess iron with chelation therapy using deferoxamine ( dfo ) ( desferal ; novartis pharma ag , basel , switzerland ) , deferiprone ( dfp ) ( ferriprox ; apotex inc . , toronto , canada ) , deferasirox ( dfx ) ( exjade ; novartis pharma ag , basel , switzerland ) , or in combination with each other ( dfo - dfp or dfp - dfx ) to avoid the serious clinical sequelae associated with iron accumulation in target organs , morbidity , and mortality . on the other hand , due to the effects of the diseases and their treatments , although dfo , which is administered intravenously or subcutaneously , is clinically effective at removing excess iron , many patients are not satisfactorily chelated by it . the reasons for the poor compliance of patients to dfo include cost of the drug , pump and tubing and its side effects ( such as local problems at the site of the infusions , hematological toxicity , allergy , shortness of breathless , headaches , dizziness , and yersinia infection ) that negatively affect patient 's health - related quality of life ( hrqol ) . dfx and dfp are orally effective iron chelators alternative to dfo that have proven their efficacy in reducing iron burden in addition to increase in patient 's compliance . however , these drugs have also unwanted side effects that negatively impact on patient 's adherence to ict ( nausea , vomiting , abdominal pain , neutropenia / agranulocytosis , arthralgia , rise in transaminases , and zinc deficiency with dfp , and diarrhea , abdominal pain , nausea , vomiting , changes in renal and liver function , auditory and ocular alterations , skin rash , headache , and dizziness with dfx ) . since each chelation regimen has a distinct safety / efficacy profile and particular costs associated with its use , when choosing a chelator regimen , physicians , patients , parents , and providers may consider a variety of factors , including the severity of iron overload , administration schedule , and adverse effect profile . dfx is one of the most widely used iron chelators despite warnings of life - threatening toxic side effects . to better understand the potential impact of current therapeutic approaches for iron overload , it is important to understand the clinical effectiveness , patient acceptability , and side effects of dfx in addition to patient 's hrqol . therefore , the aim of this single - center observational study was to evaluate the quality of life , clinical effectiveness , and satisfaction in pediatric and adult patients with btm and sca receiving dfx chelation therapy . the single - center observational study was conducted among 37 pediatric and 35 adult patients with btm or sca who received dfx ( exjade ; novartis pharma stein ag , stein , switzerland ) ( 2040 mg / kg / day ) ict at the hematology units of departments of internal medicine and pediatrics , faculty of medicine , mersin university , mersin , turkey from december 2013 to may 2014 participated . the protocol of this study was approved by mersin university clinical research ethics committee and pharmaceuticals and medical devices administration of turkey . the inclusion criteria were ( 1 ) patients willing to participate in the study , ( 2 ) patients diagnosed with btm or sca , and ( 3 ) patients receiving dfx for at least 6 months . the exclusion criteria were ( 1 ) patients not meeting the inclusion criteria , ( 2 ) patients not willing to participate in the study , ( 3 ) patients diagnosed with other types of anemia , ( 4 ) patients not receiving dfx for at least 6 months , and ( 5 ) patients having other conditions such as physical and/or mental difficulties which may affect their quality of life . at the beginning of the study , all eligible patients and/or their parents were informed of the objectives of the study and assured that all information would remain confidential . after taking signed written informed consent from the patients and/or their parents , the following data were obtained : ( 1 ) demographic and clinical characteristics of pediatric and adult patients ( using case report form and demographic data collection form ) , ( 2 ) health status of pediatric patients ( using child health questionnaire - parent form ; chq - pf50 ) , ( 3 ) quality of life of adult patients ( using life quality survey short form-36 ; [ sf-36 ] ) , ( 4 ) effectiveness of ict in pediatric and adult patients ( using case report form and ict satisfaction survey ) , and ( 5 ) compliance to ict in pediatric and adult patients ( using ict satisfaction survey ) . the chq - pf50 ( for children aged 518 years ) measures hrqol children as reported by the parent . the form contains 50 items yielding 15 subtitles : general health , physical functioning , role / social limitations - emotional / behavioral , role / social limitations - physical , bodily pain / discomfort , behavior , global behavior , emotional state , self - esteem , general health state , health transition , parent impact - emotional , parent impact - time , family activities , and family cohesion . in this study , the items were scored and the computed scores were transformed to a scale from 0 to 100 . sf-36 is a self - administered generic questionnaire that measures two major subjective health concepts ( i.e. , physical and mental health ) . the form comprises 36 multichoice questions yielding a profile of eight concepts : ( 1 ) physical functioning , ( 2 ) physical role limitation , ( 3 ) pain , ( 4 ) mental health , ( 5 ) emotional role limitation , ( 6 ) social functioning , ( 7 ) vitality , and ( 8) general health perception . the reliability and validity of sf-36 are well documented in all available language versions . in this study , responses to each of the sf-36 items were scored and summed according to a standardized scoring protocol and expressed as a score on a 0100 scale for each of the eight health concepts . the ict satisfaction survey used in the study consisted of satisfaction - specific items measuring four domains : ( 1 ) perceived effectiveness , ( 2 ) acceptance / approval , ( 3 ) burden of ict , and ( 4 ) side effects . patient responses to the items comprising these domains were scored , and the scores were transformed to a scale from 0 ( worst satisfaction ) to 100 ( best satisfaction ) . quantitative variables regarding health status of pediatric patients and compliance to ict in pediatric and adult patients were described in the form of mean standard deviation ( sd ) , median , and range . statistical analysis was based on descriptive statistic procedures , and the comparison of means between two different groups was performed with the student 's t - test and mann whitney u - test , for variables with normal and skewed distribution , respectively , stata / mp11 package ( statacorp lp , tx , usa ) or graphpad prism version 5.01 for windows ( graphpad software , inc . , ca , usa ) . the single - center observational study was conducted among 37 pediatric and 35 adult patients with btm or sca who received dfx ( exjade ; novartis pharma stein ag , stein , switzerland ) ( 2040 mg / kg / day ) ict at the hematology units of departments of internal medicine and pediatrics , faculty of medicine , mersin university , mersin , turkey from december 2013 to may 2014 participated . the protocol of this study was approved by mersin university clinical research ethics committee and pharmaceuticals and medical devices administration of turkey . the inclusion criteria were ( 1 ) patients willing to participate in the study , ( 2 ) patients diagnosed with btm or sca , and ( 3 ) patients receiving dfx for at least 6 months . the exclusion criteria were ( 1 ) patients not meeting the inclusion criteria , ( 2 ) patients not willing to participate in the study , ( 3 ) patients diagnosed with other types of anemia , ( 4 ) patients not receiving dfx for at least 6 months , and ( 5 ) patients having other conditions such as physical and/or mental difficulties which may affect their quality of life . at the beginning of the study , all eligible patients and/or their parents were informed of the objectives of the study and assured that all information would remain confidential . after taking signed written informed consent from the patients and/or their parents , the following data were obtained : ( 1 ) demographic and clinical characteristics of pediatric and adult patients ( using case report form and demographic data collection form ) , ( 2 ) health status of pediatric patients ( using child health questionnaire - parent form ; chq - pf50 ) , ( 3 ) quality of life of adult patients ( using life quality survey short form-36 ; [ sf-36 ] ) , ( 4 ) effectiveness of ict in pediatric and adult patients ( using case report form and ict satisfaction survey ) , and ( 5 ) compliance to ict in pediatric and adult patients ( using ict satisfaction survey ) . the chq - pf50 ( for children aged 518 years ) measures hrqol children as reported by the parent . the form contains 50 items yielding 15 subtitles : general health , physical functioning , role / social limitations - emotional / behavioral , role / social limitations - physical , bodily pain / discomfort , behavior , global behavior , emotional state , self - esteem , general health state , health transition , parent impact - emotional , parent impact - time , family activities , and family cohesion . in this study , the items were scored and the computed scores were transformed to a scale from 0 to 100 . sf-36 is a self - administered generic questionnaire that measures two major subjective health concepts ( i.e. , physical and mental health ) . the form comprises 36 multichoice questions yielding a profile of eight concepts : ( 1 ) physical functioning , ( 2 ) physical role limitation , ( 3 ) pain , ( 4 ) mental health , ( 5 ) emotional role limitation , ( 6 ) social functioning , ( 7 ) vitality , and ( 8) general health perception . the reliability and validity of sf-36 are well documented in all available language versions . in this study , responses to each of the sf-36 items were scored and summed according to a standardized scoring protocol and expressed as a score on a 0100 scale for each of the eight health concepts . the ict satisfaction survey used in the study consisted of satisfaction - specific items measuring four domains : ( 1 ) perceived effectiveness , ( 2 ) acceptance / approval , ( 3 ) burden of ict , and ( 4 ) side effects . patient responses to the items comprising these domains were scored , and the scores were transformed to a scale from 0 ( worst satisfaction ) to 100 ( best satisfaction ) . quantitative variables regarding health status of pediatric patients and compliance to ict in pediatric and adult patients were described in the form of mean standard deviation ( sd ) , median , and range . statistical analysis was based on descriptive statistic procedures , and the comparison of means between two different groups was performed with the student 's t - test and mann whitney u - test , for variables with normal and skewed distribution , respectively , stata / mp11 package ( statacorp lp , tx , usa ) or graphpad prism version 5.01 for windows ( graphpad software , inc . , the demographic and clinical characteristics of the pediatric and adult patients are summarized in tables 1 and 2 , respectively . the study population comprised 37 pediatric ( btm : n = 25 [ 52% female and 48% male ] ; sca : n = 12 [ 50% female and 50% male ] ) and 35 adult ( btm : n = 14 [ 43% female and 57% male ] ; sca : n = 21 [ 62% female and 38% male ] ) patients with btm or sca . all patients were receiving periodic blood transfusions . at the time of the study visit , the reasons for choosing their therapy were physician 's recommendation , poor adherence to dfo , dfp , or dfx , and the failure of reimbursement system for dfo . the percentages of pediatric patients with btm or sca who had formal education were 88% and 83% , respectively . in the adult patients with btm or sca , the percentages were 86% and 100% , respectively . eight ( 32% ) and six ( 50% ) of pediatric patients with btm or sca had undergone splenectomy . two ( 8% ) pediatric patients with btm had hepatomegaly or splenomegaly and one ( 4% ) adult patient with sca had hepatosplenomegaly . the patients also had one or more concomitant diseases / conditions and were using other drugs for their diseases in addition to the iron chelators . demographic and clinical characteristics of deferasirox - treated pediatric patients demographic and clinical characteristics of deferasirox - treated adult patients table 3 shows the chq - pf50 summary scores regarding hrqol in dfx - treated pediatric patients with btm or sca . in particular , the mean chq - pf50 scores in the patients with btm ranged from 86.0 11.0 ( n = 25 ) for the family activities to 47.2 10.6 ( n = 25 ) , the general health state . in the patients with sca , the scores ranged from 85.0 15.1 ( n = 12 ) for the general health state . the scores for general health , physical functioning , role / social limitations - physical , parent impact - time , and family activities were significantly lower , and the scores for health transition were higher in the patients with sca than the patients with btm ( p < 0.05 ) . child health questionnaire - parent form 50 summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the sf-36 summary scores regarding the quality of life in adult patients with btm or sca who received dfx are shown in table 4 . in particular , the mean sf-36 scores in the dfx - treated patients with btm ranged from 81.0 12.6 ( n = 14 ) for the physical functioning to 38.1 41.1 ( n = 14 ) the emotional role limitation . the scores in the dfx - treated patients with sca ranged from 58.3 12.5 ( n = 21 ) for the general health perception to 22.6 39.5 ( n = 21 ) the physical role limitation . when compared with the scores of the dfx - treated patients with btm , the scores for physical functioning , physical role limitation , pain , social functioning , and vitality were significantly lower in the dfx - treated patients with sca ( p < 0.05 ) . short form-36 summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 5 details hematological and biochemical parameters in dfx - treated pediatric patients with btm or sca . as a main index for effectiveness of ict , mean serum ferritin levels were higher than the normal values ( 7.0140.0 ng / ml ) in the pediatric patients receiving dfx ( btm : 2040.0 1391 ng / ml , n = 25 ; sca : 937.9 678.4 ng / ml , n = 12 ) . in the patients with btm , the values regarding platelet , platelet percentage , erythrocyte distribution width ( edw ) , total bilirubin , direct bilirubin , alanine aminotransferase ( alt ) , and aspartate transaminase ( ast ) were found to be higher than the normal values . in the patients with oral hypoglycaemic agent , the values for platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , ast , and c - reactive protein ( crp ) were higher than the normal values . however , the values regarding hemoglobin , hematocrit , and erythrocyte were lower than the normal values in both patient groups . in addition , the values regarding other hematological and biochemical parameters measured were in their normal ranges . moreover , serum ferritin levels were lower , and the values for mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , lymphocyte , monocyte , and monocyte percentage were significantly higher in the pediatric patients with sca than the patients with btm receiving dfx ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the parameters for adult patients with btm or sca who received dfx are shown in table 6 . especially , mean serum ferritin levels were significantly higher than the normal values ( 30.0400.0 ng / ml ) in the patients receiving dfx ( btm : 2199.0 1941.0 ng / ml , n = 13 ; sca : 1544.0 1470.0 ng / ml , n = 16 ) ( p < 0.05 ) . in the dfx - treated patients with btm , the values regarding platelet , edw , basophil , neutrophil , lymphocyte , total bilirubin , direct bilirubin , ast , and crp were higher than the normal values . the values regarding platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , and ast were found to be higher than the normal values in the dfx - treated patients with sca . the values regarding hemoglobin , hematocrit , and erythrocyte were lower in the dfx - treated patients with btm or sca . in the dfx - treated patients , the values for hemoglobin , hematocrit , platelet , erythrocyte , lymphocyte , lymphocyte percentage , and alt were lower , and mcv and mch were significantly higher in the patients with sca than the patients with btm ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 7 shows ict satisfaction summary scores regarding compliance level of dfx - treated pediatric patients with btm or sca . in particular , the mean scores in the patients with btm ranged from 99.7 1.2 ( n = 25 ) for the burden of ict to 85.4 14.7 ( n = 25 ) the side effects of ict . similarly , the scores ranged from 94.7 8.2 ( n = 12 ) for the burden of ict to 85.4 14.7 ( n = 25 ) for the side effects of ict in the patients with sca . when compared with the scores of the patients with btm , the scores for perceived effectiveness of ict was significantly lower in the patients with sca ( p < 0.05 ) . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the ict scores for adult patients with btm or sca who received dfx are presented in table 8 . in particular , the mean scores in the dfx - treated patients with btm ranged from 88.9 13.6 ( n = 14 ) for the burden of ict to 81.9 21.4 ( n = 14 ) for the perceived effectiveness of ict . similarly , the scores in the dfx - treated patients with sca ranged from 85.1 17.4 ( n = 21 ) for the burden of ict to 79.0 17.5 ( n = 21 ) the perceived effectiveness of ict . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia tables 9 and 10 detail the proportional distribution of side effects in the dfx - treated pediatric and adult patients with btm or sca . no serious side effects necessitating discontinuation or interruption of therapy in the patients were reported , and no patients died during the study . proportional distribution of side effects in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia proportional distribution of side effects in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia the demographic and clinical characteristics of the pediatric and adult patients are summarized in tables 1 and 2 , respectively . the study population comprised 37 pediatric ( btm : n = 25 [ 52% female and 48% male ] ; sca : n = 12 [ 50% female and 50% male ] ) and 35 adult ( btm : n = 14 [ 43% female and 57% male ] ; sca : n = 21 [ 62% female and 38% male ] ) patients with btm or sca . all patients were receiving periodic blood transfusions . at the time of the study visit , the reasons for choosing their therapy were physician 's recommendation , poor adherence to dfo , dfp , or dfx , and the failure of reimbursement system for dfo . the percentages of pediatric patients with btm or sca who had formal education were 88% and 83% , respectively . in the adult patients with btm or sca , the percentages were 86% and 100% , respectively . eight ( 32% ) and six ( 50% ) of pediatric patients with btm or sca had undergone splenectomy . two ( 8% ) pediatric patients with btm had hepatomegaly or splenomegaly and one ( 4% ) adult patient with sca had hepatosplenomegaly . the patients also had one or more concomitant diseases / conditions and were using other drugs for their diseases in addition to the iron chelators . demographic and clinical characteristics of deferasirox - treated pediatric patients demographic and clinical characteristics of deferasirox - treated adult patients table 3 shows the chq - pf50 summary scores regarding hrqol in dfx - treated pediatric patients with btm or sca . in particular , the mean chq - pf50 scores in the patients with btm ranged from 86.0 11.0 ( n = 25 ) for the family activities to 47.2 10.6 ( n = 25 ) , the general health state . in the patients with sca , the scores ranged from 85.0 15.1 ( n = 12 ) for the health transition to 42.0 16.3 ( n = 12 ) for the general health state . the scores for general health , physical functioning , role / social limitations - physical , parent impact - time , and family activities were significantly lower , and the scores for health transition were higher in the patients with sca than the patients with btm ( p < 0.05 ) . child health questionnaire - parent form 50 summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the sf-36 summary scores regarding the quality of life in adult patients with btm or sca who received dfx are shown in table 4 . in particular , the mean sf-36 scores in the dfx - treated patients with btm ranged from 81.0 12.6 ( n = 14 ) for the physical functioning to 38.1 41.1 ( n = 14 ) the emotional role limitation . the scores in the dfx - treated patients with sca ranged from 58.3 12.5 ( n = 21 ) for the general health perception to 22.6 39.5 ( n = 21 ) the physical role limitation . when compared with the scores of the dfx - treated patients with btm , the scores for physical functioning , physical role limitation , pain , social functioning , and vitality were significantly lower in the dfx - treated patients with sca ( p < 0.05 ) . the differences between patient groups for the other scales were not statistically different . short form-36 summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 5 details hematological and biochemical parameters in dfx - treated pediatric patients with btm or sca . as a main index for effectiveness of ict , mean serum ferritin levels were higher than the normal values ( 7.0140.0 ng / ml ) in the pediatric patients receiving dfx ( btm : 2040.0 1391 ng / ml , n = 25 ; sca : 937.9 678.4 ng / ml , n = 12 ) . in the patients with btm , the values regarding platelet , platelet percentage , erythrocyte distribution width ( edw ) , total bilirubin , direct bilirubin , alanine aminotransferase ( alt ) , and aspartate transaminase ( ast ) were found to be higher than the normal values . in the patients with oral hypoglycaemic agent , the values for platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , ast , and c - reactive protein ( crp ) were higher than the normal values . however , the values regarding hemoglobin , hematocrit , and erythrocyte were lower than the normal values in both patient groups . in addition , the values regarding other hematological and biochemical parameters measured were in their normal ranges . moreover , serum ferritin levels were lower , and the values for mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , lymphocyte , monocyte , and monocyte percentage were significantly higher in the pediatric patients with sca than the patients with btm receiving dfx ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the parameters for adult patients with btm or sca who received dfx are shown in table 6 . especially , mean serum ferritin levels were significantly higher than the normal values ( 30.0400.0 ng / ml ) in the patients receiving dfx ( btm : 2199.0 1941.0 ng / ml , n = 13 ; sca : 1544.0 1470.0 ng / ml , n = 16 ) ( p < 0.05 ) . in the dfx - treated patients with btm , the values regarding platelet , edw , basophil , neutrophil , lymphocyte , total bilirubin , direct bilirubin , ast , and crp were higher than the normal values . the values regarding platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , and ast were found to be higher than the normal values in the dfx - treated patients with sca . the values regarding hemoglobin , hematocrit , and erythrocyte were lower in the dfx - treated patients with btm or sca . in the dfx - treated patients , the values for hemoglobin , hematocrit , platelet , erythrocyte , lymphocyte , lymphocyte percentage , and alt were lower , and mcv and mch were significantly higher in the patients with sca than the patients with btm ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 7 shows ict satisfaction summary scores regarding compliance level of dfx - treated pediatric patients with btm or sca . in particular , the mean scores in the patients with btm ranged from 99.7 1.2 ( n = 25 ) for the burden of ict to 85.4 14.7 ( n = 25 ) the side effects of ict . similarly , the scores ranged from 94.7 8.2 ( n = 12 ) for the burden of ict to 85.4 14.7 ( n = 25 ) for the side effects of ict in the patients with sca . when compared with the scores of the patients with btm , the scores for perceived effectiveness of ict was significantly lower in the patients with sca ( p < 0.05 ) . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the ict scores for adult patients with btm or sca who received dfx are presented in table 8 . in particular , the mean scores in the dfx - treated patients with btm ranged from 88.9 13.6 ( n = 14 ) for the burden of ict to 81.9 21.4 ( n = 14 ) for the perceived effectiveness of ict . similarly , the scores in the dfx - treated patients with sca ranged from 85.1 17.4 ( n = 21 ) for the burden of ict to 79.0 17.5 iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia tables 9 and 10 detail the proportional distribution of side effects in the dfx - treated pediatric and adult patients with btm or sca . no serious side effects necessitating discontinuation or interruption of therapy in the patients were reported , and no patients died during the study . proportional distribution of side effects in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia proportional distribution of side effects in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia the purpose of this single - center observational study was to evaluate quality of life , clinical effectiveness , and satisfaction in pediatric and adult patients with btm and sca receiving dfx chelation therapy to contribute to their treatment and support process . the results of the present study indicate that the dfx - treated pediatric and adult patients with btm or sca are not achieving their target hematological marker thresholds ( mainly serum ferritin levels ) despite long - term treatment for iron overload . in addition , dfx chelation therapy appears to negatively impact their hrqol so that the patient 's satisfaction with ict is generally poor . iron overload is a major concern in patients with congenital ( e.g. , transfusion - dependent thalassemia [ including btm ] , nontransfusion - dependent thalassemia [ including beta thalassemia intermedia ] , sca , fanconi anemia , hemolytic anemia , and sideroblastic anemia ) and acquired ( e.g. , aplastic anemia , red cell aplasia , neoplastic diseases , and bone marrow transplantation ) anemias for whom regular transfusions are needed . untreated transfusional iron load results in damage to the liver , endocrine organs , and most importantly to the heart . international guidelines for management of btm and sca concur on achieving a target serum ferritin of 1.0002.500 g / l and < 1.000 g / l , respectively . in patients with iron overload as a result of regular blood transfusions because of btm and sca , oral chelating agents ( e.g. dfx and dfp ) are preferred over dfo because of their ease of administration , lesser side effects and better compliance . on the other hand , it takes months , even years , to reduce serum ferritin levels to a safe range . patients sometimes switch from one chelator to another for a variety of reasons , including worsening health status because of iron overload , side effects , and personal preference . in fact , life expectancy is directly related to the quality of chelation therapy and poor compliance to treatment increases the risk of complications and shortens survival . serum ferritin measurement is useful for close and frequent patient monitoring to indicate changes in iron burden and determine current treatment needs . in the case of increased ferritin levels , dosage may be lowered and patient preference in choice of chelator can be better accommodated . on the other hand , all forms of treatment can be inconvenient , time - consuming , and result in unpleasant side effects , all which could potentially impact physical and emotional functioning of patients . in our study , the dfx - treated pediatric and adult patients with btm or sca were heavy iron overloaded in agreement with previous reports . the increased serum ferritin levels were also associated with abnormalities in hematological and biochemical parameters . for instance , in the dfx - treated patients with btm or sca , the values regarding platelet , platelet percentage , mch , edw , basophil , neutrophil , lymphocyte , monocyte , monocyte percentage , total bilirubin , direct bilirubin , alt , ast , and crp were found to be higher than the normal values . on the other hand , the values regarding hemoglobin , hematocrit , and/or erythrocyte were lower than the normal values in the patients . furthermore , serum ferritin levels were lower , and the values for mcv , mch , lymphocyte , monocyte , and monocyte percentage were higher in the pediatric patients with sca than the patients with btm receiving dfx . in the dfx - treated adult patients , the values for hemoglobin , hematocrit , platelet , pdw , lymphocyte , lymphocyte percentage , and alt were lower , and mcv and mch were higher in the patients with sca than the patients with btm . therefore , it appears that in addition to their side effects on hematopoietic and hepatic functions , dfx is not to be effective in reducing serum ferritin levels in the pediatric and adult patients with btm or sca . hrqol involves several aspects which include domains related to emotional , physical , mental , and social functioning and focuses on the impact health status has on quality of life . supporting healthy emotional functioning is important not only to psychological well - being but also to physical health as it may impact compliance to medical regimens . the importance of the chq - pf50 and sf-36 questionnaires and the results of the individual scales on the actions that should be taken for pediatric and adult patients with thalassemia or scd have been reported . on the other hand , there is a little - published data on hrqol in dfx - treated pediatric and adult patients with btm or sca . overall , our findings regarding quality of life and satisfaction with ict indicate that the patients with btm or sca had lower scores . for instance , the highest chq - pf50 scores were found to be for the family activities ( 86.0% ) and the health transition ( 85.0% ) in the dfx - treated pediatric patients with btm or sca , respectively . the highest sf-36 score was for the physical functioning ( 81% ) in the dfx - treated adult patients with btm . in the patients with sca , the highest score was found to be for the general health perception ( 58.3% ) in the dfx group . it is also notable that the scores for general health , physical functioning , role / social limitations - physical , parent impact - time , and family activities were lower , and the scores for health transition were higher in the dfx - treated pediatric patients with sca than the patients with btm . regarding satisfaction with ict , when compared with the scores of the dfx - treated pediatric patients with btm , the scores for perceived effectiveness of ict was lower in the patients with sca . it seems that poor compliance to dfx chelation therapy in these patients is probably due to a complex combination of psychological / social / demographic factors , living with a chronic disease , concomitant diseases / conditions and drug use , and new challenges related to improved life expectancy in the diseases . it is also possible that problems with treatment regimen and side effects appear to be common causes of poor compliance to dfx chelation therapy in these patients . overall , our findings suggest that the patients are not achieving their target serum ferritin thresholds despite chronic treatment for iron overload , ict appears to negatively impact their hrqol , and compliance to ict is poor . these findings are not surprising in the context of studies on poor compliance to ict . therefore , the results are clinically meaningful and infer that changes need to be made to dfx chelation therapy for patients with iron overload . this study provides some evidence for differences in the limitations of quality of life and satisfaction among patients with btm or sca depending on the dfx chelation therapy . overall , the patients involved in the study had not achieved target serum ferritin thresholds despite long - term treatment for iron overload with dfx chelation therapy . observed compliance to dfx was generally poor , and treatment appeared to negatively impact the quality of life and satisfaction of the patients . our data also suggest that majority of the patients and/or their parents do not know about the importance of their medication . this study highlights the importance of providing pediatric and adult patients with btm or sca with the optimal chelation treatment based on their individual needs , in order to increase the hrqol and decrease the presence of metabolic , endocrine , hepatic , renal , and cardiac comorbidities in addition to side effects , leading to increased compliance , and thus resulting in optimal clinical benefit . in addition , our results emphasize the need of involvement of a multidisciplinary team ( including physicians , pharmacists , psychologists , nurse specialists , and health educators ) in the management of patients with these diseases . health care providers should be aware of the importance of monitoring iron load with timely initiation of dfx chelation therapy , and ongoing adjustments to chelation regimens and/or transfusion methods in response to these measurements . future research are needed to determine the demographic and clinical variables most likely to be associated with effective reductions in iron overload as well as strategies ( e.g. , combining two iron chelators , interventions for patient education about the tolerability of chelation , development of new oral chelators ) to decrease hospitalizations , improve compliance to ict of the patients , and reduce morbidity and mortality .

objectives : there is a need to remove excess iron with iron chelation therapy ( ict ) to avoid the serious clinical sequelae associated with iron overload in patients with beta thalassemia major ( btm ) and sickle cell anemia ( sca ) . due to the effects of the diseases and their treatments , ict is still a major reason for unsatisfactory compliance . the aim of this single - center observational study was to evaluate the quality of life , clinical effectiveness , and satisfaction in pediatric and adult patients with btm and sca receiving deferasirox ( dfx ) chelation therapy.methods:in this study , 37 pediatric and 35 adult patients with btm or sca receiving dfx for at least 6 months participated . upon receipt of informed consent form , case report form , demographic data collection form , child health questionnaire - parent form , life quality survey short form-36 , and ict satisfaction survey were used to obtain data for the effectiveness of ict and parameters that may affect compliance to treatment and life quality of the participants.results:as a main index for the effectiveness of dfx chelation therapy , serum ferritin levels were higher than the normal values in the patients receiving dfx . the increased ferritin levels were also associated with hematological and biochemical abnormalities . our findings regarding quality of life and satisfaction with dfx chelation therapy indicated that the patients with btm or sca had lower scores . overall , problems with treatment regimen and side effects appeared to be common causes of poor compliance to dfx chelation therapy.conclusions:our findings suggest that health care providers should be aware of the importance of monitoring iron load with timely initiation of dfx chelation therapy and ongoing adjustments to chelation regimens and/or transfusion methods to decrease hospitalizations and improve compliance to ict of the patients with btm and sca .

Introduction Methods Patients and study design Inclusion and exclusion criteria Study instruments and data collection Statistical analysis Results Demographic and clinical characteristics Quality of life Effectiveness of iron chelation therapy Satisfaction with iron chelation therapy Discussion Conclusions Financial support and sponsorship Conflicts of interest

the conventional approach to treatment of btm and sca is based on the correction of hemoglobin status through regular blood transfusions and iron chelation therapy ( ict ) for iron overload . thus , there is a need to remove excess iron with chelation therapy using deferoxamine ( dfo ) ( desferal ; novartis pharma ag , basel , switzerland ) , deferiprone ( dfp ) ( ferriprox ; apotex inc . , toronto , canada ) , deferasirox ( dfx ) ( exjade ; novartis pharma ag , basel , switzerland ) , or in combination with each other ( dfo - dfp or dfp - dfx ) to avoid the serious clinical sequelae associated with iron accumulation in target organs , morbidity , and mortality . on the other hand , due to the effects of the diseases and their treatments , although dfo , which is administered intravenously or subcutaneously , is clinically effective at removing excess iron , many patients are not satisfactorily chelated by it . to better understand the potential impact of current therapeutic approaches for iron overload , it is important to understand the clinical effectiveness , patient acceptability , and side effects of dfx in addition to patient 's hrqol . therefore , the aim of this single - center observational study was to evaluate the quality of life , clinical effectiveness , and satisfaction in pediatric and adult patients with btm and sca receiving dfx chelation therapy . the single - center observational study was conducted among 37 pediatric and 35 adult patients with btm or sca who received dfx ( exjade ; novartis pharma stein ag , stein , switzerland ) ( 2040 mg / kg / day ) ict at the hematology units of departments of internal medicine and pediatrics , faculty of medicine , mersin university , mersin , turkey from december 2013 to may 2014 participated . the inclusion criteria were ( 1 ) patients willing to participate in the study , ( 2 ) patients diagnosed with btm or sca , and ( 3 ) patients receiving dfx for at least 6 months . the exclusion criteria were ( 1 ) patients not meeting the inclusion criteria , ( 2 ) patients not willing to participate in the study , ( 3 ) patients diagnosed with other types of anemia , ( 4 ) patients not receiving dfx for at least 6 months , and ( 5 ) patients having other conditions such as physical and/or mental difficulties which may affect their quality of life . after taking signed written informed consent from the patients and/or their parents , the following data were obtained : ( 1 ) demographic and clinical characteristics of pediatric and adult patients ( using case report form and demographic data collection form ) , ( 2 ) health status of pediatric patients ( using child health questionnaire - parent form ; chq - pf50 ) , ( 3 ) quality of life of adult patients ( using life quality survey short form-36 ; [ sf-36 ] ) , ( 4 ) effectiveness of ict in pediatric and adult patients ( using case report form and ict satisfaction survey ) , and ( 5 ) compliance to ict in pediatric and adult patients ( using ict satisfaction survey ) . quantitative variables regarding health status of pediatric patients and compliance to ict in pediatric and adult patients were described in the form of mean standard deviation ( sd ) , median , and range . the single - center observational study was conducted among 37 pediatric and 35 adult patients with btm or sca who received dfx ( exjade ; novartis pharma stein ag , stein , switzerland ) ( 2040 mg / kg / day ) ict at the hematology units of departments of internal medicine and pediatrics , faculty of medicine , mersin university , mersin , turkey from december 2013 to may 2014 participated . the inclusion criteria were ( 1 ) patients willing to participate in the study , ( 2 ) patients diagnosed with btm or sca , and ( 3 ) patients receiving dfx for at least 6 months . the exclusion criteria were ( 1 ) patients not meeting the inclusion criteria , ( 2 ) patients not willing to participate in the study , ( 3 ) patients diagnosed with other types of anemia , ( 4 ) patients not receiving dfx for at least 6 months , and ( 5 ) patients having other conditions such as physical and/or mental difficulties which may affect their quality of life . after taking signed written informed consent from the patients and/or their parents , the following data were obtained : ( 1 ) demographic and clinical characteristics of pediatric and adult patients ( using case report form and demographic data collection form ) , ( 2 ) health status of pediatric patients ( using child health questionnaire - parent form ; chq - pf50 ) , ( 3 ) quality of life of adult patients ( using life quality survey short form-36 ; [ sf-36 ] ) , ( 4 ) effectiveness of ict in pediatric and adult patients ( using case report form and ict satisfaction survey ) , and ( 5 ) compliance to ict in pediatric and adult patients ( using ict satisfaction survey ) . quantitative variables regarding health status of pediatric patients and compliance to ict in pediatric and adult patients were described in the form of mean standard deviation ( sd ) , median , and range . the study population comprised 37 pediatric ( btm : n = 25 [ 52% female and 48% male ] ; sca : n = 12 [ 50% female and 50% male ] ) and 35 adult ( btm : n = 14 [ 43% female and 57% male ] ; sca : n = 21 [ 62% female and 38% male ] ) patients with btm or sca . child health questionnaire - parent form 50 summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the sf-36 summary scores regarding the quality of life in adult patients with btm or sca who received dfx are shown in table 4 . short form-36 summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 5 details hematological and biochemical parameters in dfx - treated pediatric patients with btm or sca . as a main index for effectiveness of ict , mean serum ferritin levels were higher than the normal values ( 7.0140.0 ng / ml ) in the pediatric patients receiving dfx ( btm : 2040.0 1391 ng / ml , n = 25 ; sca : 937.9 678.4 ng / ml , n = 12 ) . in the patients with btm , the values regarding platelet , platelet percentage , erythrocyte distribution width ( edw ) , total bilirubin , direct bilirubin , alanine aminotransferase ( alt ) , and aspartate transaminase ( ast ) were found to be higher than the normal values . in the patients with oral hypoglycaemic agent , the values for platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , ast , and c - reactive protein ( crp ) were higher than the normal values . moreover , serum ferritin levels were lower , and the values for mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , lymphocyte , monocyte , and monocyte percentage were significantly higher in the pediatric patients with sca than the patients with btm receiving dfx ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the parameters for adult patients with btm or sca who received dfx are shown in table 6 . especially , mean serum ferritin levels were significantly higher than the normal values ( 30.0400.0 ng / ml ) in the patients receiving dfx ( btm : 2199.0 1941.0 ng / ml , n = 13 ; sca : 1544.0 1470.0 ng / ml , n = 16 ) ( p < 0.05 ) . in the dfx - treated patients with btm , the values regarding platelet , edw , basophil , neutrophil , lymphocyte , total bilirubin , direct bilirubin , ast , and crp were higher than the normal values . the values regarding platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , and ast were found to be higher than the normal values in the dfx - treated patients with sca . hematological and biochemical parameters in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 7 shows ict satisfaction summary scores regarding compliance level of dfx - treated pediatric patients with btm or sca . in particular , the mean scores in the patients with btm ranged from 99.7 1.2 ( n = 25 ) for the burden of ict to 85.4 14.7 ( n = 25 ) the side effects of ict . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the ict scores for adult patients with btm or sca who received dfx are presented in table 8 . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia tables 9 and 10 detail the proportional distribution of side effects in the dfx - treated pediatric and adult patients with btm or sca . proportional distribution of side effects in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia proportional distribution of side effects in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia the demographic and clinical characteristics of the pediatric and adult patients are summarized in tables 1 and 2 , respectively . the study population comprised 37 pediatric ( btm : n = 25 [ 52% female and 48% male ] ; sca : n = 12 [ 50% female and 50% male ] ) and 35 adult ( btm : n = 14 [ 43% female and 57% male ] ; sca : n = 21 [ 62% female and 38% male ] ) patients with btm or sca . child health questionnaire - parent form 50 summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the sf-36 summary scores regarding the quality of life in adult patients with btm or sca who received dfx are shown in table 4 . short form-36 summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 5 details hematological and biochemical parameters in dfx - treated pediatric patients with btm or sca . as a main index for effectiveness of ict , mean serum ferritin levels were higher than the normal values ( 7.0140.0 ng / ml ) in the pediatric patients receiving dfx ( btm : 2040.0 1391 ng / ml , n = 25 ; sca : 937.9 678.4 ng / ml , n = 12 ) . in the patients with btm , the values regarding platelet , platelet percentage , erythrocyte distribution width ( edw ) , total bilirubin , direct bilirubin , alanine aminotransferase ( alt ) , and aspartate transaminase ( ast ) were found to be higher than the normal values . in the patients with oral hypoglycaemic agent , the values for platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , ast , and c - reactive protein ( crp ) were higher than the normal values . moreover , serum ferritin levels were lower , and the values for mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , lymphocyte , monocyte , and monocyte percentage were significantly higher in the pediatric patients with sca than the patients with btm receiving dfx ( p < 0.05 ) . hematological and biochemical parameters in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the parameters for adult patients with btm or sca who received dfx are shown in table 6 . especially , mean serum ferritin levels were significantly higher than the normal values ( 30.0400.0 ng / ml ) in the patients receiving dfx ( btm : 2199.0 1941.0 ng / ml , n = 13 ; sca : 1544.0 1470.0 ng / ml , n = 16 ) ( p < 0.05 ) . in the dfx - treated patients with btm , the values regarding platelet , edw , basophil , neutrophil , lymphocyte , total bilirubin , direct bilirubin , ast , and crp were higher than the normal values . the values regarding platelet , edw , monocyte , monocyte percentage , total bilirubin , direct bilirubin , and ast were found to be higher than the normal values in the dfx - treated patients with sca . hematological and biochemical parameters in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia table 7 shows ict satisfaction summary scores regarding compliance level of dfx - treated pediatric patients with btm or sca . in particular , the mean scores in the patients with btm ranged from 99.7 1.2 ( n = 25 ) for the burden of ict to 85.4 14.7 ( n = 25 ) the side effects of ict . iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia the ict scores for adult patients with btm or sca who received dfx are presented in table 8 . similarly , the scores in the dfx - treated patients with sca ranged from 85.1 17.4 ( n = 21 ) for the burden of ict to 79.0 17.5 iron chelation therapy satisfaction summary scores ( % ) in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia tables 9 and 10 detail the proportional distribution of side effects in the dfx - treated pediatric and adult patients with btm or sca . proportional distribution of side effects in deferasirox - treated pediatric patients with beta thalassemia major or sickle cell anemia proportional distribution of side effects in deferasirox - treated adult patients with beta thalassemia major or sickle cell anemia the purpose of this single - center observational study was to evaluate quality of life , clinical effectiveness , and satisfaction in pediatric and adult patients with btm and sca receiving dfx chelation therapy to contribute to their treatment and support process . the results of the present study indicate that the dfx - treated pediatric and adult patients with btm or sca are not achieving their target hematological marker thresholds ( mainly serum ferritin levels ) despite long - term treatment for iron overload . in fact , life expectancy is directly related to the quality of chelation therapy and poor compliance to treatment increases the risk of complications and shortens survival . in our study , the dfx - treated pediatric and adult patients with btm or sca were heavy iron overloaded in agreement with previous reports . the increased serum ferritin levels were also associated with abnormalities in hematological and biochemical parameters . for instance , in the dfx - treated patients with btm or sca , the values regarding platelet , platelet percentage , mch , edw , basophil , neutrophil , lymphocyte , monocyte , monocyte percentage , total bilirubin , direct bilirubin , alt , ast , and crp were found to be higher than the normal values . furthermore , serum ferritin levels were lower , and the values for mcv , mch , lymphocyte , monocyte , and monocyte percentage were higher in the pediatric patients with sca than the patients with btm receiving dfx . in the dfx - treated adult patients , the values for hemoglobin , hematocrit , platelet , pdw , lymphocyte , lymphocyte percentage , and alt were lower , and mcv and mch were higher in the patients with sca than the patients with btm . therefore , it appears that in addition to their side effects on hematopoietic and hepatic functions , dfx is not to be effective in reducing serum ferritin levels in the pediatric and adult patients with btm or sca . on the other hand , there is a little - published data on hrqol in dfx - treated pediatric and adult patients with btm or sca . overall , our findings regarding quality of life and satisfaction with ict indicate that the patients with btm or sca had lower scores . it is also notable that the scores for general health , physical functioning , role / social limitations - physical , parent impact - time , and family activities were lower , and the scores for health transition were higher in the dfx - treated pediatric patients with sca than the patients with btm . it seems that poor compliance to dfx chelation therapy in these patients is probably due to a complex combination of psychological / social / demographic factors , living with a chronic disease , concomitant diseases / conditions and drug use , and new challenges related to improved life expectancy in the diseases . it is also possible that problems with treatment regimen and side effects appear to be common causes of poor compliance to dfx chelation therapy in these patients . overall , our findings suggest that the patients are not achieving their target serum ferritin thresholds despite chronic treatment for iron overload , ict appears to negatively impact their hrqol , and compliance to ict is poor . therefore , the results are clinically meaningful and infer that changes need to be made to dfx chelation therapy for patients with iron overload . this study provides some evidence for differences in the limitations of quality of life and satisfaction among patients with btm or sca depending on the dfx chelation therapy . overall , the patients involved in the study had not achieved target serum ferritin thresholds despite long - term treatment for iron overload with dfx chelation therapy . observed compliance to dfx was generally poor , and treatment appeared to negatively impact the quality of life and satisfaction of the patients . this study highlights the importance of providing pediatric and adult patients with btm or sca with the optimal chelation treatment based on their individual needs , in order to increase the hrqol and decrease the presence of metabolic , endocrine , hepatic , renal , and cardiac comorbidities in addition to side effects , leading to increased compliance , and thus resulting in optimal clinical benefit . health care providers should be aware of the importance of monitoring iron load with timely initiation of dfx chelation therapy , and ongoing adjustments to chelation regimens and/or transfusion methods in response to these measurements . , combining two iron chelators , interventions for patient education about the tolerability of chelation , development of new oral chelators ) to decrease hospitalizations , improve compliance to ict of the patients , and reduce morbidity and mortality .

most incidental cartilaginous tumours are small and lack suspicious features . small cartilaginous tumours without suspicious findings may be a normal concurrent finding . they are one of the most common osseous neoplasms and are often found incidentally on radiographs , computed tomography or magnetic resonance imaging ( mri ) , especially of the knee . it is important to distinguish them from atypical cartilaginous tumour / chondrosarcoma grade 1 ( act / cs1 ) , which requires surgical treatment . enchondromas are composed of lobules of hyaline cartilage , consisting of benign chondrocytes with or without surrounding reactive bone formation . mutations of isocitrate dehydrogenase 1 ( idh1 ) and idh2 have been identified in enchondromas and chondrosarcomas , in patients with both solitary and multiple neoplasms [ 4 , 5 ] . enchondromas are typically asymptomatic , except in the case of very large lesions which may be painful or may fracture . during routine screening of knee mris for incidental findings , performed in a cohort study to investigate osteoarthritis , cartilaginous tumours because enchondromas are clinically silent , reliable data concerning their incidence and prevalence is scarce [ 1 , 6 ] . the purpose of this study was to describe the prevalence , characteristics and location of incidental cartilaginous tumours on knee mri in a population - based cohort study the netherlands epidemiology of obesity ( neo ) study is a population - based prospective cohort study that includes 6673 individuals aged 45 to 65 years , with an oversampling of individuals deemed overweight or obese . this cohort was designed to prospectively study pathways that lead to disease in such individuals . detailed information about the study design and data collection has been described elsewhere . men and women aged between 45 and 65 years with a self - reported body mass index ( bmi ) of 27 kg / m or higher and living in the greater area of leiden ( in the west of the netherlands ) were eligible to participate in the neo study . in addition , all inhabitants aged between 45 and 65 years from one nearby municipality ( leiderdorp ) were invited , irrespective of their bmi . at baseline , participants completed a questionnaire about demographic and clinical data and underwent an extensive physical examination . in random subsamples of participants without contraindications , mri of abdominal fat , pulse wave velocity of the aorta , heart , brain or right knee was performed . incidental abnormal results with potential health consequences if left undiagnosed were disclosed to the participants and their general practitioners , accompanied by advice for further work - up . the present study is a cross - sectional analysis of the baseline measurements of the 1285 participants with an mri of the knee . the local medical ethics committee approved the study and all participants gave written informed consent . all participants were asked whether they had pain in the right knee during most days of the previous month . weight and height were measured without shoes and with precision of 0.1 cm / kg and 1 kg was subtracted from the weight for clothing . bmi was calculated by dividing the weight in kilograms by the height in metres squared . mri studies were performed on a 1.5-t mri system ( philips , best , the netherlands ) with a dedicated 8-channel knee coil . the following parameters were identical for the tse images : echo train length 6 , a 150160 mm field of view and a 304 512 matrix . sequences acquired were ( 1 ) coronal proton density ( pd ) ( repetition time ( tr)/echo time ( te ) 2335/35 ms ; 3 mm slice thickness ; 0.6 mm interslice gap ) ; ( 2 ) coronal fat suppressed pd tse images ( tr / te 2334/35 ms ; 3 mm slice thickness ; 0.6 mm interslice gap ) ; ( 3 ) sagittal pd tse images ( tr / te 2338/35 ; 3.5 mm slice thickness ; 0.7 mm interslice gap ) ; ( 4 ) sagittal frequency selective fat - suppressed t1-weighted 3d gradient echo ( ge ) sequence ( tr / te 11/5.5 ; 25 flip angle ; 150 mm field of view , 272 512 matrix , 2 mm slice thickness with a 1-mm overlap between images ; no gap ) ; ( 5 ) axial fat suppressed pd ( tse ) images ( tr / te 3225/15 ; 4 mm slice thickness ; 0.8 mm interslice gap ) . total acquisition time was 30 min . a research fellow trained in reading knee mris initially screened all mris for the presence of any abnormality . all lesions possibly fulfilling the criteria set for cartilaginous tumour were assessed by two musculoskeletal radiologists with more than 10 years experience in consensus to determine lesion characteristics . cartilaginous tumours were defined as a smooth or lobulated lesion of geographic bone destruction pattern within the bone marrow with low signal on pd - weighted images and high signal on pd fat - suppressed images . subchondral lesions were excluded , because they may represent subchondral cysts , intraosseous ganglia or subchondral oedema . we included and recorded characteristics of all other osseous lesions that could be bone tumours or tumour - like lesions of bone . for all included lesions we recorded size in three dimensions , shape ( lobulated , oval or round ) , bone destruction pattern i.e. geographic with well - defined or partially ill - defined margins , permeative or moth - eaten . the location was determined to be either central or eccentric and located in the epiphysis , epi - metaphysis , metaphysis and/or diaphysis . the distance of the tumour to the growth plate was classified as a lesion being in contact with the physis , within 2 cm , or further away from the physis . the relationship with the cortex in eccentric lesions was determined ( contact or no contact ) . the presence or absence of superficial ( one - third of depth ) or deep ( at least two - thirds of depth ) scalloping ( i.e. focal resorption of the inner margin of cortical bone ) , cortical bone destruction and periosteal reaction was recorded . signal intensity was scored as low , intermediate or high compared to muscle on both pd and fat - suppressed pd - weighted images . the presence of speckled low signal intensity on both the pd and gradient - echo sequence in the lesion was scored as calcifications . presence or absence of bone marrow oedema in direct contact or within 2 cm from the lesion was noted . participants with lesions that were larger than 20 mm or that had aggressive characteristics ( i.e. cortical bone destruction , periosteal reaction , deep endosteal scalloping , ( partial ) ill definition of margin ) were contacted and referred for dynamic contrast mri to differentiate enchondroma from act / cs1 . large lesion size is a known risk factor for chondrosarcoma but no specific limit is known which optimally differentiates between enchondroma and act / cs1 . we chose a conservative cut - off of 20 mm as a trade - off between increased sensitivity and avoiding unnecessary extra diagnostic procedures . data acquisition was performed during intravenous injection of gd - dtpa ( dose 2 mg / kg body weight , power injector with 2 ml / s ) with a temporal resolution of 3 s. we recorded on electronic subtraction images the time interval between arterial enhancement and the start of lesion enhancement . lesions enhancing within 10 s were classified as early enhancement consistent with act / cs1 , while lesions that did not enhance , or enhanced after more than 10 s were classified as enchondroma . in addition we retrieved follow - up radiographs , and mri studies when these had been performed . changes of described parameters between initial and follow - up mri studies and radiographs were recorded . when the aforementioned features on radiographs or mri suggesting the presence of act / cs1 were present , material was obtained ( curettage or resection ) to allow a histological diagnosis to be made . all involved pathologists had extensive experience with bone tumour pathology owing to the hospital s function as a tertiary referral centre for orthopaedic oncology . characteristics of the lesions were expressed as number of the total number of lesions . in order to correctly estimate the population prevalence of cartilaginous tumours and represent associations in the general population , all other results were based on weighted analyses adjusting for the oversampling of persons with a bmi of 27 this was done by weighing individuals towards the bmi distribution of participants from the leiderdorp municipality , whose bmi distribution was similar to the bmi distribution of the general dutch population . weighted baseline characteristics of the study population were expressed as mean ( sd ) or as percentage . participants were categorized into four groups based on their bmi ( bmi < 25 , 25 bmi < 30 , 30 bmi < 40 and bmi 40 kg / m ) . logistic regression analysis was used to examine the associations of bmi ( both as a continuous variable and as a categorical variable using bmi < 25 kg / m as the reference group ) , age ( continuous ) , sex , and knee pain with the presence of cartilaginous tumours . the netherlands epidemiology of obesity ( neo ) study is a population - based prospective cohort study that includes 6673 individuals aged 45 to 65 years , with an oversampling of individuals deemed overweight or obese . this cohort was designed to prospectively study pathways that lead to disease in such individuals . detailed information about the study design and data collection has been described elsewhere . men and women aged between 45 and 65 years with a self - reported body mass index ( bmi ) of 27 kg / m or higher and living in the greater area of leiden ( in the west of the netherlands ) were eligible to participate in the neo study . in addition , all inhabitants aged between 45 and 65 years from one nearby municipality ( leiderdorp ) were invited , irrespective of their bmi . at baseline , participants completed a questionnaire about demographic and clinical data and underwent an extensive physical examination . in random subsamples of participants without contraindications , mri of abdominal fat , pulse wave velocity of the aorta , heart , brain or right knee was performed . incidental abnormal results with potential health consequences if left undiagnosed were disclosed to the participants and their general practitioners , accompanied by advice for further work - up . the present study is a cross - sectional analysis of the baseline measurements of the 1285 participants with an mri of the knee . the local medical ethics committee approved the study and all participants gave written informed consent . all participants were asked whether they had pain in the right knee during most days of the previous month . weight and height were measured without shoes and with precision of 0.1 cm / kg and 1 kg was subtracted from the weight for clothing . bmi was calculated by dividing the weight in kilograms by the height in metres squared . mri studies were performed on a 1.5-t mri system ( philips , best , the netherlands ) with a dedicated 8-channel knee coil . the following parameters were identical for the tse images : echo train length 6 , a 150160 mm field of view and a 304 512 matrix . sequences acquired were ( 1 ) coronal proton density ( pd ) ( repetition time ( tr)/echo time ( te ) 2335/35 ms ; 3 mm slice thickness ; 0.6 mm interslice gap ) ; ( 2 ) coronal fat suppressed pd tse images ( tr / te 2334/35 ms ; 3 mm slice thickness ; 0.6 mm interslice gap ) ; ( 3 ) sagittal pd tse images ( tr / te 2338/35 ; 3.5 mm slice thickness ; 0.7 mm interslice gap ) ; ( 4 ) sagittal frequency selective fat - suppressed t1-weighted 3d gradient echo ( ge ) sequence ( tr / te 11/5.5 ; 25 flip angle ; 150 mm field of view , 272 512 matrix , 2 mm slice thickness with a 1-mm overlap between images ; no gap ) ; ( 5 ) axial fat suppressed pd ( tse ) images ( tr / te 3225/15 ; 4 mm slice thickness ; 0.8 mm interslice gap ) . a research fellow trained in reading knee mris initially screened all mris for the presence of any abnormality . all lesions possibly fulfilling the criteria set for cartilaginous tumour were assessed by two musculoskeletal radiologists with more than 10 years experience in consensus to determine lesion characteristics . cartilaginous tumours were defined as a smooth or lobulated lesion of geographic bone destruction pattern within the bone marrow with low signal on pd - weighted images and high signal on pd fat - suppressed images . subchondral lesions were excluded , because they may represent subchondral cysts , intraosseous ganglia or subchondral oedema . we included and recorded characteristics of all other osseous lesions that could be bone tumours or tumour - like lesions of bone . for all included lesions we recorded size in three dimensions , shape ( lobulated , oval or round ) , bone destruction pattern i.e. geographic with well - defined or partially ill - defined margins , permeative or moth - eaten . the location was determined to be either central or eccentric and located in the epiphysis , epi - metaphysis , metaphysis and/or diaphysis . the distance of the tumour to the growth plate was classified as a lesion being in contact with the physis , within 2 cm , or further away from the physis . the relationship with the cortex in eccentric lesions was determined ( contact or no contact ) . the presence or absence of superficial ( one - third of depth ) or deep ( at least two - thirds of depth ) scalloping ( i.e. focal resorption of the inner margin of cortical bone ) , cortical bone destruction and periosteal reaction was recorded . signal intensity was scored as low , intermediate or high compared to muscle on both pd and fat - suppressed pd - weighted images . the presence of speckled low signal intensity on both the pd and gradient - echo sequence in the lesion was scored as calcifications . presence or absence of bone marrow oedema in direct contact or within 2 cm from the lesion was noted . participants with lesions that were larger than 20 mm or that had aggressive characteristics ( i.e. cortical bone destruction , periosteal reaction , deep endosteal scalloping , ( partial ) ill definition of margin ) were contacted and referred for dynamic contrast mri to differentiate enchondroma from act / cs1 . large lesion size is a known risk factor for chondrosarcoma but no specific limit is known which optimally differentiates between enchondroma and act / cs1 . we chose a conservative cut - off of 20 mm as a trade - off between increased sensitivity and avoiding unnecessary extra diagnostic procedures . data acquisition was performed during intravenous injection of gd - dtpa ( dose 2 mg / kg body weight , power injector with 2 ml / s ) with a temporal resolution of 3 s. we recorded on electronic subtraction images the time interval between arterial enhancement and the start of lesion enhancement . lesions enhancing within 10 s were classified as early enhancement consistent with act / cs1 , while lesions that did not enhance , or enhanced after more than 10 s were classified as enchondroma . in addition we retrieved follow - up radiographs , and mri studies when these had been performed . changes of described parameters between initial and follow - up mri studies and radiographs were recorded . when the aforementioned features on radiographs or mri suggesting the presence of act / cs1 were present , material was obtained ( curettage or resection ) to allow a histological diagnosis to be made . all involved pathologists had extensive experience with bone tumour pathology owing to the hospital s function as a tertiary referral centre for orthopaedic oncology . characteristics of the lesions were expressed as number of the total number of lesions . in order to correctly estimate the population prevalence of cartilaginous tumours and represent associations in the general population , all other results were based on weighted analyses adjusting for the oversampling of persons with a bmi of 27 kg / m or higher in the neo study . this was done by weighing individuals towards the bmi distribution of participants from the leiderdorp municipality , whose bmi distribution was similar to the bmi distribution of the general dutch population . weighted baseline characteristics of the study population were expressed as mean ( sd ) or as percentage . participants were categorized into four groups based on their bmi ( bmi < 25 , 25 bmi < 30 , 30 bmi < 40 and bmi 40 kg / m ) . logistic regression analysis was used to examine the associations of bmi ( both as a continuous variable and as a categorical variable using bmi < 25 kg / m as the reference group ) , age ( continuous ) , sex , and knee pain with the presence of cartilaginous tumours . between september 2008 and october 2012 , 6673 persons aged 45 to 65 years were included in the neo study , of whom 1285 underwent mri of the right knee . weighted mean ( sd ) age was 56 ( 6 ) years , 45 % were men and mean bmi was 27.1 ( 4.7 ) kg / m . after screening of these 1285 mris , 49 lesions in 44 participants were observed and classified as cartilaginous tumours ( estimated population prevalence 2.8 % , 95 % ci 2.04.0 % ) . four participants had more than one lesion : three participants had two and one participant had three lesions . using logistic regression analysis , we did not find any association between bmi , age or sex and the presence of cartilaginous tumours ( data not shown ) . 1small enchondroma in the distal femur located centrally in the metaphysis and with a typical lobulated appearance : a pd - weighted coronal section , b pd - weighted coronal section with fat saturation . no further imaging was obtained small enchondroma in the distal femur located centrally in the metaphysis and with a typical lobulated appearance : a pd - weighted coronal section , b pd - weighted coronal section with fat saturation . no further imaging was obtained mean lesion size was 12 ( range 2 31 ) mm in the longest axis . thirty - six lesions were lobulated , 12 had a smooth round shape and one was oval - shaped . thirty - eight were located in the distal femur , eight in the proximal tibia , two in the proximal fibula and one in the patella . nineteen tumours were located centrally in the medullary canal and 30 eccentrically ( but still within the medullary canal ) . of all tumours , 27 were located in the metaphysis , 10 in the epi - metaphysis ( crossing the growth plate ) , ten in the diaphysis and one in the epiphysis ( not applicable for the lesion in the patella ) . twenty - nine were in contact with the growth plate , nine crossed the growth plate and ten were located more than 2 cm from the growth plate ( not applicable for the lesion in the patella ) . in 11 lesions there was contact with the cortex and in one lesion there was focal destruction of the cortex . in three cases signal intensity was intermediate on pd images and heterogeneously high on pd - spir images for all lesions . eight lesions in eight participants displayed at least one of the predefined aggressive criteria and were referred for follow - up . seven had a tumour larger than 20 mm ( range 2231 mm ) , including one with deep endosteal scalloping . one participant had a smaller tumour but with focal cortical destruction . in six of the seven participants referred because of tumour size , dynamic contrast mri was performed , while one patient was subsequently lost to follow - up . in four participants there was slow lesional enhancement ( i.e. the interval between arterial enhancement and lesion enhancement was greater than 10 s ) after intravenous gd - chelate administration consistent with benign enchondroma ( fig . 2 ) ; no subsequent follow - up was performed in these patients . in the fifth patient with a lesion of 22 mm , and in the sixth patient with a larger lesion with deep endosteal scalloping , dynamic contrast mri demonstrated a fast enhancement pattern consistent with act / cs1 . curettage and en bloc resection of the tumour were performed respectively , and pathology confirmed the diagnosis of act / cs1 ( fig . 3 ) . the patient with the smaller lesion but with focal cortical destruction had radiographic follow - up and underwent curettage 2 years later , showing an act / cs1 at histology . the three act / cs1s that were found correspond to an estimated population prevalence of 0.4 % ( 95 % ci 0.20.8 % ) .fig . 2large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed slow enhancement consistent with enchondroma ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in secondsfig . 3large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed slow enhancement consistent with enchondroma ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 estimated population prevalence of cartilaginous tumours in bmi strata are shown in table 1 . univariate logistic regression analysis did not reveal associations between bmi , age , sex and the presence of cartilaginous tumour , but it was suggestive of an inverse association with knee pain ( table 2 ) . multivariate logistic regression using the same variables showed an inverse association between the presence of knee pain and the presence of cartilaginous tumour but no association with bmi ( as continuous variable ) , age or sex . compared with having a bmi < 25 kg / m , the odds ratio ( or , 95 % confidence interval ) of the presence of cartilaginous tumour associated with a bmi of 2530 kg / m was 1.79 ( 0.38 , 8.51 ) , with a bmi of 3040 kg / m it was 1.69 ( 0.38 , 7.47 ) , and with a bmi > 40 kg / m it was 3.74 ( 0.64 , 21.87).table 1participant characteristics and prevalence of cartilaginous tumours by bmi groupsbmi ( kg / m)<25(35 % ) 2530(44 % ) 3040(19 % ) 40(2 % ) age ( years)56 ( 6)56 ( 6)55 ( 6)53 ( 6)sex ( % women)63466084bmi ( kg / m)22.6 ( 1.5)27.4 ( 1.3)33.3 ( 2.7)42.9 ( 2.4)cartilaginous tumour prevalence ( % ) 1.93.23.17.1knee pain ( % yes)14172327results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentagestable 2univariate and multivariate analysis for risk factors of presence of cartilaginous tumoursvariablesunivariate analysismultivariate analysisor ( 95 % ci ) p or ( 95 % ci ) p bmi ( kg / m)1.05 ( 0.971.14)0.2021.06 ( 0.981.14)0.168age ( year)0.97 ( 0.911.04)0.4000.98 ( 0.911.05)0.482sex ( woman)1.20 ( 0.453.14)0.7171.23 ( 0.473.23)0.673knee pain ( yes)0.44 ( 0.181.10)0.0800.40 ( 0.180.93)0.034results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio participant characteristics and prevalence of cartilaginous tumours by bmi groups results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentages univariate and multivariate analysis for risk factors of presence of cartilaginous tumours results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio mean lesion size was 12 ( range 2 31 ) mm in the longest axis . thirty - six lesions were lobulated , 12 had a smooth round shape and one was oval - shaped . thirty - eight were located in the distal femur , eight in the proximal tibia , two in the proximal fibula and one in the patella . nineteen tumours were located centrally in the medullary canal and 30 eccentrically ( but still within the medullary canal ) . of all tumours , 27 were located in the metaphysis , 10 in the epi - metaphysis ( crossing the growth plate ) , ten in the diaphysis and one in the epiphysis ( not applicable for the lesion in the patella ) . twenty - nine were in contact with the growth plate , nine crossed the growth plate and ten were located more than 2 cm from the growth plate ( not applicable for the lesion in the patella ) . in 11 lesions there was contact with the cortex and in one lesion there was focal destruction of the cortex . in three cases signal intensity was intermediate on pd images and heterogeneously high on pd - spir images for all lesions . eight lesions in eight participants displayed at least one of the predefined aggressive criteria and were referred for follow - up . seven had a tumour larger than 20 mm ( range 2231 mm ) , including one with deep endosteal scalloping . one participant had a smaller tumour but with focal cortical destruction . in six of the seven participants referred because of tumour size , dynamic contrast mri was performed , while one patient was subsequently lost to follow - up . in four participants there was slow lesional enhancement ( i.e. the interval between arterial enhancement and lesion enhancement was greater than 10 s ) after intravenous gd - chelate administration consistent with benign enchondroma ( fig . 2 ) ; no subsequent follow - up was performed in these patients . in the fifth patient with a lesion of 22 mm , and in the sixth patient with a larger lesion with deep endosteal scalloping , curettage and en bloc resection of the tumour were performed respectively , and pathology confirmed the diagnosis of act / cs1 ( fig . 3 ) . the patient with the smaller lesion but with focal cortical destruction had radiographic follow - up and underwent curettage 2 years later , showing an act / cs1 at histology . the three act / cs1s that were found correspond to an estimated population prevalence of 0.4 % ( 95 % ci 0.20.8 % ) .fig . 2large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed slow enhancement consistent with enchondroma ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in secondsfig . 3large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed slow enhancement consistent with enchondroma ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . the vertical axis represents relative signal intensity and the horizontal axis represents time in seconds . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 estimated population prevalence of cartilaginous tumours in bmi strata are shown in table 1 . univariate logistic regression analysis did not reveal associations between bmi , age , sex and the presence of cartilaginous tumour , but it was suggestive of an inverse association with knee pain ( table 2 ) . multivariate logistic regression using the same variables showed an inverse association between the presence of knee pain and the presence of cartilaginous tumour but no association with bmi ( as continuous variable ) , age or sex . compared with having a bmi < 25 kg / m , the odds ratio ( or , 95 % confidence interval ) of the presence of cartilaginous tumour associated with a bmi of 2530 kg / m was 1.79 ( 0.38 , 8.51 ) , with a bmi of 3040 kg / m it was 1.69 ( 0.38 , 7.47 ) , and with a bmi > 40 kg / m it was 3.74 ( 0.64 , 21.87).table 1participant characteristics and prevalence of cartilaginous tumours by bmi groupsbmi ( kg / m)<25(35 % ) 2530(44 % ) 3040(19 % ) 40(2 % ) age ( years)56 ( 6)56 ( 6)55 ( 6)53 ( 6)sex ( % women)63466084bmi ( kg / m)22.6 ( 1.5)27.4 ( 1.3)33.3 ( 2.7)42.9 ( 2.4)cartilaginous tumour prevalence ( % ) 1.93.23.17.1knee pain ( % yes)14172327results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentagestable 2univariate and multivariate analysis for risk factors of presence of cartilaginous tumoursvariablesunivariate analysismultivariate analysisor ( 95 % ci ) p or ( 95 % ci ) p bmi ( kg / m)1.05 ( 0.971.14)0.2021.06 ( 0.981.14)0.168age ( year)0.97 ( 0.911.04)0.4000.98 ( 0.911.05)0.482sex ( woman)1.20 ( 0.453.14)0.7171.23 ( 0.473.23)0.673knee pain ( yes)0.44 ( 0.181.10)0.0800.40 ( 0.180.93)0.034results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio participant characteristics and prevalence of cartilaginous tumours by bmi groups results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentages univariate and multivariate analysis for risk factors of presence of cartilaginous tumours results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio in a cohort study including knee mri to investigate osteoarthritis , we observed incidental lesions that we classified as cartilaginous tumours on the basis of predefined imaging criteria in 2.8 % of individuals between 45 and 65 years of age . these cartilaginous tumours were all smaller than 32 mm , typically smaller than 20 mm and were located near the femoral or sometimes tibial metaphysis . the prevalence of act / cs1 in our population was low ( 0.4 % ) , but we found an act / cs1 in three out of seven participants whom we classified , on the basis of predefined criteria , as high risk of carrying an act / cs1 and had a complete data set . these act / cs1s were present in two participants with tumours exhibiting aggressive features ( deep endosteal scalloping or cortical bone destruction ) and one participant with a tumour of 22 mm and were either resected or removed by curettage . in four other lesions with a diameter of greater than 20 mm the criteria of dynamic mri were consistent with an indolent benign enchondroma . because cartilaginous tumours are typically clinically silent reliable data concerning their epidemiology are scarce . similar to our study , walden et al . reported an enchondroma prevalence of 2.9 % on clinical knee mri . in skeletally immature children undergoing knee mri for a variety of indications , enchondroma prevalence was also 2.9 % . in neither of these two studies was further follow - up performed . our study confirms these results with a similar prevalence of cartilaginous tumours of 2.8 % . however , we also showed that act / cs1s are relatively common among larger tumours and those with suspicious features . in an autopsy case series , a prevalence of enchondromas of only 0.2 % was observed , likely reflecting the higher sensitivity of mri for detecting small lesions . other asymptomatic osseous tumours were not encountered in our cohort , which means that the prevalence of these is , in our age group , less than that of cartilaginous tumours and below 0.1 % . our data did not reveal a clear association with age , sex or bmi as a continuous variable , and an inverse association with knee pain . the latter finding may merely reflect that participants with cartilaginous tumours do not suffer from knee pain . there was a clear trend towards a higher prevalence in higher bmi groups ; however , the number of detected cartilaginous tumours was probably too low to reach a significant association in the logistic regression analysis . the high sensitivity of mri in detecting small cartilaginous tumours on mri performed for other purposes can pose a dilemma to the clinician . the main differential diagnosis of enchondroma is act / cs1 . in high - grade chondrosarcoma , clinical , radiographic and pathological findings generally are apparent and treatment consists of surgical resection with a wide margin . however , differentiating act / cs1 from enchondroma is challenging , especially in the case of incidental asymptomatic lesions found on imaging . suggestions on how to address this dilemma have been made ( table 3 ) . radiographic signs are not useful in further classifying these small enchondromas . in a previous radiograph - based case series , location in the axial skeleton and size greater than 5 cm were the most reliable predictors for low - grade chondrosarcoma , while clinical symptoms and morphologic radiographic features did not have added value . only bone scintigraphy ( intense uptake on delayed images without presence of fracture ) and dynamic gd - chelate - enhanced mri ( start of enhancement within 10 s after arterial enhancement ) have been reported to add information on differentiating enchondroma from act / cs1 [ 1921 ] . we observed mri signs suggestive of chondrosarcoma such as deep endosteal scalloping , cortical destruction and soft - tissue extension in two out of three participants who indeed turned out to have act / cs1 . nevertheless , even with dynamic contrast - enhanced mri , differentiating cartilaginous tumours remains challenging with false - positive findings unavoidable .table 3imaging features reported in literature to be suggestive of chondrosarcomapain related to the tumourlocation in the axial skeletonlarge lesion size ( > 5 cm)lobulated contourill - defined marginenlargement of the medullary cavity and cortical thickeningendosteal scalloping > 2/3 of the cortical thickness or over > 2/3 of the lesion lengthneurovascular involvementliquefactionperitumoral oedema*moth - eaten or permeative osteolysis*spontaneous pathologic fracture*periosteal reaction*cortical destruction*soft - tissue extension*early ( < 10 s ) enhancement on dynamic contrast mriintense uptake on bone scintigraphyfdg - pet high uptake*imaging features reported in the literature making a diagnosis of chondrosarcoma more likely . [ 1721]*features suggestive of high - grade ( grade 1 and 2 ) chondrosarcoma imaging features reported in literature to be suggestive of chondrosarcoma imaging features reported in the literature making a diagnosis of chondrosarcoma more likely . list synthesized from refs . [ 1721 ] * features suggestive of high - grade ( grade 1 and 2 ) chondrosarcoma chondrosarcoma incidence has been estimated at 0.0005 % per year and prevalence at 0.0010.009 % , with only 7 % of those located around the knee [ 2325 ] . in our study we observed an unexpectedly high estimated population prevalence of act / cs1 of 0.4 % . as no further examinations or follow - up were performed when tumours were small and lacked suspicious features , the actual prevalence of act / cs1 may be even higher . thus knee mri performed in participants should be carefully reviewed for the presence of cartilaginous tumours . only small lesions lacking suspicious features may be regarded as a normal concurrent finding on mri . in patients with any of the described suspicious features , the chance of having an atc / cs1 is quite high ( three out of seven in our population ) . the present study was conducted in a cohort study of volunteers . compared to the general population , some may have had a healthier lifestyle , while others may have had more health complaints and participated because of the diagnostic examinations performed as part of the study . furthermore , a large proportion of participants were overweight or obese . to estimate the population prevalence we weighted the analyses towards the bmi distribution of a population comparable to the general dutch population . radiographs were not available for the study group , making it impossible to determine the discrepancy between prevalence on radiographs and mri . however , because the estimated population prevalence in our study was similar to that in earlier studies , we are confident that our results are robust . because of the benign nature of enchondromas , there are no histopathological studies confirming the origin of the otherwise unsuspicious lesions and they are classified as enchondromas solely on imaging findings . as we did not routinely do resections and do not yet have 10-year follow - up , the proof of excluding act / cs1 in the three unsuspicious larger lesions was incomplete . the estimated population prevalence of incidental asymptomatic cartilaginous tumours around the knee was 2.8 % . typically the lesions were less than 20 mm in size , were located within 2 cm of the growth plate and did not cause cortical abnormalities . the prevalence of act / cs1 may be higher than previously thought ( 0.4 % ) . given the high prevalence of enchondromas , as a practical rule we do not advocate further imaging examination or follow - up of accidentally found cartilaginous tumours in patients up to 65 years when the tumours are small ( at most 20 mm ) and suspicious features such as deep endosteal scalloping or cortical destruction are absent . however , when any of these suspicious features are present , or when the lesion is larger than 20 mm in diameter , further analysis is indicated .

objectivesthe purpose was to determine prevalence of enchondromas and atypical cartilaginous tumour / chondrosarcoma grade 1 ( act / cs1 ) of the knee on mri in a large cohort study , namely the netherlands epidemiology of obesity ( neo ) study.methodsparticipants aged 45 to 65 years were prospectively included , oversampling overweight and obese persons . within a subgroup of participants , mri of the right knee was performed and screened for incidental cartilaginous tumours , as defined by their characteristic location and appearance.resultsforty-nine cartilaginous tumours were observed in 44 out of 1285 participants ( estimated population prevalence 2.8 % , 95 % ci 2.04.0 % ) . mean largest tumour diameter was 12 mm ( range 231 mm ) . eight participants with a tumour larger than 20 mm or a tumour with aggressive features were referred to rule out low - grade chondrosarcoma . one was lost to follow - up , three had histologically proven act / cs1 and four had dynamic contrast mri findings consistent with benign enchondroma.conclusionsincidental cartilaginous tumours were relatively common on knee mri and may be regarded as a normal concurrent finding . however , more tumours than expected were act / cs1 . because further examination was performed only when suspicion of chondrosarcoma was high , the actual prevalence might be even higher.key points incidental cartilaginous tumours are relatively common on knee mri. most incidental cartilaginous tumours are small and lack suspicious features. small cartilaginous tumours without suspicious findings may be a normal concurrent finding. large tumours and/or those with suspicious findings should be further investigated. atypical cartilaginous tumour / chondrosarcoma grade 1 was found more often than expected .

None Introduction Materials and methods Study design and study population MRI of the right knee Lesion analysis Dynamic contrast MR Histological diagnosis Statistical analyses Results Lesion characteristics Association of BMI, age, sex and knee pain with presence of cartilaginous tumours Discussion Limitations Conclusion

most incidental cartilaginous tumours are small and lack suspicious features . small cartilaginous tumours without suspicious findings may be a normal concurrent finding . it is important to distinguish them from atypical cartilaginous tumour / chondrosarcoma grade 1 ( act / cs1 ) , which requires surgical treatment . during routine screening of knee mris for incidental findings , performed in a cohort study to investigate osteoarthritis , cartilaginous tumours because enchondromas are clinically silent , reliable data concerning their incidence and prevalence is scarce [ 1 , 6 ] . the purpose of this study was to describe the prevalence , characteristics and location of incidental cartilaginous tumours on knee mri in a population - based cohort study the netherlands epidemiology of obesity ( neo ) study is a population - based prospective cohort study that includes 6673 individuals aged 45 to 65 years , with an oversampling of individuals deemed overweight or obese . men and women aged between 45 and 65 years with a self - reported body mass index ( bmi ) of 27 kg / m or higher and living in the greater area of leiden ( in the west of the netherlands ) were eligible to participate in the neo study . in random subsamples of participants without contraindications , mri of abdominal fat , pulse wave velocity of the aorta , heart , brain or right knee was performed . the present study is a cross - sectional analysis of the baseline measurements of the 1285 participants with an mri of the knee . participants with lesions that were larger than 20 mm or that had aggressive characteristics ( i.e. cortical bone destruction , periosteal reaction , deep endosteal scalloping , ( partial ) ill definition of margin ) were contacted and referred for dynamic contrast mri to differentiate enchondroma from act / cs1 . in order to correctly estimate the population prevalence of cartilaginous tumours and represent associations in the general population , all other results were based on weighted analyses adjusting for the oversampling of persons with a bmi of 27 this was done by weighing individuals towards the bmi distribution of participants from the leiderdorp municipality , whose bmi distribution was similar to the bmi distribution of the general dutch population . the netherlands epidemiology of obesity ( neo ) study is a population - based prospective cohort study that includes 6673 individuals aged 45 to 65 years , with an oversampling of individuals deemed overweight or obese . men and women aged between 45 and 65 years with a self - reported body mass index ( bmi ) of 27 kg / m or higher and living in the greater area of leiden ( in the west of the netherlands ) were eligible to participate in the neo study . in random subsamples of participants without contraindications , mri of abdominal fat , pulse wave velocity of the aorta , heart , brain or right knee was performed . the present study is a cross - sectional analysis of the baseline measurements of the 1285 participants with an mri of the knee . participants with lesions that were larger than 20 mm or that had aggressive characteristics ( i.e. cortical bone destruction , periosteal reaction , deep endosteal scalloping , ( partial ) ill definition of margin ) were contacted and referred for dynamic contrast mri to differentiate enchondroma from act / cs1 . between september 2008 and october 2012 , 6673 persons aged 45 to 65 years were included in the neo study , of whom 1285 underwent mri of the right knee . after screening of these 1285 mris , 49 lesions in 44 participants were observed and classified as cartilaginous tumours ( estimated population prevalence 2.8 % , 95 % ci 2.04.0 % ) . eight lesions in eight participants displayed at least one of the predefined aggressive criteria and were referred for follow - up . seven had a tumour larger than 20 mm ( range 2231 mm ) , including one with deep endosteal scalloping . in six of the seven participants referred because of tumour size , dynamic contrast mri was performed , while one patient was subsequently lost to follow - up . in the fifth patient with a lesion of 22 mm , and in the sixth patient with a larger lesion with deep endosteal scalloping , dynamic contrast mri demonstrated a fast enhancement pattern consistent with act / cs1 . the patient with the smaller lesion but with focal cortical destruction had radiographic follow - up and underwent curettage 2 years later , showing an act / cs1 at histology . the three act / cs1s that were found correspond to an estimated population prevalence of 0.4 % ( 95 % ci 0.20.8 % ) .fig . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 estimated population prevalence of cartilaginous tumours in bmi strata are shown in table 1 . compared with having a bmi < 25 kg / m , the odds ratio ( or , 95 % confidence interval ) of the presence of cartilaginous tumour associated with a bmi of 2530 kg / m was 1.79 ( 0.38 , 8.51 ) , with a bmi of 3040 kg / m it was 1.69 ( 0.38 , 7.47 ) , and with a bmi > 40 kg / m it was 3.74 ( 0.64 , 21.87).table 1participant characteristics and prevalence of cartilaginous tumours by bmi groupsbmi ( kg / m)<25(35 % ) 2530(44 % ) 3040(19 % ) 40(2 % ) age ( years)56 ( 6)56 ( 6)55 ( 6)53 ( 6)sex ( % women)63466084bmi ( kg / m)22.6 ( 1.5)27.4 ( 1.3)33.3 ( 2.7)42.9 ( 2.4)cartilaginous tumour prevalence ( % ) 1.93.23.17.1knee pain ( % yes)14172327results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentagestable 2univariate and multivariate analysis for risk factors of presence of cartilaginous tumoursvariablesunivariate analysismultivariate analysisor ( 95 % ci ) p or ( 95 % ci ) p bmi ( kg / m)1.05 ( 0.971.14)0.2021.06 ( 0.981.14)0.168age ( year)0.97 ( 0.911.04)0.4000.98 ( 0.911.05)0.482sex ( woman)1.20 ( 0.453.14)0.7171.23 ( 0.473.23)0.673knee pain ( yes)0.44 ( 0.181.10)0.0800.40 ( 0.180.93)0.034results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio participant characteristics and prevalence of cartilaginous tumours by bmi groups results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentages univariate and multivariate analysis for risk factors of presence of cartilaginous tumours results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio mean lesion size was 12 ( range 2 31 ) mm in the longest axis . eight lesions in eight participants displayed at least one of the predefined aggressive criteria and were referred for follow - up . seven had a tumour larger than 20 mm ( range 2231 mm ) , including one with deep endosteal scalloping . in six of the seven participants referred because of tumour size , dynamic contrast mri was performed , while one patient was subsequently lost to follow - up . in the fifth patient with a lesion of 22 mm , and in the sixth patient with a larger lesion with deep endosteal scalloping , curettage and en bloc resection of the tumour were performed respectively , and pathology confirmed the diagnosis of act / cs1 ( fig . the patient with the smaller lesion but with focal cortical destruction had radiographic follow - up and underwent curettage 2 years later , showing an act / cs1 at histology . the three act / cs1s that were found correspond to an estimated population prevalence of 0.4 % ( 95 % ci 0.20.8 % ) .fig . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 large cartilaginous tumour centrally located in the distal femur , referred for follow - up : a axial and b sagittal t1-weighted images , c axial t2-weighted image , d axial and e sagittal t1-weighted images after gadolinium administration with fat suppression . the time signal intensity curve of the dynamic mri ( e ) showed fast enhancement ( < 10 s ) , interpreted as most likely act / cs1 , although chondrosarcoma grade 2 could not be excluded due to extensive presence of mucoid ( pink artery , orange tumour , and blue bone marrow as reference tissue ) . curettage was performed and histology ( f ) confirmed the presence of cartilaginous tumour with increased cellularity and occasional binucleated cells as well as focal mucomyxoid matrix changes ( left lower area ) , diagnosed as atypical cartilaginous tumour / chondrosarcoma grade 1 estimated population prevalence of cartilaginous tumours in bmi strata are shown in table 1 . compared with having a bmi < 25 kg / m , the odds ratio ( or , 95 % confidence interval ) of the presence of cartilaginous tumour associated with a bmi of 2530 kg / m was 1.79 ( 0.38 , 8.51 ) , with a bmi of 3040 kg / m it was 1.69 ( 0.38 , 7.47 ) , and with a bmi > 40 kg / m it was 3.74 ( 0.64 , 21.87).table 1participant characteristics and prevalence of cartilaginous tumours by bmi groupsbmi ( kg / m)<25(35 % ) 2530(44 % ) 3040(19 % ) 40(2 % ) age ( years)56 ( 6)56 ( 6)55 ( 6)53 ( 6)sex ( % women)63466084bmi ( kg / m)22.6 ( 1.5)27.4 ( 1.3)33.3 ( 2.7)42.9 ( 2.4)cartilaginous tumour prevalence ( % ) 1.93.23.17.1knee pain ( % yes)14172327results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentagestable 2univariate and multivariate analysis for risk factors of presence of cartilaginous tumoursvariablesunivariate analysismultivariate analysisor ( 95 % ci ) p or ( 95 % ci ) p bmi ( kg / m)1.05 ( 0.971.14)0.2021.06 ( 0.981.14)0.168age ( year)0.97 ( 0.911.04)0.4000.98 ( 0.911.05)0.482sex ( woman)1.20 ( 0.453.14)0.7171.23 ( 0.473.23)0.673knee pain ( yes)0.44 ( 0.181.10)0.0800.40 ( 0.180.93)0.034results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio participant characteristics and prevalence of cartilaginous tumours by bmi groups results are based on weighted analysis of the study population ( n = 1285 ) . values are presented as mean ( sd ) or percentages univariate and multivariate analysis for risk factors of presence of cartilaginous tumours results are based on weighted analysis of the study population ( n = 1285 ) ci confidence interval , or odds ratio in a cohort study including knee mri to investigate osteoarthritis , we observed incidental lesions that we classified as cartilaginous tumours on the basis of predefined imaging criteria in 2.8 % of individuals between 45 and 65 years of age . these cartilaginous tumours were all smaller than 32 mm , typically smaller than 20 mm and were located near the femoral or sometimes tibial metaphysis . the prevalence of act / cs1 in our population was low ( 0.4 % ) , but we found an act / cs1 in three out of seven participants whom we classified , on the basis of predefined criteria , as high risk of carrying an act / cs1 and had a complete data set . these act / cs1s were present in two participants with tumours exhibiting aggressive features ( deep endosteal scalloping or cortical bone destruction ) and one participant with a tumour of 22 mm and were either resected or removed by curettage . in four other lesions with a diameter of greater than 20 mm the criteria of dynamic mri were consistent with an indolent benign enchondroma . our study confirms these results with a similar prevalence of cartilaginous tumours of 2.8 % . however , we also showed that act / cs1s are relatively common among larger tumours and those with suspicious features . the high sensitivity of mri in detecting small cartilaginous tumours on mri performed for other purposes can pose a dilemma to the clinician . in a previous radiograph - based case series , location in the axial skeleton and size greater than 5 cm were the most reliable predictors for low - grade chondrosarcoma , while clinical symptoms and morphologic radiographic features did not have added value . we observed mri signs suggestive of chondrosarcoma such as deep endosteal scalloping , cortical destruction and soft - tissue extension in two out of three participants who indeed turned out to have act / cs1 . nevertheless , even with dynamic contrast - enhanced mri , differentiating cartilaginous tumours remains challenging with false - positive findings unavoidable .table 3imaging features reported in literature to be suggestive of chondrosarcomapain related to the tumourlocation in the axial skeletonlarge lesion size ( > 5 cm)lobulated contourill - defined marginenlargement of the medullary cavity and cortical thickeningendosteal scalloping > 2/3 of the cortical thickness or over > 2/3 of the lesion lengthneurovascular involvementliquefactionperitumoral oedema*moth - eaten or permeative osteolysis*spontaneous pathologic fracture*periosteal reaction*cortical destruction*soft - tissue extension*early ( < 10 s ) enhancement on dynamic contrast mriintense uptake on bone scintigraphyfdg - pet high uptake*imaging features reported in the literature making a diagnosis of chondrosarcoma more likely . [ 1721 ] * features suggestive of high - grade ( grade 1 and 2 ) chondrosarcoma chondrosarcoma incidence has been estimated at 0.0005 % per year and prevalence at 0.0010.009 % , with only 7 % of those located around the knee [ 2325 ] . in our study we observed an unexpectedly high estimated population prevalence of act / cs1 of 0.4 % . as no further examinations or follow - up were performed when tumours were small and lacked suspicious features , the actual prevalence of act / cs1 may be even higher . only small lesions lacking suspicious features may be regarded as a normal concurrent finding on mri . in patients with any of the described suspicious features , the chance of having an atc / cs1 is quite high ( three out of seven in our population ) . as we did not routinely do resections and do not yet have 10-year follow - up , the proof of excluding act / cs1 in the three unsuspicious larger lesions was incomplete . the estimated population prevalence of incidental asymptomatic cartilaginous tumours around the knee was 2.8 % . the prevalence of act / cs1 may be higher than previously thought ( 0.4 % ) . given the high prevalence of enchondromas , as a practical rule we do not advocate further imaging examination or follow - up of accidentally found cartilaginous tumours in patients up to 65 years when the tumours are small ( at most 20 mm ) and suspicious features such as deep endosteal scalloping or cortical destruction are absent . however , when any of these suspicious features are present , or when the lesion is larger than 20 mm in diameter , further analysis is indicated .

although nitric oxide ( no ) has been shown to be involved in many physiological processes and to affect several organ systems , it is best known for its role in vasodilation and related cardiovascular health.1 vascular dysfunction and damage have been shown to be associated with impaired endothelial no metabolism and function . this impairment is considered to be the primary step in the development of metabolic syndrome and atherosclerosis.24 therefore , maintaining no homeostasis is critical for optimal health and disease prevention . furthermore , it has been suggested that an increase in no production may enhance oxygen and nutrient delivery to active muscles , thus offering promise for the potential to improve exercise performance and recovery.5 supplementation of l - arginine has a vasodilatory effect in systemic and pulmonary vascular beds and can correct abnormal endothelium - dependent vasodilation seen in hypertensive patients.6 no is synthesized endogenously in various cells from the recirculation of nitrites and from the nonessential amino acid l - arginine.7 enterosalivary recirculation whereby nitrate from the diet and from endogenous no production is reduced to nitrite contributes to the maintenance of no . the recent recognition of this enterosalivary recirculation opens up the potential for using saliva as a biomarker for no status.8 there has been extensive investigation into the use of supplemental l - arginine to enhance no production in order to enhance vascular function and prevent associated diseases . there is limited evidence to support the efficacy of supplemental l - arginine in cardiovascular disease.9 it has been suggested that the lack of efficacy may be explained by alterations in bioavailability as a result of the disease state . this was supported by evidence demonstrating that results are more promising when l - arginine is combined with other substances , such as silicon , that enhance absorption and bioavailability.1014 silicon is an abundant trace element found in many plant based foods as well as many antacids and analgesics . it plays an important role in skin and bone health.15 additionally , there is some evidence to suggest that silicon , especially as silicic acid , may protect vascular integrity during age - related vascular diseases.16 it has been demonstrated in rat models that arginine silicate inositol complex is more effective in increasing serum arginine levels than the commonly used arginine hydrochloride.17 very little is known about the pharmacokinetics ( pks ) and dynamics of arginine or its availability relative to an ingested dose , and even less is known about arginine combined with silicon.18 therefore , it is the purpose of this study to : 1 ) characterize the short - term ( 6-hour ) single - dose pks of plasma l - arginine and serum silicon with an oral arginine silicate dietary supplement , before and after 14 days supplementation , based on cmax , tmax , and t1/2 and 2 ) determine the effects of a single dose and 14 days use of an oral arginine silicate dietary supplement on no availability / production as measured as salivary nitrite . ten healthy nonsmoking males of age 1840 with a body mass index ( bmi ) between 18 kg / m and 29.9 kg / m were included in this study . they were recruited from a group of 34 phone - screened subjects from whom 15 were brought in for on - site screening and five did not meet the inclusion criteria . subjects were eligible if they met the abovementioned criteria and were not taking any supplements at the time of the study . elevated blood pressure ( 140/90 ) , taking silicate supplements or medications containing silicate , elevated serum creatinine , having a significant medical history , or any medical condition deemed significant by the principal investigator were all exclusion criteria . the study protocol was approved by aspire independent review board , august 15 , 2013 . this was a prospective , single - product , open - label , pk / pharmacodynamic ( pd)/safety clinical trial . subjects reported to the laboratory for two pk test visits , with visits separated by 14 days 2 days . subjects took three 500 mg ( total of 1,500 mg / day ) capsules of the arginine silicate dietary supplement , nitrosigine ( nutrition 21 , llc , purchase , ny , usa ) a food and drug administration generally recognized as safe substance , for 14 days between the two pk test visits . the purpose of the 14-day supplementation period was to address concerns that the dose of arginine silicate in a single dose might be too small for the assessment of arginine bioavailability . prior to each pk test visit , subjects were asked not to consume foods high in arginine or silica for 24 hours , not to exercise for 12 hours , and to fast for at least 10 hours before testing . upon reporting to the laboratory on days 1 and 14 , subjects received three 500 mg ( 1,500 mg ) capsules of the arginine silicate dietary supplement and had blood collected nine times over a 6-hour test period . blood collections were drawn prior to taking the test product ( for baseline assessment ) by subjects and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration . all blood samples , for both pk test visits , were analyzed for serum silicon and plasma l - arginine to determine the bioavailability and pk parameters of silicon and arginine . saliva samples were collected prior to subjects taking the test product ( for baseline assessment ) and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration for the assessment of salivary nitrite ( for both pk test visits ) . subjects were not provided standardized meals during the pk test visits but were provided standard quantities of ultra - high purity water for hydration purposes . safety analysis consisted of adverse events and subjective comments , and all analytics for plasma and serum samples were analyzed by a third party laboratory , keystone bioanalytical ( north wales , pa , usa ) . plasma l - arginine concentrations were determined by liquid chromatography ( lc)-tandem mass spectrometry ( ms ) analysis . briefly , a 50-l aliquot of plasma was spiked with stable isotope - labeled l - arginine , which served as internal standards . protein was precipitated with 100 l of methanol , filtrated through a 0.22 m hydrophilic membrane ( multiscreen hts ; emd millipore , billerica , ma , usa ) and analyzed by lc - tandem ms . quantification was performed by selected reaction monitoring of the respective daughter ions of analytes and internal standards ( waters , eschborn , germany ) . quantitative determination of silicon in human plasma was by inductively coupled plasma mass spectrometry ( icp - ms ) . the diluted solution was then spiked with internal standard solution ( vanadium ) and pumped to icp - ms system ( aglient 7500cx ) for analysis . concentration was calculated based on ratio of si to v signals with calibration range of 0.120 mcg / ml . salivary nitrite testing was done via the colorimetric test strip method ( neogenis labs , austin , tx , usa ) . pk and pd variables consisted of plasma l - arginine , serum silicon , and salivary nitrite at preingestion and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postingestion . pk endpoints consisted of the standard noncompartmental pk parameters ( cmax , tmax , and t1/2 ) for plasma l - arginine and serum silicon . pd endpoints consisted of the changes in salivary nitrite from baseline to 6 hours postproduct administration after a single dose and after 14 days use . all pk endpoints , with and without adjustment for body weight , were evaluated , along with their standard errors and 95% confidence intervals . changes from preingestion to each postingestion time point and changes between the two testing visits ( before and after 14 days of supplementation ) were tested for significance by paired student s t - test or repeated - measures analysis of variance . ten healthy nonsmoking males of age 1840 with a body mass index ( bmi ) between 18 kg / m and 29.9 kg / m were included in this study . they were recruited from a group of 34 phone - screened subjects from whom 15 were brought in for on - site screening and five did not meet the inclusion criteria . subjects were eligible if they met the abovementioned criteria and were not taking any supplements at the time of the study . elevated blood pressure ( 140/90 ) , taking silicate supplements or medications containing silicate , elevated serum creatinine , having a significant medical history , or any medical condition deemed significant by the principal investigator were all exclusion criteria . the study protocol was approved by aspire independent review board , august 15 , 2013 . this was a prospective , single - product , open - label , pk / pharmacodynamic ( pd)/safety clinical trial . subjects reported to the laboratory for two pk test visits , with visits separated by 14 days 2 days . subjects took three 500 mg ( total of 1,500 mg / day ) capsules of the arginine silicate dietary supplement , nitrosigine ( nutrition 21 , llc , purchase , ny , usa ) a food and drug administration generally recognized as safe substance , for 14 days between the two pk test visits . the purpose of the 14-day supplementation period was to address concerns that the dose of arginine silicate in a single dose might be too small for the assessment of arginine bioavailability . prior to each pk test visit , subjects were asked not to consume foods high in arginine or silica for 24 hours , not to exercise for 12 hours , and to fast for at least 10 hours before testing . upon reporting to the laboratory on days 1 and 14 , subjects received three 500 mg ( 1,500 mg ) capsules of the arginine silicate dietary supplement and had blood collected nine times over a 6-hour test period . blood collections were drawn prior to taking the test product ( for baseline assessment ) by subjects and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration . all blood samples , for both pk test visits , were analyzed for serum silicon and plasma l - arginine to determine the bioavailability and pk parameters of silicon and arginine . saliva samples were collected prior to subjects taking the test product ( for baseline assessment ) and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration for the assessment of salivary nitrite ( for both pk test visits ) . subjects were not provided standardized meals during the pk test visits but were provided standard quantities of ultra - high purity water for hydration purposes . all analytics for plasma and serum samples were analyzed by a third party laboratory , keystone bioanalytical ( north wales , pa , usa ) . plasma l - arginine concentrations were determined by liquid chromatography ( lc)-tandem mass spectrometry ( ms ) analysis . briefly , a 50-l aliquot of plasma was spiked with stable isotope - labeled l - arginine , which served as internal standards . protein was precipitated with 100 l of methanol , filtrated through a 0.22 m hydrophilic membrane ( multiscreen hts ; emd millipore , billerica , ma , usa ) and analyzed by lc - tandem ms . quantification was performed by selected reaction monitoring of the respective daughter ions of analytes and internal standards ( waters , eschborn , germany ) . quantitative determination of silicon in human plasma was by inductively coupled plasma mass spectrometry ( icp - ms ) . the diluted solution was then spiked with internal standard solution ( vanadium ) and pumped to icp - ms system ( aglient 7500cx ) for analysis . concentration was calculated based on ratio of si to v signals with calibration range of 0.120 mcg / ml . salivary nitrite testing was done via the colorimetric test strip method ( neogenis labs , austin , tx , usa ) . pk and pd variables consisted of plasma l - arginine , serum silicon , and salivary nitrite at preingestion and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postingestion . pk endpoints consisted of the standard noncompartmental pk parameters ( cmax , tmax , and t1/2 ) for plasma l - arginine and serum silicon . pd endpoints consisted of the changes in salivary nitrite from baseline to 6 hours postproduct administration after a single dose and after 14 days use . all pk endpoints , with and without adjustment for body weight , were evaluated , along with their standard errors and 95% confidence intervals . changes from preingestion to each postingestion time point and changes between the two testing visits ( before and after 14 days of supplementation ) were tested for significance by paired student s t - test or repeated - measures analysis of variance . all ten participants completed the study and were healthy male nonsmokers , age 1840 , with a bmi between 18 kg / m and 29.9 kg / m . therefore , the product was found to be safe within the protocol of this study . day 1 pk parameters for arginine were cmax 30.067.80 g / ml , tmax 1.130.52 hours , and t1/2 15.939.55 hours and for silicon were cmax 2.990.63 g / ml , tmax 2.442.05 hours , and t1/2 34.5616.56 hours . there were no significant changes in pk parameters with 14 days of use ( with and without adjustment for body weight ) . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use indicating a predictable pk profile . for day 1 and day 14 , there were statistically significant increases in plasma arginine from baseline at 0.5 hours , 1 hour , and 1.5 hours ( p=<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p=<0.05 ) . baseline plasma arginine was higher at day 14 than day 1 , and the difference trended toward statistical significance ( p=0.0645 ) . additionally , 4-hour plasma arginine was significantly higher at day 14 than day 1 ( p=0.0488 ) , figure 1 . for day 1 and day 14 , there were statistically significant increases in serum silicon from baseline at 1 hour and 1.5 hours ( p<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p<0.01 ) . there were no significant differences in serum silicon levels at any time point over the 6-hour postdose period between day 1 and day 14 , figure 2 . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 0.5 hours after the first dose ( p=0.125 ) . after 14 days of use , baseline salivary nitrite levels increased in six subjects and stayed the same in four subjects ; this shift was significant ( p=0.031 ) . day 1 pk parameters for arginine were cmax 30.067.80 g / ml , tmax 1.130.52 hours , and t1/2 15.939.55 hours and for silicon were cmax 2.990.63 g / ml , tmax 2.442.05 hours , and t1/2 34.5616.56 hours . there were no significant changes in pk parameters with 14 days of use ( with and without adjustment for body weight ) . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use indicating a predictable pk profile . for day 1 and day 14 , there were statistically significant increases in plasma arginine from baseline at 0.5 hours , 1 hour , and 1.5 hours ( p=<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p=<0.05 ) . baseline plasma arginine was higher at day 14 than day 1 , and the difference trended toward statistical significance ( p=0.0645 ) . additionally , 4-hour plasma arginine was significantly higher at day 14 than day 1 ( p=0.0488 ) , figure 1 . for day 1 and day 14 , there were statistically significant increases in serum silicon from baseline at 1 hour and 1.5 hours ( p<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p<0.01 ) . there were no significant differences in serum silicon levels at any time point over the 6-hour postdose period between day 1 and day 14 , figure 2 . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 0.5 hours after the first dose ( p=0.125 ) . after 14 days of use , baseline salivary nitrite levels increased in six subjects and stayed the same in four subjects ; this shift was significant ( p=0.031 ) . it has been suggested that adding substances shown to enhance arginine absorption , such as silicon , may be an effective method for addressing issues with arginine bioavailability.914 therefore , in this study , we tested an arginine silicate supplement to determine if it increased plasma l - arginine and serum silicon as well as no production . our results indicate that an arginine silicate dietary supplement increased blood levels of arginine and silicon in both an acute and chronic dosing regimen . after a single 1,500 mg dose of an arginine silicate supplement , levels of arginine increased significantly for up to 5 hours and levels of silicon for up to 1.5 hours . similarly , a study by proctor et al demonstrated in rat models that arginine silicate inositol complex is more effective in increasing serum arginine levels than the commonly used arginine hydrochloride product.17 this was supported by evidence demonstrating that results in clinical populations are more promising when l - arginine is combined with other substances such as silicon that enhance absorption and bioavailability.1014 the increase observed in the current study indicates that the product we tested does increase levels of both arginine and silicon . the peak concentration of plasma arginine ( cmax ) was 30.067.80 g / ml , ~1 hour ( tmax ) after an oral dose of 1,500 mg of the arginine silicate dietary supplement and t1/2 15.939.55 hours . there is very little reported in the literature regarding the pk parameters for arginine.19,20 tangphao et al reported a peak concentration ( cmax ) for arginine of 5013.4 g / ml 1 hour after oral administration of 10 g l - arginine.20 schwedhelm et al reported peak concentrations of 49.06 g / ml 3.7 hours after 7 days of a 3.2 g daily oral dose of slow release l - arginine and 84.09.7 hours after a 3 g daily dose of immediate release arginine.21 the higher peak values measured in the tangphao et al and schwedhelm et al studies could be explained by the higher doses ( 10 g and 3 g over 7 days vs 681 mg ) or by individual differences influencing a small subject population . for example , schwedhelm et al demonstrated that plasma arginine and the associated no - dependent signaling is increased in a dose - dependent manner when they compared the pk parameters of different doses of l - arginine , indicating the dose does influence peak serum values . tangphao et al measured plasma arginine in 20 subjects consuming standardized meals over 8 hours and reported significant individual variations . the authors suggested that individual differences in bioavailability and clearance make clarifying pk parameters for arginine challenging.20 it is promising to note that although our peak values were slightly lower than those seen in the other studies , we reported similar tmax values . furthermore , the peak serum arginine was only slightly lower considering the much lower dose used in our study , which was 50% of the dose used in the schwedhelm et al study and 15% of the dose used in the tangphao et al study . this is possibly explained by the addition of silicon in the product used in our study . in our study , the peak concentration ( cmax ) for silicon was 2.990.63 g / ml approximately 2 hours and 44 minutes after the 1,500 mg dose of the product and t1/2 34.5616.56 hours . defining the pk parameters for silicon is difficult due to the variation in absorption rates for different forms of silicon.22,23 a study by sripanyakorn et al22 attempted to define the silicon absorption of various foods and common dietary supplemental sources . sripanyakorn et al reported peak serum silicon concentrations after ingestion of various common supplemental forms of silicon ranging from 30 minutes to 4 hours , with peak concentrations dependent on the source of silicon . therefore , it can be hypothesized that increased serum levels of l - arginine can increase no production and under healthy normal conditions , no production typically proceeds quite efficiently.24 the recognition of an enterosalivary cycle whereby nitrate and nitrite are reduced to no makes it reasonable to use saliva as a potential biomarker for no status.8 therefore , we used salivary nitrite as a biomarker for no production . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 30 minutes after the first dose . after 14 days of use , baseline salivary nitrite levels increased in six of the ten subjects . this is supporting evidence that there was some improvement in no production after ingesting the dietary arginine silicate supplement . similarly , schwedhelm et al showed evidence of increased no production after supplementation of 3.2 g daily of l - arginine for 1 week as measured by the ratio of plasma l - arginine over asymmetric dimethylarginine , an endogenous inhibitor of no synthase ( arginine / asymmetric dimethylarginine ratio ) . schwedhelm et al did not report any effect from lower doses of l - arginine . our study used a lower dose and saw evidence suggesting an increase in no production most likely due to the addition of silicate in the product tested , whereas in the schwedhelm et al study , arginine hcl was tested . while the use of salivary nitrite as a biomarker for no production offers an inexpensive noninvasive method for measuring no production , its limitations need to be considered . individuals do not have the same communities of oral bacteria that are responsible for reducing salivary nitrate to nitrite . it can be altered by the use of antiseptic mouthwash , overuse of antibiotics , reduced gastric acidity , decreased swallowing , and by different degrees of oral hygiene . all efforts were made to standardize salivary collections to minimize chances of contamination or non - optimal sample collection . second , steady - state concentrations of nitrite in the saliva are dependent upon salivary flow rates and production . therefore , patients with various clinical conditions and/or surgeries will potentially have a breakdown in this pathway making accurate assessment challenging.8 the administration of performance - enhancing supplements is often rationalized on the basis of a chain of plausibility arguments . arginine has been studied extensively for 15 years as a natural metabolic donor of no ; so arginine - containing products should increase circulating arginine levels , which should increase ( or at least maintain ) relatively high no levels , which should promote relaxation of smooth muscle in blood vessels , which should increase blood flow to the muscles , which should lead to improved muscle function and stamina , and which should show up as improved athletic performance . the results of this study support two links in the above chain : the first link , the arginine silicate product increased circulating arginine levels , and the second link , this increase in circulating arginine was associated with a tendency of baseline salivary nitrite to increase which could suggest increased no levels . studies exploring the remaining links of this plausibility chain are warranted as the potential for the arginine silicate product to enhance athletic performance is promising . the arginine silicate dietary supplement increases blood levels of arginine after a single dose within 30 minutes for up to 5 hours and increases blood levels of silicon for up to 1.5 hours . blood levels of arginine silicon and no ( salivary nitrite ) were elevated consistently after 14 days of daily use . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use , indicating a predictable pk profile . the observed increase in baseline salivary nitrite is supporting information that there was some improvement in no production . further study on the effect of this supplement on no production and the resulting physiological effect is warranted .

purposethe purpose of this study was to characterize the pharmacokinetics ( pks ) and pharmacodynamics ( pds ) of an oral inositol - stabilized arginine silicate dietary supplement.subjects and methodsten healthy males , 26.75.4 years , took three 500 mg arginine silicate capsules ( active product ) for 14 days . the subjects attended test visits on days 1 and 14 . fasting blood and saliva collections were performed predose and at 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postdose for plasma arginine , serum silicon , and salivary nitric oxide ( no ) + nitrite.resultsday 1 pk parameters ( adjusted for body weight ) for arginine were peak serum concentration ( cmax ) 30.067.80 g / ml , time it takes to reach peak serum concentration ( tmax ) 1.130.52 hours , and time required to reach half its original concentration ( t1/2 ) 15.939.55 hours and for silicon were cmax 2.990.63 g / ml , tmax 2.442.05 hours , and t1/2 34.5616.56 hours . after day 1 dose , arginine levels increased at 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , and 5 hours ( p<0.01 ) and silicon levels increased at 1 hour and 1.5 hours ( p<0.05 ) . after day 14 dose , arginine levels increased at 0.5 hours , 1 hour , and 1.5 hours ( p<0.05 ) and silicon levels increased at 1 hour , 1.5 hours , 2 hours , and 3 hours ( p<0.01 ) . after 14 days of use , baseline arginine trended toward being higher than baseline day 1 ( p=0.0645 ) , and 4-hour postdose plasma arginine was significantly higher ( p=0.0488 ) at day 14 than day 1 . although not a significant difference , no , as measured as salivary nitrate , increased in four subjects and stayed the same in six subjects at 0.5 hours after the first dose ( p=0.125 ) . after 14 days of use , baseline no levels increased in six subjects and stayed the same in four subjects ; this shift was significant ( p=0.031).conclusionthe arginine silicate dietary supplement increases blood levels of arginine after a single dose within 30 minutes and blood levels of silicon for up to 1.5 hours . blood levels of arginine , silicon , and no ( salivary nitrite ) were elevated consistently after 14 days of use . the observed increase in baseline salivary nitrite is supporting information that there was some improvement in no production . further study on the effect of this supplement on no production and the resulting physiological effect is warranted . within the specific protocol of this study , the product was found to be safe .

Introduction Material and methods Subjects Study design Biochemical analyses PK/PD analyses Statistical analyses Results PK parameters Plasma arginine Serum silicon Salivary nitrite Discussion Conclusion

although nitric oxide ( no ) has been shown to be involved in many physiological processes and to affect several organ systems , it is best known for its role in vasodilation and related cardiovascular health.1 vascular dysfunction and damage have been shown to be associated with impaired endothelial no metabolism and function . furthermore , it has been suggested that an increase in no production may enhance oxygen and nutrient delivery to active muscles , thus offering promise for the potential to improve exercise performance and recovery.5 supplementation of l - arginine has a vasodilatory effect in systemic and pulmonary vascular beds and can correct abnormal endothelium - dependent vasodilation seen in hypertensive patients.6 no is synthesized endogenously in various cells from the recirculation of nitrites and from the nonessential amino acid l - arginine.7 enterosalivary recirculation whereby nitrate from the diet and from endogenous no production is reduced to nitrite contributes to the maintenance of no . the recent recognition of this enterosalivary recirculation opens up the potential for using saliva as a biomarker for no status.8 there has been extensive investigation into the use of supplemental l - arginine to enhance no production in order to enhance vascular function and prevent associated diseases . it plays an important role in skin and bone health.15 additionally , there is some evidence to suggest that silicon , especially as silicic acid , may protect vascular integrity during age - related vascular diseases.16 it has been demonstrated in rat models that arginine silicate inositol complex is more effective in increasing serum arginine levels than the commonly used arginine hydrochloride.17 very little is known about the pharmacokinetics ( pks ) and dynamics of arginine or its availability relative to an ingested dose , and even less is known about arginine combined with silicon.18 therefore , it is the purpose of this study to : 1 ) characterize the short - term ( 6-hour ) single - dose pks of plasma l - arginine and serum silicon with an oral arginine silicate dietary supplement , before and after 14 days supplementation , based on cmax , tmax , and t1/2 and 2 ) determine the effects of a single dose and 14 days use of an oral arginine silicate dietary supplement on no availability / production as measured as salivary nitrite . elevated blood pressure ( 140/90 ) , taking silicate supplements or medications containing silicate , elevated serum creatinine , having a significant medical history , or any medical condition deemed significant by the principal investigator were all exclusion criteria . subjects took three 500 mg ( total of 1,500 mg / day ) capsules of the arginine silicate dietary supplement , nitrosigine ( nutrition 21 , llc , purchase , ny , usa ) a food and drug administration generally recognized as safe substance , for 14 days between the two pk test visits . the purpose of the 14-day supplementation period was to address concerns that the dose of arginine silicate in a single dose might be too small for the assessment of arginine bioavailability . prior to each pk test visit , subjects were asked not to consume foods high in arginine or silica for 24 hours , not to exercise for 12 hours , and to fast for at least 10 hours before testing . upon reporting to the laboratory on days 1 and 14 , subjects received three 500 mg ( 1,500 mg ) capsules of the arginine silicate dietary supplement and had blood collected nine times over a 6-hour test period . blood collections were drawn prior to taking the test product ( for baseline assessment ) by subjects and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration . all blood samples , for both pk test visits , were analyzed for serum silicon and plasma l - arginine to determine the bioavailability and pk parameters of silicon and arginine . saliva samples were collected prior to subjects taking the test product ( for baseline assessment ) and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration for the assessment of salivary nitrite ( for both pk test visits ) . the diluted solution was then spiked with internal standard solution ( vanadium ) and pumped to icp - ms system ( aglient 7500cx ) for analysis . pk and pd variables consisted of plasma l - arginine , serum silicon , and salivary nitrite at preingestion and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postingestion . pk endpoints consisted of the standard noncompartmental pk parameters ( cmax , tmax , and t1/2 ) for plasma l - arginine and serum silicon . pd endpoints consisted of the changes in salivary nitrite from baseline to 6 hours postproduct administration after a single dose and after 14 days use . all pk endpoints , with and without adjustment for body weight , were evaluated , along with their standard errors and 95% confidence intervals . changes from preingestion to each postingestion time point and changes between the two testing visits ( before and after 14 days of supplementation ) were tested for significance by paired student s t - test or repeated - measures analysis of variance . elevated blood pressure ( 140/90 ) , taking silicate supplements or medications containing silicate , elevated serum creatinine , having a significant medical history , or any medical condition deemed significant by the principal investigator were all exclusion criteria . subjects reported to the laboratory for two pk test visits , with visits separated by 14 days 2 days . subjects took three 500 mg ( total of 1,500 mg / day ) capsules of the arginine silicate dietary supplement , nitrosigine ( nutrition 21 , llc , purchase , ny , usa ) a food and drug administration generally recognized as safe substance , for 14 days between the two pk test visits . the purpose of the 14-day supplementation period was to address concerns that the dose of arginine silicate in a single dose might be too small for the assessment of arginine bioavailability . prior to each pk test visit , subjects were asked not to consume foods high in arginine or silica for 24 hours , not to exercise for 12 hours , and to fast for at least 10 hours before testing . upon reporting to the laboratory on days 1 and 14 , subjects received three 500 mg ( 1,500 mg ) capsules of the arginine silicate dietary supplement and had blood collected nine times over a 6-hour test period . blood collections were drawn prior to taking the test product ( for baseline assessment ) by subjects and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration . all blood samples , for both pk test visits , were analyzed for serum silicon and plasma l - arginine to determine the bioavailability and pk parameters of silicon and arginine . saliva samples were collected prior to subjects taking the test product ( for baseline assessment ) and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postproduct administration for the assessment of salivary nitrite ( for both pk test visits ) . pk and pd variables consisted of plasma l - arginine , serum silicon , and salivary nitrite at preingestion and 0.5 hours , 1 hour , 1.5 hours , 2 hours , 3 hours , 4 hours , 5 hours , and 6 hours postingestion . pk endpoints consisted of the standard noncompartmental pk parameters ( cmax , tmax , and t1/2 ) for plasma l - arginine and serum silicon . pd endpoints consisted of the changes in salivary nitrite from baseline to 6 hours postproduct administration after a single dose and after 14 days use . all pk endpoints , with and without adjustment for body weight , were evaluated , along with their standard errors and 95% confidence intervals . changes from preingestion to each postingestion time point and changes between the two testing visits ( before and after 14 days of supplementation ) were tested for significance by paired student s t - test or repeated - measures analysis of variance . therefore , the product was found to be safe within the protocol of this study . day 1 pk parameters for arginine were cmax 30.067.80 g / ml , tmax 1.130.52 hours , and t1/2 15.939.55 hours and for silicon were cmax 2.990.63 g / ml , tmax 2.442.05 hours , and t1/2 34.5616.56 hours . there were no significant changes in pk parameters with 14 days of use ( with and without adjustment for body weight ) . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use indicating a predictable pk profile . for day 1 and day 14 , there were statistically significant increases in plasma arginine from baseline at 0.5 hours , 1 hour , and 1.5 hours ( p=<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p=<0.05 ) . baseline plasma arginine was higher at day 14 than day 1 , and the difference trended toward statistical significance ( p=0.0645 ) . additionally , 4-hour plasma arginine was significantly higher at day 14 than day 1 ( p=0.0488 ) , figure 1 . for day 1 and day 14 , there were statistically significant increases in serum silicon from baseline at 1 hour and 1.5 hours ( p<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p<0.01 ) . there were no significant differences in serum silicon levels at any time point over the 6-hour postdose period between day 1 and day 14 , figure 2 . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 0.5 hours after the first dose ( p=0.125 ) . after 14 days of use , baseline salivary nitrite levels increased in six subjects and stayed the same in four subjects ; this shift was significant ( p=0.031 ) . day 1 pk parameters for arginine were cmax 30.067.80 g / ml , tmax 1.130.52 hours , and t1/2 15.939.55 hours and for silicon were cmax 2.990.63 g / ml , tmax 2.442.05 hours , and t1/2 34.5616.56 hours . there were no significant changes in pk parameters with 14 days of use ( with and without adjustment for body weight ) . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use indicating a predictable pk profile . for day 1 and day 14 , there were statistically significant increases in plasma arginine from baseline at 0.5 hours , 1 hour , and 1.5 hours ( p=<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p=<0.05 ) . baseline plasma arginine was higher at day 14 than day 1 , and the difference trended toward statistical significance ( p=0.0645 ) . additionally , 4-hour plasma arginine was significantly higher at day 14 than day 1 ( p=0.0488 ) , figure 1 . for day 1 and day 14 , there were statistically significant increases in serum silicon from baseline at 1 hour and 1.5 hours ( p<0.05 ) . for day 14 , there were also statistically significant increases from baseline at 2 hours and 3 hours ( p<0.01 ) . there were no significant differences in serum silicon levels at any time point over the 6-hour postdose period between day 1 and day 14 , figure 2 . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 0.5 hours after the first dose ( p=0.125 ) . after 14 days of use , baseline salivary nitrite levels increased in six subjects and stayed the same in four subjects ; this shift was significant ( p=0.031 ) . it has been suggested that adding substances shown to enhance arginine absorption , such as silicon , may be an effective method for addressing issues with arginine bioavailability.914 therefore , in this study , we tested an arginine silicate supplement to determine if it increased plasma l - arginine and serum silicon as well as no production . our results indicate that an arginine silicate dietary supplement increased blood levels of arginine and silicon in both an acute and chronic dosing regimen . after a single 1,500 mg dose of an arginine silicate supplement , levels of arginine increased significantly for up to 5 hours and levels of silicon for up to 1.5 hours . similarly , a study by proctor et al demonstrated in rat models that arginine silicate inositol complex is more effective in increasing serum arginine levels than the commonly used arginine hydrochloride product.17 this was supported by evidence demonstrating that results in clinical populations are more promising when l - arginine is combined with other substances such as silicon that enhance absorption and bioavailability.1014 the increase observed in the current study indicates that the product we tested does increase levels of both arginine and silicon . the peak concentration of plasma arginine ( cmax ) was 30.067.80 g / ml , ~1 hour ( tmax ) after an oral dose of 1,500 mg of the arginine silicate dietary supplement and t1/2 15.939.55 hours . there is very little reported in the literature regarding the pk parameters for arginine.19,20 tangphao et al reported a peak concentration ( cmax ) for arginine of 5013.4 g / ml 1 hour after oral administration of 10 g l - arginine.20 schwedhelm et al reported peak concentrations of 49.06 g / ml 3.7 hours after 7 days of a 3.2 g daily oral dose of slow release l - arginine and 84.09.7 hours after a 3 g daily dose of immediate release arginine.21 the higher peak values measured in the tangphao et al and schwedhelm et al studies could be explained by the higher doses ( 10 g and 3 g over 7 days vs 681 mg ) or by individual differences influencing a small subject population . for example , schwedhelm et al demonstrated that plasma arginine and the associated no - dependent signaling is increased in a dose - dependent manner when they compared the pk parameters of different doses of l - arginine , indicating the dose does influence peak serum values . tangphao et al measured plasma arginine in 20 subjects consuming standardized meals over 8 hours and reported significant individual variations . the authors suggested that individual differences in bioavailability and clearance make clarifying pk parameters for arginine challenging.20 it is promising to note that although our peak values were slightly lower than those seen in the other studies , we reported similar tmax values . furthermore , the peak serum arginine was only slightly lower considering the much lower dose used in our study , which was 50% of the dose used in the schwedhelm et al study and 15% of the dose used in the tangphao et al study . this is possibly explained by the addition of silicon in the product used in our study . in our study , the peak concentration ( cmax ) for silicon was 2.990.63 g / ml approximately 2 hours and 44 minutes after the 1,500 mg dose of the product and t1/2 34.5616.56 hours . defining the pk parameters for silicon is difficult due to the variation in absorption rates for different forms of silicon.22,23 a study by sripanyakorn et al22 attempted to define the silicon absorption of various foods and common dietary supplemental sources . sripanyakorn et al reported peak serum silicon concentrations after ingestion of various common supplemental forms of silicon ranging from 30 minutes to 4 hours , with peak concentrations dependent on the source of silicon . therefore , it can be hypothesized that increased serum levels of l - arginine can increase no production and under healthy normal conditions , no production typically proceeds quite efficiently.24 the recognition of an enterosalivary cycle whereby nitrate and nitrite are reduced to no makes it reasonable to use saliva as a potential biomarker for no status.8 therefore , we used salivary nitrite as a biomarker for no production . although not a significant difference , salivary nitrite increased in four subjects and stayed the same in six subjects at 30 minutes after the first dose . after 14 days of use , baseline salivary nitrite levels increased in six of the ten subjects . this is supporting evidence that there was some improvement in no production after ingesting the dietary arginine silicate supplement . similarly , schwedhelm et al showed evidence of increased no production after supplementation of 3.2 g daily of l - arginine for 1 week as measured by the ratio of plasma l - arginine over asymmetric dimethylarginine , an endogenous inhibitor of no synthase ( arginine / asymmetric dimethylarginine ratio ) . our study used a lower dose and saw evidence suggesting an increase in no production most likely due to the addition of silicate in the product tested , whereas in the schwedhelm et al study , arginine hcl was tested . while the use of salivary nitrite as a biomarker for no production offers an inexpensive noninvasive method for measuring no production , its limitations need to be considered . arginine has been studied extensively for 15 years as a natural metabolic donor of no ; so arginine - containing products should increase circulating arginine levels , which should increase ( or at least maintain ) relatively high no levels , which should promote relaxation of smooth muscle in blood vessels , which should increase blood flow to the muscles , which should lead to improved muscle function and stamina , and which should show up as improved athletic performance . the results of this study support two links in the above chain : the first link , the arginine silicate product increased circulating arginine levels , and the second link , this increase in circulating arginine was associated with a tendency of baseline salivary nitrite to increase which could suggest increased no levels . studies exploring the remaining links of this plausibility chain are warranted as the potential for the arginine silicate product to enhance athletic performance is promising . the arginine silicate dietary supplement increases blood levels of arginine after a single dose within 30 minutes for up to 5 hours and increases blood levels of silicon for up to 1.5 hours . blood levels of arginine silicon and no ( salivary nitrite ) were elevated consistently after 14 days of daily use . pk data indicate that absorption and active kinetics of the product remain consistent over 14 days of daily use , indicating a predictable pk profile . the observed increase in baseline salivary nitrite is supporting information that there was some improvement in no production . further study on the effect of this supplement on no production and the resulting physiological effect is warranted .

originally described as a pre - b cell growth factor expressed in bone marrow stromal cells ( 1 ) , interleukin-7 ( il-7 ) was subsequently identified as an essential and non - redundant cytokine in t cells ( 2,3 ) . in fact , il-7 plays a critical role in every aspect of t cell biology , including early t cell development in the thymus , cd4/cd8 lineage choice during positive selection as well as maintaining the survival and homeostasis of nave and memory t cells in the periphery ( 2 - 6 ) . genetic deficiencies in il-7 or in any component of the il-7 receptor ( il-7r ) complex , namely the il-7r alpha ( il-7r ) chain and the cytokine receptor shared common gamma ( c)-chain , result in severely impaired thymopoiesis and t cell survival . il-7 deficiency is manifested in humans as t(-)b(+)nk(- ) scid ( severe combined immunodeficiency ) ( 7,8 ) and in mice as severe deficiency in both b and t cells ( 2,3,9 ) . the requirement of il-7 in t cell development and survival is chiefly due to its anti - apoptotic effects by upregulating expression of bcl-2 and mcl-1 ( 10,11 ) , but also because of its trophic effects , such as inducing expression of glucose transporter-1 ( 12 ) . in mature nave t cells , il-7 is a non - redundant survival cytokine that can not be replaced by other pro - survival cytokines ( 4,5,13,14 ) . this non - redundancy is also partly the case for memory t cell maintenance ( 15 ) . interestingly , antigen specific memory cd8 t cells are still largely dependent on il-7 for cell survival but intermittent and basal homeostatic proliferation of these cells seems to be more dependent on il-15 ( 4,16 - 18 ) . this is in contrast to the bulk of memory phenotype cd8 t cells which are dependent on il-15 but not il-7 ( 19 ) . nevertheless , it remains evident that il-7 is a key molecule for maintaining the fitness and size of the peripheral t cell pool by providing essential survival signals . il-7 signaling is transduced by the il-7r heterodimer , which is composed of the il-7-specific il-7r -chain and the c - chain , which is a shared component with other members of the c - cytokine family , i.e. il-2 , il-4 , il-7 , il-9 , il-15 and il-21 ( 20 ) . ligand - induced heterodimerization of the il-7r/c - chains leads to juxtaposition and trans - phosphorylation of receptor - associated tyrosine kinases jak1 and jak3 ( 21 ) . activated jaks then phosphorylate specific tyrosine residues in the il-7r cytosolic domain that attract stat5 ( signal transducers and activators of transcription 5 ) and pi-3 kinases for jak - mediated phosphorylation and further downstream signaling . known downstream molecules of il-7 signaling include among others bcl-2 , bcl - xl , mcl-1 , glut-1 , cdc25a , il-7r , runx3 , and cd8 coreceptors , which are all essential for functional fitness and survival of t cells ( 6,10,12,22 - 25 ) . notably , multiple studies have documented that il-7 functions beyond serving as a pro - survival factor . for example , il-7 provides specific differentiation signals in immature lymphocytes by opening up the tcr-chain and the immunoglobulin heavy chain locus for antigen receptor recombination ( 26,27 ) . in this line , we recently showed that il-7 is necessary to specify cd8 lineage differentiation during cd4/cd8 cell fate choice in the thymus by inducing expression of the transcription factor runx3 ( 6 ) . additionally , il-7 is involved in the development of secondary lymphoid tissues since impaired il-7 signaling results in defects in lymph node organogenesis ( 28 ) . here , il-7 seems to regulate the size of the fetal lymphoid tissue inducer ( lti ) cell pool that interacts with stromal organizer cells to form lymphoid tissues ( 29 ) . thus , il-7 is a critical player in cell lineage choice and differentiation during lymphocyte as well as lymphoid tissue development . furthermore , il-7 also controls homeostasis of several innate lymphoid cell subsets ( 30 ) . within the t cell compartment , il-7 controls the tcr activation threshold of nave cd8 t cells by tuning cd8 coreceptor expression levels to the self - specificity of the tcr . in this way , il-7 maintains a self - reactive but not autoimmune t cell pool ( 31 ) . finally , il-7 downregulates expression of its own receptor . il-7 signaled cells express lower surface levels of il-7r so that unsignaled cells outcompete il-7 signaled t cells for remaining il-7 . we previously showed that maintaining this negative feedback loop is necessary for maximizing the size of the nave t cell pool ( 32 ) . collectively , il-7 is a multi - faceted pro - survival cytokine that governs the development , differentiation , and maintenance of t cells in a non - redundant manner . despite playing such critical roles , in fact , the identity of il-7 producing cells in the thymus is not fully understood as well as the precise location of il-7 expression in peripheral tissues remains veiled . recently , much light was shed on the in vivo source and regulation of il-7 expression by a series of four independent publications reporting the generation and characterization of il-7 reporter mice . using fluorescent proteins , cre recombinase and other reporter proteins , il-7 transcriptional activities were monitored in vivo and so depicted a spatiotemporal expression profile of il-7 expression during ontogeny and steady state conditions . here we review the experimental results and their findings in context of il-7 mediated t cell development and peripheral homeostasis . adoptive transfer of nave t cells into an il-7 deficient environment fail to induce homeostatic proliferation and transferred cells succumb to programmed cell death ( 4,5 ) . even as all t lineage cells are critically dependent on il-7 , no lymphocytes including t cells themselves produce il-7 ( 33 ) . importantly , the anatomical locations where immature thymocytes and t cells encounter il-7 for development and homeostasis are not entirely mapped . previously , il-7 message and proteins have been reported in various tissues including the thymus and secondary lymphoid organs such as spleen ( 34,35 ) and lymph nodes ( 36 ) . also , other tissues were implicated as sources of in vivo il-7 which comprise the intestine ( 37 ) , human liver ( 38 ) , skin ( 39,40 ) , murine ovaries ( 41 ) , human brain ( 42 ) , and human prostate ( 43 ) . but the precise identities of il-7 expressing cells remain still obscure . while il-7 is generally considered as a secreted cytokine , its expression levels in serum are extremely low suggesting that il-7 in vivo is limiting and that il-7 would be consumed locally . in fact , serum il-7 was never convincingly detected in mice sera until most recently , when mackall and colleagues were able to determine serum il-7 in wild type mice at levels of 10 pg / ml ( 44 ) . scarce il-7 in the circulation on the one hand suggests that il-7 would be produced only at very low levels , but on the other hand there is also the possibility that il-7 is sequestered from serum by being complexed to the extracellular matrix ( ecm ) . potentially , such immobilized il-7 could be the actual bioactive form of il-7 in vivo . indeed , mhc class ii thymic epithelial cells do express the ecm components fibronectin and heparin sulfate , and il-7 signaling in immature thymocytes is likely mediated by an ecm - associated form of il-7 ( 45 ) . moreover , il-7 can also act as a heterodimer when bound to a variant -chain of the hepatocyte growth factor ( hgf ) , which is then known as the pre - pro - b cell growth - stimulating factor ( ppbsf ) . both heterodimerization of il-7 and hgf and the biological activity of ppbsf are dependent on low levels of heparin sulfate - derived oligosaccharides . hgf is a cytokine homologous to plasminogen , and these data suggest that hgf -chains could allow interaction of il-7 with the stromal cell surface and thus deliver il-7 signaling as a membrane bound cytokine ( 46 ) . if this is indeed the case , then identifying the actual il-7 producer cells in vivo would be even more critical to understand where and when il-7 signaling is happening in t cells . without identifying il-7 producers , however generally , il-7 production in vivo is considered to be constitutive and unaffected by external signals , so that il-7 availability is a limiting factor in determining the size of the peripheral t cell pool ( 47 ) . on the other hand , there is a great body of evidence that il-7 expression in vivo can be modulated . serum il-7 concentration is elevated in lymphopenic humans and other primates ( 48,49 ) , and in the mouse , a novel il-7r mediated il-7 expression feedback loop was recently reported , further indicating that il-7 production is actively regulated in vivo ( 44 ) . furthermore , il-6 and tlr signaling in the liver have been shown to induce il-7 expression in hepatocytes suggesting that expression of immunoregulatory il-7 is broader and more dynamically regulated than currently presumed ( 50 ) . because of the lack of adequate reagents , however , the whole scope of il-7 expression remained largely unaddressed . recently , four independent studies reported a total of five different il-7 reporter mice whose detailed characterizations will be discussed below ( 51 - 54 ) . all il-7 reporter mice were generated by basically the same strategy employing bac transgenesis and insertion of a reporter gene into the il-7 locus . , il-7 bac transgenes were generated by insertion of a reporter gene immediately after the atg start codon of il-7 in exon 1 , which effectively disrupts the bac - encoded il-7 gene ( fig . . the choices of the il-7 reporter genes , however , were distinct between each study explaining possible variations among the reports ( table i ) . the first il-7 reporter mouse was generated and reported by di santo and colleagues ( 51 ) . the bac reporter construct expressed a yellow fluorescence protein ( yfp ) cdna downstream of the atg translational start codon of il-7 exon 1 , thus inactivating the bac il-7 open reading frame . consequently , endogenous il-7 levels are not perturbed and transgenic mice did not show any significant differences in the cellular composition of primary and secondary lymphoid organs . il-7 reporter activities were monitored either by flow cytometry for yfp expression or by immunohistochemistry using anti - yfp antibodies . both fetal and adult thymic stromal cells were found to contain yfp ( il-7 ) positive cells which were further identified as cd45 mhcii thymic epithelial cells ( tec ) in the fetal thymus and as either medullary or cortical tec - specific markers bearing cells in the adult thymus . interestingly , il-7 reporter activity was not detected in either endothelial cells or fibroblasts , suggesting that only a subset of specialized tecs produce this cytokine . of note , yfp expression was also not detected in peripheral lymphoid organs including the bone marrow , lymph node and spleen . this observation , however , does not agree with the general idea of peripheral il-7 being necessary for t cell survival ( 4,5 ) . a convincing explanation for this discrepancy , however , was provided when quantifying il-7 mrna transcripts in gp38 fibroblastic reticular cells in the lymph node and comparing those to the il-7 mrna expression in yfp tecs . fibroblastic reticular cells are an established source of peripheral il-7 ( 36 ) , but their il-7 mrna expression levels were found to be significantly lower than in yfp tecs . therefore , il-7 reporter activity in this particular reporter mouse identifies only cells with the highest levels of il-7 transcription in vivo , which is clearly the case for thymic stromal cells as confirmed in subsequent studies by others . richie and colleagues chose to generate two different il-7 reporter transgenes with either the human cd25 or the cre recombinase cdna inserted into nucleotide position 1 of the il-7 exon 1 ( 52 ) . the constructs were designed to replace the entire coding region of il-7 exon 1 of the bac clone rpci-295a3 that contained the entire 43 kb il-7 locus and its 5 ' and 3 ' flanking sequences ( 96 kb of the 5 ' sequence and 17 kb of the 3 ' of the il7 locus ) to include as much of the il7 regulatory elements as possible . il-7 reporter activities were monitored using either anti - human cd25 antibodies in il-7.hcd25 transgenes or by crossing il-7.cre transgenic mice with rosa26r ( 55 ) and r26yfp ( 56 ) reporter strains to induce expression of either -galactosidase or yfp , respectively ( 52 ) . reporter gene expression was detected either by flow cytometry or in whole mount staining by x - gal and by anti - yfp antibodies . in these reporter strains , il-7 expression faithfully reproduced the results from a previous study where in situ hybridization detected il-7 expression in early thymic rudiments as early as at embryonic day 13.5 ( 57 ) and also in thymic stromal cells . additionally , il-7 expression was mapped to lymph nodes , bone marrow , liver , small intestine and skin . durum and colleagues generated an il-7 reporter mouse , il7promecfp , by introducing an enhanced cyan fluorescence protein ( ecfp ) immediately after the translational initiation sequence of an il-7 bac clone ( 53 ) . since direct ecfp fluorescence was very weak in these mice , the reporter signal was further enhanced by an anti - ecfp antibody followed by fluorescent secondary antibodies . in two - photon microscopy , however , ecfp signals were sufficiently strong to perform intravital imaging . il-7 reporter activities were detected in both cortex and medulla of the thymus , and further detailed analysis with antibodies for stromal cell subsets identified ly51-positive cortical epithelial cells and epcam ( epithelial cell adhesion molecule)-positive cells , in both the medulla and cortex , as the il-7 producers . in agreement with ( 51 ) , fibroblasts , endothelial cells and also dendritic and myeloid derived cells in the thymus did not show il-7 reporter expression . and as before , no ecfp - positive cells were detected in secondary lymphoid tissues and in no other additional organs including gut , lung and skin . similarly to the yfp reporter mice ( 51 ) , the il7promecfp mouse is likely to report il-7 expression only in il-7 high expressing cells , and accordingly , ecfp - positive cells were found in the cervical thymus and also in the bone marrow . collectively , because of the extremely low level of il-7 transcription in peripheral tissues , monitoring reporter activity using il-7 promoter dependent fluorescence signals turned out to be ineffective outside of the thymus . rather , linking enzymatic activities downstream of il-7 so that they can either accumulate or be enhanced by secondary reporter activity could provide greater sensitivity in reporting il-7 expression in vivo . schuler and colleagues have employed precisely such an approach when they established a new il-7 reporter line ( 54 ) . here , they subcloned four genes linked to each other as a single reporter construct into an il-7 bac transgene . the four reporter genes were interlinked with p2a peptide sequences that allow translation of polycistronic mrna in mammalian cells ( 58 ) . this resulted in a transgenic mouse line ( il-7gcdl ) that simultaneously expressed enhanced green fluorescent protein ( egfp ) ( g ) , cre recombinase ( c ) , the human diphtheria toxin receptor ( d ) , and click beetle green luciferase 99 ( l ) . again , the reporter construct replaced the il-7 atg initiation site so that bac - encoded il-7 expression was abolished . reporter activity was then determined either by injection of luciferin and measuring in vivo bioluminescence or by anti - gfp antibodies . similar to ecfp and yfp reporter expression ( 51,53 ) , peripheral lymphoid tissues expressed gfp mrna but failed to exhibit any significant amounts of gfp as assessed by flow cytometry or by fluorescence microscopy . these results reaffirm that fluorescence proteins are not optimal reagents for detecting the low promoter activity of il-7 in vivo . importantly , in il-7gcdl mice , even cre - mediated activation of gfp reporter genes was still insufficient to accurately report il-7 expression when compared to in vivo bioluminescence assays . specifically , while luciferase activity was detected in the thymus , skin , small intestine , and to a lower level in lymph nodes , fluorescent protein expression was not detectable in any of these organs except for the thymus . notably , even with bioluminescence assays , only low or undetectable levels of il-7 expression were found in the heart , kidney , liver , and spleen ( 54 ) . the where and when of il-7 expression in vivo has been a hotly contended issue as much as the question of whether il-7 expression is actively regulated or constitutively expressed ( 49,59 ) . the current series of il-7 reporter mice are now providing insights into the anatomical distribution and in vivo signals that control il-7 expression during development and homeostasis . this is especially important since , in a striking demonstration , durum and colleagues documented that the conventional methods of immunohistochemistry and in situ hybridization are quite inadequate to monitor il-7 expression in mice ( 53 ) . using cre - recombinase expressing reporter mice that mark cells which have expressed and/or are currently expressing il-7 , and also using fluorescent proteins or luciferase expressing reporter mice that report the actual il-7 expression levels in situ , there is now an emerging consensus on il-7 expression in vivo ( table ii ) . the thymus , because of il-7 producing thymic stromal cells , is likely to be the organ with the highest level of il-7 expression . specifically , alves et al . ( 51 ) , describe a distribution of il-7 producing cells that closely matches observations from zamisch et al . ( 57 ) in that il-7 cells are mostly concentrated around the corticomedullary junction of an adult thymus . the phenotypes of il-7 cells were heterogeneous in that they bear markers for both cortical and medullary tecs but they lacked markers of hematopoietic cells , endothelial cells and fibroblasts . ( 53 ) further extended this observation by detailed morphological analysis and found that in the cortex , il-7 cells appear to envelop thymocytes in a " basket - like " shape , while in the medulla , an extensive network of processes extended from il-7 cells that made contact with thymocytes . ontogenically , il-7 reporter expression was first detectable in e12.5 fetal thymuses ( 60 ) and fully expressed throughout the thymus at e13.5 ( 57 ) . interestingly , il-7 tec frequency steadily declined with increasing numbers of thymocytes and thymic maturation , and was augmented when thymocyte development was intrinsically blocked , suggesting a potential negative feedback of thymocytes on il-7 tecs ( 60 ) . indeed , il-7 tec frequencies were significantly increased in immunocompromised mice such as in rag2il2rg mice whereas bone marrow transfer of either tcra or wt cells markedly reduced their frequencies . these data suggest that thymocyte - derived signals modulate il-7 expression by remodeling the thymic stroma , and it uncovers a novel thymocyte - tec cross - talk mechanism in the development of t cells ( 60 ) . outside of the thymus , identification of il-7 reporter positive cells all reporter mice using fluorescent proteins to monitor il-7 expression fail to detect il-7 expression in any other peripheral organ than the bone marrow ( 51,53 ) . moreover , even mice in which il-7 expression was mapped by cre or luciferase activity did not completely agree on the tissue distribution of il-7 producer cells ( 52,54 ) . nevertheless , it seems that every study agrees that il-7 expression was below detectable levels in spleen and kidney , while the skin and intestine showed strong signals of il-7 reporter expression . investigating the role of il-7 in these organs will be clearly the subject of future studies . however , harnessing the power of newly generated il-7 reporter mice , a series of questions have been already addressed . as mentioned above , thymic il-7 expression was developmentally regulated and not fixed . in fact , frequency of il-7 tecs declined with age , and there was a marked reduction of il-7 cell frequency in 5~7 weeks aged mice compared to neonatal and 1~2 weeks old mice ( 51 ) . furthermore , in addition to il-7 , the thymic stromal lymphopoietin ( tslp ) also utilizes the il-7r and stat5 for signaling in t cells , and tslp has been proposed to be a partially redundant pro - survival cytokine in immature thymocytes ( 61 ) . however , using il-7 reporter mice , il-7 and tslp expression was now shown to occupy different cellular and anatomical niches suggesting that il-7 plays a distinct role to tslp in thymopoiesis . such analyses at single cell levels were difficult to do before the availability of il-7 reporter mice . another interesting observation from il-7 reporter mice is the reduced frequency of il-7 expressing tecs by tcr-selected thymocytes ( 60 ) . , the authors observed that introduction of a tcr transgene into rag2-deficient mice largely restored thymic cellularity and also the generation of dp thymocytes but that it paradoxically resulted in diminished frequencies of il-7 tecs . similarly , il-7 tecs are retained in the thymus of mice with profound and early block in thymocyte development ( rag2il2rg ) and reconstitution of alymphoid reporter thymus with tcra hematopoietic progenitors diminishes the frequency of il-7 tecs ( 60 ) . together , these data revealed that thymocytes negatively regulate thymic il-7 production , but the biological significance of this feedback mechanism still needs to be addressed . with the ability to visualize il-7 producing cells , it is now possible to track their biological activities in vivo and in real time . a compelling study by durum and colleagues ( 53 ) assessed the interactions of t cells with ecfp il-7 producing stromal cells using intravital microscopy . central memory t cells ( tcm ) are known to home to the bone marrow , and it had been suggested that il-7 signaling by bone marrow resident stromal cells could be involved ( 62 ) . indeed , two photon microscopy of cfse - labeled and intravenously injected tcms displayed a preferential interaction with il-7 producing cells in the bone marrow , but subsequent studies showed that such preferential encounters was independent of il-7 or the chemokine cxcl12 ( 53 ) . thus , while the exact identity of il-7 producing cells in the bone marrow is unknown and also why tcm preferentially interact with these cells is unclear , these data confirm the power of il-7-reporter mice in assessing unresolved questions on il-7 biology in vivo . finally , il-7 expression was shown to be expressed by intestinal epithelial cells ( iec ) in the gut ( 37 ) . il-7 reporter mice confirmed this observation by displaying strong bioluminescence in the intestine and more specifically in epcam iecs . since in vitro data suggested that il-7 expression in iec is regulated by ifn- ( 63 ) , il-7 reporter activity was assessed in ifng mice and a significant reduction in il-7 expression was found in the colon ( 54 ) . these observations suggest a novel regulatory mechanism for mucosal il-7 expression : ifn- expression is induced by the microflora in the gut , and ifn- directly upregulates il-7 expression in iec which then supports the survival of intestinal t cells . these findings provide a powerful clue in understanding the role of the commensal microbiota in the cytokine - mediated homeostasis of the mucosal immune system . the availability of new il-7 reporter mice provided new information on the in vivo expression and regulation of il-7 . these studies also enabled the identification and precise phenotyping of il-7 producing cells in the thymus as thymic epithelial cells whose development and maintenance were actively regulated during ontogeny and aging . in peripheral tissues , il-7 producing cells were identified in a number of different tissues including lymph node , skin and intestine , but reporter activity also allowed the exclusion of spleen as an il-7 site . importantly , il-7 expression by iecs in the intestine was found to be dependent on the commensal microflora and regulated by ifn- , which suggest that il-7 expression can be actively modulated and is locally controlled . the new information gained from these il-7 reporter mice greatly improved our understanding on il-7 function by illuminating the source of in vivo il-7 . we anticipate that these new tools will provide us with further insights in future studies .

interleukin-7 ( il-7 ) is an essential cytokine for t cells . however , il-7 is not produced by t cells themselves such that t cells are dependent on extrinsic il-7 . in fact , in the absence of il-7 , t cell development in the thymus as well as survival of naive t cells in the periphery is severely impaired . furthermore , modulating il-7 availability in vivo either by genetic means or other experimental approaches determines the size , composition and function of the t cell pool . consequently , understanding il-7 expression is critical for understanding t cell immunity . until most recently , however , the spatiotemporal expression of in vivo il-7 has remained obscured . shortage of such information was partly due to scarce expression of il-7 itself but mainly due to the lack of adequate reagents to monitor il-7 expression in vivo . this situation dramatically changed with a recent rush of four independent studies that describe the generation and characterization of il-7 reporter mice , all utilizing bacterial artificial chromosome transgene technology . the emerging consensus of these studies confirmed thymic stromal cells as the major producers of il-7 but also identified il-7 reporter activities in various peripheral tissues including skin , intestine and lymph nodes . strikingly , developmental and environmental cues actively modulated il-7 reporter activities in vivo suggesting that il-7 regulation might be a new mechanism of shaping t cell development and homeostasis . collectively , the availability of these new tools opens up new venues to assess unanswered questions in il-7 biology in t cells and beyond .

INTRODUCTION OUTSTANDING QUESTIONS ON IL-7 EXPRESSION GENERATION AND CHARACTERIZATION OF IL7 REPORTER MICE SPATIOTEMPORAL EXPRESSION OF IL-7 CONCLUSION

originally described as a pre - b cell growth factor expressed in bone marrow stromal cells ( 1 ) , interleukin-7 ( il-7 ) was subsequently identified as an essential and non - redundant cytokine in t cells ( 2,3 ) . in fact , il-7 plays a critical role in every aspect of t cell biology , including early t cell development in the thymus , cd4/cd8 lineage choice during positive selection as well as maintaining the survival and homeostasis of nave and memory t cells in the periphery ( 2 - 6 ) . genetic deficiencies in il-7 or in any component of the il-7 receptor ( il-7r ) complex , namely the il-7r alpha ( il-7r ) chain and the cytokine receptor shared common gamma ( c)-chain , result in severely impaired thymopoiesis and t cell survival . the requirement of il-7 in t cell development and survival is chiefly due to its anti - apoptotic effects by upregulating expression of bcl-2 and mcl-1 ( 10,11 ) , but also because of its trophic effects , such as inducing expression of glucose transporter-1 ( 12 ) . in mature nave t cells , il-7 is a non - redundant survival cytokine that can not be replaced by other pro - survival cytokines ( 4,5,13,14 ) . interestingly , antigen specific memory cd8 t cells are still largely dependent on il-7 for cell survival but intermittent and basal homeostatic proliferation of these cells seems to be more dependent on il-15 ( 4,16 - 18 ) . this is in contrast to the bulk of memory phenotype cd8 t cells which are dependent on il-15 but not il-7 ( 19 ) . nevertheless , it remains evident that il-7 is a key molecule for maintaining the fitness and size of the peripheral t cell pool by providing essential survival signals . known downstream molecules of il-7 signaling include among others bcl-2 , bcl - xl , mcl-1 , glut-1 , cdc25a , il-7r , runx3 , and cd8 coreceptors , which are all essential for functional fitness and survival of t cells ( 6,10,12,22 - 25 ) . in this line , we recently showed that il-7 is necessary to specify cd8 lineage differentiation during cd4/cd8 cell fate choice in the thymus by inducing expression of the transcription factor runx3 ( 6 ) . additionally , il-7 is involved in the development of secondary lymphoid tissues since impaired il-7 signaling results in defects in lymph node organogenesis ( 28 ) . here , il-7 seems to regulate the size of the fetal lymphoid tissue inducer ( lti ) cell pool that interacts with stromal organizer cells to form lymphoid tissues ( 29 ) . thus , il-7 is a critical player in cell lineage choice and differentiation during lymphocyte as well as lymphoid tissue development . within the t cell compartment , il-7 controls the tcr activation threshold of nave cd8 t cells by tuning cd8 coreceptor expression levels to the self - specificity of the tcr . in this way , il-7 maintains a self - reactive but not autoimmune t cell pool ( 31 ) . we previously showed that maintaining this negative feedback loop is necessary for maximizing the size of the nave t cell pool ( 32 ) . collectively , il-7 is a multi - faceted pro - survival cytokine that governs the development , differentiation , and maintenance of t cells in a non - redundant manner . despite playing such critical roles , in fact , the identity of il-7 producing cells in the thymus is not fully understood as well as the precise location of il-7 expression in peripheral tissues remains veiled . recently , much light was shed on the in vivo source and regulation of il-7 expression by a series of four independent publications reporting the generation and characterization of il-7 reporter mice . using fluorescent proteins , cre recombinase and other reporter proteins , il-7 transcriptional activities were monitored in vivo and so depicted a spatiotemporal expression profile of il-7 expression during ontogeny and steady state conditions . here we review the experimental results and their findings in context of il-7 mediated t cell development and peripheral homeostasis . even as all t lineage cells are critically dependent on il-7 , no lymphocytes including t cells themselves produce il-7 ( 33 ) . importantly , the anatomical locations where immature thymocytes and t cells encounter il-7 for development and homeostasis are not entirely mapped . previously , il-7 message and proteins have been reported in various tissues including the thymus and secondary lymphoid organs such as spleen ( 34,35 ) and lymph nodes ( 36 ) . also , other tissues were implicated as sources of in vivo il-7 which comprise the intestine ( 37 ) , human liver ( 38 ) , skin ( 39,40 ) , murine ovaries ( 41 ) , human brain ( 42 ) , and human prostate ( 43 ) . while il-7 is generally considered as a secreted cytokine , its expression levels in serum are extremely low suggesting that il-7 in vivo is limiting and that il-7 would be consumed locally . in fact , serum il-7 was never convincingly detected in mice sera until most recently , when mackall and colleagues were able to determine serum il-7 in wild type mice at levels of 10 pg / ml ( 44 ) . scarce il-7 in the circulation on the one hand suggests that il-7 would be produced only at very low levels , but on the other hand there is also the possibility that il-7 is sequestered from serum by being complexed to the extracellular matrix ( ecm ) . potentially , such immobilized il-7 could be the actual bioactive form of il-7 in vivo . moreover , il-7 can also act as a heterodimer when bound to a variant -chain of the hepatocyte growth factor ( hgf ) , which is then known as the pre - pro - b cell growth - stimulating factor ( ppbsf ) . both heterodimerization of il-7 and hgf and the biological activity of ppbsf are dependent on low levels of heparin sulfate - derived oligosaccharides . if this is indeed the case , then identifying the actual il-7 producer cells in vivo would be even more critical to understand where and when il-7 signaling is happening in t cells . without identifying il-7 producers , however generally , il-7 production in vivo is considered to be constitutive and unaffected by external signals , so that il-7 availability is a limiting factor in determining the size of the peripheral t cell pool ( 47 ) . on the other hand , there is a great body of evidence that il-7 expression in vivo can be modulated . serum il-7 concentration is elevated in lymphopenic humans and other primates ( 48,49 ) , and in the mouse , a novel il-7r mediated il-7 expression feedback loop was recently reported , further indicating that il-7 production is actively regulated in vivo ( 44 ) . furthermore , il-6 and tlr signaling in the liver have been shown to induce il-7 expression in hepatocytes suggesting that expression of immunoregulatory il-7 is broader and more dynamically regulated than currently presumed ( 50 ) . because of the lack of adequate reagents , however , the whole scope of il-7 expression remained largely unaddressed . recently , four independent studies reported a total of five different il-7 reporter mice whose detailed characterizations will be discussed below ( 51 - 54 ) . all il-7 reporter mice were generated by basically the same strategy employing bac transgenesis and insertion of a reporter gene into the il-7 locus . , il-7 bac transgenes were generated by insertion of a reporter gene immediately after the atg start codon of il-7 in exon 1 , which effectively disrupts the bac - encoded il-7 gene ( fig . the choices of the il-7 reporter genes , however , were distinct between each study explaining possible variations among the reports ( table i ) . il-7 reporter activities were monitored either by flow cytometry for yfp expression or by immunohistochemistry using anti - yfp antibodies . both fetal and adult thymic stromal cells were found to contain yfp ( il-7 ) positive cells which were further identified as cd45 mhcii thymic epithelial cells ( tec ) in the fetal thymus and as either medullary or cortical tec - specific markers bearing cells in the adult thymus . interestingly , il-7 reporter activity was not detected in either endothelial cells or fibroblasts , suggesting that only a subset of specialized tecs produce this cytokine . this observation , however , does not agree with the general idea of peripheral il-7 being necessary for t cell survival ( 4,5 ) . a convincing explanation for this discrepancy , however , was provided when quantifying il-7 mrna transcripts in gp38 fibroblastic reticular cells in the lymph node and comparing those to the il-7 mrna expression in yfp tecs . therefore , il-7 reporter activity in this particular reporter mouse identifies only cells with the highest levels of il-7 transcription in vivo , which is clearly the case for thymic stromal cells as confirmed in subsequent studies by others . richie and colleagues chose to generate two different il-7 reporter transgenes with either the human cd25 or the cre recombinase cdna inserted into nucleotide position 1 of the il-7 exon 1 ( 52 ) . the constructs were designed to replace the entire coding region of il-7 exon 1 of the bac clone rpci-295a3 that contained the entire 43 kb il-7 locus and its 5 ' and 3 ' flanking sequences ( 96 kb of the 5 ' sequence and 17 kb of the 3 ' of the il7 locus ) to include as much of the il7 regulatory elements as possible . il-7 reporter activities were monitored using either anti - human cd25 antibodies in il-7.hcd25 transgenes or by crossing il-7.cre transgenic mice with rosa26r ( 55 ) and r26yfp ( 56 ) reporter strains to induce expression of either -galactosidase or yfp , respectively ( 52 ) . in these reporter strains , il-7 expression faithfully reproduced the results from a previous study where in situ hybridization detected il-7 expression in early thymic rudiments as early as at embryonic day 13.5 ( 57 ) and also in thymic stromal cells . additionally , il-7 expression was mapped to lymph nodes , bone marrow , liver , small intestine and skin . in two - photon microscopy , however , ecfp signals were sufficiently strong to perform intravital imaging . il-7 reporter activities were detected in both cortex and medulla of the thymus , and further detailed analysis with antibodies for stromal cell subsets identified ly51-positive cortical epithelial cells and epcam ( epithelial cell adhesion molecule)-positive cells , in both the medulla and cortex , as the il-7 producers . in agreement with ( 51 ) , fibroblasts , endothelial cells and also dendritic and myeloid derived cells in the thymus did not show il-7 reporter expression . similarly to the yfp reporter mice ( 51 ) , the il7promecfp mouse is likely to report il-7 expression only in il-7 high expressing cells , and accordingly , ecfp - positive cells were found in the cervical thymus and also in the bone marrow . collectively , because of the extremely low level of il-7 transcription in peripheral tissues , monitoring reporter activity using il-7 promoter dependent fluorescence signals turned out to be ineffective outside of the thymus . rather , linking enzymatic activities downstream of il-7 so that they can either accumulate or be enhanced by secondary reporter activity could provide greater sensitivity in reporting il-7 expression in vivo . schuler and colleagues have employed precisely such an approach when they established a new il-7 reporter line ( 54 ) . reporter activity was then determined either by injection of luciferin and measuring in vivo bioluminescence or by anti - gfp antibodies . these results reaffirm that fluorescence proteins are not optimal reagents for detecting the low promoter activity of il-7 in vivo . importantly , in il-7gcdl mice , even cre - mediated activation of gfp reporter genes was still insufficient to accurately report il-7 expression when compared to in vivo bioluminescence assays . specifically , while luciferase activity was detected in the thymus , skin , small intestine , and to a lower level in lymph nodes , fluorescent protein expression was not detectable in any of these organs except for the thymus . notably , even with bioluminescence assays , only low or undetectable levels of il-7 expression were found in the heart , kidney , liver , and spleen ( 54 ) . the where and when of il-7 expression in vivo has been a hotly contended issue as much as the question of whether il-7 expression is actively regulated or constitutively expressed ( 49,59 ) . the current series of il-7 reporter mice are now providing insights into the anatomical distribution and in vivo signals that control il-7 expression during development and homeostasis . this is especially important since , in a striking demonstration , durum and colleagues documented that the conventional methods of immunohistochemistry and in situ hybridization are quite inadequate to monitor il-7 expression in mice ( 53 ) . using cre - recombinase expressing reporter mice that mark cells which have expressed and/or are currently expressing il-7 , and also using fluorescent proteins or luciferase expressing reporter mice that report the actual il-7 expression levels in situ , there is now an emerging consensus on il-7 expression in vivo ( table ii ) . the thymus , because of il-7 producing thymic stromal cells , is likely to be the organ with the highest level of il-7 expression . ( 53 ) further extended this observation by detailed morphological analysis and found that in the cortex , il-7 cells appear to envelop thymocytes in a " basket - like " shape , while in the medulla , an extensive network of processes extended from il-7 cells that made contact with thymocytes . ontogenically , il-7 reporter expression was first detectable in e12.5 fetal thymuses ( 60 ) and fully expressed throughout the thymus at e13.5 ( 57 ) . these data suggest that thymocyte - derived signals modulate il-7 expression by remodeling the thymic stroma , and it uncovers a novel thymocyte - tec cross - talk mechanism in the development of t cells ( 60 ) . outside of the thymus , identification of il-7 reporter positive cells all reporter mice using fluorescent proteins to monitor il-7 expression fail to detect il-7 expression in any other peripheral organ than the bone marrow ( 51,53 ) . nevertheless , it seems that every study agrees that il-7 expression was below detectable levels in spleen and kidney , while the skin and intestine showed strong signals of il-7 reporter expression . however , harnessing the power of newly generated il-7 reporter mice , a series of questions have been already addressed . in fact , frequency of il-7 tecs declined with age , and there was a marked reduction of il-7 cell frequency in 5~7 weeks aged mice compared to neonatal and 1~2 weeks old mice ( 51 ) . furthermore , in addition to il-7 , the thymic stromal lymphopoietin ( tslp ) also utilizes the il-7r and stat5 for signaling in t cells , and tslp has been proposed to be a partially redundant pro - survival cytokine in immature thymocytes ( 61 ) . however , using il-7 reporter mice , il-7 and tslp expression was now shown to occupy different cellular and anatomical niches suggesting that il-7 plays a distinct role to tslp in thymopoiesis . such analyses at single cell levels were difficult to do before the availability of il-7 reporter mice . another interesting observation from il-7 reporter mice is the reduced frequency of il-7 expressing tecs by tcr-selected thymocytes ( 60 ) . , the authors observed that introduction of a tcr transgene into rag2-deficient mice largely restored thymic cellularity and also the generation of dp thymocytes but that it paradoxically resulted in diminished frequencies of il-7 tecs . similarly , il-7 tecs are retained in the thymus of mice with profound and early block in thymocyte development ( rag2il2rg ) and reconstitution of alymphoid reporter thymus with tcra hematopoietic progenitors diminishes the frequency of il-7 tecs ( 60 ) . central memory t cells ( tcm ) are known to home to the bone marrow , and it had been suggested that il-7 signaling by bone marrow resident stromal cells could be involved ( 62 ) . indeed , two photon microscopy of cfse - labeled and intravenously injected tcms displayed a preferential interaction with il-7 producing cells in the bone marrow , but subsequent studies showed that such preferential encounters was independent of il-7 or the chemokine cxcl12 ( 53 ) . thus , while the exact identity of il-7 producing cells in the bone marrow is unknown and also why tcm preferentially interact with these cells is unclear , these data confirm the power of il-7-reporter mice in assessing unresolved questions on il-7 biology in vivo . finally , il-7 expression was shown to be expressed by intestinal epithelial cells ( iec ) in the gut ( 37 ) . il-7 reporter mice confirmed this observation by displaying strong bioluminescence in the intestine and more specifically in epcam iecs . since in vitro data suggested that il-7 expression in iec is regulated by ifn- ( 63 ) , il-7 reporter activity was assessed in ifng mice and a significant reduction in il-7 expression was found in the colon ( 54 ) . these observations suggest a novel regulatory mechanism for mucosal il-7 expression : ifn- expression is induced by the microflora in the gut , and ifn- directly upregulates il-7 expression in iec which then supports the survival of intestinal t cells . the availability of new il-7 reporter mice provided new information on the in vivo expression and regulation of il-7 . these studies also enabled the identification and precise phenotyping of il-7 producing cells in the thymus as thymic epithelial cells whose development and maintenance were actively regulated during ontogeny and aging . in peripheral tissues , il-7 producing cells were identified in a number of different tissues including lymph node , skin and intestine , but reporter activity also allowed the exclusion of spleen as an il-7 site . importantly , il-7 expression by iecs in the intestine was found to be dependent on the commensal microflora and regulated by ifn- , which suggest that il-7 expression can be actively modulated and is locally controlled . the new information gained from these il-7 reporter mice greatly improved our understanding on il-7 function by illuminating the source of in vivo il-7 .

limb traumas are among the most painful and stressful events , that anybody may experience during his or her life . so long as pain control is concerned , morphine remains the gold standard for analgesia . it has been the standard analgesic for many years , and the effects of newer analgesics are often expressed in comparison with the effect of morphine . the search for good analgesics , however , should not only focus on analgesia . as far as analgesia is concerned , morphine is an excellent drug ; but morphine lacks quality of an ideal analgesia due to its side effects . these side effects include sedation , respiratory suppression , nausea and vomiting and pruritus , which may be avoided by substituting other drugs or combinations with the same effects and lower or no adverse effects such as ketamine with or without midazolam . first synthesized in 1963 during the search for the ideal anesthetic , ket - amine was so named because it is a derivative of an amine . and compared with the r ( - ) isomer , the s ( + ) isomer has a fourfold greater affinity for the nmda receptor , twice the analgesic potency , and fewer psychomimetic effects . ketamine is useful as a general anesthetic for trauma patients , because it preserves sympathetic reflexes that help support blood pressure in patients who have lost blood . because it does not interfere with respiratory drive , it is also widely used in resource - poor set - tings where intubation and intraoperative mechanical ventilation are unfeasible . the analgesic action of low , subanesthetic doses of ketamine predominantly de - rives from its activity - dependent , noncompetitive blockade of the glutaminergic nmda - receptor channel complex , through binding at phen - cyclidine ( pcp)-binding sites in the ion channel . experimental and clinical evidence indicates that ketamine reduces opioid - induced tolerance and hyperalgesia ; however , the mechanisms involved are only partially understood . low - dose ketamine decreased morphine - resistant pain , reduced dosage requirements in opioid - tolerant patients , decreased hyperalgesia after remifentanil infusion , and reduced hyperalgesia and allodynia along surgical incisions . traditionally , they have been considered to lack analgesic action ; however , benzodiazepines reduce the minimum alveolar anesthetic concentration of inhaled anesthetics . this is of interest that the neurophysiological mechanisms of the effects of benzodiazepines ( i.e. midazolam ) on nociception have been studied in animals , but the conclusions often are conflicting . according to these studies midazolam may suppress pain pathways of the spinal cord by its specific action and that effect is not due to the action of the drug on supraspinal systems . in this study we had two main objectives : ( 1 ) to compare the efficacy of two analgesic regimens regarding pain control in trauma patients ( closed fractures : defined as fractures with intact skin and soft tissue above the fracture site ) ; ( 2 ) to compare the incidence of adverse events , categorized as respiratory , nausea , and vomiting , and agitation in conjunction with hemodynamic alterations between groups receiving low dose iv ketamine plus midazolam versus iv morphine . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . quantitative variables were expressed as mean sd and qualitative variables as number ( percentage ) . two independent samples t - test was used as the main statistical method for comparing the main outcomes . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . within group comparisons chi - square test also was used for comparing the qualitative variables between two studied groups . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the main reason for which we considered this study as a single blind was the possibility of a physician bias resulting from ketamine - induced nystagmus . the study was approved by the hospital ethics committee , and written informed consent was obtained from each patient . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . patients , who had already received an opioid analgesic ( either by self - administration or by another attending physician or emergency medicine service ( ems ) ) , were also not included in our study . a table of random numbers determined the randomization sequence , using a restricted randomization scheme to ensure roughly equal numbers in each group . the threshold for administration of analgesics in this study was severe acute pain , defined as a vas of at least 6 . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the latter regimen was also repeated as needed , until pain relief was obtained as defined by a vas score not exceeding 30/100 . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . pain intensity was verified using a 10 cm ( 100 mm ) vas , anchored by no pain at one end and by worst possible pain at the opposite end . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . in this situation the patient was considered as having adequate pain relief and was assigned a vas score of 0 . when pain was initially too severe to obtain a vas ( patient refusal ) , it was scored 100 . quantitative variables were expressed as mean sd and qualitative variables as number ( percentage ) . two independent samples t - test was used as the main statistical method for comparing the main outcomes . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . within group comparisons chi - square test also was used for comparing the qualitative variables between two studied groups . two hundred and thirty - six patients were selected , among whom were 207 males ( 87.3% ) , and 29 females ( 12.2% ) . the average age was 32.6 12.8 with extremes of 60 years and 18 years . the patients were divided into two groups : g1 : 116 patients receiving ketamine - midazolam ( 33.8 14.1 ) and g2 : 120 patients receiving morphine alone ( 31.5 11.4 ) . the median for body weight among g1 patients was 69 kg , and among g2 patients it was 70 kg [ table 1 ] . demographic characteristics of two studied groups the vas score at t30 was significantly decreased compared with vas score at t0 , in both groups ( p < 0.0001 ) . no statistically significant difference , however , was observed between two groups ( p < 0.16 ) . the mean value of the vaps between two groups showed no meaningful difference ; in other words the efficacy of both regimens was the same in terms of pain control . the mean value of dbp revealed no significant difference in both groups ; whereas regarding the mean value of rr and spo2 a meaningful difference was encountered between two groups . furthermore , the mean value of the sbp and hr depicted a meaningful difference ; this means that sbp and hr mean values increased 30 minutes after taking ketamine - midazolam but with morphine these values were decreased [ table 2 ] . nausea , vomiting , agitation , or hallucination was not noted in both groups . with reference to satisfaction from pain control process , no significant difference was seen between two groups [ table 3 ] . comparing the mean of analgesic in two groups in terms of main studied outcome variables satisfaction rates among physicians , nurses , and patients the most significant finding achieved in this study is that there is no meaningful difference between low - dose ketamine - midazolam and morphine with respect to pain control in patients sustaining closed limb fracture(s ) . we found that hypoventilation and increased heart rate / blood pressure were more prominent in group morphine and group ketamine plus midazolam , respectively . the changes noted in cardiopulmonary system , however , were congruent with what we already knew according to the previous evidence - based researches . the emergency atmosphere and ed are somehow stressful per se ; so ketamine - midazolam regimen could be a better choice in terms of pain control in limb trauma patients , while it imposes a minimal risk on cardiopulmonary systems ( like respiratory depression and apnea ) . it also makes itself a reasonable choice in opium - addicted patients , in group ketamine - midazolam inflicted with drug tolerance because of its morphine - sparing effects . regarding the effects of ketamine and morphine on pain on the one hand and their interaction , on the other , so many studies have been yet accomplished . in 2003 , fj et al published an article as to comparing the quality of intravenous patient controlled analgesia ( pcia ) of low - dose morphine plus ketamine with morphine in a group of patients scheduled for elective abdominal hysterectomy . contrary to our research , this study compared morphine consumption and morphine sparing effect of ketamine . they concluded that morphine plus ketamine pcia , in doses used in this study ( 7 mg / kg morphine plus 14 mg / kg ketamine as a bolus ) provided analgesia inferior to that of morphine pcia , but may improve the respiratory side effect profile of morphine . one reason for this conclusion , of course , could be the ultra - low dose of ketamine . laeben et al reported a study about low - dose ketamine for analgesia in the emergency department ( ed ) . however , there was concern about the patient group in this study which may interfere with their conclusion when applied to the general population . compared to our study , the majority of the patients in this research had the record of chronic pain medication use or illicit drug abuse . tolerance or dependence of opioids or opioid - like agents was frequently seen in these patients , which make them become a drug seeker in the ed , and thus , analgesia or so - called improvement of pain for them is more complicated . in our study , however , anybody with history of chronic drug abuse was excluded . galinski assessed management of severe acute pain in emergency settings in an article titled as the aim of the study was to compare in emergency settings two analgesic regimens , morphine with ketamine ( k group ) or morphine with placebo ( p group ) , for severe acute pain in trauma patients . at t30 , the main problem with this study , nonetheless , seems to be possible ketamine - induced nystagmus , and a resultant bias pursued , which we tried to resolve it , as mentioned earlier . similarly , babak garaei and coworkers in iran did a randomized , placebo - controlled clinical trial , in opioid abusers based on their daily opioid consumption . lithotripsy was performed under moderate sedation with intermittent bolus doses of remifentanil ( 0.2 g / kg ) to alleviate pain . the incidences of bradypnea , apnea , nausea , vomiting , and hemodynamic changes were not statistically different between the ketamine and placebo groups . they concluded that preemptive low - dose ketamine ( 0.1 mg / kg ) as a bolus has opioid - sparing effects in opioid abusers undergoing moderate sedation . what has not been studied in the above mentioned researches , however , is the comparison of morphine versus ketamine regarding ed pain control . they all used placebo in their studies but this intervention did not necessarily create an unethical issue in these studies , because all the patients received analgesic irrespective of taking ketamine . regarding midazolam , our goals were to decrease pain as toshinobu sumida et al followed in their study , beside its already known effects on anxiety and ketamine induced agitation , known as emergence phenomenon. of course , probable analgesic effects of midazolam are yet to be investigated by more precise , double - blind studies in the future . another issue that makes our sample more different from its predecessors is that it is a noninferiority trial . the term noninferiority trial is commonly used to refer to a randomized clinical trial in which a new test treatment is compared with a standard active treatment rather than a placebo or untreated control group . one starting principle is that no patient is denied a known effective treatment by entering a clinical trial . in our study we noted no adverse pulmonary effects like hypoventilation as caused by morphine , in group ketamine - midazolam . the effect of ketamine on respiratory rate is in accordance with the findings of presson et al . alfentanil induced a decrease in respiratory rate , without affecting tidal volume and respiratory drive . firstly , ketamine caused subjective side effects in all subjects ( e.g. , strange feeling , body feels tight , arms and legs strange , body feels heavy , etc ) that might have caused general arousal , thereby stimulating respiration indirectly . secondly , being an nmda receptor antagonist , ketamine may antagonize the effect of opioids on ventilation as the effect of opioids on the control of breathing may be through inhibition of glutaminergic transmission . the ketamine dose was , therefore , high enough to have an analgesic effect , but lower than a dose that causes hallucinations . finally , we recommend more extensive , double - blinded studies with lower therapeutic range of ketamine , in the future which may clarify more exactly the real effect and safety of ketamine plus midazolam compared with morphine . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. , femoral head , intertrochanteric or distal radius fractures in the elderly and suprachondylar humeral fractures in children , both of them not involved in the study ) . closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . we could not , unfortunately , trust his or her real intent , despite receiving analgesics and being assigned for this study . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. , femoral head , intertrochanteric or distal radius fractures in the elderly and suprachondylar humeral fractures in children , both of them not involved in the study ) . closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . we could not , unfortunately , trust his or her real intent , despite receiving analgesics and being assigned for this study . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we concluded that low - dose iv ketamine - midazolam has the same analgesic effects as iv morphine concerning pain control in adult patients sustaining closed limb fracture(s ) , with less pulmonary adverse events . whether midazolam has significant analgesic effects , however mehdi nasr isfahani , awat feizi : substantial contributions to the conception or design of the work ; or the acquisition , analysis , or interpretation of data for the work . mehdi nasr isfahani : drafting the work or revising it critically for important intellectual content . omid ahmadi , mehdi nasr isfahani : final approval of the version to be published . omid ahmadi , mehdi nasr isfahani : agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved .

background : we assessed the effects of low - dose iv ketamine - midazolam versus morphine on pain control in patients with closed limb fracture(s ) ; and also compared the incidence of adverse events ( cardio - pulmonary ) between two groups.materials and methods : this prospective , single - blind , non - inferiority trial randomized consecutive emergency department ( ed ) patients aged 18 - 60 years to two groups : receiving 300 - 500 mcg / kg ketamine plus 0.03 mg / kg midazolam , or 0.05 - 0.1 mg / kg morphine . visual analogue score ( vas ) and adverse events were verified during an interval of 30 minutes.results:two hundred and thirty six patients were selected , among whom 207 were males ( 87.3% ) . the average age was 29 2 , ( range , 18 - 60 years ) . the vas score at t30 ( i.e. , 30 minutes after initial analgesic dose ) was significantly decreased compared with vas score at t0 , in both groups . no statistically significant difference , however , was observed between the two groups ( 6.1 1.1 versus 6.2 1.0 ; p = 0.16 ) . with regard to systolic blood pressure and respiratory rate , however , a meaningful difference was noted between the two groups ( 1.5 6.4 versus 2.1 6.6 ; p = 0.000 for sbp , and 0.2 1.1 versus 1.1 6.1 ; p = 0.048 for rr).conclusion : low - dose intravenous ketamine plus midazolam has the same analgesic effects as morphine on pain control in trauma patients with closed limb fracture(s ) , in addition to less respiratory adverse events .

INTRODUCTION MATERIALS AND METHODS Study procedures and outcome measure Study design and Patients Study analysis RESULTS DISCUSSION AND CONCLUSIONS Limitations CONCLUSIONS AUTHOR'S CONTRIBUTIONS

it has been the standard analgesic for many years , and the effects of newer analgesics are often expressed in comparison with the effect of morphine . the search for good analgesics , however , should not only focus on analgesia . and compared with the r ( - ) isomer , the s ( + ) isomer has a fourfold greater affinity for the nmda receptor , twice the analgesic potency , and fewer psychomimetic effects . ketamine is useful as a general anesthetic for trauma patients , because it preserves sympathetic reflexes that help support blood pressure in patients who have lost blood . low - dose ketamine decreased morphine - resistant pain , reduced dosage requirements in opioid - tolerant patients , decreased hyperalgesia after remifentanil infusion , and reduced hyperalgesia and allodynia along surgical incisions . this is of interest that the neurophysiological mechanisms of the effects of benzodiazepines ( i.e. in this study we had two main objectives : ( 1 ) to compare the efficacy of two analgesic regimens regarding pain control in trauma patients ( closed fractures : defined as fractures with intact skin and soft tissue above the fracture site ) ; ( 2 ) to compare the incidence of adverse events , categorized as respiratory , nausea , and vomiting , and agitation in conjunction with hemodynamic alterations between groups receiving low dose iv ketamine plus midazolam versus iv morphine . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . the vas , after teaching the patient as to determining their pain score visually or numerically , was used to evaluate pain , with the patient attributing a value that corresponded to the level of his or her pain . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . we performed a prospective , randomized , single - blind , non - inferiority clinical trial in the emergency department ( ed ) of a university medical center from december 2011 until february 2013 ( clinicaltrials.gov identifier : nct01807429 ) . the sample size was determined about 236 patients ; considering type 1 error rate as 0.05 , statistical power as 80% and common variance as 1.8 for detecting = 1 as the fixed non - inferiority margin ( pain score in terms of visual analogue score ( vas ) ) . two hundred and thirty - six patients of both genders sustaining closed fracture of the extremities ( i.e. , long bones ) who were between the ages of 18 and 60 years , and in good health or with only mild systemic diseases ( american society of anesthesiologists grade 1 or 2 ) were recruited for this study as a convenience sample because the availability of such a population in the ed . patients were eligible for inclusion if they presented a trauma with a severe acute pain defined as a vas score of at least 60/100 ( or 6 ) ; were aged between 18 and 60 years ; and were without acute respiratory , hemodynamic , or neurologic compromise ( respiratory distress signs , systolic blood pressure < 90 mmhg , glasgow coma score < 15 ) . the patients with a psychiatric history ; chronic respiratory , and acute pulmonary infection ; renal , or hepatic failure ; known ketamine sensitivity ; known opioid allergies ; treatment of chronic pain or treatment with opioids ; incapacity to understand the vas ; the use of drugs that affect the central nervous system ; chemical substance abuse ; chronic pain ; pregnancy ; morbid obesity ; increased intracranial pressure ; cardiovascular , hepatic , renal or ocular pathology ; thyroid disease , and pregnancy were not included in the study . eligible patients were randomly allocated to receive either morphine ( 0.05 - 0.1 mg / kg ) followed by additional doses of 3 mg every 5 - 10 minutes or ketamine - midazolam ( 300 - 500 mcg / kg ketamine and 0.03 mg / kg midazolam ) . the consort flow diagram the patients were assessed at the time of arrival at the ed and every 10 minutes for a total interval of 30 minutes , and the following variables were carefully monitored : visual analogue pain score ( vaps ) , pulse oxymetry ( spo2 ) , respiratory rate ( rr ) , blood pressure ( sbp , dbp ) , heart rate ( hr ) , nausea and vomiting , screaming , cursing , nightmares , and unpleasant hallucinations . the threshold for efficient analgesia the statistically significant analgesia , however , was held to be as a vas score of 30/100 or lower . respiratory adverse events were defined as oxygen desaturation ( spo2 < 90% ) , apnea ( a minimum 20 seconds transient cessation of breathing ) , or laryngospasm . thirty minutes after the first injection , patients , physicians , and nurses were queried about their level of satisfaction , from 1 ( least satisfied ) to 5 ( most satisfied ) , with a likert scale . the responses were categorized as satisfied ( likert scale score of 4 or 5 ) and not satisfied ( score of 1 , 2 , or 3 ) . primary outcome measure was the percentage of patients with pain relief ( with a vas score of 30/100 or lower ) 30 minutes ( t30 ) after the first injection ( t0 ) . secondary outcomes included pain score comparisons every 10 minutes within the first 30 minutes and comparison of adverse events as mentioned above . thirty minutes after initial analgesic dose , if the patient still did not reach a visual analogue score of 30 mm or less , he or she was excluded from the study . for all main outcomes the percentage changes from baseline values were calculated and compared between two groups . two hundred and thirty - six patients were selected , among whom were 207 males ( 87.3% ) , and 29 females ( 12.2% ) . the average age was 32.6 12.8 with extremes of 60 years and 18 years . the patients were divided into two groups : g1 : 116 patients receiving ketamine - midazolam ( 33.8 14.1 ) and g2 : 120 patients receiving morphine alone ( 31.5 11.4 ) . demographic characteristics of two studied groups the vas score at t30 was significantly decreased compared with vas score at t0 , in both groups ( p < 0.0001 ) . no statistically significant difference , however , was observed between two groups ( p < 0.16 ) . the mean value of the vaps between two groups showed no meaningful difference ; in other words the efficacy of both regimens was the same in terms of pain control . the mean value of dbp revealed no significant difference in both groups ; whereas regarding the mean value of rr and spo2 a meaningful difference was encountered between two groups . furthermore , the mean value of the sbp and hr depicted a meaningful difference ; this means that sbp and hr mean values increased 30 minutes after taking ketamine - midazolam but with morphine these values were decreased [ table 2 ] . nausea , vomiting , agitation , or hallucination was not noted in both groups . with reference to satisfaction from pain control process , no significant difference was seen between two groups [ table 3 ] . comparing the mean of analgesic in two groups in terms of main studied outcome variables satisfaction rates among physicians , nurses , and patients the most significant finding achieved in this study is that there is no meaningful difference between low - dose ketamine - midazolam and morphine with respect to pain control in patients sustaining closed limb fracture(s ) . we found that hypoventilation and increased heart rate / blood pressure were more prominent in group morphine and group ketamine plus midazolam , respectively . the changes noted in cardiopulmonary system , however , were congruent with what we already knew according to the previous evidence - based researches . the emergency atmosphere and ed are somehow stressful per se ; so ketamine - midazolam regimen could be a better choice in terms of pain control in limb trauma patients , while it imposes a minimal risk on cardiopulmonary systems ( like respiratory depression and apnea ) . it also makes itself a reasonable choice in opium - addicted patients , in group ketamine - midazolam inflicted with drug tolerance because of its morphine - sparing effects . regarding the effects of ketamine and morphine on pain on the one hand and their interaction , on the other , so many studies have been yet accomplished . in 2003 , fj et al published an article as to comparing the quality of intravenous patient controlled analgesia ( pcia ) of low - dose morphine plus ketamine with morphine in a group of patients scheduled for elective abdominal hysterectomy . they concluded that morphine plus ketamine pcia , in doses used in this study ( 7 mg / kg morphine plus 14 mg / kg ketamine as a bolus ) provided analgesia inferior to that of morphine pcia , but may improve the respiratory side effect profile of morphine . laeben et al reported a study about low - dose ketamine for analgesia in the emergency department ( ed ) . in our study , however , anybody with history of chronic drug abuse was excluded . galinski assessed management of severe acute pain in emergency settings in an article titled as the aim of the study was to compare in emergency settings two analgesic regimens , morphine with ketamine ( k group ) or morphine with placebo ( p group ) , for severe acute pain in trauma patients . at t30 , the main problem with this study , nonetheless , seems to be possible ketamine - induced nystagmus , and a resultant bias pursued , which we tried to resolve it , as mentioned earlier . the incidences of bradypnea , apnea , nausea , vomiting , and hemodynamic changes were not statistically different between the ketamine and placebo groups . they concluded that preemptive low - dose ketamine ( 0.1 mg / kg ) as a bolus has opioid - sparing effects in opioid abusers undergoing moderate sedation . what has not been studied in the above mentioned researches , however , is the comparison of morphine versus ketamine regarding ed pain control . of course , probable analgesic effects of midazolam are yet to be investigated by more precise , double - blind studies in the future . in our study we noted no adverse pulmonary effects like hypoventilation as caused by morphine , in group ketamine - midazolam . alfentanil induced a decrease in respiratory rate , without affecting tidal volume and respiratory drive . finally , we recommend more extensive , double - blinded studies with lower therapeutic range of ketamine , in the future which may clarify more exactly the real effect and safety of ketamine plus midazolam compared with morphine . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we studied only adults between the ages of 18 and 60 years , and thus our data can not apply to elderly patients who are perhaps more likely to experience the sympathomimetic effects of ketamine . furthermore , according to the available data and medical textbooks , the incidence of the closed fractures of the extremities tends to be more at the extremes of ages ( e.g. closed limb fracture because of its low - energy nature ( vas of less than 6 ) , most of the times does not need to be managed with analgesics ; and if a patient who has been probably a drug seeker would apply for analgesic or declared that his pain score was above 6 . another limitation was the ketamine induced nystagmus that made us perform this study as a single - blind trial , in fear for the probable physician bias while monitoring the patients . we concluded that low - dose iv ketamine - midazolam has the same analgesic effects as iv morphine concerning pain control in adult patients sustaining closed limb fracture(s ) , with less pulmonary adverse events . whether midazolam has significant analgesic effects , however mehdi nasr isfahani , awat feizi : substantial contributions to the conception or design of the work ; or the acquisition , analysis , or interpretation of data for the work .

within and beyond the world of nanoscience , images abound . there are lots of colourful images of structures at the nanoscale , based on visualizations of digital data created by scanning probe microscopy or other imaging instruments ( as are made available to colleague nanoscientists ) . there are also artist s impressions , ranging from impressions of entities at the nanoscale to envisioning nanoachievements to be realized in the near or long - term future , including science - fiction type images of nanobots . one can distinguish imaging , that is , creating images based on data and aiming at resemblance , from imagining , where imagination is mobilized to create a vision of the nanoworld . actually , imaging and imagining are always entangled , already in so - called scientific visualizations where expectations about how the nanoscale could look like steer the choices involved in making images . a subsequent question then is what such entangled images stand for , not only representing in the sense of resemblance , but also in terms of what images do when representing the nanoscale . a clear example are images which demonstrate of technoscientific achievements . in practice , there is a continuum ranging from visualizations of the nanoscale adapted from original instrument - based data to so - called artist s impressions ( which need not be produced by artists ; the phrase is used to indicate some freedom in visualizing ) of how the nanoscale could look like , or how audiences may expect that it looks . this occurs already within science , for example when images indicate possible functions of complex molecular structures . to capture the continuum of imaging and imagining , we will write imag(in)ing.1 we will start by analyzing occurrences of imag(in)ing and the tensions involved . while imag(in)ing has to do with representation , which is often focused on making something present through resemblance , we emphasize representation as standing for and being able to political representation ( cf . ) . standing for and being able to act for , is somewhat independent of what is being stood for. thus , this notion of representation is broader than the epistemological puzzles , and debates , about reality ( out there and/or in contrast with fiction ) . in the final part of our paper right representation is , in terms of images that become iconic , and in that sense stand for , and are allowed to speak for , nanotechnology . the nanoscale , invisible to the naked eye , is visualized through the use of a variety of imaging instruments . at the outset , it is not clear what the right imaging tools would be , and this relates to the basic problem how one can know whether the visualizations obtained actually visualize the nanoscale harry collins highlighted the problem of choice between instruments that would be adequate to the task of studying unknown phenomena a problem which he introduced as an experimenter s regress because there is no independent check of the correctness of the choice . for the production of visualizations there is the additional question about the ways to produce images / pictures that lead to reliable representations of the invisible . just as the experimenter s regress can ( and will ) be stopped in practice when expectations about the unknown phenomenon become shared , expectations about how the invisible phenomena should look like , can provisionally stop the visualization regress (: 5 ) . a subsequent imaging challenge is to reach audiences which are not familiar with the production processes of the images . there will be ( .. ) a diffuse message about the nanoscale which has to be presented as clearly and convincingly as possible (: 9 ) to intended audiences . artist s impressions may do such a job better than data - based images . in visualization practices expectations about what can be seen at the nanoscale and how the nanoscale should be visualized are to some extent internalized as rules about how to do it . the data - based images are manipulated to highlight the information they should convey , which is an accepted practice in the domain of science , as long as the scientific content remains unchanged . artist s impressions of how the nanoscale could look like are similar , in the sense that imaging and imagining are also entangled . in other words visualizations of the nanoscale as well as artist s impressions are used in scientific journals to show research results.2 such practices are not uncontested . correctly , make it to the cover of scientific journals.3 when they do so , they come to stand for scientific achievements , and cover pictures are often presented on research groups websites next to their list of publications . 1artist s impression of an array of nanotubes fets overlaid with gold source and drain electrodes . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view courtesy of : c. dekker , tu delft / tremani artist s impression of an array of nanotubes fets overlaid with gold source and drain electrodes . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view as an emerging technology , nanotechnology comprises present achievements and visions of what it may become . while such combinations occur for all sorts of sciences and technologies ( see van lente on promising technologies ) this appears particularly striking in nanotechnology . mody argues that nanotechnology , in contrast to , for example , physics and chemistry , seems decidedly non - presentist and that nanotechnologists work as much in this future world as in the present (: 108 ) . then , there is the design orientation in nanotechnology , where a possible future is projected to be realized by a present design . with computer simulations playing an important role , much of nanotechnology s created reality thus has a virtual , yet - to - be - realized quality . kaiser adds another twist:they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . from a modal logical point of view , they do nt represent possible worlds in the fictional universe ; on the contrary , they constitute future worlds in ours (: 667 ) . they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . from a modal logical point of view , they do nt represent possible worlds in the fictional universe ; on the contrary , they constitute future worlds in ours (: 667 ) . if one wants to use categories like reality and fiction ( as actors often do ) , one can say that boundaries between reality and fiction are blurred in the nanorealm , and this will be reflected in images of nanotechnology . scientists produce images equivalent to artist s impressions that show what could be possible applications for nanotechnology . images of bucky balls , molecular machines , new uses of carbon nanotubes , are created to make a possible future present . artists offer their ( own or commissioned ) visions of nanotechnology s potential future applications . as soon as the future is incorporated , there is no sharp boundary between images that articulate directions of further research and more open - ended visions of new performances which then merge with futuristic visions . some actors will attempt to separate science ( reality ) from science fiction. this can be a tactic to have their project prevail over that of others ( this tactic has been deployed by critics of drexler s visions of molecular manufacturing , cf . ) . or it can be a defensive move to avoid being called to account for possible negative implications of investing in nanotechnology research ( that s only fiction ) . arguments for disentangling the future from the present also occur in relation to lay - audiences . such an argument would run like : it should be made clear which images are realistic and which are fictive , so as to avoid giving the false impression that ( say ) nanobots have already been built . ottino extended his crusade for correctness to the nanolouse image ( see fig . 2 ) : fig . 2the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . source : ottino the image looks so real that it is easy to imagine a viewer being fooled into believing it has already been built . this is important in terms of public reaction , especially in the backdrop of scenarios such as in michael crichton s new novel prey , which portrays swarms of self - replicating nanomachines destroying all other life forms that they encounter (: 476 ) . the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . source : ottino the image looks so real that it is easy to imagine a viewer being fooled into believing it has already been built . this is important in terms of public reaction , especially in the backdrop of scenarios such as in michael crichton s new novel prey , which portrays swarms of self - replicating nanomachines destroying all other life forms that they encounter (: 476 ) . ottino links his criticism with a concern about public acceptance of nanotechnology , if such images should come to stand for nanotechnology in the public eye . the public , however , may well be more discerning than he projects it to be . still , the image does contain an ambivalence , which is brought out in how it has been presented on the bbc website ( www.bbc.co.uk ) . on one occasion it was positioned as the 2002 winner of the visions of science award : the more over - hyped applications see tiny machines roaming the body to cure diseases. on another occasion , realistic possibilities were emphasized : the nanolouse image intends to show one of the possible applications of nanotechnology in medicine in the future microscopic machines roaming the body , injecting or taking samples for tests. continuing with this example , it is interesting that the nanolouse image has been often used in the world of science . in the period 20052007 , the image was used in presentations about drug delivery , nano - probes and ( n)mems , as an illustration and occasionally as a possible future application .4 in general terms , images of nanotechnology that import the future into the present are used to communicate what nanotechnology is as well as to indicate nanotechnology s potential . in doing so , within science and beyond , such images are used strategically to put audiences in desired positions and at the same time to strengthen one s own position . an interesting example is how the foresight nanotech institute used the nanogear image to position itself . on their website , they present the nanogear image ( fig . 5 ) ( and the image of the strained - shell sleeve bearing ) to indicate that 1 day , surely , productive nanosystems will be realized . respirocytes ( artificial blood cells able to carry 236 times more oxygen than human s red blood ) as designs , implying that they can ( and thus will ) be built in the future . what is new in nanotechnology is that these images are about aimed - for possibilities rather than an actual construction plan of such a future . the nanogear image is an extreme example , but these kind of images and their use are widespread . nanoscientists play with the entanglement of data - based imaging and impressionistic imagining , and with the entanglement resulting from projecting of the future into the present . they produce and use images to illustrate scientific breakthroughs and to create expectations about the potential realization of scientific achievements , or to highlight socio - political debates . in addition , the aesthetic appeal of the images that are produced as visualizations of the nanoscale , is actively pursued , for example through impressive colouring , as in ibm s image of the quantum corral ( fig . 7 ) . the fact that images are now given a name , and an evocative name at that , is an indication that they are seen as works of art , which have titles.5 this link with the world of art , at least with the world of being creative in making images , is not just an excursion of scientists . an interesting development is how artists make use of , or even get involved in , the production of images through imaging instruments . as chris orfescu noted , there appear to be two ways to do so : nanolandscapes in which given structures of matter at the nanoscale are depicted , and nanosculptures , which are created through the manipulation of matter at the nanoscale by nanoscientists , and then visualized.6 one sees how such images offer a view to a new world that is in the process of being explored . many of the nano- and microsculptures like the nano - guitar , nano - opera house , nano - toilet and the nano - bull ( see fig . interestingly , the methods which are used to create such nanosculptures are of scientific interest . fig . a team of japanese engineers has created the smallest sculpture of a bull , which could only be viewed with a scanning electron microscope . a team of japanese engineers has created the smallest sculpture of a bull , which could only be viewed with a scanning electron microscope . source : kawata et al . for example , kawata et al . created a model bull , measuring 10 by 7 m , which is about the size of a red blood cell . the nano - bull was used to show the potential of two - photon photopolymerization . but the fact that it was a bull was the immediate message , and kawata et al.s claim about their technoscientific achievement piggy - backed ( if we may say so ) on this message . another example comes from a nanoart project in which imaging tools like the scanning tunneling microscope ( stm ) and atomic force microscope ( afm ) are deliberately used for artistic purposes . scali and goode produced the smallest map ever of the continent africa , created through the use of a scanning electron microscope ( sem ) and visualized with an afm ( see fig . the actual size is 200 m 130 m , and it is meant to highlight the paradox of africa being geographically and anthropological central to our world , yet unrecognizable , unexplored , and invisible (: 408 ) . apart from the political message that requires text to be conveyed , a visualization is produced to create an aesthetic paradox : a piece of art that you can never see . yet that exists and carries a message.7 at the same time , it shows the research group s abilities to work at the nanoscale and to visualize it , and so illustrate / demonstrate the innovative character of nanotech developments . the artwork is a small sliver of silicon , visualized with an afm , and only visible by using the same instrument . the artwork is a small sliver of silicon , visualized with an afm , and only visible by using the same instrument . source : http://www.nanoarte.it in this way , nanoart also becomes a showcase for nanoscience . by crafting impressive nanosculptures , scientists capacities to manipulate matter at the nanoscale are highlighted , and expectations are created about the possible applications of the new methods . then there are artists ( often graphic artists ) who create impressions of how the nanoscale could look , or give their impressions of possible future applications for nanotechnology developments . they can be commissioned to do so by scientists to enhance the visual appeal of their achievements . the aesthetic appeal of images becomes more relevant when images are intended to circulate more widely and to reach broader audiences . this happens when imaging contests are organized where beautiful visualizations of the nanoscale are selected , and which then may become linked to more general issues and foreground nanotechnology developments and science in general , and , importantly , the beauty of the nanoscale . such images might then stand for nanotechnology . the image of the nanoflower , for example , is a nanosculpture , it was created in a laboratory , and its colouring was intentionally chosen to enhance its appeal to wider audiences.8 it was indeed used as a general illustration in texts explaining nanotechnology . how can images of nanotechnology become iconic , in the sense of standing for nanotechnology and its further development ? there are two parts to this question about the process and what is involved . the overall process is their reception and further circulation , where the message that is conveyed also plays a role . at some moment , the image has so much standing that it can stand for nanotechnology in its own right . secondly , there are struggles between actors with an interest in which image will eventually come to stand for nanotechnology . in a sense , whether an image becomes iconic or not is the outcome of such struggles . on the basis of these two processes 2 ) , the nanogear ( see fig . 5 ) and the ibm - nanologo ( see fig . 6).9fig . 5the nanogear image . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . this structure is designed through molecular construction software developed by nanorex . it stands for the idea that 1 day atoms could be used as the ultimate building blocks . source : http://nanoengineer-1.com/content/index.php?option = com_content&task = view&id = 52&itemid = 62fig . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . this structure is designed through molecular construction software developed by nanorex . it stands for the idea that 1 day atoms could be used as the ultimate building blocks . source : http://nanoengineer-1.com/content/index.php?option = com_content&task = view&id = 52&itemid = 62 ibm nano - logo . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . eigler and schweizer in the case of the nanolouse image , we have traced its circulation and reception ( see ) . after winning the science visions award in 2002 , the nanolouse image draws on , and reinforces , ideas about magic bullets redressing our sorrows . while contested within science that it might create wrong ideas about nanotechnology when presented to lay audiences ( cf . ) it was actually nanoscientists who continued to use the nanolouse image in their presentations , making it available on their websites . there is a reversal in its use : first , the nanolouse was accompanied by text explaining what the image depicted , but later , such explanations were not needed anymore ; the image could speak for itself and came to stand for the future of nanomedicine . in the case of the nanogear image there was a general appreciation of the image in the 1990s , and a widespread uptake which made it iconic according to our definition . but with the downturn in the standing of the drexlerian vision , it is not generally accepted anymore as standing for nanotechnology . the nanogear image builds on ideas that atoms can be used to engineer products from the bottom - up , and is used as standing for molecular manufacturing , the drexlerian vision . the image was designed by the foresight ( nanotech ) institute to carry that message , and was used almost as a logo . when the vision of molecular manufacturing lost its general standing in the early / mid 2000s , at least within the nanotechnology establishment , the use of the nanogear image as standing for nanotechnology became contested . but it continued to perform outside the immediate world of nanotechnology , up to appearing in official publications and brochures of research institutes wanting to show they were into nanotechnology as well.10 our third iconic image , the ibm nanologo ( see fig . [ it ] became visually compelling evidence of the human capacity to manipulate the world atom - by - atom and to build various molecular devices in the future (: 54 ; cf . ) . the ibm logo seems to suggest progression toward the full potential of nanotech (: 272 ) , and stimulated imaginations of the discovery and conquest of new spaces . writing of the ibm logo with the use of the american stars and stripes flag to mark territory.11 other images produced by ibm almaden like the elliptical corral of cobalt atoms on a copper substrate , with its judicious use of colours to create an aesthetically pleasing image ( fig . 7 ) , were used as illustrations , but never became iconic . fig . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . source : http://www.almaden.ibm.com/vis/stm/images/stm_small.gif a large variety of images of nanotechnology circulates inside as well as outside the world of nanotechnology , but most of them do not become iconic . that depends on their reception and if and how they take on a life of their own , including the struggles linked to their being the three images that did become iconic all depict actual or imagined technoscientific objects , and are widely seen as representing technoscientific achievements , even when they only offer a promise . the entanglement of imaging and imagining occurs all the time , and it can stabilize into specific patterns and established rules . this is very clear in practices of visualizing the nanoscale . while imaging is what these practices are about , imagining is an integral part , both in expectations about how the nanoscale could / should look like , and in the highlighting of the images , e.g. with colours , to enhance their visual appeal within the world of nanotechnology and for wider audiences . how to do this is broadly accepted , even if there are some debates . in concrete situations , there will always be choices to be made which require judgment rather than following a stabilized rule . incorporating the future in the present occurs in various ways , already in how images are used to visualize a design that might / should be realized . while there are debates about the plausibility of the futures that are imagined , there is widespread acceptance of such imagining , e.g. in the use of the nanolouse image . the cross - traffic between the world of nanotechnology and the wider world is premised on promises , and images embodying such promises are an integral part of this cross - traffic . while there is interest from both sides , there is little stabilization ( and perhaps there should nt be ) . seen from the world of nanotechnology , art is peripheral , so there is no pressure to stabilize the entanglements . this is different from the situation of visualization of the nanoscale , where the entanglement of imaging and imagining is addressed daily , and stabilization is necessary to work productively . for the entanglement of the present and the future , there is some pressure to stabilize , but also opportunism : if images like the nanolouse appear to work , they are used even if they could be ( and had been ) criticized . there are tensions in the entanglements and their stabilization . already within scientific practices , struggles become clearly visible when images are used to reach out to broader audiences . because the future and the present are entangled in nanotechnology and its images , there are always possibilities to emphasize one side over the other , in terms of simple actor s categories of the example of the nanolouse image shows that its fictional character is often downplayed in order to profit from the associations ( like magic bullet ) elicited by the image . this went as far as the image being featured in the documents of the european technology platform nanomedicine . at some moment , there may be a backlash , however , when actors in the world of nanomedicine get frustrated by the expectations that were induced by the wide use of the nanolouse image . for nano and art , there may be tensions between nano - actors thinking in terms of aesthetics , while artists focus on creating a new reality.12 there are no antagonisms , because the worlds of artists and of nanoscientists are far apart , and the interactions are seen merely as intriguing curiosities . there is a gray area where graphic designers produce images , and nanoscientists like chris ewels dabble in graphic design . at the moment , there is space for all of these ventures . the umbrella term this will change when images ( of whatever provenance ) might come to stand for nanotechnology as a whole.13 then there is something at stake : nanotechnology is not something given it lives on promises . so , the struggle will not be about what nanotechnology is now ( even if that is one element ) , but about what nanotechnology could be and should be . these struggles occur in a variety of arenas , like funding programs , national science and technology priority setting , and eligibility of who is allowed to speak for nanotechnology . images play a role as resources in these struggles , but also as agents in their own right when they have a life of their own . not as an active force , but as an affordance or a repertoire which will shape what happens without being determinant . it is the entangled imag(in)ing which creates such affordances and repertoires , not the force of images as such .

images , ranging from visualizations of the nanoscale to future visions , abound within and beyond the world of nanotechnology . rather than the contrast between imaging , i.e. creating images that are understood as offering a view on what is out there , and imagining , i.e. creating images offering impressions of how the nanoscale could look like and images presenting visions of worlds that might be realized , it is the entanglement between imaging and imagining which is the key to understanding what images do . three main arenas of entanglement of imag(in)ing and the tensions involved are discussed : production practices and use of visualizations of the nanoscale ; imag(in)ing the future and the present ; and entanglements of nanoscience and art . in these three arenas one sees struggles about which images might stand for nanotechnology , but also some stabilization of the entanglement of imag(in)ing , for example in established rules in the practices of visualizing the nanoscale . three images have become iconic , through the combination of their wide reception and further circulation . all three , the ibm logo , the foresight institute s nanogear image , and the so - called nanolouse , depict actual or imagined technoscientific objects and are thus seen as representing technoscientific achievements while marking out territory .

Introduction Imag(in)ing the Invisible Nanoscale Practices of Entangling the Future and the Present Entanglements of Nanoscience and Arts Images Standing for Nanotechnology In Conclusion

within and beyond the world of nanoscience , images abound . there are lots of colourful images of structures at the nanoscale , based on visualizations of digital data created by scanning probe microscopy or other imaging instruments ( as are made available to colleague nanoscientists ) . there are also artist s impressions , ranging from impressions of entities at the nanoscale to envisioning nanoachievements to be realized in the near or long - term future , including science - fiction type images of nanobots . one can distinguish imaging , that is , creating images based on data and aiming at resemblance , from imagining , where imagination is mobilized to create a vision of the nanoworld . actually , imaging and imagining are always entangled , already in so - called scientific visualizations where expectations about how the nanoscale could look like steer the choices involved in making images . a subsequent question then is what such entangled images stand for , not only representing in the sense of resemblance , but also in terms of what images do when representing the nanoscale . in practice , there is a continuum ranging from visualizations of the nanoscale adapted from original instrument - based data to so - called artist s impressions ( which need not be produced by artists ; the phrase is used to indicate some freedom in visualizing ) of how the nanoscale could look like , or how audiences may expect that it looks . this occurs already within science , for example when images indicate possible functions of complex molecular structures . to capture the continuum of imaging and imagining , we will write imag(in)ing.1 we will start by analyzing occurrences of imag(in)ing and the tensions involved . while imag(in)ing has to do with representation , which is often focused on making something present through resemblance , we emphasize representation as standing for and being able to political representation ( cf . ) thus , this notion of representation is broader than the epistemological puzzles , and debates , about reality ( out there and/or in contrast with fiction ) . in the final part of our paper right representation is , in terms of images that become iconic , and in that sense stand for , and are allowed to speak for , nanotechnology . the nanoscale , invisible to the naked eye , is visualized through the use of a variety of imaging instruments . at the outset , it is not clear what the right imaging tools would be , and this relates to the basic problem how one can know whether the visualizations obtained actually visualize the nanoscale harry collins highlighted the problem of choice between instruments that would be adequate to the task of studying unknown phenomena a problem which he introduced as an experimenter s regress because there is no independent check of the correctness of the choice . for the production of visualizations there is the additional question about the ways to produce images / pictures that lead to reliable representations of the invisible . just as the experimenter s regress can ( and will ) be stopped in practice when expectations about the unknown phenomenon become shared , expectations about how the invisible phenomena should look like , can provisionally stop the visualization regress (: 5 ) . a subsequent imaging challenge is to reach audiences which are not familiar with the production processes of the images . there will be ( .. ) a diffuse message about the nanoscale which has to be presented as clearly and convincingly as possible (: 9 ) to intended audiences . in visualization practices expectations about what can be seen at the nanoscale and how the nanoscale should be visualized are to some extent internalized as rules about how to do it . the data - based images are manipulated to highlight the information they should convey , which is an accepted practice in the domain of science , as long as the scientific content remains unchanged . artist s impressions of how the nanoscale could look like are similar , in the sense that imaging and imagining are also entangled . in other words visualizations of the nanoscale as well as artist s impressions are used in scientific journals to show research results.2 such practices are not uncontested . correctly , make it to the cover of scientific journals.3 when they do so , they come to stand for scientific achievements , and cover pictures are often presented on research groups websites next to their list of publications . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view courtesy of : c. dekker , tu delft / tremani artist s impression of an array of nanotubes fets overlaid with gold source and drain electrodes . this image was used for a cover of science , and was subsequently criticized by ottino , who argued that if the carbon atoms are visible , then the much larger gold atoms in the structure should also be on view as an emerging technology , nanotechnology comprises present achievements and visions of what it may become . mody argues that nanotechnology , in contrast to , for example , physics and chemistry , seems decidedly non - presentist and that nanotechnologists work as much in this future world as in the present (: 108 ) . then , there is the design orientation in nanotechnology , where a possible future is projected to be realized by a present design . kaiser adds another twist:they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . they [ future visions ] have been invented and are presented with the strong probability claim that they will become reality in the future . if one wants to use categories like reality and fiction ( as actors often do ) , one can say that boundaries between reality and fiction are blurred in the nanorealm , and this will be reflected in images of nanotechnology . artists offer their ( own or commissioned ) visions of nanotechnology s potential future applications . as soon as the future is incorporated , there is no sharp boundary between images that articulate directions of further research and more open - ended visions of new performances which then merge with futuristic visions . arguments for disentangling the future from the present also occur in relation to lay - audiences . 2the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . the nanolouse image ; over - hyped or a possible application of nanotechnology in medicine in the future . this is important in terms of public reaction , especially in the backdrop of scenarios such as in michael crichton s new novel prey , which portrays swarms of self - replicating nanomachines destroying all other life forms that they encounter (: 476 ) . ottino links his criticism with a concern about public acceptance of nanotechnology , if such images should come to stand for nanotechnology in the public eye . still , the image does contain an ambivalence , which is brought out in how it has been presented on the bbc website ( www.bbc.co.uk ) . on one occasion it was positioned as the 2002 winner of the visions of science award : the more over - hyped applications see tiny machines roaming the body to cure diseases. on another occasion , realistic possibilities were emphasized : the nanolouse image intends to show one of the possible applications of nanotechnology in medicine in the future microscopic machines roaming the body , injecting or taking samples for tests. continuing with this example , it is interesting that the nanolouse image has been often used in the world of science . in the period 20052007 , the image was used in presentations about drug delivery , nano - probes and ( n)mems , as an illustration and occasionally as a possible future application .4 in general terms , images of nanotechnology that import the future into the present are used to communicate what nanotechnology is as well as to indicate nanotechnology s potential . an interesting example is how the foresight nanotech institute used the nanogear image to position itself . 5 ) ( and the image of the strained - shell sleeve bearing ) to indicate that 1 day , surely , productive nanosystems will be realized . respirocytes ( artificial blood cells able to carry 236 times more oxygen than human s red blood ) as designs , implying that they can ( and thus will ) be built in the future . what is new in nanotechnology is that these images are about aimed - for possibilities rather than an actual construction plan of such a future . the nanogear image is an extreme example , but these kind of images and their use are widespread . nanoscientists play with the entanglement of data - based imaging and impressionistic imagining , and with the entanglement resulting from projecting of the future into the present . in addition , the aesthetic appeal of the images that are produced as visualizations of the nanoscale , is actively pursued , for example through impressive colouring , as in ibm s image of the quantum corral ( fig . the fact that images are now given a name , and an evocative name at that , is an indication that they are seen as works of art , which have titles.5 this link with the world of art , at least with the world of being creative in making images , is not just an excursion of scientists . an interesting development is how artists make use of , or even get involved in , the production of images through imaging instruments . as chris orfescu noted , there appear to be two ways to do so : nanolandscapes in which given structures of matter at the nanoscale are depicted , and nanosculptures , which are created through the manipulation of matter at the nanoscale by nanoscientists , and then visualized.6 one sees how such images offer a view to a new world that is in the process of being explored . many of the nano- and microsculptures like the nano - guitar , nano - opera house , nano - toilet and the nano - bull ( see fig . but the fact that it was a bull was the immediate message , and kawata et al.s claim about their technoscientific achievement piggy - backed ( if we may say so ) on this message . scali and goode produced the smallest map ever of the continent africa , created through the use of a scanning electron microscope ( sem ) and visualized with an afm ( see fig . the actual size is 200 m 130 m , and it is meant to highlight the paradox of africa being geographically and anthropological central to our world , yet unrecognizable , unexplored , and invisible (: 408 ) . yet that exists and carries a message.7 at the same time , it shows the research group s abilities to work at the nanoscale and to visualize it , and so illustrate / demonstrate the innovative character of nanotech developments . by crafting impressive nanosculptures , scientists capacities to manipulate matter at the nanoscale are highlighted , and expectations are created about the possible applications of the new methods . then there are artists ( often graphic artists ) who create impressions of how the nanoscale could look , or give their impressions of possible future applications for nanotechnology developments . this happens when imaging contests are organized where beautiful visualizations of the nanoscale are selected , and which then may become linked to more general issues and foreground nanotechnology developments and science in general , and , importantly , the beauty of the nanoscale . such images might then stand for nanotechnology . the image of the nanoflower , for example , is a nanosculpture , it was created in a laboratory , and its colouring was intentionally chosen to enhance its appeal to wider audiences.8 it was indeed used as a general illustration in texts explaining nanotechnology . how can images of nanotechnology become iconic , in the sense of standing for nanotechnology and its further development ? the overall process is their reception and further circulation , where the message that is conveyed also plays a role . at some moment , the image has so much standing that it can stand for nanotechnology in its own right . secondly , there are struggles between actors with an interest in which image will eventually come to stand for nanotechnology . on the basis of these two processes 2 ) , the nanogear ( see fig . 5 ) and the ibm - nanologo ( see fig . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . the nanogear image features a complex molecular structure which is envisioned to be used to build products from the bottom - up . the ibm logo was created at the nanoscale by moving 35 xenon atoms on a nickel surface . this image showed that atoms could not only be visualized , but also be moved through the use of an stm . eigler and schweizer in the case of the nanolouse image , we have traced its circulation and reception ( see ) . after winning the science visions award in 2002 , the nanolouse image draws on , and reinforces , ideas about magic bullets redressing our sorrows . there is a reversal in its use : first , the nanolouse was accompanied by text explaining what the image depicted , but later , such explanations were not needed anymore ; the image could speak for itself and came to stand for the future of nanomedicine . in the case of the nanogear image there was a general appreciation of the image in the 1990s , and a widespread uptake which made it iconic according to our definition . but with the downturn in the standing of the drexlerian vision , it is not generally accepted anymore as standing for nanotechnology . the nanogear image builds on ideas that atoms can be used to engineer products from the bottom - up , and is used as standing for molecular manufacturing , the drexlerian vision . the image was designed by the foresight ( nanotech ) institute to carry that message , and was used almost as a logo . when the vision of molecular manufacturing lost its general standing in the early / mid 2000s , at least within the nanotechnology establishment , the use of the nanogear image as standing for nanotechnology became contested . but it continued to perform outside the immediate world of nanotechnology , up to appearing in official publications and brochures of research institutes wanting to show they were into nanotechnology as well.10 our third iconic image , the ibm nanologo ( see fig . [ it ] became visually compelling evidence of the human capacity to manipulate the world atom - by - atom and to build various molecular devices in the future (: 54 ; cf . ) the ibm logo seems to suggest progression toward the full potential of nanotech (: 272 ) , and stimulated imaginations of the discovery and conquest of new spaces . writing of the ibm logo with the use of the american stars and stripes flag to mark territory.11 other images produced by ibm almaden like the elliptical corral of cobalt atoms on a copper substrate , with its judicious use of colours to create an aesthetically pleasing image ( fig . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . ring of atoms are shown in light shade of green , in which the ripples ( colored in blue ) are wave patterns of some of the electrons trapped in the corral . source : http://www.almaden.ibm.com/vis/stm/images/stm_small.gif a large variety of images of nanotechnology circulates inside as well as outside the world of nanotechnology , but most of them do not become iconic . that depends on their reception and if and how they take on a life of their own , including the struggles linked to their being the three images that did become iconic all depict actual or imagined technoscientific objects , and are widely seen as representing technoscientific achievements , even when they only offer a promise . the entanglement of imaging and imagining occurs all the time , and it can stabilize into specific patterns and established rules . this is very clear in practices of visualizing the nanoscale . while imaging is what these practices are about , imagining is an integral part , both in expectations about how the nanoscale could / should look like , and in the highlighting of the images , e.g. with colours , to enhance their visual appeal within the world of nanotechnology and for wider audiences . incorporating the future in the present occurs in various ways , already in how images are used to visualize a design that might / should be realized . while there are debates about the plausibility of the futures that are imagined , there is widespread acceptance of such imagining , e.g. in the use of the nanolouse image . the cross - traffic between the world of nanotechnology and the wider world is premised on promises , and images embodying such promises are an integral part of this cross - traffic . seen from the world of nanotechnology , art is peripheral , so there is no pressure to stabilize the entanglements . this is different from the situation of visualization of the nanoscale , where the entanglement of imaging and imagining is addressed daily , and stabilization is necessary to work productively . for the entanglement of the present and the future , there is some pressure to stabilize , but also opportunism : if images like the nanolouse appear to work , they are used even if they could be ( and had been ) criticized . there are tensions in the entanglements and their stabilization . because the future and the present are entangled in nanotechnology and its images , there are always possibilities to emphasize one side over the other , in terms of simple actor s categories of the example of the nanolouse image shows that its fictional character is often downplayed in order to profit from the associations ( like magic bullet ) elicited by the image . this went as far as the image being featured in the documents of the european technology platform nanomedicine . at some moment , there may be a backlash , however , when actors in the world of nanomedicine get frustrated by the expectations that were induced by the wide use of the nanolouse image . for nano and art , there may be tensions between nano - actors thinking in terms of aesthetics , while artists focus on creating a new reality.12 there are no antagonisms , because the worlds of artists and of nanoscientists are far apart , and the interactions are seen merely as intriguing curiosities . there is a gray area where graphic designers produce images , and nanoscientists like chris ewels dabble in graphic design . the umbrella term this will change when images ( of whatever provenance ) might come to stand for nanotechnology as a whole.13 then there is something at stake : nanotechnology is not something given it lives on promises . so , the struggle will not be about what nanotechnology is now ( even if that is one element ) , but about what nanotechnology could be and should be . these struggles occur in a variety of arenas , like funding programs , national science and technology priority setting , and eligibility of who is allowed to speak for nanotechnology . images play a role as resources in these struggles , but also as agents in their own right when they have a life of their own . not as an active force , but as an affordance or a repertoire which will shape what happens without being determinant . it is the entangled imag(in)ing which creates such affordances and repertoires , not the force of images as such .

ruthenium compounds belong to the most promising candidates of non - platinum containing metal complexes for cancer therapy . compared to pt drugs ru complexes cause less side effects and resistances against the drug are less likely . the overall chemical and pharmacokinetic behavior of ru is quite different from that of pt compounds , as reflected in extensive protein binding . imidazolium trans-[tetrachloro(dimethylsulfoxide)(imidazole)ruthenate(iii ) ] ( 13 , nami - a ) , indazolium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 1 , kp1019 ) , its sodium analogue sodium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 2 , kp1339 ) , and ru(ii)-arene complexes like [ ru(-p - cymene)cl2(pta ) ] ( 14 , rapta - c , pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decanephosphine ) are the most promising investigational drug candidates up to now . phase i clinical trials in patients with refractory solid malignancies have successfully been completed for both 13(19 ) and 1 , the latter yielding disease stabilizations in more than half of the patients , and further clinical studies have commenced recently . in general , it is assumed that the ru(iii ) complexes serve as prodrugs that are reduced under pathophysiological conditions to ru(ii).13 is a ru(iii ) complex mainly active against metastases , and it is assumed that its main site of action is located in the extracellular matrix . the ru(ii)-arenes are organometallic complexes that are quite stable in the bloodstream , hydrolyzed in the nucleus , and bind to dna . the ru complexes 1 ( figure 1 ) and its na salt analogue 2 ( figure 1 ) are the subject of investigation in this work . 1 and 2 differ only in their countercations , indazolium for 1 and na for 2 , exhibiting the same ru(iii ) coordination sphere [ tetrachlorobis(1h - indazole)ruthenate(iii ) ] . therefore , their main binding partners in vivo and their mode of action are thought to be very similar . both are showing antitumor activity exceeding that of pt compounds or other cytostatic agents , e.g. , in colorectal carcinomas in vivo and a variety of primary explanted human tumors in vitro . fast uptake of 2 by the cell and initiation of apoptosis ( indicated by caspase activation ) were observed . the uptake mechanism of the two drugs may involve human serum albumin ( hsa ) , the iron transport protein transferrin ( tf ) , and the tf receptor , which is overexpressed in tumor cells to meet their increased demand for fe . a prodrug function is supposed involving reduction of ru(iii ) to ru(ii ) , which is supported by solution studies in the presence of different reductants . the low oxygen level favors the reduction of ru(iii ) and other redox - active substances . the electrochemical potential of ru(iii)/ru(ii ) is physiologically accessible , and glutathione ( gsh ) and single - electron transfer proteins are able to reduce ru(iii ) in the presence of nadh . however , the direct determination of the oxidation state , the coordination of the ru active site , and the mechanism of action in vivo for these and other ru drugs remains elusive . x - ray absorption spectroscopy ( xas ) is an element specific method that allows the investigation of the questioned element in disordered environments missing any long - range order . xas has proven to be a valuable tool for the speciation of metal centers . by application of x - ray absorption near edge structure ( xanes ) analysis , it is possible to determine the oxidation state and coordination of the ru metal center , whereas extended x - ray absorption fine structure ( exafs ) analysis opens the opportunity to specify the identity , number , and distances of the adjacent atoms . xas is one method of choice to determine the oxidation state of the metal site in model compounds and especially of metal coordination sites in vivo . for instance hambley et al . could monitor activation by reduction of anticancer pt(iv ) compounds inside cells . new reduction pathways of pt(iv ) to pt(ii ) species were monitored with xanes by nemirovski et al . , and the distribution and relative inertness of a ga(iii ) compound in biological tissue were investigated in a previous publication . the main obstacle for bio - xas is the low concentration of the metal in the targeted tissue . to generate spectra with a sufficiently good signal - to - noise ratio , x - ray sources like synchrotrons , with a sufficiently high photon flux in the range of 1 10 to 4 10 photons per second , are necessary . this high brilliance may cause the reduction of the probed metal through secondary electrons and the degradation of the biological tissue . these effects can be minimized using low temperature sample environments like helium cooled cryostats at around 20 k. ascone et al . studied the reaction of bovine serum albumin ( bsa ) with 13 and the formation of the respective 13/bsa adducts by means of the sulfur and chlorine k - edges and the ru k- and l3-edges . ru k- and l3-edge spectra proved unambiguously that the ru center remains in oxidation state + 3 after protein binding . comparative analysis of the cl k - edge xas spectra of 13 and 13/bsa revealed that the cl environment is greatly perturbed upon protein binding . in a subsequent study of liu et al . the binding behavior of 13 to bsa was analyzed by fitting a series of model compounds with known coordination to the unknown ru(iii)-bsa spectrum . the coordination environment of the adduct turned out to be completely different from that of the parent complex 13 with about 60% n and 40% o ligands coordinated to the ru(iii ) center . with a combination of s k - edge and ru k - edge xas and density functional theory ( dft ) techniques studied the activation of ru(ii)-arene complexes through oxygenation and subsequent protonation of the thiolate ( sr ) ligand . the sr ligand is oxygenated to sulfinate ( so2r ) via sulfenate ( sor ) and activated through protonation under acidic conditions . the protonation turned out to be a crucial step , which facilitates the dissociation of the so2r ligand and the dna binding . the ru metal center stayed in its ru(ii ) state throughout this process and was not affected by the ligand oxygenation . sadler and co - workers studied the photodissocation of ru bipyridine complexes and their nucleobase binding with a combination of exafs and dft calculations . used combined cl k - edge and ru l3-edge xas measurements together with dft calculations to describe the electronic structure properties of a set of four ru(iii ) and six ru(ii ) complexes with mixed cl / s / n coordination spheres , among them 1 and 13 . the effect of differing amounts of s ( from dimethyl sulfoxide , dmso ) and n ( from indazole / imidazole ) ligands onto the electronic configuration within each ru series ( iii and ii ) was quite low , and the ligand - to - metal donations reach a limit at around two s , two cl , and two n ligands . this was ascribed to a so - called trans effect where the -donor and -accepting effects of dmso s ligands counteract each other . for 1 it was found that the indazole ligands are weaker donors toward ru than imidazole ones , which is congruent with the proposed activation by reduction mechanism stated for 1 and its easier reduction to ru(ii ) compared to the imidazole analogue trans-[tetrachlorobis(1h - imidazole)ruthenate(iii ) ] ( kp418 ) . getty et al . assigned ru k - edge pre - edge peaks to the centrosymmetric structure of ru carbene complexes for olefin metathesis . with increasing symmetry of the complexes the pre - edge peaks got weakened , which was ascribed to a lowered ru 4d5p orbital mixing . even though 1 and 2 are known to undergo hydrolysis ( facilitating further ligand exchange reactions ) and to be redox - active , both of which are features proposed to be significant for the mechanism of action , neither coordination nor redox condition of 1/2 after biotransformation in biological systems has been examined because of the lack of appropriate analytical methods . xas is the method of choice that possesses a high value as a method for elucidation of the structure of metal coordination centers in biological environments . the aim of this work is to apply xas to investigate the tumor - inhibiting ru compound 1 in citrate saline buffer ( cs buffer ) at ph 3.5 within different time intervals , in the presence of the reducing agent gsh , in carbonate buffer in the presence of the possible transport protein apo - transferrin ( apotf ) , and as a powder . xanes spectra were recorded on tissue samples from mice treated with 1 and 2 . micro x - ray fluorescence ( micro - xrf ) maps were taken from tumor tissue samples taken from mice treated with 2 . additionally model compounds representing possible coordinations and oxidation states in vivo were measured as references . this study is designed to make a contribution toward the fundamental understanding of the biotransformation of ru compounds in vivo and the underlying principle of activation by reduction which has to be evaluated . the following model compounds were investigated with xas : 1 ( with first coordination shell ru(iii)cl4n2 ) , ruthenium(iii ) acetylacetonate ( 3 , ru(iii)o6 , sigma aldrich , cas 14284 - 93 - 6 , 97% ) , hexammineruthenium(iii ) trichloride ( 4 , ru(iii)n6 , sigma aldrich , cas 14282 - 91 - 8 , 99%),mer , trans - aquatrichloridobis(indazole)ruthenium(iii ) ( 5 , kasc003 , ru(iii)cl3n2o),trans , trans - dichloridotetrakis(indazole)ruthenium(iii ) chloride ( 6 , gabu129 , ru(iii)cl2n4),mer - trichloridotris(indazole)ruthenium(iii ) ( 7 , gupl328 , ru(iii)cl3n3 ) , hexammineruthenium(ii ) dichloride ( 8 , ru(ii)n6 , sigma aldrich , cas 15305 - 72 - 3 , 99.9%),mer , trans - trichlorido(dimethylsulfide)bis(indazole)ruthenium(iii ) ( 9 , flan005 , ru(iii)cl3n2s),trans , trans - dichloridotetrakis(indazole)ruthenium(ii ) ( 10 , gabu128 , ru(ii)cl2n4),mer - trichloridotris(ethylphenylsulfide)ruthenium(iii ) ( 11 , flan006 , ru(iii)cl3s3),trans , trans , trans - dichloridobis(dimethylsulfide)bis(indazole)ruthenium(ii ) ( 12 , flan004 , ru(ii)cl2n2s2 ) . all spectra were collected at 20 k. the structural formulas are shown in figure 1 . the xanes spectra of the model compounds and their corresponding first derivatives are presented in figure 2 . in figure 2a the ru(ii ) figure 2b exhibits ru(iii ) compounds with mixed cl / n first shell ligand atoms ( including 1 ) . figure 2c displays compounds with o or s containing first shells . within the same ru oxidation state the edge energies increase with increasing electronegativity of the first shell atoms ( see table 1 ) . model compounds with the same first shells show an edge shift of about + 2 ev from ru(ii ) to ru(iii ) ( see 8/4 and 10/6 ) . the absence of any distinct pre - edge feature implies a centrosymmetric coordination of the here investigated ru compounds . the edge energies observed in this study span a range of about 6 ev , with 1 occupying an energy position right in the middle . the bottom limit is marked by the s containing 12 in its oxidation state + 2 , and the upper boundary is represented by 3 with six o ligands and an oxidation state of + 3 . comprising the highest amount of four cl atoms , 1 occupies the lowest energy position within the ru(iii ) models , exhibiting only cl / n atoms in the first shells . the models 1 , 3 , and the ru hexammine compounds 8 and 4 , in both oxidation states , exhibit a characteristic edge shoulder . normalized spectra of the model compounds and their corresponding first derivatives with a ru(ii ) center ( a ) , mixed cl / n first shells ( b ) , and o or s containing first shells ( c ) . the k weighted exafs spectra and the fourier transforms ( ft ) of the ru model compounds are shown in figure 3 . in figure 3a the fine structures and the non - phase - shifted ft of the models containing a ru(ii ) center figure 3b shows the fine structures and ft of the ru(iii ) compounds with mixed cl / n first shells , and figure 3c shows the one for the models containing o or s in their first shell around the ru(iii ) center . the amplitudes of the fine structure oscillations and the ft magnitudes increase with a growing amount of heavy cl and s scatterers . 11 shows the strongest k - space oscillations and ft magnitudes . for compounds with mixed n / o / cl / s first shells and increasing number of n and/or o ligands a splitting of the first peak in the ft is observable . the ru(ii ) compounds 10 and 12 have the largest cl / n bond length differences and show the strongest peak splitting . the backscattering amplitudes of the heavy scatterers like s and cl and the light scatterers like n and o are out of phase . for 10 this cancellation leads to a node between 7 and 9 seen in figure 3a ( red dashed curve ) . the ru(iii ) analogue 6 has the same features between 8 and 10 seen in figure 3b ( red dashed curve ) . the shift of this feature is mainly attributed to the shortened cl bond lengths of the higher oxidized 6 . the fitting analysis using feff was restricted to the first coordination shell extracted from the first peak in the ft . the identity and number of backscatterers were fixed to the crystallographic values not to exceed the number of fitting parameters , and the known distances were taken as a starting point for the fitting analysis . the results of the first shell fits of the model compounds using theoretical amplitudes and phases provided by the feff code are presented in the supporting information ( table s2 ) , as well as the results for the dl - excurv fits ( table s3 ) . the distances are given as the average fitted distances for each atom type / shell ( cl / s , o / n , and n / c ) . for 6 and 10 the second shell scatterers 4 n and 4 c at about 3 were included in the feff fit , as they had a significant influence on the first shell spectral features . the curve fitting results obtained by feff and dl - excurv are both in good agreement with the crystallographic data . thus , the reliability of the amplitude and phase shift extracted from the model compounds is confirmed . extracted fine structures and fourier transforms of the model compounds with a ru(ii ) center ( a ) , a mixed cl / n first shell ( b ) , and a o or s containing first coordination shell ( c ) . in previous articles a correlation between the coordination charge and a distinct spectral edge feature could be shown . in this study the edge position determined over the first maximum in the first derivative was chosen as the point of reference . the electronegativity values according to the allred / rochow tables were used in the calculations . the calculated ionicities and corresponding degrees of covalence of the elements occurring in the first shells of the models are presented in table 2 . in table 1 the ruthenium model compounds are listed in order of their observed edge positions . in figure 4 the calculated coordination charges versus the experimentally determined ru k - edge positions are shown . the edge energy of 1 in boron nitride ( bn ) was set as an arbitrary origin and lies right in the middle of the observed edge energy range . a straight line was regressed with a coefficient of determination r = 0.95 , demonstrating a linear correlation between the coordination charge and the edge positions . the compounds containing ru(ii ) and/or s are on the left ( lower energy ) side , and the compounds containing ru(iii ) , n , and o are on the right ( higher energy ) side . this correlation is the basis for the further interpretation of the coordination mode of ru compounds in biological samples and in the presence of potential transport proteins . z , atomic number ; ar , electronegativity according to allred rochow ; iar , calculated ionicity ; car calculated covalency . calculated coordination charge ar according to the allred rochow scale in comparison to the observed edge energies of the xanes spectra . in figure 5a the xanes spectra of 1 in bn , suspended in cs buffer and in the presence of gsh ( 5-fold excess , cs buffer , ph 3.5 ) , are shown . 1 was incubated at 37 c for 30 min , 4 h , 8 h , and 5 h in the presence of gsh . the spectra of 1 after 30 min and 4 h of incubation are shifted by + 1.1 ev to higher energies than 1 in bn . after 8 h of incubation of 1 in cs buffer and 5 h in the presence of gsh , both spectra are shifted significantly to lower energies by 1.4 and 1.3 ev , respectively . the cs buffer spectra , after 30 min and 4 h , lost the edge shoulder at 22 130 ev present in the solid sample of 1 . the nearest model compound edge positions to these solution spectra ( e = + 1.1 ev ) are those with ru(iii)cl2n4 and ru(iii)cl3on2 first coordination shells . the shift in energy suggests that an average exchange of one cl ligand with an o atom arising from the buffer solution or a n atom from indazole ( deprotonated indazolium ) took place . the indazolium cation has a pka of 1.25 which enables deprotonation and opens the possibility to act as a n donor at ph 3.5 . this coordination environment proved to be stable up to 4 h , and the reduction to ru(ii ) is not assumed . in the ru(ii ) state an edge shift of + 1.1 ev would only be possible through the exchange of all ligands by o arising from the buffer solution . the spectrum of 1 , after 8 h , is shifted to lower energies by 1.4 ev . in comparison to the solution spectrum of 1 after 4 h a shift of 2.5 ev this demonstrates fundamental changes in the first coordination environments around the ru center . in the presence of gsh the spectrum is shifted to the low energy positions in the region of the ru(ii ) and ru(iii ) s containing model compounds . least square fits ( lsf ) were performed to suggest the most likely first coordination modes . ( a ) normalized xanes spectra and first derivative of 1 in bn , 1 in cs buffer ( after 30 min , 4 h , and 8 h ) and in cs buffer in the presence of the reducing agent gsh ( after 5 h ) . ( b ) 1 in cs buffer ( 30 min ) and carbonate buffer ( 30 min ) and in carbonate buffer in the presence of apotf ( 30 min ) . ( c ) normalized xanes spectra and first derivatives of the tumor and liver samples . lsf to 1 in cs buffer ( 30 min , 4 h ) and in the presence of gsh ( 5 h ) was performed using the solid state spectra of 1 , 4 , 5 , 6 , 9 , 10 , and 12 . the fit results are shown in table 3 and were analyzed according to the goodness of fit parameters and red , the shift in energy e , and the scaling factor c . the best fits for each sample are highlighted in bold - face . the best fits to 1 ( 30 min , 4 h ) could be achieved with 5 . an exchange of an average of one cl atom for an o from the buffer solution or n from indazole ( deprotonated countercation ) are two possible scenarios . in the presence of gsh the spectra of 9 and 12 gave similar results in the goodness of fit parameters but with opposite edge shift variances . this points toward a ru(ii)(cl / s)4n2 coordinated species , which would best match the experimental data , but a ru(iii)cls3n2 can not be ruled out . c is the sum of the fitted components and is the scaling factor applied to the fitted spectrum . the best fit is highlighted in boldface . in figure 5b the normalized xanes spectra and first derivatives of 1 suspended in carbonate buffer ( 30 min , 37 c ) and in carbonate buffer in the presence of apotf ( 30 min , 37 c ) are shown . for comparison the spectrum of 1 in cs buffer ( ph 3.5 ) is shown as well . the spectrum of 1 in carbonate buffer is shifted by + 1.7 ev to higher energies , which is slightly more than 1 in cs buffer ( + 1.1 ev ) . the spectrum of 1 in the presence of apotf is shifted even further to a higher energy position ( + 3.3 ev ) . this immense shift to positive energies is mostly due to the change in edge shape . when introducing a horizontal line at the ordinate value corresponding to the edge energy of the carbonate buffer spectra , a change to the apotf spectrum results in an energy displacement of about + 0.5 ev . this suggests the exchange of additional cl ligands by o or n , thus resulting in a first shell with a high content in o / n in the presence of apotf ( see figure 7 ) . lsf on 1 in carbonate buffer ( 30 min ) and in the presence of apotf ( 30 min ) was done in the same way as described for 1 dissolved in cs buffer . the best fits to 1 in the carbonate buffer solution were achieved with 5 and 6 . in the presence of apotf proposed structural motifs are presented in figure 7 . in a subsequent step linear combination analysis ( lca ) lsf of 3 , 4 , and 11 and combinations of them has been performed on 1 in bn , 1 in cs buffer at ph 3.5 ( 4 and 5 h with gsh ) , and 1 in carbonate buffer at ph 7.5 ( 30 min with and without apotf ) . the lca with ruo6 , run6 , and rucl3s3 environments resembles the known first coordination shell of solid 1 very well . after 4 h in cs buffer at 37 c a decrease to an overall coordination of ru to about 3 n and 3 cl in solution is seen by lca . after an incubation of 5 h in cs buffer in the presence of gsh a ratio of 2 n to 4 cl / s gave the best fitting results . in the presence of apotf the best lca fits were obtained with a first shell environment dominated by o / n and a small proportion of cl . the proposed first shell coordinations are presented in figure 7 , and the fitting results are shown in the supporting information ( table s4 ) . xanes spectra of tumor and liver samples from mice treated with 1 or 2 were collected in fluorescence mode at 20 k with an unfocused beam . because of the low concentration of ru in the biological tissue , data collection was restricted to the xanes region . the normalized xanes spectra and the corresponding first derivatives of the tumor and liver samples from mice are shown in figure 5c . the samples were taken from mice treated with different concentrations and application schemes of 1 and 2 ( details for drug administration see supporting information ) . the edge positions of the xanes spectra taken from all tumor and liver material are around 22 124.4 ev within 0.1 ev deviation ( see table 1 ) . because of the low signal strength , the changes in the edge positions are within the statistical error and there is no evidence for a change in comparison of all measured tissue spectra . the ru k - edge xanes spectra taken from liver and tumor samples are highly similar , which indicates the same local coordination around the ru center . the apparent coordination charge of the ru center of 1 and 2 in the tissue is similar to 1 in cs buffer after 30 min and 4 h of incubation at 37 c . principal component analysis ( pca ) was performed to confirm the high similarities seen within the probed tissue samples . lsf was used to propose a possible coordination and oxidation state pairing for the chemical constitution in tissue . in the following tissue xanes spectra were studied applying pca to give a statistical foundation for possible coordinations in vivo . the variance within all tissue samples could be described to an extent of 98% with the first principal component . the target transforms of the model compounds 1 , 4 , 5 , 6 , and 7 to the vector subspace of the first principal component are presented in the supporting information ( figure s1 , not for 4 ) . obviously 1 and 4 gave the worst results with spoil factors of 10.22 and 9.00 , which are well above an acceptable value . , a spoil factor below 6 is acceptable and below 3 would be a good result . the model compounds 7 , 5 , and 6 reach values of 4.95 , 5.31 , and 5.49 , respectively ( see table 4 ) . all other reference compounds measured in this study are far away from an acceptable result . on basis of the target transformation results , lsf to each tissue sample the outcomes of the lsf of 4 , 5 , 6 , and 7 to the tissue sample spectra are summarized in table 5 . fits based on solid 1 to each tissue sample are included in this study as well . the best values based on the statistical misfit , red , and energy shift e were achieved with 5 and 7 . these two fits are nearly identical , whereas 5 shows the smallest edge shift and 7 a lower statistical misfit . the fits with 6 are slightly worse in all fitting parameters . during the fits , the shift in energy e of the fitted samples was between 0.39 and + 0.39 ev . the fits of 5 to b2-liver and b2-tumor are coplotted in the supporting information ( figure s2 ) . the edge position of the tissue samples is indicated by the vertical blue line in figure 4 . this shift is slightly above the one for 5 and 6 . for the tissue samples ru(iii)cl3n2(o / n ) is proposed as a possible first shell environment for the majority of the 1 and 2 molecules , which are dominating the xas signals . nevertheless a ru(ii ) species with a high o and/or n content in the first coordination sphere as in ru(ii)cln2(o / n)3 can not be ruled out . spoil factor is an arbitrary defined value for the goodness of fit and should be below 6 . is the goodness of fit parameter scaled to the estimated uncertainty , red . e accounts for the energy shift of the sample data , and c is the sum of the fitted components and is the scaling factor applied to the fitted spectrum . the best fit is highlighted in boldface . tumor ( sw480 ) samples of mice treated with 2 were scanned with a x - ray beam focused to 10 m . the resulting maps give an overview of the elemental distribution patterns of ruthenium , iron , copper , and zinc in a defined region of the tumor . in figure 6 the 924 660 m micro - xrf maps of ru , fe , cu , and zn of one sw480 tumor ( mouse 148 - 1 ) sample are presented . the scanned region is indicated by a rectangle in the pictures in the top row . picture a is the scanned 10 m thin section on ultralene film , and picture b is the consecutively cut hematoxylin and eosin ( h & e ) stained 5 m thin section mounted on a glass slide . the concentrations of all four elements are highest in the region of the blood vessels and in the edge regions of the tissue . in contrast to the fe , cu , and zn distribution , ru shows a more disperse pattern with hotspots apart from the predominantly higher concentrated areas . the correlation between the ru distribution pattern and the fe one is of the same order of magnitude as between ru / cu or ru / zn . this opens the question of whether physiological pathways other than the fe - dependent ones are involved in the ru transport as well . the scanned regions are indicated by the rectangle in the pictures in the top row . the scatter peak and the transmitted signal ( the data show a total variation of 3% ) are shown as well . in the top left is shown a picture of the scanned 10 m thin section . in the top right is shown a picture of the consecutively cut h & e stained 5 m thin section . xanes analysis has been applied to propose a first coordination environment of an investigational anticancer drug in cs buffer ( ph 3.5 ) and carbonate buffer ( ph 7.4 ) , in carbonate buffer in the presence of apotf , in cs buffer in the presence of gsh , and in the target tissue . the concept of the coordination charge was applied to the model compounds , and a linear correlation between the coordination / oxidation state and the observed edge energies was established . this was the basis for the further interpretation of the spectra of 1 in solution and in biological samples . proposed oxidation state and first shell coordination of 1 in cs buffer ( 4 h ) , in liver and tumor tissue , in cs buffer in the presence of gsh and in carbonate buffer in the presence of apotf ( top ) . the spectra collected on 1 suspended in cs buffer ( 30 min , 4 h at 37 c , ph 3.5 ) suggest the dissociation of an average of one cl ligand and a change of the first shell environment from ru(iii)cl4n2 to ru(iii)cl3on2 . this coordination turned out to be prevalent up to 4 h of incubation , and 1 is suggested to remain in its oxidation state ru(iii ) . this can be interpreted by an average exchange of one cl ligand for an o atom from the buffer solution or a n atom from indazole formed by deprotonation of the indazolium cation at ph 3.5 . a possible n coordination is unique for 1 because of its chemical composition including an indazolium countercation . after 8 h in cs buffer at 37 c a significant change in the xanes spectra was seen , which is proof of a further transformation of the compound in solution . at ph 7.4 and 37 c in carbonate buffer , the results of this xas study are pointing toward an average exchange of at least one cl ligand after 30 min of incubation as well . the cl ligand can be exchanged for an o atom arising from water , an o atom from carbonate , or a n ligand from indazole . compared to the 1 mm concentrated indazolium countercation , the possible o donors are much higher concentrated in the buffer solution . the hydrolyzing reactions of 1 were studied previously by high performance capillary electrophoresis ( hpce ) , high performance liquid chromatography mass spectrometry ( hplc ms ) , and capillary zone electrophoresis inductively coupled plasma mass spectrometry ( cze - icp - ms).1 turned out to be the most labile complex in comparison to 13 and 14 . the half - life of 1 in buffer solution ( 10 mm phosphate buffer , 100 mm nacl , ph 7.4 ) was determined to be 17.1 min . in water 1 was hydrolyzed to about 2% within 2 h. the half - life in buffered solutions at 37 c turned out to be much smaller with values of 5.5 h ( ph 6.0 ) and < 0.5 h ( ph 7.4 ) . altogether the stability of 1 is strongly dependent on the ph of the buffer used , with the highest stability in solutions of low ph . ru(iii ) , like pt(iv ) , can be reduced by gsh under physiological conditions , and the resulting ru(ii ) complexes maintain their octahedral ligand set . an energy shift of 1.4 ev in comparison to solid 1 was found after 5 h of incubation in cs buffer in the presence of gsh ( 5-fold excess , 37 c , ph 3.5 ) . because of the 5-fold excess of gsh and the shift to lower energies , an adduct formation between 1 and gsh accompanied by a reduction to ru(ii ) is very likely , with a first coordination environment of ru(ii)cl3sn2 . an unchanged ru(iii ) center with a ru(iii)cls3n2 coordination would also match the seen shift in energy ( see figure 7 ) . reduction by gsh has previously been reported for [ cr(vi)o4 ] to a gsh bis - ligated chromium(v)-gsh complex . 1 binds to tf and is released from the protein supposedly after reduction to ru(ii ) by biological reductants , and the interference with the fe metabolism is assumed . in the presence of apotf , 1 incubated for 30 min in carbonate buffer ( ph 7.4 ) shows a slightly higher edge position than 1 in carbonate buffer without apotf . this is explainable by the exchange of additional cl through o and/or n ligands , whereas the ru center stays in its ru(iii ) state ( see figure 7 ) . it can not be stated without any doubt if the exchanged ligands are arising from the buffer solution or are due to the binding of apotf . in view of the more positive shift in energy in the presence of apotf at the same incubation conditions the ligand exchange is possibly due to an interaction with apotf . a consecutive binding of carbonate and apotf to the ru(iii ) center is a possible pathway , resulting in a complete dissociation of the four cl ligands . because of the changes in the xanes edge shape , both postulated binding modes are feasible and should be taken into consideration . in 2009 an electron paramagnetic resonance ( epr ) study on the interaction of 1 with human serum apotf showed a slow binding of 1 via ligand exchange reactions to the protein . in plasma samples of patients the fraction of 1/apotf adducts was smaller than 1% , but in vitro in the absence of other serum proteins the 1/apotf adduct formation took place within minutes . the strong binding of 1 to the histidine residues of apotf was reported from circular dichroism ( cd ) spectroscopy , electrospray mass spectrometry ( esi - ms ) , and gel filtration studies . in a x - ray diffraction study the interaction with lactoferrin , a protein closely related to apotf , could be shown . the xanes spectra collected on tissue samples from mice treated with 1 and 2 are the same ( see figure 5c ) . 2 is more soluble in water because of its sodium ( na ) countercation , and although the pharmacologically active complex anion is identical and biological targets are therefore likely to be the same , it is somewhat less cytotoxic than 1 and differences in their intracellular distribution have been recognized in the past years . the spectral features of the xanes spectra from all tissue samples are clearly different from that of solid 1 . their edge shifts fall into the same energy region as seen for 1 in cs buffer ( ph 3.5 ) up to 4 h of incubation at 37 c . the xanes spectra bear resemblance to the reference spectra of ru complexes with ru(iii)cl2on3 or ru(iii)cl2n4 active sites . a s ligation in the tissue samples can be ruled out , since all s containing and ru(ii ) model compounds are at lower energies . the two possible first shells ru(iii)cl3n2(o / n ) , based on model compound data , and ru(ii)cln2(o / n)3 , derived from theoretical considerations , are the best matching structural motifs . the stepwise release of the cl ligands throughout the course of application from the original ru(iii)cl4n2 over a ru(iii)cl3n2(o / n ) to a ru(ii)cln2(o / n)3 first shell environment is reasonable . a ru(ii ) state would be in agreement with the physiological accessibility of the ru(iii)/ru(ii ) redox pair in the presence of a reducing agent and with the activation by reduction mechanism suggested for ru and other transition metals in cancer therapy like pt(iv)/pt(ii ) . the favored major average coordination of ru in tissue is ru(iii)cl3n2(o / n ) , which is also in agreement with the possible interaction with apotf in the ru(iii ) oxidation state . the micro - xrf distribution maps of ru in tumor revealed the very good penetration of the drug into all regions of the tissue . notably , a recent micro - xrf study on 1 and 13 in single sh - sy5y cells showed a colocalization of the ru and fe concentrations inside the cells treated with 1 but not for 13 treated cells . furthermore , a change in iron distribution after treatment with 1 was seen , which provides further evidence for the interaction of 1 with the fe homeostasis . xas spectroscopy and micro - xrf were applied to study 1 in vitro and 1/2 in vivo . a xas database of ru model compounds representing possible coordinations and oxidation states of the ru metal center was established . the results obtained by fitting theoretical exafs amplitudes and phases to the model compounds were in good agreement with the known crystallographic data . the concept of the coordination charge was adopted to correlate the coordination / oxidation environment with the measured energy shifts . this linear correlation was the basis for the assignment of coordination / oxidation states in solution and tissue spectra . xanes analysis on 1 in cs buffer ( ph 3.5 , 37 c ) suggests the replacement of an average of one cl ligand through an o atom arising from the buffer solution or a n atom from the deprotonated indazolium countercation . the complex stayed predominantly in this coordination mode up to 4 h. after 8 h of incubation in cs buffer ( ph 3.5 ) a change in the xanes spectra and a strong shift in the edge position by 2.5 ev were observed , pointing toward subsequent changes in the coordination / oxidation state of the ru center . there is evidence for the release of one or more cl ligands in carbonate buffer ( ph 7.4 ) to a first coordination shell rich in o and/or n ligands . under the same incubation conditions , in the presence of apotf , a further dissociation of cl ligands is indicated , and this is a hint for a possible interaction with apotf . in the presence of gsh xanes spectra measured on liver and tumor samples , taken from mice treated with 1 and 2 , revealed identical first coordination environments in vivo . the two most probable coordinations are ru(iii)cl3n2(o / n ) and ru(ii)cln2(o / n)3 . the results were independent of the dosages and schedules tested . pca resulted in a first principal component describing 98% of the variance in all tissue samples . micro - xrf studies confirmed the high penetration depth of 2 into the closer packed tissue regions . this is the first time coordination / oxidation pairs could be assigned to the drug directly in target tissue . the model compounds were diluted in bn ( sigma aldrich , cas 10043 - 11 - 5 , 99.5% ) , placed into aluminum sample holders , and sealed with kapton foil . the bn preparations were prepared for a calculated absorption of about 1 absorbance unit according to standard methods . the solution samples were prepared by suspending 1 ( 1 mm ) , 1 and gsh ( 0.5:2.5 mm ) in 5 mm cs buffer ( ph 3.5 ) . 1 in the presence of apotf ( 0.5:0.5 mm ) and 1 ( 1 mm ) as a reference were suspended in carbonate buffer ( ph 7.4 ) , as carbonate is necessary in the binding process to tf . the resulting suspensions were placed into aluminum sample holders and sealed with kapton foil . 1 in cs buffer samples were incubated at 37 c for 30 min , 4 h , and 8 h , respectively . 1 in cs buffer in the presence of gsh was incubated for 5 h at 37 c . 1 in carbonate buffer ( ph 7.4 ) without and with the xas spectra were collected on the flash frozen samples at 20 k. the tissue samples were taken from experiments performed in accordance with the european community guidelines for the use of experimental animals in the animal facility at the cancer research institute , slovak academy of sciences , bratislava , slovak republic , and at the cancer research institute at the medical university of vienna , austria . the tissue material was placed into aluminum sample holders , sealed with kapton foil , flash frozen in liquid nitrogen , and stored at 80 c . the following tissue samples were measured : c1-tumor / liver ( treated with 15 mg / kg 1 ) , b2-tumor / liver ( 7.5 mg / kg 1 ) , 148 - 1-tumor / liver ( 40 mg / kg 2 ) , b1/f1-liver ( 40 mg / kg 2 ) . details for the buffer preparations and animal tests are given in the supporting information . part of the tumor ( sw480 , mouse 148 - 1 ) samples from mice treated with 2 were fixed in ethanol and embedded in paraffin according to standard procedures . the 5 m thick sections were mounted on glass slides and stained with h & e. the 10 m thick sections were mounted on 4 m thick ultralene foil fixed on aluminum frames and used for the micro - xrf measurements . the xas experiments were carried out at beamline bm26a at the european synchrotron radiation facility ( esrf ) in grenoble , france . the model compounds were measured in transmission mode , and the tissue samples and the drug solutions were collected in fluorescence mode . all experiments were conducted at cryogenic temperatures at 20 k. the energy dispersive micro - xrf experiments were conducted at the fluo beamline at the angstrmquelle karlsruhe synchrotron ( anka , germany ) with a spot size of 10 m . the program packages athena , artemis , ifeffit , feff , pyspline , dl - excurv , sixpack , and pymca were applied for the xas and micro - xrf data analysis . the normalization was accomplished by fitting a quadratic polynomial ( models ) or a straight line ( tissue and solution samples ) to the postedge region . the exafs analysis followed standard procedures described in refs ( 82 ) , ( 85 ) , and ( 93 ) . the ab initio amplitude and phases were provided by the program packages feff7 and dl - excurv , respectively . the dl - excurv fits were performed on the k - weighted exafs signals , and the feff7 fits were performed on the back - transform of the first peak in the ft . the program sixpack was used to perform the pca , lsf , and lca in the energy range of 2210022200 ev . the detailed descriptions of the experimental setups and data extraction and analysis procedures are given in the supporting information . the edge positions of xanes spectra are a combination of several features like valence , electronegativity of the first shell atoms , their coordination number , and the formal oxidation state of the central atom . batsanov introduced the concept of the coordination charge to account for these factors . with increasing electronegativity of the neighboring atoms the edge positions are shifted to higher energies when the number of ligands stays the same . the coordination charge is defined as depicted in eq 1.1where m is the formal oxidation state of the central metal , ck is the degree of covalence of a bond k , and nk is the number of such bonds . the degree of covalence ck is defined as 1 ik , where ik is the ionicity of that bond . the ionicity is calculated using pauling s formula ( eq 2)2and depends on the electronegativity of the central ru atom m and the electronegativity of the ligand atom l . the coordination charge is calculated as in eq 1 applying ik calculated as in eq 2 . the change in electronegativity due to changes in hybridization state was not considered in these calculations .

indazolium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 1 , kp1019 ) and its analogue sodium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 2 , kp1339 ) are promising redox - active anticancer drug candidates that were investigated with x - ray absorption near edge structure spectroscopy . the analysis was based on the concept of the coordination charge and ruthenium model compounds representing possible coordinations and oxidation states in vivo . 1 was investigated in citrate saline buffer ( ph 3.5 ) and in carbonate buffer ( ph 7.4 ) at 37 c for different time intervals . interaction studies on 1 with glutathione in saline buffer and apo - transferrin in carbonate buffer were undertaken , and the coordination of 1 and 2 in tumor tissues was studied too . the most likely coordinations and oxidation states of the compound under the above mentioned conditions were assigned . microprobe x - ray fluorescence of tumor thin sections showed the strong penetration of ruthenium into the tumor tissue , with the highest concentrations near blood vessels and in the edge regions of the tissue samples .

Introduction Results Discussion Conclusions Experimental Section

imidazolium trans-[tetrachloro(dimethylsulfoxide)(imidazole)ruthenate(iii ) ] ( 13 , nami - a ) , indazolium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 1 , kp1019 ) , its sodium analogue sodium trans-[tetrachlorobis(1h - indazole)ruthenate(iii ) ] ( 2 , kp1339 ) , and ru(ii)-arene complexes like [ ru(-p - cymene)cl2(pta ) ] ( 14 , rapta - c , pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decanephosphine ) are the most promising investigational drug candidates up to now . phase i clinical trials in patients with refractory solid malignancies have successfully been completed for both 13(19 ) and 1 , the latter yielding disease stabilizations in more than half of the patients , and further clinical studies have commenced recently . the ru complexes 1 ( figure 1 ) and its na salt analogue 2 ( figure 1 ) are the subject of investigation in this work . 1 and 2 differ only in their countercations , indazolium for 1 and na for 2 , exhibiting the same ru(iii ) coordination sphere [ tetrachlorobis(1h - indazole)ruthenate(iii ) ] . the uptake mechanism of the two drugs may involve human serum albumin ( hsa ) , the iron transport protein transferrin ( tf ) , and the tf receptor , which is overexpressed in tumor cells to meet their increased demand for fe . however , the direct determination of the oxidation state , the coordination of the ru active site , and the mechanism of action in vivo for these and other ru drugs remains elusive . x - ray absorption spectroscopy ( xas ) is an element specific method that allows the investigation of the questioned element in disordered environments missing any long - range order . by application of x - ray absorption near edge structure ( xanes ) analysis , it is possible to determine the oxidation state and coordination of the ru metal center , whereas extended x - ray absorption fine structure ( exafs ) analysis opens the opportunity to specify the identity , number , and distances of the adjacent atoms . , and the distribution and relative inertness of a ga(iii ) compound in biological tissue were investigated in a previous publication . to generate spectra with a sufficiently good signal - to - noise ratio , x - ray sources like synchrotrons , with a sufficiently high photon flux in the range of 1 10 to 4 10 photons per second , are necessary . for 1 it was found that the indazole ligands are weaker donors toward ru than imidazole ones , which is congruent with the proposed activation by reduction mechanism stated for 1 and its easier reduction to ru(ii ) compared to the imidazole analogue trans-[tetrachlorobis(1h - imidazole)ruthenate(iii ) ] ( kp418 ) . even though 1 and 2 are known to undergo hydrolysis ( facilitating further ligand exchange reactions ) and to be redox - active , both of which are features proposed to be significant for the mechanism of action , neither coordination nor redox condition of 1/2 after biotransformation in biological systems has been examined because of the lack of appropriate analytical methods . the aim of this work is to apply xas to investigate the tumor - inhibiting ru compound 1 in citrate saline buffer ( cs buffer ) at ph 3.5 within different time intervals , in the presence of the reducing agent gsh , in carbonate buffer in the presence of the possible transport protein apo - transferrin ( apotf ) , and as a powder . xanes spectra were recorded on tissue samples from mice treated with 1 and 2 . micro x - ray fluorescence ( micro - xrf ) maps were taken from tumor tissue samples taken from mice treated with 2 . additionally model compounds representing possible coordinations and oxidation states in vivo were measured as references . the following model compounds were investigated with xas : 1 ( with first coordination shell ru(iii)cl4n2 ) , ruthenium(iii ) acetylacetonate ( 3 , ru(iii)o6 , sigma aldrich , cas 14284 - 93 - 6 , 97% ) , hexammineruthenium(iii ) trichloride ( 4 , ru(iii)n6 , sigma aldrich , cas 14282 - 91 - 8 , 99%),mer , trans - aquatrichloridobis(indazole)ruthenium(iii ) ( 5 , kasc003 , ru(iii)cl3n2o),trans , trans - dichloridotetrakis(indazole)ruthenium(iii ) chloride ( 6 , gabu129 , ru(iii)cl2n4),mer - trichloridotris(indazole)ruthenium(iii ) ( 7 , gupl328 , ru(iii)cl3n3 ) , hexammineruthenium(ii ) dichloride ( 8 , ru(ii)n6 , sigma aldrich , cas 15305 - 72 - 3 , 99.9%),mer , trans - trichlorido(dimethylsulfide)bis(indazole)ruthenium(iii ) ( 9 , flan005 , ru(iii)cl3n2s),trans , trans - dichloridotetrakis(indazole)ruthenium(ii ) ( 10 , gabu128 , ru(ii)cl2n4),mer - trichloridotris(ethylphenylsulfide)ruthenium(iii ) ( 11 , flan006 , ru(iii)cl3s3),trans , trans , trans - dichloridobis(dimethylsulfide)bis(indazole)ruthenium(ii ) ( 12 , flan004 , ru(ii)cl2n2s2 ) . the bottom limit is marked by the s containing 12 in its oxidation state + 2 , and the upper boundary is represented by 3 with six o ligands and an oxidation state of + 3 . the models 1 , 3 , and the ru hexammine compounds 8 and 4 , in both oxidation states , exhibit a characteristic edge shoulder . extracted fine structures and fourier transforms of the model compounds with a ru(ii ) center ( a ) , a mixed cl / n first shell ( b ) , and a o or s containing first coordination shell ( c ) . in previous articles a correlation between the coordination charge and a distinct spectral edge feature could be shown . the edge energy of 1 in boron nitride ( bn ) was set as an arbitrary origin and lies right in the middle of the observed edge energy range . a straight line was regressed with a coefficient of determination r = 0.95 , demonstrating a linear correlation between the coordination charge and the edge positions . the compounds containing ru(ii ) and/or s are on the left ( lower energy ) side , and the compounds containing ru(iii ) , n , and o are on the right ( higher energy ) side . this correlation is the basis for the further interpretation of the coordination mode of ru compounds in biological samples and in the presence of potential transport proteins . in figure 5a the xanes spectra of 1 in bn , suspended in cs buffer and in the presence of gsh ( 5-fold excess , cs buffer , ph 3.5 ) , are shown . 1 was incubated at 37 c for 30 min , 4 h , 8 h , and 5 h in the presence of gsh . after 8 h of incubation of 1 in cs buffer and 5 h in the presence of gsh , both spectra are shifted significantly to lower energies by 1.4 and 1.3 ev , respectively . the indazolium cation has a pka of 1.25 which enables deprotonation and opens the possibility to act as a n donor at ph 3.5 . in the presence of gsh the spectrum is shifted to the low energy positions in the region of the ru(ii ) and ru(iii ) s containing model compounds . ( a ) normalized xanes spectra and first derivative of 1 in bn , 1 in cs buffer ( after 30 min , 4 h , and 8 h ) and in cs buffer in the presence of the reducing agent gsh ( after 5 h ) . ( b ) 1 in cs buffer ( 30 min ) and carbonate buffer ( 30 min ) and in carbonate buffer in the presence of apotf ( 30 min ) . lsf to 1 in cs buffer ( 30 min , 4 h ) and in the presence of gsh ( 5 h ) was performed using the solid state spectra of 1 , 4 , 5 , 6 , 9 , 10 , and 12 . in figure 5b the normalized xanes spectra and first derivatives of 1 suspended in carbonate buffer ( 30 min , 37 c ) and in carbonate buffer in the presence of apotf ( 30 min , 37 c ) are shown . for comparison the spectrum of 1 in cs buffer ( ph 3.5 ) is shown as well . the spectrum of 1 in carbonate buffer is shifted by + 1.7 ev to higher energies , which is slightly more than 1 in cs buffer ( + 1.1 ev ) . lsf on 1 in carbonate buffer ( 30 min ) and in the presence of apotf ( 30 min ) was done in the same way as described for 1 dissolved in cs buffer . in a subsequent step linear combination analysis ( lca ) lsf of 3 , 4 , and 11 and combinations of them has been performed on 1 in bn , 1 in cs buffer at ph 3.5 ( 4 and 5 h with gsh ) , and 1 in carbonate buffer at ph 7.5 ( 30 min with and without apotf ) . after 4 h in cs buffer at 37 c a decrease to an overall coordination of ru to about 3 n and 3 cl in solution is seen by lca . because of the low concentration of ru in the biological tissue , data collection was restricted to the xanes region . the apparent coordination charge of the ru center of 1 and 2 in the tissue is similar to 1 in cs buffer after 30 min and 4 h of incubation at 37 c . the target transforms of the model compounds 1 , 4 , 5 , 6 , and 7 to the vector subspace of the first principal component are presented in the supporting information ( figure s1 , not for 4 ) . the edge position of the tissue samples is indicated by the vertical blue line in figure 4 . for the tissue samples ru(iii)cl3n2(o / n ) is proposed as a possible first shell environment for the majority of the 1 and 2 molecules , which are dominating the xas signals . the resulting maps give an overview of the elemental distribution patterns of ruthenium , iron , copper , and zinc in a defined region of the tumor . the concentrations of all four elements are highest in the region of the blood vessels and in the edge regions of the tissue . xanes analysis has been applied to propose a first coordination environment of an investigational anticancer drug in cs buffer ( ph 3.5 ) and carbonate buffer ( ph 7.4 ) , in carbonate buffer in the presence of apotf , in cs buffer in the presence of gsh , and in the target tissue . the concept of the coordination charge was applied to the model compounds , and a linear correlation between the coordination / oxidation state and the observed edge energies was established . this was the basis for the further interpretation of the spectra of 1 in solution and in biological samples . proposed oxidation state and first shell coordination of 1 in cs buffer ( 4 h ) , in liver and tumor tissue , in cs buffer in the presence of gsh and in carbonate buffer in the presence of apotf ( top ) . the spectra collected on 1 suspended in cs buffer ( 30 min , 4 h at 37 c , ph 3.5 ) suggest the dissociation of an average of one cl ligand and a change of the first shell environment from ru(iii)cl4n2 to ru(iii)cl3on2 . after 8 h in cs buffer at 37 c a significant change in the xanes spectra was seen , which is proof of a further transformation of the compound in solution . at ph 7.4 and 37 c in carbonate buffer , the results of this xas study are pointing toward an average exchange of at least one cl ligand after 30 min of incubation as well . the half - life of 1 in buffer solution ( 10 mm phosphate buffer , 100 mm nacl , ph 7.4 ) was determined to be 17.1 min . in water 1 was hydrolyzed to about 2% within 2 h. the half - life in buffered solutions at 37 c turned out to be much smaller with values of 5.5 h ( ph 6.0 ) and < 0.5 h ( ph 7.4 ) . altogether the stability of 1 is strongly dependent on the ph of the buffer used , with the highest stability in solutions of low ph . an energy shift of 1.4 ev in comparison to solid 1 was found after 5 h of incubation in cs buffer in the presence of gsh ( 5-fold excess , 37 c , ph 3.5 ) . because of the 5-fold excess of gsh and the shift to lower energies , an adduct formation between 1 and gsh accompanied by a reduction to ru(ii ) is very likely , with a first coordination environment of ru(ii)cl3sn2 . in the presence of apotf , 1 incubated for 30 min in carbonate buffer ( ph 7.4 ) shows a slightly higher edge position than 1 in carbonate buffer without apotf . their edge shifts fall into the same energy region as seen for 1 in cs buffer ( ph 3.5 ) up to 4 h of incubation at 37 c . a s ligation in the tissue samples can be ruled out , since all s containing and ru(ii ) model compounds are at lower energies . the favored major average coordination of ru in tissue is ru(iii)cl3n2(o / n ) , which is also in agreement with the possible interaction with apotf in the ru(iii ) oxidation state . the micro - xrf distribution maps of ru in tumor revealed the very good penetration of the drug into all regions of the tissue . furthermore , a change in iron distribution after treatment with 1 was seen , which provides further evidence for the interaction of 1 with the fe homeostasis . a xas database of ru model compounds representing possible coordinations and oxidation states of the ru metal center was established . the concept of the coordination charge was adopted to correlate the coordination / oxidation environment with the measured energy shifts . xanes analysis on 1 in cs buffer ( ph 3.5 , 37 c ) suggests the replacement of an average of one cl ligand through an o atom arising from the buffer solution or a n atom from the deprotonated indazolium countercation . the complex stayed predominantly in this coordination mode up to 4 h. after 8 h of incubation in cs buffer ( ph 3.5 ) a change in the xanes spectra and a strong shift in the edge position by 2.5 ev were observed , pointing toward subsequent changes in the coordination / oxidation state of the ru center . there is evidence for the release of one or more cl ligands in carbonate buffer ( ph 7.4 ) to a first coordination shell rich in o and/or n ligands . in the presence of gsh xanes spectra measured on liver and tumor samples , taken from mice treated with 1 and 2 , revealed identical first coordination environments in vivo . the solution samples were prepared by suspending 1 ( 1 mm ) , 1 and gsh ( 0.5:2.5 mm ) in 5 mm cs buffer ( ph 3.5 ) . 1 in the presence of apotf ( 0.5:0.5 mm ) and 1 ( 1 mm ) as a reference were suspended in carbonate buffer ( ph 7.4 ) , as carbonate is necessary in the binding process to tf . 1 in cs buffer samples were incubated at 37 c for 30 min , 4 h , and 8 h , respectively . 1 in carbonate buffer ( ph 7.4 ) without and with the xas spectra were collected on the flash frozen samples at 20 k. the tissue samples were taken from experiments performed in accordance with the european community guidelines for the use of experimental animals in the animal facility at the cancer research institute , slovak academy of sciences , bratislava , slovak republic , and at the cancer research institute at the medical university of vienna , austria . the model compounds were measured in transmission mode , and the tissue samples and the drug solutions were collected in fluorescence mode . the dl - excurv fits were performed on the k - weighted exafs signals , and the feff7 fits were performed on the back - transform of the first peak in the ft . the edge positions of xanes spectra are a combination of several features like valence , electronegativity of the first shell atoms , their coordination number , and the formal oxidation state of the central atom . batsanov introduced the concept of the coordination charge to account for these factors . the coordination charge is defined as depicted in eq 1.1where m is the formal oxidation state of the central metal , ck is the degree of covalence of a bond k , and nk is the number of such bonds .

efficient delivery of drugs to their targets can significantly improve their therapeutic potency . drug delivery systems ( ddss ) have been introduced as novel tools to improve the pharmacodynamics and pharmacokinetics properties of drugs . to increase the stability , bioavailability , and intracellular uptake and to reduce the toxicity , a wide range of ddss were constructed recently from different materials including polymers , peptides , and liposomes . nanodrug delivery systems ( nano - ddss ) have offered several advantages compared to larger ddss , such as lower toxicity and improved cellular uptake . the highly active surface area of nanoparticles provides maximized loading capacity of cargo molecules . furthermore , when small sized nanoparticles get functionalized properly , they can go through rapid dissolution and enhanced adhesion to the biological target surfaces providing great bioavailability at the site of the action . nano - dds containing metal nanoparticles ( metal nano - ddss ) have emerged as effective tools for the treatment of various diseases . metal nano - ddss containing hydrophobic and hydrophilic segments have been investigated for carrier - mediated drug delivery through entrapping hydrophobic and hydrophilic cargo molecules because of their amphiphilic properties . moreover , metal nano - dds has become a subject of major attention because of their nonimmunogenicity and biocompatibility . among various metal nanoparticles , selenium nanoparticles ( senps ) have not been investigated extensively . selenium ( se ) is an essential micronutrient with a recommended dietary intake of 55 g / day . although selenium is required for normal cellular function , high doses of selenium in the form of senps could selectively kill malignant cells . thus , there is a compelling need to design novel senps that could be used as nontoxic nano - ddss . two parameters including the chemical entity of selenium and the concentration are responsible for its toxicity and anticancer activity . selenium can be found in different oxidation states , i.e. , selenite ( seo3 ) oxyanions , selenite ( seo4 ) , and elemental selenium ( se ) where the oxidation states are + 4 , + 6 , and 0 , respectively . it has been previously reported that changing the redox state of the metal ions can reduce or eliminate their toxicity . senps have been coated or functionalized with other compounds to improve their biological profile . previous studies have shown that bovine serum albumin coated with senps ( bsa senps ) exhibit similar activity to selenomethionine ( sem ) and se - methylselenocysteine ( semsc ) in increasing the activity of selenoenzymes and se levels in tissue , and showed less toxicity than organic se compounds . thus , the dose and chemical form of se have become critical to reduce the toxicity and to increase the therapeutic effects . furthermore , the size of nanoparticles is a critical element that can alter their biological properties . recently , liu and co - workers developed a facile procedure for the preparation of 5-fluorouracil surface - decorated senps with improved anticancer activity . it was found that the surface functionalization of spherical senps with 5-fluorouracil through physical adsorption increased the uptake of the senps by cells . furthermore , to improve the cellular uptake of senps , their surface has been decorated by various ligands , such as atp , spirulina polysaccharide , mushroom polysaccharides - protein , and chitosan . although these methods disclosed new aspects of senps application , they suffer from multistep synthesis , purifications , and inherent toxicity of nano - ddss . furthermore , the majority of carriers were found to be energy - dependent since they use endocytic pathways to cross the membrane . thus , there is a need to develop new biomaterials for the functionalization of the surface of senps . peptides are commonly used as nanoscaled systems for carrier - mediated drug delivery because of their ability to carry a wide range of cargo molecules through encapsulation . moreover , peptides have been used as a component in the structure of nano - ddss by taking advantage of a broad range of amino acids with different physicochemical properties . for example , peptide - functionalized gold nanoparticles have been employed as one of the prior biocompatible systems in drug delivery and improved the cellular delivery of several drugs via noncovalent complexation significantly . the design and synthesis of homochiral l - cyclic peptides and their application for the nuclear targeting delivery of anti - hiv drugs and biomolecules have been reported by us . moreover , physical mixing of the cyclic peptide containing arginine , tryptophan , and haucl4 resulted in the generation of peptide - capped gold nanoparticles and increased the cellular delivery of drugs and biomolecules dramatically . we report here synthesis and evaluation of a cyclic cell - penetrating peptide composed of ten amino acids including arginine , tryptophan , and cysteine . cysteine was selected to improve binding to se through s se bond formation . the ability of the peptide for in situ synthesis of cyclic peptide - capped selenium nanoparticles ( cp senps ) was investigated . subsequently , cp senps were evaluated as a nanometal dds for the delivery of a model anticancer drug , dasatinib , and a negatively charged cell - impermeable phosphopeptide . furthermore , cp senps were used to improve the antiproliferative activity of a number of anticancer agents . this work is distinct from the previous work since the double - barreled novel delivery system that includes cyclic peptides and senps significantly improves the antiproliferative activity of anticancer agents . peptide synthesis reagents , including coupling reagents , h - arg(pbf)-2-chlorotrityl resin , and fmoc - amino acids , were purchased from chempep ( miami , fl ) . the other reagents were obtained from sigma - aldrich chemical co. ( milwaukee , wi ) . the cyclic peptide [ w5r4c ] was synthesized based on our previously reported methodology . the synthesis was performed in quark glass vessels by mixing through bubbling nitrogen gas into the mixture at room temperature unless otherwise mentioned . fmoc / tbu solid phase procedure was used for the synthesis of the peptide . the coupling and activating reagents were 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) and n , n - diisopropylethylamine ( dipea ) , respectively , in anhydrous n , n - dimethylformamide ( dmf ) . the fmoc group was deprotected using piperidine in dmf ( 20% v / v ) . the h - arg(pbf)-2-chlorotrityl resin ( 730 mg , 0.3 mmol , 0.41 mmol / g ) was added to quark glass vessels followed by the addition of dmf ( 50 ml ) and mixing through bubbling nitrogen gas into the mixture for 30 min . the solvent was drained followed by coupling of fmoc - trp(boc)-oh ( 473.9 mg , 0.9 mmol ) using hbtu ( 341.6 mg , 0.9 mmol ) and dipea ( 313.4 l , 1.8 mmol ) in anhydrous dmf ( 10 ml ) for 1.5 h. the resin was washed using dmf ( 15 ml 3 ) followed by deprotection of fmoc group with 20% piperidine in dmf . the resin was washed with dmf ( 15 ml 3 ) and coupled with fmoc - arg(pbf)-oh ( 583.9 mg , 0.9 mmol ) and followed by deprotection of the fmoc group . the coupling and deprotection cycles were repeated to complete the synthesis of protected peptide sequence on resin . the last coupling was performed with fmoc - cys(trt)-oh ( 583.9 mg , 0.9 mmol ) followed by deprotection of fmoc group and washing the resin . the protected peptide was cleaved from the resin by agitating the resin with a cleavage cocktail of trifluoroethanol ( tfe)/acetic acid / dcm ( 2:2:6 v / v / v , 15 ml ) for 2 h. the resin was filtered off , and the cleavage cocktail was removed using a rotatory evaporator to provide solid linear protected peptide for cyclization . the solid linear protected peptide was dissolved in dmf / dcm ( 150 ml , 2:1 v / v ) under nitrogen atmosphere followed by the addition of coupling reagents n , n-diisopropylcarbodiimide ( dic , 93.5 l , 0.6 mmol ) and 1-hydroxy-7-azabenzotriazole ( hoat , 81.6 mg , 0.6 mmol ) . the cleavage of side chain protecting group was conducted by using cleavage cocktail trifluoroacetic acid ( tfa)/thioanisole / anisole / ethanedithiol ( edt ) ( 10 ml , 90:5:2:3 v / v / v / v ) for 2 h. the crude peptide was precipitated with diethyl ether followed by centrifugation to provide solid crude cyclized peptide . the crude peptide was purified using reverse phase chromatography using a hitachi lachrom elite reversed - phase high - performance liquid chromatograph ( rp - hplc ) on a phenomenax gemini axia packed c18 ( 250 21.2 mm , 10 m ) and using flow rate of 10 ml / min with a gradient system of 0100% acetonitrile ( 0.1% tfa ) and water ( 0.1% tfa ) over 60 min to elute the pure fraction . evaporation and lyophilization afforded pure powdered cyclic peptide . the purity of the final product ( 95% ) was confirmed by analytical rp - hplc that was carried out on hitachi analytical hplc system using a shimadzu premier c18 column ( 150 4.6 mm , 3 m ) and with a gradient system ( h2o / ch3cn ) at a flow rate of 1 ml / min and 220 nm . the peptide structure was confirmed by high - resolution matrix - assisted laser desorption ionization time - of - flight ( maldi tof / tof ) mass spectrometer ( shimadzu biotech ) . maldi - tof ( m / z ) [ c82h103n27o10s ] : calcd , 1657.8080 ; found , 1658.7102 ; [ m + h ] , 1680.4571 [ m + na ] , 1696.7428 [ m + k ] . the synthesis of senps was carried out by reacting the selenium solution with the cyclic peptide . the stock solution of the peptide ( 10 mm ) was mixed with na2seo3 solution in water ( 10 mm ) at 50 c . after the formation of [ w5r4c]senps , the stock solution was diluted into desired concentration for further experiments . to synthesize the fluorescence - labeled dasatinib ( f-das ) , the drug was conjugated through using a glycine linker as elaborated in the supporting information ( scheme s1 ) , where f = fluorescein . tem sample was prepared by using an aqueous solution of [ w5r4c]senps ( 5 l of 5 mm ) according to our previously reported procedure and analyzed . to load [ w5r4c]senps by doxorubicin ( dox ) , an aqueous solution of [ w5r4c]senps ( 5 mm ) and dox ( 500 m ) were mixed in water to end up with 10:1 ratio . all experiments on the samples including [ w5r4c ] ( 50 m , h2o ) , [ w5r4c]senps ( 50 m , h2o ) , and camptothecin ( cpt , 5 m , dmso ) were performed by using a ratio of 10:1 between the carrier and the drug at room temperature . ovarian adenocarcinoma ( sk - ov-3 , atcc no htb-77 ) , human leukemia ( ccrf - cem , atcc no . ccl-119 ) , and human embryonic kidney epithelial ( hek-293 t , atcc no . rpmi-16 medium was used for ccrf - cem cells , and emem medium was used for sk - ov-3 and hek-293 t cells , containing fetal bovine serum ( fbs , 10% ) , and the penicillin streptomycin solution ( 1% , 10 000 units of penicillin and streptomycin ( 10 mg in 0.9% nacl ) ) in a humidified atmosphere of co2 ( 5% ) and air ( 95% ) at 37 c . the cytotoxicity assay was performed by using sk - ov-3 ( 5000 ) , hek-293 t ( 4000 ) , and ccrf - cem ( 40 000 ) cells based on our reported procedure . the only modification was that the old medium was not replaced in the case of ccrf - cem by treatments . antiproliferative activities of several anticancer drugs including dox , irinotecan , gemcitabine , epirubicin , dasatinib , etoposide , paclitaxel , camptothecin , fludarabine , and clofarabine were evaluated in sk - ov-3 with [ w5r4c]senps , and the results were compared with those of drugs alone after 48 h. the antiproliferative assay was performed by employing our previously reported method and reagents . furthermore , the entrapment of the drugs was also carried out by using our previously reported procedure . six - well plates with opti - mem were used for seeding ccrf - cem cells ( 1 10 ) . in the next step , the fluorescence - labeled dasatinib ( f-dasatinib ) or f-pepylgld ( 5 m ) was added to each well containing [ w5r4c ] ( 50 m ) and [ w5r4c]senps ( 50 m ) in opti - mem . the rest of the assay was performed under a similar condition as described earlier . sk - ov-3 cells were grown on coverslips in 6-well plates ( 1 10 cells per well ) overnight . the fluorescence - labeled treatments with either f-dasatinib or f-pepylgld ( 5 m ) in the presence and absence of [ w5r4c]senps ( 50 m ) were added to cells in opti - mem for 2 h at 37 c . the procedures for washing the slides and staining the cell nuclei by 4,6-diamidino-2-phenylindole ( dapi ) were previously reported by us . dox solution ( 100 l of 200 m ) was mixed with [ w5r4c]senps solution in water ( 400 l of 500 m ) . the concentration ratio of dox and peptide - capped senps was maintained to be 1:10 . a dialysis membrane ( 1 ml and molecular weight cutoff of 1000 d ; float - a - lyzer g2 , spectrum laboratories ) was used for nanoparticle - loaded dox . the methods for hplc and quantifying the dox amount was previously reported by us . the following equation was used : to calculate the loading capacity , the quantity of senps in the dialysis membrane was analyzed by using inductively coupled plasma mass spectrometry ( icp ms ) after 24 h. the icp ms results exhibited that 2.94 g of senps were accountable for dox encapsulation . the loading capacity equation was used for the calculation as shown below : dox intracellular release was investigated by using an hplc technique as described previously . at the beginning , the cells were seeded into 6-well plates ( 1 10 in 2 ml of medium per well ) . the dox - loaded [ w5r4c]senps ( 5:50 m ) camptothecin solution ( dmso , 10 l of 10 m ) was mixed with the peptide - capped senps solution in water ( 100 l of 10 m ) to adjust the molar ratio to be 1:1 . the cyclic decapeptide [ w5r4c ] was synthesized by employing fmoc / tbu - based solid phase chemistry ( scheme 1 ) as described previously . in brief , the linear protected peptide was first synthesized , followed by cleavage of side chain - protected peptide from the resin and cyclization in dilute conditions using dic / hoat in dmf / dcm solution for 12 h. after cyclization , the side chain of protected peptide was deprotected by using cleavage cocktail followed by reverse phase hplc purification to afford pure cyclic peptide . the reaction was carried out via physical mixing of the peptide solution ( 10 mm ) with the solution of na2seo4 ( 10 mm ) in water at 50 c . however , after 6 h of incubation at 50 c , the color was turned to orange showing the formation of senps . this strategy is a one - pot reaction without any surface functionalization of senps . tryptophan ( w ) is known to have the most efficient reducing capability compared to the other 19 amino acids . positively charged arginine ( r ) residues enhanced the reducing activity of the peptide via the appropriate ionic interactions with the negatively charged selenite anions . cysteine facilitated the formation of senps possibly through stabilizing the formed nanoparticles by favorable s to investigate the morphology and size of [ w5r4c]senps , further studies were conducted using tem . tem images exhibited that [ w5r4c]senps formed spherical nanostructures with a size in the range of 110150 nm after 1 day of incubation of the sample ( figure 1 ) . we assume the presence of some noncovalent and/or covalent interactions between senps and peptide as binding ligands . positively charged arginines and hydrophobic tryptophans contribute to intermolecular interactions and this morphology . the induced hydrophobic interactions by tryptophan residues possibly lead to the formation of peptide - coated senps . moreover , cysteine is probably involved in the formation of this morphology presumably through strong interaction with the surface of senps . the reaction of the sulfur group in glutathione peroxidase with selenium has been previously reported . moreover , previous reports have shown that the presence of sh can stabilize selenium in the chemical environment . thus , the formation of selenium sulfur covalent binding between selenium nanoparticles and sh group of te peptide was likely . thus , we assume that both covalent and noncovalent interactions are involved in the formation of peptide - capped selenium nanoparticles . in addition to [ w5r4c]senps characterization , tem microscopy was used to visualize the change on the size and morphology of nanoparticles after incubation with dox . as shown in figure 2 , the shape of senps was changed to multilayer ball - shape nanoparticles , suggesting the surface capping of senps by peptides or interactions with dox . the size range of nanoparticles was increased after incubation with dox to 250270 nm , showing a relatively significant change in the diameter of nanoparticles due to entrapping or interaction with the drug . we speculate that dox interacts with [ w5r4c]senps by covering the surface of peptide - capped senps through intermolecular interactions ( figure 2 ) . however , dox ( white cover ) was not able to cover up the whole surface of the particles . cd was used to determine whether the senps formation can change the peptide secondary structure through intermolecular interactions . a comparative cd experiment was performed by using an aqueous solution of the peptide and its corresponding cp senps ( 50 m ) . the cd spectra of [ w5r4c]senps showed a very similar cd pattern with a minimum peak at 216 nm . however , the maxima peak disappeared possibly because of the interaction of the peptide with senps . to determine whether [ w5r4c]senps can entrap marketed available drugs , cpt was used as a model drug in cd . cpt is a hydrophobic anticancer drug that works through inhibiting topoisomerase i activity . the effect of cpt encapsulation by [ w5r4c]senps was investigated using cd to determine whether the secondary structure of the peptide changes in this process . as exhibited in figure 3 , [ w5r4c]senp - loaded cpt demonstrated a significantly different cd pattern compared to that of cpt and [ w5r4c]senps alone . the results demonstrate that the interactions between the [ w5r4c]senps and cpt generates the new orientation of amino acids in the secondary structure , presumably through their involvement in the encapsulation process . comparative cd of cyclic [ w5r4c ] and [ w5r4c]senps in the presence and absence of cpt . the cd spectra of [ w5r4c]senp - loaded cpt ( 5:1 ) showed a blue shift with a maxima at 209 nm , suggesting significant interaction of cpt with peptide - capped metal nanoparticles . these results were consistent with previously reported methods where conjugation and interaction of nanoparticles with their stabilizing ligands caused change in their cd pattern . furthermore , change in cd patterns have been observed through an interaction between peptides and various types of drugs . fluorescence spectroscopy was used to validate the entrapment of cpt by [ w5r4c]senps . a significant blue shift in maxima s emission of cpt fluorescence spectra was found following the incubation of cpt by [ w5r4c]senps . the characteristic cpt maximum peak at 442 nm was shifted to 431 nm after interaction of cpt with [ w5r4c]senps ( figure 4 ) . the distribution of cpt in a hydrophobic region and hydrophobic forces are possibly responsible for the observed blue shift of cpt s peak in the presence of [ w5r4c]senps . we postulate that this is because of the self - quenching of bounded compound and the distribution and entrapment in the hydrophobic region formed by the cp senps . these studies suggested that the cyclic peptide senps were capable of entrapment of cpt in the hydrophobic region possibly generated by involved tryptophans . we have previously reported the formation of hydrophobic regions through interactions of tryptophan residues . moreover , extended investigations showed that hydrophobic interactions occur between dox and the self - assembled cyclic peptide . fluorescence of cpt in the presence of [ w5r4c]senps ( 1:1 molar ratio ) after 4 h of incubation . dox was chosen as a representative drug because of its high stability and inherent uv vis properties to investigate the quantity of the loaded drug by [ w5r4c]senps . aqueous solution of dox ( 100 l of 200 m ) was mixed with the solution of [ w5r4c]senps in water ( 400 l of 500 m ) to maintain a 1 to 10 molar ratio . after 24 h of incubation , the free dox was collected by using the dialysis method . the efficiency of [ w5r4c]senps to load dox after 24 h was 45.6% when the ratio of dox of [ w5r4c]senps by weight in feed was 1 to 10 . furthermore , loading capacity was found to be 16 1% considering the weight ratio of dox to [ w5r4c]senps ( 1:10 ) when the amount of senps was determined using icp fresh synthesized [ w5r4c]senps ( 50 m ) did not exhibit significant toxicity after 2 h in human ovarian adenocarcinoma ( sk - ov-3 ) , human leukemia ( ccrf - cem ) cancer , and human embryonic kidney 293 ( hek-293 t ) cells . these data revealed that [ w5r4c]senps ( 50 m ) has minimal toxicity ( 810% ) in sk - ov-3 , ccrf - cem , and hek-293 t cells ( figure 5 ) . therefore , [ w5r4c]senps concentration was maintained at 50 m for further flow cytometry studies . [ wr]4 cyclic structure contributes significantly to its cell - penetrating ability . to determine whether cp senps can act as drug carriers , a clinically used anticancer drug , dasatinib ( das ) , dasatinib is a nonselective tyrosine kinase inhibitor against src , abl , and bcr . dasatinib is used clinically for the treatment of philadelphia chromosome - positive acute lymphoblastic leukemia ( ph all ) and chronic myeloid leukemia ( cml ) . to detect the transportation of the drug by cp senps for cell - based studies , we synthesized a carboxyfluorescein conjugate of das ( f-das ) . then the incubation of ccrf - cem cells and f-das ( 5 m ) was conducted for 2 h with or without diluted [ w5r4c]senps ( 50 m ) and the corresponding parent peptide [ w5r4c ] ( 50 m ) . after 2 h , the cell surface - attached fluorescence - labeled drug was washed with trypsin . f-das ( 5 m ) intracellular uptake was evaluated by a flow cytometry technique . flow cytometry results indicated that cells incubated with f-drug mixed with [ w5r4c]senps and f-drug mixed with [ w5r4c ] ( 50 m ) have notably higher fluorescence intensity compared to that in cells incubated with f-das alone . [ w5r4c]senps and [ w5r4c ] improved the intracellular uptake of f-das by 9.5- and 4.3-folds when compared with the cells treated with f-das alone ( figure 6 ) . uptake of f-das with [ w5r4c ] and [ w5r4c]senps by cells after 2 h. the assay was performed in triplicate ( n = 3 ) . furthermore , flow cytometry results demonstrated that the cellular uptake of the fluorescently labeled drug was improved by 2.2-fold with peptide - capped senps compared to that with the peptide . the data reveal that both the peptide and senps in [ w5r4c]senps contribute to the improvement of the cellular uptake of the drug in a higher degree . the enhancement of cellular delivery of f-das with [ w5r4c ] and [ w5r4c]senps indicates that the peptide alone has the potential to work as a drug carrier . however , the transporting efficiency of the peptide was enhanced after interaction with senps presumably due to a change in the peptide secondary structure leading to higher efficiency in drug entrapment . confocal microscopy was employed to visualize the enhancement of f-das ( 5 m ) uptake by skov-3 cells . however , high fluorescence intracellular intensity was observed in the presence of f-das - loaded [ w5r4c]senps ( 50 m ) when compared with that of drug after 2 h ( figure 7 ) . confocal microscopy images displayed that f-das was mostly localized in the nucleus of sk - ov-3 cells when mixed with [ w5r4c]senps after 2 h incubation . images of the cellular uptake of f-das in sk - ov-3 cells with or without [ w5r4c]senps after 2 h of incubation . after evaluating [ w5r4c]senps as a molecular transporter of a small - molecule drug , its potential was examined for transporting a relatively large negatively charged phosphopeptide with limited cellular permeability . having negatively charged phosphate groups and relatively large size of phosphopeptides make their intracellular delivery a challenging task . here , a model phosphopeptide , pepylgld , was used for further studies . the sequence of amino acid in pepylgld mimics the phosphotyrosine 1246 of erbb2 that binds to the chk sh2 domain . senps were examined for the cellular delivery of fluorescence conjugate of pepylgld ( f-pepylgld ) using ccrf - cem cells . the analysis of flow cytometry results showed that the f-pepylgld ( 5 m ) uptake was improved by 25-fold when mixed with [ w5r4c]senps ( 50 m ) compared to that of f-pepylgld alone , suggesting that [ w5r4c]senps may work as a f-pepylgld carrier . [ w5r4c]senps increased the f-pepylgld uptake by 1.4-fold when compared to that of [ w5r4c ] ( figure 8) . these data propose that senps have a significant effect on enhancing the cellular uptake of the phosphopeptide . f-pepylgld uptake by cells when used in combination with [ w5r4c]senps and [ w5r4c ] after 2 h of incubation . the intracellular uptake enhancement of f-pepylgld by [ w5r4c]senps was also confirmed via confocal microscopy . a comparative microscopy investigation was performed by comparing f-pepylgld ( 5 m)-loaded [ w5r4c]senp ( 50 m ) fluorescence intensity with that of f-pepylgld ( 5 m ) alone in sk - ov-3 cells . no green fluorescence was seen in cells when they were incubated with f-pepylgld alone . this part of our findings suggests that the phosphopeptide alone could not cross the cellular membrane . however , when [ w5r4c]senps were used , a significantly higher fluorescence intensity was observed , showing that cp senps can work as a transporter system for the fluorescein - labeled phosphopeptide . merged images revealed that the phosphopeptide in combination with peptide senps was mostly localized in the nuclei of sk - ov-3 cells ( figure 9 ) . the imaging results showed that [ w5r4c]senps presence is critical to increase the cellular delivery of the cell impermeable phosphopeptide . microscopy images of f-pepylgld with and without [ w5r4c]senps in sk - ov-3 cells after 2 h. molecular cargos can employ various mechanisms for cellular entry . these mechanisms include micropinocytosis , phagocytosis , and receptor - mediated endocytosis ( rme ) pathways . caveolae - mediated , clathrin - mediated , and clathrin / caveolae independent endocytosis are examples of rme . to investigate possible transportation mechanism of cp senps cellular uptake , the fluorescence intensity of f-pepylgld loaded [ w5r4c]senps ( 1:10 ) was quantified in combination with several endocytic inhibitors like nystatin , chloroquine , chlorpromazine , 5-(n - ethyl - n - isopropyl)-amiloride ( eia ) , and methyl--cyclodextrin by using flow cytometry . figure 10 shows the uptake of f-pepylgld loaded [ w5r4c]senps by cells did not significantly decrease in the presence of chloroquine , chlorpromazine , and methyl--cyclodextrin after 2 h incubation at 37 c in sk - ov-3 cells . these data suggest that clathrin - mediated or caveolae - mediated endocytosis and phagocytosis are not the only mechanisms of cellular uptake . however , the uptake of the cargo molecule was inhibited by 52% and 40% when nystatin and eia were used , respectively , showing that caveolae - mediated and macropinocytosis pathways could be two of the involved mechanisms for the internalization of cargo molecules by functionalized senps . moreover , as it is evident in figure 10 , these two inhibitors did not block the uptake of cargo completely , meaning that other endocytic pathways are also involved in the delivery of f-pepylgld - loaded [ w5r4c]senps . in addition to endocytic inhibitors , sodium azide was employed as an atp depleting agent . the results revealed that f-pepylgld uptake did not alter in combination with sodium azide showing that atp depletion is not involved in the mechanism of uptake . flow cytometry showing the uptake of f-pepylgld loaded [ w5r4c]senps with or without various endocytic inhibitors in sk - ov-3 cells after 2 h. the assay was performed in triplicate ( n = 3 ) . the surface functionalization of senps by using the peptide could improve the primary interactions of involved amino acids , such as tryptophan and arginine , with the lipid bilayer s hydrophobic groups and negatively charged phospholipids present in the cell membrane . this electrostatic / hydrophobic interactions could trigger the complex to cross the phospholipid bilayer . tryptophan residues can generate hydrophobic forces , disturb the available phopholipids , and cause distortion of the exterior phospholipid monolayer . subsequently , peptide internalizes into the membrane and improves the cargo cellular entry . the surface decoration of senps by the peptide structure can improve nanoparticle stability and influence the cellular uptake mechanism . however , to get a detailed understanding of the uptake mechanism by these cp senps , further investigations are required . to explore the drug cellular release profile by the carrier , a hplc technique was used to investigate the intracellular release of dox by [ w5r4c]senps dox complex in ccrf cem cells . ccrf cem cells ( 1.2 10 ) were incubated with [ w5r4c]senps ( 50 m ) loaded with dox ( 5 m ) for various times ( 1248 h ) . the quantity of the released dox was calculated by analyzing hplc data at 490 nm after different time intervals . the data showed that [ w5r4c]senps dox releases dox following a time - dependent pattern . the hplc profile showed that dox appeared in a retention time of 15.815.9 min ( figure s2 , supporting information ) . hplc results exhibited that , after 12 , 24 , and 48 h of incubation in cells , 10.5 , 20 , and 40% of dox was released , respectively . the hplc profile showed that a sustained / slow release of dox play an important role in the overall activity of the [ w5r4c]senps loaded with dox as a potential prodrug . the antiproliferative activity of several anticancer drugs including dox , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel were examined in sk - ov-3 cells with and without [ w5r4c]senps in a time - dependent pattern . the experiments were used to investigate the impact of using [ w5r4c]senps in the biological doses of anticancer drugs in cells . the antiproliferative results revealed that the activity of all anticancer drugs ( 5 m ) , dox , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel was enhanced when combined with [ w5r4c]senps ( 50 m ) after 48 h in sk - ov-3 cells by 38% , 49% , 36% , 36% , 31% , 30% , 30% , 28% , 24% , and 17% , respectively . this effect could be due to improving the cellular uptake of the drugs ( figure 11 ) . in addition , the degradation of peptide - capped senps complex could generate aggregated toxic selenium leading to the synergistic effect in cancer cells . the antiproliferative results showed the sustained release of drugs in cells in a time - dependent pattern in sk - ov-3 cells . time - dependent antiproliferative assay of ( a ) irinotecan , ( b ) gemcitabine , ( c ) epirubicin , ( d ) dasatinib , ( e ) doxorubicin , ( f ) etoposide , ( g ) paclitaxel , ( h ) camptothecin , ( i ) fludarabine , and ( j ) clofarabine in the absence and presence of [ w5r4c]senps . a novel group of ddss was synthesized from senps and cell - penetrating peptide having tryptophan , arginine , and cysteine residues . [ w5r4c]senps showed an ability to interact with cpt , a hydrophobic drug , possibly via noncovalent forces . this system transported a fluorescence conjugate of labeled dasatinib and a negatively charged cell - impermeable phosphopeptide ( f-pepylgld ) into cells . the antiproliferative activities of several anticancer drugs , such as doxorubicin , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel , were significantly enhanced in the presence of [ w5r4c]senps . this work provides insights for the design of peptide metal nanoparticle novel drug delivery systems .

a cyclic peptide composed of five tryptophan , four arginine , and one cysteine [ w5r4c ] was synthesized . the peptide was evaluated for generating cyclic peptide - capped selenium nanoparticles ( cp senps ) in situ . a physical mixing of the cyclic peptide with seo32 solution in water generated [ w5r4c]senps via the combination of reducing and capping properties of amino acids in the peptide structure . transmission electron microscopy ( tem ) images showed that [ w5r4c]senps were in the size range of 110150 nm . flow cytometry data revealed that a fluorescence - labeled phosphopeptide ( f-pepylgld , where f = fluorescein ) and an anticancer drug ( f-dasatinib ) exhibited approximately 25- and 9-times higher cellular uptake in the presence of [ w5r4c]senps than those of f-pepylgld and dasatinib alone in human leukemia ( ccrf - cem ) cells after 2 h of incubation , respectively . confocal microscopy also exhibited higher cellular delivery of f-pepylgld and f-dasatinib in the presence of [ w5r4c]senps compared to the parent fluorescence - labeled drug alone in human ovarian adenocarcinoma ( sk - ov-3 ) cells after 2 h of incubation at 37 c . the antiproliferative activities of several anticancer drugs doxorubicin , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel were improved in the presence of [ w5r4c]senps ( 50 m ) by 38% , 49% , 36% , 36% , 31% , 30% , 30% , 28% , 24% , and 17% , respectively , after 48 h incubation in sk - ov-3 cells . the results indicate that cp senps can be potentially used as nanosized delivery tools for negatively charged biomolecules and anticancer drugs .

Introduction Experimental Section Results and Discussion Conclusions

previous studies have shown that bovine serum albumin coated with senps ( bsa senps ) exhibit similar activity to selenomethionine ( sem ) and se - methylselenocysteine ( semsc ) in increasing the activity of selenoenzymes and se levels in tissue , and showed less toxicity than organic se compounds . moreover , peptides have been used as a component in the structure of nano - ddss by taking advantage of a broad range of amino acids with different physicochemical properties . for example , peptide - functionalized gold nanoparticles have been employed as one of the prior biocompatible systems in drug delivery and improved the cellular delivery of several drugs via noncovalent complexation significantly . moreover , physical mixing of the cyclic peptide containing arginine , tryptophan , and haucl4 resulted in the generation of peptide - capped gold nanoparticles and increased the cellular delivery of drugs and biomolecules dramatically . we report here synthesis and evaluation of a cyclic cell - penetrating peptide composed of ten amino acids including arginine , tryptophan , and cysteine . the ability of the peptide for in situ synthesis of cyclic peptide - capped selenium nanoparticles ( cp senps ) was investigated . subsequently , cp senps were evaluated as a nanometal dds for the delivery of a model anticancer drug , dasatinib , and a negatively charged cell - impermeable phosphopeptide . the cyclic peptide [ w5r4c ] was synthesized based on our previously reported methodology . the stock solution of the peptide ( 10 mm ) was mixed with na2seo3 solution in water ( 10 mm ) at 50 c . to synthesize the fluorescence - labeled dasatinib ( f-das ) , the drug was conjugated through using a glycine linker as elaborated in the supporting information ( scheme s1 ) , where f = fluorescein . to load [ w5r4c]senps by doxorubicin ( dox ) , an aqueous solution of [ w5r4c]senps ( 5 mm ) and dox ( 500 m ) were mixed in water to end up with 10:1 ratio . all experiments on the samples including [ w5r4c ] ( 50 m , h2o ) , [ w5r4c]senps ( 50 m , h2o ) , and camptothecin ( cpt , 5 m , dmso ) were performed by using a ratio of 10:1 between the carrier and the drug at room temperature . ovarian adenocarcinoma ( sk - ov-3 , atcc no htb-77 ) , human leukemia ( ccrf - cem , atcc no . rpmi-16 medium was used for ccrf - cem cells , and emem medium was used for sk - ov-3 and hek-293 t cells , containing fetal bovine serum ( fbs , 10% ) , and the penicillin streptomycin solution ( 1% , 10 000 units of penicillin and streptomycin ( 10 mg in 0.9% nacl ) ) in a humidified atmosphere of co2 ( 5% ) and air ( 95% ) at 37 c . the cytotoxicity assay was performed by using sk - ov-3 ( 5000 ) , hek-293 t ( 4000 ) , and ccrf - cem ( 40 000 ) cells based on our reported procedure . antiproliferative activities of several anticancer drugs including dox , irinotecan , gemcitabine , epirubicin , dasatinib , etoposide , paclitaxel , camptothecin , fludarabine , and clofarabine were evaluated in sk - ov-3 with [ w5r4c]senps , and the results were compared with those of drugs alone after 48 h. the antiproliferative assay was performed by employing our previously reported method and reagents . in the next step , the fluorescence - labeled dasatinib ( f-dasatinib ) or f-pepylgld ( 5 m ) was added to each well containing [ w5r4c ] ( 50 m ) and [ w5r4c]senps ( 50 m ) in opti - mem . the fluorescence - labeled treatments with either f-dasatinib or f-pepylgld ( 5 m ) in the presence and absence of [ w5r4c]senps ( 50 m ) were added to cells in opti - mem for 2 h at 37 c . dox solution ( 100 l of 200 m ) was mixed with [ w5r4c]senps solution in water ( 400 l of 500 m ) . the dox - loaded [ w5r4c]senps ( 5:50 m ) camptothecin solution ( dmso , 10 l of 10 m ) was mixed with the peptide - capped senps solution in water ( 100 l of 10 m ) to adjust the molar ratio to be 1:1 . the cyclic decapeptide [ w5r4c ] was synthesized by employing fmoc / tbu - based solid phase chemistry ( scheme 1 ) as described previously . the reaction was carried out via physical mixing of the peptide solution ( 10 mm ) with the solution of na2seo4 ( 10 mm ) in water at 50 c . however , after 6 h of incubation at 50 c , the color was turned to orange showing the formation of senps . positively charged arginine ( r ) residues enhanced the reducing activity of the peptide via the appropriate ionic interactions with the negatively charged selenite anions . tem images exhibited that [ w5r4c]senps formed spherical nanostructures with a size in the range of 110150 nm after 1 day of incubation of the sample ( figure 1 ) . thus , we assume that both covalent and noncovalent interactions are involved in the formation of peptide - capped selenium nanoparticles . the size range of nanoparticles was increased after incubation with dox to 250270 nm , showing a relatively significant change in the diameter of nanoparticles due to entrapping or interaction with the drug . a comparative cd experiment was performed by using an aqueous solution of the peptide and its corresponding cp senps ( 50 m ) . the results demonstrate that the interactions between the [ w5r4c]senps and cpt generates the new orientation of amino acids in the secondary structure , presumably through their involvement in the encapsulation process . comparative cd of cyclic [ w5r4c ] and [ w5r4c]senps in the presence and absence of cpt . the distribution of cpt in a hydrophobic region and hydrophobic forces are possibly responsible for the observed blue shift of cpt s peak in the presence of [ w5r4c]senps . fluorescence of cpt in the presence of [ w5r4c]senps ( 1:1 molar ratio ) after 4 h of incubation . aqueous solution of dox ( 100 l of 200 m ) was mixed with the solution of [ w5r4c]senps in water ( 400 l of 500 m ) to maintain a 1 to 10 molar ratio . furthermore , loading capacity was found to be 16 1% considering the weight ratio of dox to [ w5r4c]senps ( 1:10 ) when the amount of senps was determined using icp fresh synthesized [ w5r4c]senps ( 50 m ) did not exhibit significant toxicity after 2 h in human ovarian adenocarcinoma ( sk - ov-3 ) , human leukemia ( ccrf - cem ) cancer , and human embryonic kidney 293 ( hek-293 t ) cells . these data revealed that [ w5r4c]senps ( 50 m ) has minimal toxicity ( 810% ) in sk - ov-3 , ccrf - cem , and hek-293 t cells ( figure 5 ) . to determine whether cp senps can act as drug carriers , a clinically used anticancer drug , dasatinib ( das ) , dasatinib is a nonselective tyrosine kinase inhibitor against src , abl , and bcr . then the incubation of ccrf - cem cells and f-das ( 5 m ) was conducted for 2 h with or without diluted [ w5r4c]senps ( 50 m ) and the corresponding parent peptide [ w5r4c ] ( 50 m ) . after 2 h , the cell surface - attached fluorescence - labeled drug was washed with trypsin . f-das ( 5 m ) intracellular uptake was evaluated by a flow cytometry technique . flow cytometry results indicated that cells incubated with f-drug mixed with [ w5r4c]senps and f-drug mixed with [ w5r4c ] ( 50 m ) have notably higher fluorescence intensity compared to that in cells incubated with f-das alone . uptake of f-das with [ w5r4c ] and [ w5r4c]senps by cells after 2 h. the assay was performed in triplicate ( n = 3 ) . furthermore , flow cytometry results demonstrated that the cellular uptake of the fluorescently labeled drug was improved by 2.2-fold with peptide - capped senps compared to that with the peptide . the data reveal that both the peptide and senps in [ w5r4c]senps contribute to the improvement of the cellular uptake of the drug in a higher degree . the enhancement of cellular delivery of f-das with [ w5r4c ] and [ w5r4c]senps indicates that the peptide alone has the potential to work as a drug carrier . however , the transporting efficiency of the peptide was enhanced after interaction with senps presumably due to a change in the peptide secondary structure leading to higher efficiency in drug entrapment . however , high fluorescence intracellular intensity was observed in the presence of f-das - loaded [ w5r4c]senps ( 50 m ) when compared with that of drug after 2 h ( figure 7 ) . confocal microscopy images displayed that f-das was mostly localized in the nucleus of sk - ov-3 cells when mixed with [ w5r4c]senps after 2 h incubation . images of the cellular uptake of f-das in sk - ov-3 cells with or without [ w5r4c]senps after 2 h of incubation . senps were examined for the cellular delivery of fluorescence conjugate of pepylgld ( f-pepylgld ) using ccrf - cem cells . the analysis of flow cytometry results showed that the f-pepylgld ( 5 m ) uptake was improved by 25-fold when mixed with [ w5r4c]senps ( 50 m ) compared to that of f-pepylgld alone , suggesting that [ w5r4c]senps may work as a f-pepylgld carrier . [ w5r4c]senps increased the f-pepylgld uptake by 1.4-fold when compared to that of [ w5r4c ] ( figure 8) . f-pepylgld uptake by cells when used in combination with [ w5r4c]senps and [ w5r4c ] after 2 h of incubation . a comparative microscopy investigation was performed by comparing f-pepylgld ( 5 m)-loaded [ w5r4c]senp ( 50 m ) fluorescence intensity with that of f-pepylgld ( 5 m ) alone in sk - ov-3 cells . however , when [ w5r4c]senps were used , a significantly higher fluorescence intensity was observed , showing that cp senps can work as a transporter system for the fluorescein - labeled phosphopeptide . merged images revealed that the phosphopeptide in combination with peptide senps was mostly localized in the nuclei of sk - ov-3 cells ( figure 9 ) . the imaging results showed that [ w5r4c]senps presence is critical to increase the cellular delivery of the cell impermeable phosphopeptide . microscopy images of f-pepylgld with and without [ w5r4c]senps in sk - ov-3 cells after 2 h. molecular cargos can employ various mechanisms for cellular entry . to investigate possible transportation mechanism of cp senps cellular uptake , the fluorescence intensity of f-pepylgld loaded [ w5r4c]senps ( 1:10 ) was quantified in combination with several endocytic inhibitors like nystatin , chloroquine , chlorpromazine , 5-(n - ethyl - n - isopropyl)-amiloride ( eia ) , and methyl--cyclodextrin by using flow cytometry . figure 10 shows the uptake of f-pepylgld loaded [ w5r4c]senps by cells did not significantly decrease in the presence of chloroquine , chlorpromazine , and methyl--cyclodextrin after 2 h incubation at 37 c in sk - ov-3 cells . moreover , as it is evident in figure 10 , these two inhibitors did not block the uptake of cargo completely , meaning that other endocytic pathways are also involved in the delivery of f-pepylgld - loaded [ w5r4c]senps . flow cytometry showing the uptake of f-pepylgld loaded [ w5r4c]senps with or without various endocytic inhibitors in sk - ov-3 cells after 2 h. the assay was performed in triplicate ( n = 3 ) . the surface functionalization of senps by using the peptide could improve the primary interactions of involved amino acids , such as tryptophan and arginine , with the lipid bilayer s hydrophobic groups and negatively charged phospholipids present in the cell membrane . ccrf cem cells ( 1.2 10 ) were incubated with [ w5r4c]senps ( 50 m ) loaded with dox ( 5 m ) for various times ( 1248 h ) . hplc results exhibited that , after 12 , 24 , and 48 h of incubation in cells , 10.5 , 20 , and 40% of dox was released , respectively . the hplc profile showed that a sustained / slow release of dox play an important role in the overall activity of the [ w5r4c]senps loaded with dox as a potential prodrug . the antiproliferative activity of several anticancer drugs including dox , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel were examined in sk - ov-3 cells with and without [ w5r4c]senps in a time - dependent pattern . the antiproliferative results revealed that the activity of all anticancer drugs ( 5 m ) , dox , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel was enhanced when combined with [ w5r4c]senps ( 50 m ) after 48 h in sk - ov-3 cells by 38% , 49% , 36% , 36% , 31% , 30% , 30% , 28% , 24% , and 17% , respectively . the antiproliferative results showed the sustained release of drugs in cells in a time - dependent pattern in sk - ov-3 cells . time - dependent antiproliferative assay of ( a ) irinotecan , ( b ) gemcitabine , ( c ) epirubicin , ( d ) dasatinib , ( e ) doxorubicin , ( f ) etoposide , ( g ) paclitaxel , ( h ) camptothecin , ( i ) fludarabine , and ( j ) clofarabine in the absence and presence of [ w5r4c]senps . a novel group of ddss was synthesized from senps and cell - penetrating peptide having tryptophan , arginine , and cysteine residues . this system transported a fluorescence conjugate of labeled dasatinib and a negatively charged cell - impermeable phosphopeptide ( f-pepylgld ) into cells . the antiproliferative activities of several anticancer drugs , such as doxorubicin , gemcitabine , clofarabine , etoposide , camptothecin , irinotecan , epirubicin , fludarabine , dasatinib , and paclitaxel , were significantly enhanced in the presence of [ w5r4c]senps .

we aimed at an early and full correction of all the components of the deformity by gentle manipulation and well molded , thinly padded plaster casts which were changed every four to seven days . the plaster cast was applied in two sections , the first section extended from the toes to just below the knee and the second covered the knee and thigh . the knee was immobilized at a right angle while the leg was gently rotated outward to correct tibial torsion . a clear understanding of the club - foot deformity is possible after identifying by palpation the position of the bones in the foot and their relationship to one another and to the leg . the head of the talus is palpable on the lateral aspect of the dorsum of the foot , owing to the inward and backward displacement of the navicular . the calcaneus is in severe equinus deformity with its anterior portion lying directly beneath the head of the talus . this displacement is responsible for the severe varus deformity of the heel ( fig . the medial displacements of the navicular , cuboid , cuneiforms , and metatarsals contribute in different degrees to the severe adduction deformity of the club foot . the varus deformity of the calcaneus and the adduction of the mid - tarsometatarsal bones together are responsible for the inversion . the fore part of the foot , although adducted and inverted , is not as severely inverted as the hind part . as a result , the front of the foot is somewhat pronated with respect to the back of the foot , and this relationship causes the cavus deformity . the cavus deformity is thus produced by the slight downward displacement of the cuneiforms and by the fact that the first metatarsal is plantar flexed to a greater degree than the fifth metatarsal . the cavus deformity is sometimes erroneously designated as equinus of the fore part of the foot . excessive plantar flexion of the anterior part of the foot occurs primarily on its inner aspect . the plantar flexion of the outer aspect of the front part of the foot may be normal , as evidenced by the fact that the calcaneus , cuboid , and fifth metatarsal are in a straight line , even though the club foot deformity is severe ( fig . b(a ) in club foot , the anterior portion of the calcaneus lies beneath the head of the talus . ( b ) lateral displacement of the anterior portion of the calcaneus to its normal relationship with the talus will correct the heel varus deformity of the club foot.fig . f(a ) severe bilateral club - foot deformity in a six - week - old infant . the cavus deformity results from the slightly pronated position of the fore part of the foot in relation with the heel . the fore part of the foot is slightly supinated to be placed in proper alignment with the hind part of the foot . this maneuver increases the cavus deformity and fails to correct the varus deformity of the heel . the head of the talus ( t ) was palpable in the dorsolateral aspect of the foot in front of the ankle joint . the navicular ( s ) was displaced medially and its tuberosity was palpable just anterior to the medial malleolus . ( e ) and ( f ) manipulation to correct the inversion : outward pressure is exerted on the first metatarsal and counter pressure on the lateral aspect of the head of the talus ( t ) . when the navicular and cuboid and the entire fore part of the foot are displaced laterally in relation with the head of the talus , the anterior portion of the calcaneus follows ; thus , the heel varus deformity is corrected . while the inversion is corrected the fore part of the foot should not be pronated to prevent recurrence of the cavus deformity . ( a ) in club foot , the anterior portion of the calcaneus lies beneath the head of the talus . ( b ) lateral displacement of the anterior portion of the calcaneus to its normal relationship with the talus will correct the heel varus deformity of the club foot . ( a ) severe bilateral club - foot deformity in a six - week - old infant . the cavus deformity results from the slightly pronated position of the fore part of the foot in relation with the heel . the fore part of the foot is slightly supinated to be placed in proper alignment with the hind part of the foot . this maneuver increases the cavus deformity and fails to correct the varus deformity of the heel . the head of the talus ( t ) was palpable in the dorsolateral aspect of the foot in front of the ankle joint . the navicular ( s ) was displaced medially and its tuberosity was palpable just anterior to the medial malleolus . ( e ) and ( f ) manipulation to correct the inversion : outward pressure is exerted on the first metatarsal and counter pressure on the lateral aspect of the head of the talus ( t ) . when the navicular and cuboid and the entire fore part of the foot are displaced laterally in relation with the head of the talus , the anterior portion of the calcaneus follows ; thus , the heel varus deformity is corrected . while the inversion is corrected the fore part of the foot should not be pronated to prevent recurrence of the cavus deformity . brockman noted that in a club foot there is subluxation of the talocalcaneonavicular joint and alterations in position of the navicular and calcaneus with respect to the talus like those which occur in the normal foot when it is adducted , inverted , and plantar flexed , but they are exaggerated in degree . however , a normal foot can not adopt a true club - foot position because even in the extreme degrees of plantar flexion and inversion , the fore part of the foot moves with the hind part , and a cavus deformity does not develop since there is no discrepancy in the degree of inversion of the front and back part of the foot . since the cavus deformity is related to the pronation of the fore part of the foot with respect to the hind part , the cavus is corrected by placing the fore part of the foot in supination in proper alignment with the hind part . an attempt to correct the inversion of the foot by forcible pronation of the anterior part of the foot will increase the cavus deformity as the first metatarsal is further plantar flexed . this common maneuver is harmful because it hinders greatly any correction of the club - foot deformity by increasing the pronation of the fore part of the foot and thus making it very difficult to mobilize the navicular and displace it laterally in relation to the head of the talus . the navicular , cuneiforms , and metatarsals should be placed in straight alignment to form the lever arm needed for the correction of the inversion ( fig . 2b c ) . to correct the inversion of the foot , all of the foot distal to the talus must be made to rotate laterally underneath the talus which is fixed in the ankle mortise . a thumb placed on the lateral aspect of the head of the talus is used as a fulcrum while outward pressure is exerted on the first metatarsal and first cuneiform . during this manipulation an attempt is made to realign properly and simultaneously the calcaneocuboid , the talocalcaneonavicular , and the posterior talocalcaneal joints . when the navicular and cuboid are displaced laterally , the anterior portion of the calcaneus will be displaced outward and upward from its initial position underneath the head of the talus , and thus the varus deformity of the heel will be corrected ( fig . care is taken not to pronate the fore part of the foot during this manipulation to prevent recurrence of the cavus deformity ( fig . the manipulations should be gentle and followed by the application of a well molded thinly padded light plaster cast . four to five plaster - cast changes are usually sufficient to correct the inversion of the foot . the equinus deformity is corrected next by dorsiflexing the foot with the heel in a neutral or slight valgus position . this is the most difficult deformity to correct because of the great shortening of the tendo achillis which resists stretching . two to three casts are often applied after manipulations in an attempt to correct equinus deformity . if it then becomes evident that many more casts will be necessary for a complete correction , a simple subcutaneous tenotomy of the tendo achillis is performed with the patient under general anesthesia . a toe - to - groin cast with the foot in maximum dorsiflexion and the knee at a right angle the equinus deformity is thus immediately corrected , obviating a rocker - bottom deformity which often results from prolonged forceful manipulation . when the plaster cast is removed three weeks later , the defect in the tendon is healed . the scar in the tendon after this procedure is minimum , as observed in several instances where a tendo achillis lengthening was performed several years later to correct a recurrence . medial tibial torsion of variable degree is present in most patients with club feet and is a tenacious deformity if below - the - knee casts are used during treatment . tibial torsion can be gradually corrected when toe - to - groin casts are applied with the knee in 90 degrees of flexion . to do this , the leg portion of the cast which includes the foot is held in slight outward rotation while the thigh portion hardens . from five to ten ( average 7.6 ) plaster casts worn for periods of from five to twelve weeks ( average 9.5 weeks ) were necessary for the correction of all the club - foot components in our cases . a subcutaneous section of the tendo achillis was done in seventy - four of the ninety - four feet . to prevent recurrences of the deformity , denis browne splints with high - top shoes with well molded heels these splints were left on full time for an average period of three months and at night for an average of twenty - one and a half months more ( ranging from ten to thirty months).1 ordinary high - top shoes were used for walking . the deformity recurred in fifty - three feet ( 56 per cent ) ( table 1 ) at ages ranging from ten months to five years , with an average of two and one - half years . some authors stated that recurrences only occur when the club - foot deformity is not completely corrected at the initial treatment . however , when we reviewed the roentgenograms made at the end of the primary treatment , we found that the relationship of the talus to the calcanceus had not been completely corrected in only five cases . the causes for the recurrences are difficult to determine . in the forty - one feet permanently corrected with the first treatment the deformity tended to be less rigid , the leg muscles better developed , and the length of denis browne splint treatment longer than in the feet with recurrent deformities . about half of the recurrences occurred from two to four months after the denis browne splints were discarded , usually on the family s own initiative ; the recurrences could be blamed on the neglect of follow - up treatment with these splints . in other patients the recurrence was associated with a severe initial deformity and apparently poorly developed leg muscles ; these recurrences seemed to be related to the severity of the primary aberration which caused the deformity.table 1treatment of recurrencesfirstsecondthirdfourthno . ( years)21/2341/27treatmentno . of feetno . of feetno . of feetno . of feetplaster casts47 ( 6.4 wks.)17 ( 8 wks.)9 ( 7 wks.)1 ( 6 wks.)denis browne splints6 ( 11 mos.)subcutaneous section , tendo achillis5 * 4**1*tendo achillis lengthening5**11*anterior tibial transfer27561recession , extensor hallucis longus311recession , extensor digitorum longus1subcutaneous plantar fasciotomy111medial release12lisfranc capsulotomy1 * one foot had subcutaneous tendo achillis tenotomy with the initial treatment . treatment of recurrences * one foot had subcutaneous tendo achillis tenotomy with the initial treatment . * * two feet had subcutaneous tendo achillis tenotomy with the initial treatment . in six patients the recurrence was treated with the denis browne splint worn at night and during napping hours . in forty - seven patients , the recurrence was more severe and was treated with manipulation and toe - to - groin plaster casts changed each week . the cast treatment lasted from three to twelve weeks ( an average of 6.4 weeks ) . in the majority of recurrences , the equinus deformity was mild and responded to conservative treatment . of the seventy - four feet treated with heel - cord section at primary treatment three required further surgery . the tendo achillis was sectioned subcutaneously again in one patient and was lengthened through a short medial longitudinal skin incision in the other two patients . the tendo achillis was sectioned subcutaneously in four of these and lengthened in the other three . a subcutaneous section is preferred in patients under one year of age and tendo achillis lengthening in the older patients . in many of the recurrences , the varus deformity of the heel was more resistant to conservative treatment than the equinus deformity . a transfer of the anterior tibial tendon to the third cuneiform was done in twenty - seven feet in which there was a tendency for this muscle to supinate the foot strongly after the correction . the tendon was transferred to the third cuneiform in nineteen feet and to the cuboid in eight . the tendon was attached to the bone through a drill hole using a bunnell pull - out suture in twenty - one feet and a silk suture and osteoperiosteal flap in six feet . two skin incisions were made , one along the distal one or one and one - half inches of the anterior tibial tendon , the other shorter incision on the dorsum of the foot at the level of the third cuneiform . the tendon was transferred to its new attachment without changing its position underneath the ankle retinaculum . the foot was immobilized in a toe - to groin plaster cast for four weeks . an over - correction of the club - foot deformity after this procedure was not observed in this series . in three feet in addition to the anterior tibial transfer the extensor hallucis longus was recessed to the neck of the first metatarsal after suturing its distal stump to the tendon of the short extensor of the big toe . the recession of the extensor hallucis longus was done in cases with severe plantar flexion of the first metatarsal and hyperextension of the first metatarsophalangeal joint . in one foot with severe cavus deformity , a subcutaneous plantar fasciotomy was performed . in another , a lisfranc capsulotomy was done to correct a severe metatarsus adductus . a second recurrence was observed in seventeen feet ( 18 per cent ) at ages ranging from fourteen months to five years , the average being three years . in five of these seventeen feet the initial club - foot deformity was very rigid and the leg muscles were atrophic . the first recurrence in these four feet and the first recurrence in three other feet with deformities of average severity had been incompletely corrected by the application of only two to four plaster casts . in the five remaining feet the second recurrence was observed shortly after the denis browne splint was discarded prematurely . the second recurrence was treated with reapplication of toe - to - groin plaster casts changed every one to two weeks for periods ranging from four to twelve weeks ( average , eight weeks ) . this was followed by a subcutaneous section of the tendo achillis in four feet , a lengthening of the tendo achillis in one foot , and a transfer of the anterior tibial to the third cuneiform in five feet . in one foot , the extensor hallucis longus was recessed to the neck of the first metatarsal , all the tendons of the extensor digitorum longus were recessed to the third cuneiform , and a subcutaneous plantar fasciotomy was done to relieve the cavus deformity . a medial release operation was necessary in another foot with a severe recurrence which had been treated previously by plaster - cast applications and transfer of the anterior tibial tendon . the deformity recurred for the third time in nine feet ( 10 per cent ) at an age ranging from three to eight years ( average , four and a half years ) . in five of these feet , the recurrence was mild and was apparently caused by the strong supinatory action of the anterior tibial muscle . these recurrences were permanently corrected by lateral transfer of the anterior tibial tendon after the application of two or three plaster casts . the other four feet were somewhat rigid and the leg muscles were very atrophic . in one of these , a medial release operation , as well as a subcutaneous section of the tendo achillis , was necessary ; in another , a medial release was combined with a transfer of the anterior tibial tendon to the third cuneiform ; in the third , a tendo achillis lengthening was performed ; and in the fourth treatment consisted in five plaster - cast changes which were followed by a recurrence of the deformity . the last foot , just mentioned , was the only fourth recurrence observed ( 1 per cent ) . treatment consisted in the application of one plaster cast followed by a subcutaneous plantar fasciotomy , a transfer of the anterior tibial tendon to the third cuneiform , and a recession of the extensor hallucis longus to the neck of the first metatarsal . the deformity recurred in fifty - three feet ( 56 per cent ) ( table 1 ) at ages ranging from ten months to five years , with an average of two and one - half years . some authors stated that recurrences only occur when the club - foot deformity is not completely corrected at the initial treatment . however , when we reviewed the roentgenograms made at the end of the primary treatment , we found that the relationship of the talus to the calcanceus had not been completely corrected in only five cases . the causes for the recurrences are difficult to determine . in the forty - one feet permanently corrected with the first treatment the deformity tended to be less rigid , the leg muscles better developed , and the length of denis browne splint treatment longer than in the feet with recurrent deformities . about half of the recurrences occurred from two to four months after the denis browne splints were discarded , usually on the family s own initiative ; the recurrences could be blamed on the neglect of follow - up treatment with these splints . in other patients the recurrence was associated with a severe initial deformity and apparently poorly developed leg muscles ; these recurrences seemed to be related to the severity of the primary aberration which caused the deformity.table 1treatment of recurrencesfirstsecondthirdfourthno . of patients371271no . of feetplaster casts47 ( 6.4 wks.)17 ( 8 wks.)9 ( 7 wks.)1 ( 6 wks.)denis browne splints6 ( 11 mos.)subcutaneous section , tendo achillis5 * 4**1*tendo achillis lengthening5**11*anterior tibial transfer27561recession , extensor hallucis longus311recession , extensor digitorum longus1subcutaneous plantar fasciotomy111medial release12lisfranc capsulotomy1 * one foot had subcutaneous tendo achillis tenotomy with the initial treatment . treatment of recurrences * one foot had subcutaneous tendo achillis tenotomy with the initial treatment . * * two feet had subcutaneous tendo achillis tenotomy with the initial treatment . in six patients the recurrence was treated with the denis browne splint worn at night and during napping hours . in forty - seven patients , the recurrence was more severe and was treated with manipulation and toe - to - groin plaster casts changed each week . the cast treatment lasted from three to twelve weeks ( an average of 6.4 weeks ) . in the majority of recurrences , the equinus deformity was mild and responded to conservative treatment . of the seventy - four feet treated with heel - cord section at primary treatment three required further surgery . the tendo achillis was sectioned subcutaneously again in one patient and was lengthened through a short medial longitudinal skin incision in the other two patients . the tendo achillis was sectioned subcutaneously in four of these and lengthened in the other three . a subcutaneous section is preferred in patients under one year of age and tendo achillis lengthening in the older patients . in many of the recurrences , the varus deformity of the heel was more resistant to conservative treatment than the equinus deformity . a transfer of the anterior tibial tendon to the third cuneiform was done in twenty - seven feet in which there was a tendency for this muscle to supinate the foot strongly after the correction . the tendon was transferred to the third cuneiform in nineteen feet and to the cuboid in eight . the tendon was attached to the bone through a drill hole using a bunnell pull - out suture in twenty - one feet and a silk suture and osteoperiosteal flap in six feet . two skin incisions were made , one along the distal one or one and one - half inches of the anterior tibial tendon , the other shorter incision on the dorsum of the foot at the level of the third cuneiform . the tendon was transferred to its new attachment without changing its position underneath the ankle retinaculum . the foot was immobilized in a toe - to groin plaster cast for four weeks . an over - correction of the club - foot deformity after this procedure was not observed in this series . in three feet in addition to the anterior tibial transfer the extensor hallucis longus was recessed to the neck of the first metatarsal after suturing its distal stump to the tendon of the short extensor of the big toe . the recession of the extensor hallucis longus was done in cases with severe plantar flexion of the first metatarsal and hyperextension of the first metatarsophalangeal joint . in one foot with severe cavus deformity , a subcutaneous plantar fasciotomy was performed . in another , a lisfranc capsulotomy was done to correct a severe metatarsus adductus . a second recurrence was observed in seventeen feet ( 18 per cent ) at ages ranging from fourteen months to five years , the average being three years . in five of these seventeen feet the initial club - foot deformity was very rigid and the leg muscles were atrophic . four other feet were short and stubby and hence difficult to treat . the first recurrence in these four feet and the first recurrence in three other feet with deformities of average severity had been incompletely corrected by the application of only two to four plaster casts . in the five remaining feet the second recurrence was treated with reapplication of toe - to - groin plaster casts changed every one to two weeks for periods ranging from four to twelve weeks ( average , eight weeks ) . this was followed by a subcutaneous section of the tendo achillis in four feet , a lengthening of the tendo achillis in one foot , and a transfer of the anterior tibial to the third cuneiform in five feet . in one foot , the extensor hallucis longus was recessed to the neck of the first metatarsal , all the tendons of the extensor digitorum longus were recessed to the third cuneiform , and a subcutaneous plantar fasciotomy was done to relieve the cavus deformity . a medial release operation was necessary in another foot with a severe recurrence which had been treated previously by plaster - cast applications and transfer of the anterior tibial tendon . the deformity recurred for the third time in nine feet ( 10 per cent ) at an age ranging from three to eight years ( average , four and a half years ) . in five of these feet , the recurrence was mild and was apparently caused by the strong supinatory action of the anterior tibial muscle . these recurrences were permanently corrected by lateral transfer of the anterior tibial tendon after the application of two or three plaster casts . in one of these , a medial release operation , as well as a subcutaneous section of the tendo achillis , was necessary ; in another , a medial release was combined with a transfer of the anterior tibial tendon to the third cuneiform ; in the third , a tendo achillis lengthening was performed ; and in the fourth treatment consisted in five plaster - cast changes which were followed by a recurrence of the deformity . the last foot , just mentioned , was the only fourth recurrence observed ( 1 per cent ) . treatment consisted in the application of one plaster cast followed by a subcutaneous plantar fasciotomy , a transfer of the anterior tibial tendon to the third cuneiform , and a recession of the extensor hallucis longus to the neck of the first metatarsal . the correction obtained in each of the components of the club - foot deformity was evaluated clinically and roentgenographically . both evaluations correlated closely with respect to ankle dorsiflexion , heel varus , and adduction of the fore part of the foot . therefore only the clinical measurements of these components are charted ( table 2 ) . all the clinical measurements were performed by the senior author for the sake of uniformity.table 2resultsankle dorsiflexion ( degrees)heel varus ( degrees)adduction of the fore part of the foot ( degrees)tibial torsion ( degrees)result > 1000100good : 67 feet ( 71 per cent)0100101020moderateacceptable : 26 feet ( 28 per cent)0over 10over 20severepoor : 1 foot ( 1 per cent ) on the anteroposterior roentgenograms , the degree of heel varus deformity was estimated by measuring the angle formed by the long axis of the talus and the calcaneus . a 30-degree angle was considered normal and was classified as 0 degree of heel varus deformity . thus a measured talocalcaneal angle of 20 degrees corresponded to 10 degrees of heel varus deformity . the adduction of the fore part of the foot was also estimated on the anteroposterior roentgenograms by measuring the angle between the long axis of the calcaneus and that of the fifth metatarsal . on the lateral roentgenograms , the cavus deformity was estimated by measuring the angle between the long axis of the calcaneus and that of the first metatarsal ( fig . the correction of the equinus deformity was estimated by measuring the degree of ankle dorsiflexion . the degree of tibial torsion was estimated clinically by having the patient seated on the edge of the examining table with the knees at 90 degrees of flexion and the feet in slight plantar flexion . in the normal foot , the head of the talus can be palpated in front of the ankle mortise in the same plane as the axis of the thigh . medial or lateral tibial torsion is indicated by the orientation of the head of the talus in respect to the patella and axis of the thigh.fig . 3a lpart i. ( a d ) anteroposterior and lateral roentgenograms of the feet of a six - week - old baby boy with severe congenital club feet . in the anteroposterior roentgenograms , the talus and calcaneus are superimposed and their axes coincide indicating a severe heel varus deformity . the angle between the axis of the calcaneus and that of the fifth metatarsal ( adduction of the fore part of the foot ) measures 74 degrees . in the lateral views , the angle formed by the long axis of the calcaneus and that of the first metatarsal ( cavus ) measures 86 degrees . the calcaneus is in severe equinus and the talusis subluxated forward in relation with the tibial mortise . treatment consisted in manipulation and application of five plaster casts for the correction of the cavus , the adduction , and the heel varus deformities . a bilateral tenotomy of the tendo achillis was then performed followed by a plaster cast applied for three weeks . ( e h ) anteroposterior and lateral roentgenograms made after removal of the last plaster cast , seven weeks after onset of treatment . in the anteroposterior roentgenograms the angle between the long axis of the talus and that of the calcaneus measures 33 degrees , indicating correction of the heel varus deformity , and the angle between the calcaneus and the fifth metatarsal measures 13 degrees , indicating correction of the adduction of the fore part of the foot . in the lateral roentgenograms the equinus deformity appears corrected and the angle between the long axis of the calcaneus and that of the fifth metatarsal measures 7 degrees , indicating correction of the cavus deformity . denis browne splints on shoes were worn full time for ten months and at night until four and one - half years of age . ( i l ) anteroposterior and lateral roentgenograms made when the boy was seven years old . a good result was obtained in the right foot ( i and k ) and an acceptable result in the left ( j and l ) . in the anteroposterior roentgenograms , the angle between the long axis of the calcaneus and that of the talus measures 28 degrees on the right and 22 degrees on the left , indicating correction of the heel varus deformity on the right and incomplete correction on the left . the angle between the calcaneus and fifth metatarsal measures 7 degrees on the right and 0 degree on the left , indicating correction of the adduction of the fore part of both feet . in the lateral roentgenograms the angle between the long axis of the calcaneus and that of the first metatarsal measures 32 degrees on the right and 24 degrees on the left , indicating correction of the cavus deformity in both feet . part i. ( a d ) anteroposterior and lateral roentgenograms of the feet of a six - week - old baby boy with severe congenital club feet . in the anteroposterior roentgenograms , the talus and calcaneus are superimposed and their axes coincide indicating a severe heel varus deformity . the angle between the axis of the calcaneus and that of the fifth metatarsal ( adduction of the fore part of the foot ) measures 74 degrees . in the lateral views , the angle formed by the long axis of the calcaneus and that of the first metatarsal ( cavus ) measures 86 degrees . the calcaneus is in severe equinus and the talusis subluxated forward in relation with the tibial mortise . treatment consisted in manipulation and application of five plaster casts for the correction of the cavus , the adduction , and the heel varus deformities . a bilateral tenotomy of the tendo achillis was then performed followed by a plaster cast applied for three weeks . ( e h ) anteroposterior and lateral roentgenograms made after removal of the last plaster cast , seven weeks after onset of treatment . in the anteroposterior roentgenograms the angle between the long axis of the talus and that of the calcaneus measures 33 degrees , indicating correction of the heel varus deformity , and the angle between the calcaneus and the fifth metatarsal measures 13 degrees , indicating correction of the adduction of the fore part of the foot . in the lateral roentgenograms the equinus deformity appears corrected and the angle between the long axis of the calcaneus and that of the fifth metatarsal measures 7 degrees , indicating correction of the cavus deformity . denis browne splints on shoes were worn full time for ten months and at night until four and one - half years of age . ( i l ) anteroposterior and lateral roentgenograms made when the boy was seven years old . a good result was obtained in the right foot ( i and k ) and an acceptable result in the left ( j and l ) . in the anteroposterior roentgenograms , the angle between the long axis of the calcaneus and that of the talus measures 28 degrees on the right and 22 degrees on the left , indicating correction of the heel varus deformity on the right and incomplete correction on the left . the angle between the calcaneus and fifth metatarsal measures 7 degrees on the right and 0 degree on the left , indicating correction of the adduction of the fore part of both feet . in the lateral roentgenograms the angle between the long axis of the calcaneus and that of the first metatarsal measures 32 degrees on the right and 24 degrees on the left , indicating correction of the cavus deformity in both feet . the cavus deformity was corrected in most feet by the first plaster - cast application . the recurrences of this deformity were usually mild and responded to manipulation and plaster - cast applications with upward pressure on the first metatarsal head . however , a subcutaneous plantar fasciotomy was necessary in three feet with severe cavus deformity . in two of the three feet , this operation was combined with recession of the extensor hallucis longus tendon to the neck of the first metatarsal . recession of this tendon was done in three other feet with severe plantar flexion of the first metatarsal . all these operations were successful , and in the final examination the cavus deformity was corrected in all cases . the heel varus deformity was completely corrected in seventy feet ( 74 per cent ) . a slight degree of heel varus deformity of less than 10 degrees persisted in twenty - four feet ( 26 per cent ) . the heel varus deformity was corrected after the initial treatment in all feet but it recurred in fifty - two . anterior tibial transfer was done after plaster - cast correction in thirty - nine of these feet . the operation was successful in thirty feet but from 1 to 10 degrees of heel varus deformity persisted in nine feet . in six of these feet , the tendon pulled loose from its insertion ( in three the wire broke and in the other three the silk stitches apparently came loose ) . in three feet the tendon was transferred to the third cuneiform and probably should have been transferred to the cuboid . adduction of the fore part of the foot was completely corrected in seventy - two feet ( 77 per cent ) , was less than 20 degrees in twenty - one feet ( 22 per cent ) , and was severe in one foot . in one foot with severe adduction , of the twenty - two feet with residual adduction , seven had an anterior tibial transfer and fifteen did not . it appears then that anterior tibial transfer may help to correct not only the heel varus deformity but also the adduction of the fore part of the foot . tibial torsion was completely corrected in seventy - eight feet ( 83 per cent ) . in fifteen feet ( 16 per cent ) a moderate residual medial tibial torsion ( if less than 10 degrees was observed , and in one there was tibial torsion of 20 degrees . in no instance dorsiflexion of the ankle of more than 10 degrees above a right angle with the knee in extension was observed in seventy - five feet ( 80 per cent ) . in this group are included ten feet in which neither section nor lengthening of the tendo achillis was performed and sixty - five feet which had this tendon sectioned at the initial treatment . in eighteen feet ( 19 per cent ) dorsiflexion of the ankle was limited to from 0 to 10 degrees . a second section of the tendon was done in four to treat a recurrence , and in three the tendon was lengthened . the other ten feet of this group had either a section or a lengthening of the tendo achillis at the time of the first or second recurrence . one foot in which the heel cord was sectioned at the initial treatment and sectioned again at the third recurrence had a 5-degree residual equinus deformity . mild flattening of the superior articular surface of the talus was observed in the final roentgenograms of this last foot and in fourteen feet of the preceding group . good results were obtained in 71 per cent of the feet ( figs . 4 and 5 ) , acceptable in 28 per cent ( figs . 6 and 7 ) , and poor in 1 per cent.fig . photographs made after removal of the plaster casts which had been applied three days previously . cavus deformity is completely corrected on the right side and partially corrected on the left side . 5above , left : bilateral club - foot deformities in a one - month - old female infant . six plaster casts were applied in a period of one month , followed by bilateral subcutaneous tendo achillis tenotomy . above , right : at three months of age both feet were corrected . at fourteen months of age , the deformity recurred on the left and was treated by application of two toe - to - groin plaster casts . a bilateral recurrence of the deformity occurred when the child was five years old and was treated by transfer of the anterior tibial tendon to the third cuneiform after the application of three plaster casts . 6bilateral club - foot deformities in a five - day - old male infant treated with the application of ten plaster casts in a period of two months . a recurrence of the equinus deformity at one year of age was treated by the application of two toe - to - groin plaster casts and subcutaneous tenotomy . all the components of the club - foot deformity recurred at three years of age . three plaster casts were then applied , followed by the transfer of the anterior tibial tendon to the third cuneiform . when the child was eight years old the left foot was well corrected . on the right a 20-degree metatarsus adductus and 10-degree heel varus deformity persisted.fig . 7bilateral club - foot deformities in a female infant treated at two months with six plaster casts in a period of six weeks , followed by a bilateral subcutaneous tendo achillis tenotomy . photographs made when she was five months old , and later , when she was two and one - half years old , show all the components of the deformity well corrected . a mild recurrence of the deformity occurred when she was five years old , which was treated by the application of four plaster casts which were changed every ten days . the left foot was treated by lengthening of the heel cord and transfer of the anterior tibial to the third cuneiform . below , right : in the photographs made four months after operation , the deformity is well corrected on the left side . a 15-degree varus deformity of the heel and 5-degree equinus deformity persisted on the right . subsequently , a lengthening of the heel cord and transfer of the anterior tibial tendon was done on the right foot . above : club foot in a ten - day - old male infant . photographs made after removal of the plaster casts which had been applied three days previously . cavus deformity is completely corrected on the right side and partially corrected on the left side . below : at five years of age both feet were well corrected . above , left : bilateral club - foot deformities in a one - month - old female infant . six plaster casts were applied in a period of one month , followed by bilateral subcutaneous tendo achillis tenotomy . above , right : at three months of age both feet were corrected . at fourteen months of age , the deformity recurred on the left and was treated by application of two toe - to - groin plaster casts . a bilateral recurrence of the deformity occurred when the child was five years old and was treated by transfer of the anterior tibial tendon to the third cuneiform after the application of three plaster casts . bilateral club - foot deformities in a five - day - old male infant treated with the application of ten plaster casts in a period of two months . a recurrence of the equinus deformity at one year of age was treated by the application of two toe - to - groin plaster casts and subcutaneous tenotomy . all the components of the club - foot deformity recurred at three years of age . three plaster casts were then applied , followed by the transfer of the anterior tibial tendon to the third cuneiform . when the child was eight years old the left foot was well corrected . on the right a 20-degree metatarsus adductus and 10-degree heel varus deformity persisted . bilateral club - foot deformities in a female infant treated at two months with six plaster casts in a period of six weeks , followed by a bilateral subcutaneous tendo achillis tenotomy . photographs made when she was five months old , and later , when she was two and one - half years old , show all the components of the deformity well corrected . a mild recurrence of the deformity occurred when she was five years old , which was treated by the application of four plaster casts which were changed every ten days . the left foot was treated by lengthening of the heel cord and transfer of the anterior tibial to the third cuneiform . below , right : in the photographs made four months after operation , the deformity is well corrected on the left side . a 15-degree varus deformity of the heel and 5-degree equinus deformity persisted on the right . subsequently , a lengthening of the heel cord and transfer of the anterior tibial tendon was done on the right foot . although the treatment of a mild congenital club foot may be easy , the complete and permanent correction of a severe and rigid club foot is often difficult . in this study the early months of life offer a golden opportunity for the correction of club feet since the skeleton , which is to a great extent cartilaginous , is little deformed , and the joint capsules , ligaments , tendons , and muscles can be stretched without damage . early correction of all the components of the deformity in the shortest possible time is necessary for the proper development of the foot , since plaster - cast treatment prolonged for many months interferes with growth and may cause stiffness of the joints . correction of a severe equinus deformity can be radically shortened by subcutaneous section of the heel cord followed by the application of a plaster cast ( with the foot in maximum dorsiflexion ) for three weeks . after this procedure , the tendon always heals with little scarring ; and , if it is done early , a posterior capsulotomy of the ankle joint is unnecessary and rocker bottom and flattening of the upper articular surface of the talus are prevented . however , the heel cord should he sectioned only after the other components of the club - foot deformity are completely corrected . of seventy - four feet with severe equinus deformity treated with a subcutaneous section of the heel cord in the primary treatment , only eight required further surgery to treat a recurrent equinus deformity . recurrence of the heel varus deformity and adduction of the fore part of the foot are common even after complete correction . denis browne splints on shoes worn full time for the first two or three months after correction , and part time thereafter until the child is from three to five years of age , are useful to prevent recurrences in many cases . however , only half of the recurrences could be blamed on the neglect of follow - up treatment ; often these could be corrected by the reapplication of a few plaster casts . some form of surgical treatment was necessary to prevent further recurrence in the more severe cases . a transfer of the anterior or posterior tibial tendons to the third cuneiform or to the cuboid in these feet seems to be the most effective procedure to prevent further recurrence of the heel varus deformity . the tendon transfer should be performed only after the foot is well corrected either by the application of several corrective plaster casts or , if necessary , by a medial release operation . however , the medial release operation often leaves extensive scarring and stiffness in the mid - tarsal joints and , when possible , should be avoided . early transfer of the anterior tibial tendon in the very severe cases reduces greatly the need for this operation . the anterior tibial tendon was preferred over the posterior tibial for transfer because the operation is easier to perform and the anterior tibial functions in phase with the foot dorsiflexors , thereby making unnecessary postoperative training . excessive plantar flexion on the first metatarsal and cock - up of the big toe was observed in only six of our feet ; three of them before and three after anterior tibial transfer . this deformity was corrected by the transfer of the long extensor of the big toe to the neck of the first metatarsal . the nine failures of the anterior tibial transfer to correct permanently the varus deformity of the heel could he blamed on surgical errors . tibial osteotomy was not necessary in our patients because medial tibial torsion was corrected during the application of toe - to - groin plaster casts followed by the use of denis browne splints . tibial torsion will remain uncorrected if below - the - knee plaster casts are inadvisably used in the treatment . the results of treatment in sixty - seven patients with a total of ninety - four severe congenital club feet were evaluated five to thirteen years after the initial treatment . the primary treatment consisted in the application of several plaster casts changed frequently for an average period of 9.5 weeks . in many instances a subcutaneous tendo achillis tenotomy was performed in the primary treatment to obtain a complete correction of the equinus deformity . denis browne splints were used in the follow - up care in all patients . in fifty - three feet a transfer of the anterior tibial tendon to the dorsolateral aspect of the foot was performed in thirty - nine feet to prevent further recurrences of the heel varus deformity . medial release operations were necessary in only three feet . in no case was bone surgery performed . the results in 71 per cent of the feet were good ; in 28 per cent a slight residual deformity persisted ; and in one foot a poor result was obtained .

this classic article is a reprint of the original work by ignacio v. ponseti and eugene n. smoley , congenital club foot : the results of treatment . an accompanying biographical sketch on ignacio v. ponseti , md , is available at doi 10.1007/s11999 - 009 - 0719 - 8 and a second classic article is available at 10.1007/s11999 - 009 - 0721 - 1 . this article is 1963 by the journal of bone and joint surgery , inc . , and is reprinted with permission from ponseti iv , smoley en . congenital club foot : the results of treatment . j bone joint surg am . 1963;45:261344 .

Method of Treatment First Recurrence Second Recurrence Third Recurrence Fourth Recurrence Results Discussion Summary

we aimed at an early and full correction of all the components of the deformity by gentle manipulation and well molded , thinly padded plaster casts which were changed every four to seven days . the head of the talus is palpable on the lateral aspect of the dorsum of the foot , owing to the inward and backward displacement of the navicular . this displacement is responsible for the severe varus deformity of the heel ( fig . the medial displacements of the navicular , cuboid , cuneiforms , and metatarsals contribute in different degrees to the severe adduction deformity of the club foot . the varus deformity of the calcaneus and the adduction of the mid - tarsometatarsal bones together are responsible for the inversion . the fore part of the foot , although adducted and inverted , is not as severely inverted as the hind part . as a result , the front of the foot is somewhat pronated with respect to the back of the foot , and this relationship causes the cavus deformity . the cavus deformity is thus produced by the slight downward displacement of the cuneiforms and by the fact that the first metatarsal is plantar flexed to a greater degree than the fifth metatarsal . excessive plantar flexion of the anterior part of the foot occurs primarily on its inner aspect . the plantar flexion of the outer aspect of the front part of the foot may be normal , as evidenced by the fact that the calcaneus , cuboid , and fifth metatarsal are in a straight line , even though the club foot deformity is severe ( fig . b(a ) in club foot , the anterior portion of the calcaneus lies beneath the head of the talus . ( b ) lateral displacement of the anterior portion of the calcaneus to its normal relationship with the talus will correct the heel varus deformity of the club foot.fig . this maneuver increases the cavus deformity and fails to correct the varus deformity of the heel . the head of the talus ( t ) was palpable in the dorsolateral aspect of the foot in front of the ankle joint . ( e ) and ( f ) manipulation to correct the inversion : outward pressure is exerted on the first metatarsal and counter pressure on the lateral aspect of the head of the talus ( t ) . when the navicular and cuboid and the entire fore part of the foot are displaced laterally in relation with the head of the talus , the anterior portion of the calcaneus follows ; thus , the heel varus deformity is corrected . ( a ) in club foot , the anterior portion of the calcaneus lies beneath the head of the talus . ( b ) lateral displacement of the anterior portion of the calcaneus to its normal relationship with the talus will correct the heel varus deformity of the club foot . this maneuver increases the cavus deformity and fails to correct the varus deformity of the heel . ( e ) and ( f ) manipulation to correct the inversion : outward pressure is exerted on the first metatarsal and counter pressure on the lateral aspect of the head of the talus ( t ) . when the navicular and cuboid and the entire fore part of the foot are displaced laterally in relation with the head of the talus , the anterior portion of the calcaneus follows ; thus , the heel varus deformity is corrected . brockman noted that in a club foot there is subluxation of the talocalcaneonavicular joint and alterations in position of the navicular and calcaneus with respect to the talus like those which occur in the normal foot when it is adducted , inverted , and plantar flexed , but they are exaggerated in degree . however , a normal foot can not adopt a true club - foot position because even in the extreme degrees of plantar flexion and inversion , the fore part of the foot moves with the hind part , and a cavus deformity does not develop since there is no discrepancy in the degree of inversion of the front and back part of the foot . the navicular , cuneiforms , and metatarsals should be placed in straight alignment to form the lever arm needed for the correction of the inversion ( fig . a thumb placed on the lateral aspect of the head of the talus is used as a fulcrum while outward pressure is exerted on the first metatarsal and first cuneiform . during this manipulation an attempt is made to realign properly and simultaneously the calcaneocuboid , the talocalcaneonavicular , and the posterior talocalcaneal joints . when the navicular and cuboid are displaced laterally , the anterior portion of the calcaneus will be displaced outward and upward from its initial position underneath the head of the talus , and thus the varus deformity of the heel will be corrected ( fig . the manipulations should be gentle and followed by the application of a well molded thinly padded light plaster cast . if it then becomes evident that many more casts will be necessary for a complete correction , a simple subcutaneous tenotomy of the tendo achillis is performed with the patient under general anesthesia . medial tibial torsion of variable degree is present in most patients with club feet and is a tenacious deformity if below - the - knee casts are used during treatment . to prevent recurrences of the deformity , denis browne splints with high - top shoes with well molded heels these splints were left on full time for an average period of three months and at night for an average of twenty - one and a half months more ( ranging from ten to thirty months).1 ordinary high - top shoes were used for walking . in the forty - one feet permanently corrected with the first treatment the deformity tended to be less rigid , the leg muscles better developed , and the length of denis browne splint treatment longer than in the feet with recurrent deformities . in other patients the recurrence was associated with a severe initial deformity and apparently poorly developed leg muscles ; these recurrences seemed to be related to the severity of the primary aberration which caused the deformity.table 1treatment of recurrencesfirstsecondthirdfourthno . of the seventy - four feet treated with heel - cord section at primary treatment three required further surgery . in many of the recurrences , the varus deformity of the heel was more resistant to conservative treatment than the equinus deformity . an over - correction of the club - foot deformity after this procedure was not observed in this series . in three feet in addition to the anterior tibial transfer the extensor hallucis longus was recessed to the neck of the first metatarsal after suturing its distal stump to the tendon of the short extensor of the big toe . this was followed by a subcutaneous section of the tendo achillis in four feet , a lengthening of the tendo achillis in one foot , and a transfer of the anterior tibial to the third cuneiform in five feet . in one foot , the extensor hallucis longus was recessed to the neck of the first metatarsal , all the tendons of the extensor digitorum longus were recessed to the third cuneiform , and a subcutaneous plantar fasciotomy was done to relieve the cavus deformity . in five of these feet , the recurrence was mild and was apparently caused by the strong supinatory action of the anterior tibial muscle . these recurrences were permanently corrected by lateral transfer of the anterior tibial tendon after the application of two or three plaster casts . treatment consisted in the application of one plaster cast followed by a subcutaneous plantar fasciotomy , a transfer of the anterior tibial tendon to the third cuneiform , and a recession of the extensor hallucis longus to the neck of the first metatarsal . however , when we reviewed the roentgenograms made at the end of the primary treatment , we found that the relationship of the talus to the calcanceus had not been completely corrected in only five cases . in the forty - one feet permanently corrected with the first treatment the deformity tended to be less rigid , the leg muscles better developed , and the length of denis browne splint treatment longer than in the feet with recurrent deformities . the tendon was attached to the bone through a drill hole using a bunnell pull - out suture in twenty - one feet and a silk suture and osteoperiosteal flap in six feet . two skin incisions were made , one along the distal one or one and one - half inches of the anterior tibial tendon , the other shorter incision on the dorsum of the foot at the level of the third cuneiform . an over - correction of the club - foot deformity after this procedure was not observed in this series . the recession of the extensor hallucis longus was done in cases with severe plantar flexion of the first metatarsal and hyperextension of the first metatarsophalangeal joint . the first recurrence in these four feet and the first recurrence in three other feet with deformities of average severity had been incompletely corrected by the application of only two to four plaster casts . this was followed by a subcutaneous section of the tendo achillis in four feet , a lengthening of the tendo achillis in one foot , and a transfer of the anterior tibial to the third cuneiform in five feet . in one foot , the extensor hallucis longus was recessed to the neck of the first metatarsal , all the tendons of the extensor digitorum longus were recessed to the third cuneiform , and a subcutaneous plantar fasciotomy was done to relieve the cavus deformity . in five of these feet , the recurrence was mild and was apparently caused by the strong supinatory action of the anterior tibial muscle . these recurrences were permanently corrected by lateral transfer of the anterior tibial tendon after the application of two or three plaster casts . in one of these , a medial release operation , as well as a subcutaneous section of the tendo achillis , was necessary ; in another , a medial release was combined with a transfer of the anterior tibial tendon to the third cuneiform ; in the third , a tendo achillis lengthening was performed ; and in the fourth treatment consisted in five plaster - cast changes which were followed by a recurrence of the deformity . treatment consisted in the application of one plaster cast followed by a subcutaneous plantar fasciotomy , a transfer of the anterior tibial tendon to the third cuneiform , and a recession of the extensor hallucis longus to the neck of the first metatarsal . both evaluations correlated closely with respect to ankle dorsiflexion , heel varus , and adduction of the fore part of the foot . all the clinical measurements were performed by the senior author for the sake of uniformity.table 2resultsankle dorsiflexion ( degrees)heel varus ( degrees)adduction of the fore part of the foot ( degrees)tibial torsion ( degrees)result > 1000100good : 67 feet ( 71 per cent)0100101020moderateacceptable : 26 feet ( 28 per cent)0over 10over 20severepoor : 1 foot ( 1 per cent ) on the anteroposterior roentgenograms , the degree of heel varus deformity was estimated by measuring the angle formed by the long axis of the talus and the calcaneus . the adduction of the fore part of the foot was also estimated on the anteroposterior roentgenograms by measuring the angle between the long axis of the calcaneus and that of the fifth metatarsal . in the normal foot , the head of the talus can be palpated in front of the ankle mortise in the same plane as the axis of the thigh . medial or lateral tibial torsion is indicated by the orientation of the head of the talus in respect to the patella and axis of the thigh.fig . ( a d ) anteroposterior and lateral roentgenograms of the feet of a six - week - old baby boy with severe congenital club feet . in the lateral views , the angle formed by the long axis of the calcaneus and that of the first metatarsal ( cavus ) measures 86 degrees . treatment consisted in manipulation and application of five plaster casts for the correction of the cavus , the adduction , and the heel varus deformities . ( e h ) anteroposterior and lateral roentgenograms made after removal of the last plaster cast , seven weeks after onset of treatment . in the anteroposterior roentgenograms the angle between the long axis of the talus and that of the calcaneus measures 33 degrees , indicating correction of the heel varus deformity , and the angle between the calcaneus and the fifth metatarsal measures 13 degrees , indicating correction of the adduction of the fore part of the foot . in the anteroposterior roentgenograms , the angle between the long axis of the calcaneus and that of the talus measures 28 degrees on the right and 22 degrees on the left , indicating correction of the heel varus deformity on the right and incomplete correction on the left . ( a d ) anteroposterior and lateral roentgenograms of the feet of a six - week - old baby boy with severe congenital club feet . in the lateral views , the angle formed by the long axis of the calcaneus and that of the first metatarsal ( cavus ) measures 86 degrees . treatment consisted in manipulation and application of five plaster casts for the correction of the cavus , the adduction , and the heel varus deformities . ( e h ) anteroposterior and lateral roentgenograms made after removal of the last plaster cast , seven weeks after onset of treatment . in the anteroposterior roentgenograms the angle between the long axis of the talus and that of the calcaneus measures 33 degrees , indicating correction of the heel varus deformity , and the angle between the calcaneus and the fifth metatarsal measures 13 degrees , indicating correction of the adduction of the fore part of the foot . in the lateral roentgenograms the equinus deformity appears corrected and the angle between the long axis of the calcaneus and that of the fifth metatarsal measures 7 degrees , indicating correction of the cavus deformity . in the lateral roentgenograms the angle between the long axis of the calcaneus and that of the first metatarsal measures 32 degrees on the right and 24 degrees on the left , indicating correction of the cavus deformity in both feet . the cavus deformity was corrected in most feet by the first plaster - cast application . recession of this tendon was done in three other feet with severe plantar flexion of the first metatarsal . adduction of the fore part of the foot was completely corrected in seventy - two feet ( 77 per cent ) , was less than 20 degrees in twenty - one feet ( 22 per cent ) , and was severe in one foot . it appears then that anterior tibial transfer may help to correct not only the heel varus deformity but also the adduction of the fore part of the foot . in fifteen feet ( 16 per cent ) a moderate residual medial tibial torsion ( if less than 10 degrees was observed , and in one there was tibial torsion of 20 degrees . in eighteen feet ( 19 per cent ) dorsiflexion of the ankle was limited to from 0 to 10 degrees . a second section of the tendon was done in four to treat a recurrence , and in three the tendon was lengthened . good results were obtained in 71 per cent of the feet ( figs . photographs made after removal of the plaster casts which had been applied three days previously . a recurrence of the equinus deformity at one year of age was treated by the application of two toe - to - groin plaster casts and subcutaneous tenotomy . three plaster casts were then applied , followed by the transfer of the anterior tibial tendon to the third cuneiform . photographs made when she was five months old , and later , when she was two and one - half years old , show all the components of the deformity well corrected . a mild recurrence of the deformity occurred when she was five years old , which was treated by the application of four plaster casts which were changed every ten days . a recurrence of the equinus deformity at one year of age was treated by the application of two toe - to - groin plaster casts and subcutaneous tenotomy . three plaster casts were then applied , followed by the transfer of the anterior tibial tendon to the third cuneiform . photographs made when she was five months old , and later , when she was two and one - half years old , show all the components of the deformity well corrected . a mild recurrence of the deformity occurred when she was five years old , which was treated by the application of four plaster casts which were changed every ten days . the left foot was treated by lengthening of the heel cord and transfer of the anterior tibial to the third cuneiform . subsequently , a lengthening of the heel cord and transfer of the anterior tibial tendon was done on the right foot . although the treatment of a mild congenital club foot may be easy , the complete and permanent correction of a severe and rigid club foot is often difficult . in this study the early months of life offer a golden opportunity for the correction of club feet since the skeleton , which is to a great extent cartilaginous , is little deformed , and the joint capsules , ligaments , tendons , and muscles can be stretched without damage . correction of a severe equinus deformity can be radically shortened by subcutaneous section of the heel cord followed by the application of a plaster cast ( with the foot in maximum dorsiflexion ) for three weeks . however , the heel cord should he sectioned only after the other components of the club - foot deformity are completely corrected . of seventy - four feet with severe equinus deformity treated with a subcutaneous section of the heel cord in the primary treatment , only eight required further surgery to treat a recurrent equinus deformity . however , only half of the recurrences could be blamed on the neglect of follow - up treatment ; often these could be corrected by the reapplication of a few plaster casts . this deformity was corrected by the transfer of the long extensor of the big toe to the neck of the first metatarsal . the nine failures of the anterior tibial transfer to correct permanently the varus deformity of the heel could he blamed on surgical errors . the results of treatment in sixty - seven patients with a total of ninety - four severe congenital club feet were evaluated five to thirteen years after the initial treatment . in many instances a subcutaneous tendo achillis tenotomy was performed in the primary treatment to obtain a complete correction of the equinus deformity . the results in 71 per cent of the feet were good ; in 28 per cent a slight residual deformity persisted ; and in one foot a poor result was obtained .

many adolescents are insufficiently active for enhanced health and weight control . in order to effectively promote physical activity among adolescents , it is necessary to understand factors that influence adolescents ' physical activity levels . ecological models such as the social ecological theory and the social cognitive theory recognize the importance of simultaneously investigating demographic , psychosocial , and environmental factors to understand physical activity behaviors . in addition , it has been demonstrated that relationships between personal or environmental factors and physical activity levels differ according to the type of physical activity behavior and the context in which it takes place . although a recent review showed that participation in extracurricular activities at school considerably contributes to adolescents overall engagement in physical activities of moderate - to - vigorous intensity , few studies looked at this specific type of behavior typically related to the school context [ 2 , 8 ] . at the same time , schools are considered as preferred environments for promoting physical activity among adolescents [ 9 , 10 ] . schools offer many opportunities for adolescents to engage in physical activities ( i.e. , physical education classes , extracurricular physical activities , and recess periods ) and adolescents spend large amounts of time at school . although it is recommended to promote physical activity during physical education classes , physical education time is limited and even the best school physical education programs do not provide enough physical activity to meet health - related recommendations . therefore , it has been suggested that the organization of physical activities at school outside physical education classes , such as during the lunch breaks , during recesses , and during after school hours , might be important to reach the recommended daily physical activity time of one hour . one study in american middle schools found that schools that organize extracurricular activities might increase physical activity levels among boys ( grades 68 ) . the same study pointed out that the objectively measured school environment ( area type , area size , supervision , equipment , improvements ) explains the largest amount of variance in the number of boys and girls engaging in mvpa at school . boys were most active on outdoor courts with high levels of supervision or when both equipment and supervision were provided . both boys and girls were more active when schools had improvements ( e.g. , basketball hoops , volleyball nets , tennis courts ) and high levels of supervision . two studies among norwegian adolescents showed that self - reported activity levels during recess were positively related to the number of available outdoor spaces [ 12 , 13 ] and the availability of playground equipment . however , the authors argued that more in - depth research simultaneously addressing the impact of individual , sociocultural , environmental factors , and the interaction between them is needed to get a better understanding of activity levels at school . therefore , the first purpose of this explorative study was to investigate the relationship between environmental features of the school environment such as accommodation and supervision and participation in extracurricular physical activities at school , while simultaneously including psychosocial determinants . next to school - based activities , also leisure time sports take a significant place in the activity culture of many adolescents . for engagement in leisure time sports , the home and neighborhood environment might be important contexts to consider . studies in adults extensively investigated the relationship between the neighborhood environment ( e.g. , access to facilities ) and participation in leisure time physical activity [ 1517 ] . the results of a recent review showed that among adolescents , only four neighborhood environmental factors ( access to facilities , availability of facilities , availability of equipment , crime incidence ) were examined in more than three samples . the same review furthermore revealed that only one adolescent study looked at the influence of distance to destinations and residential density , suggesting that studies including these variables are needed . with regard to the home environment , most prior studies explored sociocultural ( i.e. , family structure , modelling and support from family and friends ) correlates of physical activity among adolescents . only seven studies investigated the effects of availability of physical activity equipment and most of these studies were conducted in the us and canada . availability of exercise equipment was found to be unrelated to adolescents ' physical activity levels . additionally , none of these studies included a measure of availability of sedentary equipment ( e.g. , play stations and televisions ) . therefore , a second purpose of this explorative study was to investigate the relationship between adolescents ' engagement in leisure time sports and neighborhood and home environmental features , while simultaneously including psychosocial determinants and the interactions between the two . finally , as health - related guidelines specifically address engagement in physical activity of moderate - to - vigorous intensity ( mvpa ) , the final purpose of this study was to investigate psychosocial and environmental correlates of objectively measured mvpa by means of accelerometers . participants were recruited from four randomly selected middle schools offering technical - vocational education in west - flanders ( belgium ) . parents of all students in seventh and eight grade ( n = 667 ) received a letter seeking informed consent for each child to complete measurements . parents of 634 ( 95% ) of those adolescents , 523 completed all questionnaires ; missing data were due to absence on the day of measurements or questionnaires filled out inaccurately , yielding in a final response rate of 78% . from each of the four schools , one class of seventh graders was randomly selected for more in - depth physical activity measurements with accelerometers , which resulted in a subsample of 62 adolescents . data on demographics like gender , birth date , and occupation of father and mother were collected in the first part of the questionnaire . an estimate of higher and lower social economic status ( ses ) was obtained by classifying occupation of father and mother into white and blue collar . participation in extracurricular activities at school and leisure time sports was determined in a second part of the questionnaire using a selection of questions from the flemish physical activity questionnaire ( fpaq , see supplementary material available online at doi:10.1155/2009/320372 ) . one question addressed time spent in extracurricular physical activities at school ( question 4 , see supplementary material ) . three questions asked for frequency and duration of time spent in leisure time sports ( question 7 , see supplementary material ) . validity and reliability of a computerized version of the questionnaire used in the present study was investigated in a separate study with different participants aged 1218 . moderate - to - high reliability of the indexes derived from the fpaq was reported . for all types of physical activity test - retest , icc 's exceeded 0.70 . to obtain validity measures , data from questionnaires were correlated to data derived from accelerometers . pearson correlations were significant and ranged between 0.43 ( total activity levels ) and 0.78 ( minutes spent in vigorous activities ) , indicating acceptable validity of the instrument used in the present study . ecological models recognize the importance of the interactions between personal - level factors and the environment . to investigate these interactions , measures of students ' general - affective attitudes , that is , social support , self - efficacy , perceived benefits , and barriers , were assessed by 29 items with a 5-point scale . questions were selected and adopted from previous studies with adolescents and adults [ 2022 ] . general - affective attitudes ( 4 items ) toward physical activity were assessed using bipolar adjectives . participants were asked whether sports and physical activity are not pleasant - pleasant , bad - good , healthy - unhealthy , , and dangerous - safe ( cronbach 's = 0.79 ) . social support ( 4 items ) was assessed by asking respondents how frequently their parents , brothers and sisters , friends , and teachers encouraged them to be physically active ( cronbach 's = 0.79 ) . self - efficacy ( 2 items ) was measured by asking how easy or difficult it is to be active at their school or at their home ( cronbach 's = 0.38 ) . perceived benefits and barriers with regard to physical activity were investigated by asking respondents to rate their agreement with possible effects of sports and physical activity ( 8 items : weight and physical appearance , health and fitness , social interaction , pleasure , competition , stress and depression , admiration of others , relaxation from ( school ) work , cronbach 's = 0.85 ) , and the frequency with which barriers prevented them from exercising ( 11 items : lack of time , lack of discipline , lack of interest , health problems , personal problems , not skilled enough , too expensive , no transportation , not liking to sweat , fear of being laughed at , lack of facilities at school , cronbach 's = 0.88 ) . for measures of psychosocial determinants physical activity levels were also assessed using accelerometers ( model 7164 , computer science application , inc . , shalimar , fla , usa ) . accelerometers are considered as valid and reliable tools for assessment of physical activity among adolescents . accelerometer measurements could only be conducted in a subsample of one class per school ( n = 62 adolescents ) because given the expense of these instruments , only a limited number of instruments were available . independent sample t - tests with self - reported physical activity levels as dependent variables indicated that there were no significant differences between those students with and without accelerometer data ( all t 0.9 ) . adolescents wore the accelerometer during six days above the right hipbone , underneath the clothes . accelerometer data were used to determine engagement in physical activity of moderate to vigorous intensity ( mvpa ) . in agreement with most recently published guidelines , the cut - off point for mvpa was 3200 counts per minute [ 23 , 24 ] . to measure potential environmental correlates of physical activity among adolescents , a modified version of a questionnaire validated in adults was used . the scale composition , scale items , response categories , reliability , and validity data are all represented in table 1 . 15 minutes cycling from home to more than 30 minutes cycling from home . to measure neighborhood residential density adolescents rated on a three - point scale ( none - some - much ) , how many detached single - family residences , row houses , and apartments there were in their neighborhood . the number of tv 's , computers , and playstations was questioned to get a measure of the availability of sedentary equipment at home . finally , home availability of physical activity equipment ( 13 items ) was questioned ( see table 1 ) . test - retest reliability was analyzed by subjects completing the questionnaires twice within a 2-week interval . to test validity , all parents of participating pupils were contacted by telephone to verbally answer the same questionnaire as their child . the lowest icc was found for residential density ( 0.49 ) , for all other indexes , icc 's significantly ranged between 0.63 and 0.95 . validity coefficients significantly ranged between 0.46 and 0.95 . a questionnaire for measuring the school environment related to physical activity accessibility of sports facilities and sports materials availability of supervision and extracurricular activities ( during breaks and after school hours ) was measured using a three - point answering scale ( daily - weekly - never ) . preliminary analyses consisted of descriptive statistics of sample characteristics . multiple linear hierarchical regression analyses with extracurricular physical activity , leisure time sports , and mvpa as dependent variables were conducted to investigate the relationship between personal , environmental factors , and these physical activity measurements . all analyses were controlled for age and ses by entering these factors into the first block . in the first series of regression analyses ( model 1 ) , neighborhood ( convenience of facilities ) , home ( sedentary and sports equipment ) , and school ( availability of physical activities , accessibility of sports accommodation and sports materials , supervision ) environmental variables were entered into the second block . due to its low reliability ( 0.49 ) residential density was excluded from the analyses . for analyses on time spent in extracurricular physical activity only neighborhood and home environmental factors were entered into the second block . for mvpa school , in the second series of regression analyses ( model 2 ) psychosocial determinants ( attitude , self - efficacy , social support , perceived benefits , and perceived barriers ) were entered into the second block . in the third series of regression analyses ( model 3 ) , interactions between environmental and psychosocial determinants were entered into the second block . to investigate the interaction effects , the product of two variables was computed after these were mean centred . in a first phase , hierarchical regression analyses with one environmental correlate , and the interaction terms between that specific correlate and the psychosocial determinants were conducted , to test which variables needed to be included in the final models . in a second phase , only significant correlates were included in the regression analyses ( see tables 37 ) . finally , all variables were added together to estimate the total variance explained . in all of the models , variation inflation factors were below 10 , indicating that there were no problems of multicollinearity . previous studies showed gender differences in levels of physical activity [ 1 , 26 ] or intervention effects [ 2729 ] . preliminary analyses furthermore revealed differences in physical activity levels and correlates of physical activity for boys and girls of the present sample . therefore , all analyses were conducted in boys and girls separately . however , due to the small sample size ( n = 62 ) , analyses on mvpa were conducted in boys and girls together . hierarchical regression analyses on participation in extracurricular physical activities are presented in table 3 for boys and table 4 for girls . among boys , the total model explained 28% of the variance in participation in extracurricular physical activity . availability of extra physical activities was the only significant correlate within the second block . in the second model , the psychosocial determinants explained 19% of the variance . self - efficacy was the only significant correlate within the second block . in the third model , the interaction terms explained 10% of the variance . the interaction term between supervision and perceived benefits was significant . among girls , the total model explained 17% of the variance in participation in extracurricular physical activity . supervision was the only significant correlate within the second block . in the second model , psychosocial determinants explained 7% of the variance . self - efficacy and perceived barriers were significant correlates of extracurricular physical activity . in the third model , the interactions terms explained 4.0% of the variance . hierarchical regression analyses on self - reported leisure time sports levels are presented in table 5 for boys and table 6 for girls . among boys , the entire model explained 32% of the variance in leisure time sports . perceived benefits were significantly positively related to leisure time sports among boys . in the third block , the interactions terms between sedentary equipment and social support and sedentary equipment and barriers were significant . among girls , the entire model explained 27% of the variance in leisure time sports . availability of pa equipment was the only significant environmental correlate . in the second model , psychosocial determinants explained 21% of the variance . attitude and self - efficacy were significantly positively related to leisure time sports among girls . in the third block , the entire model explained 51% of the variance in mvpa . in the first model , environmental factors explained 1% of the variance in mvpa . in the second model , the psychosocial determinants explained 18% of the variance . perceived benefits were positively related to mvpa , whereas the attitude was negatively correlated to mvpa . in the third model , the first purpose of this explorative study was to investigate the relationship between environmental features of the school environment and participation in extracurricular activities at school . the time spent in extracurricular activities at school contributes considerably to overall physical activity levels . hence , in order to be able to design effective school - based intervention to increase this type of behavior , it is important to investigate influencing factors . findings of the present study showed that adolescents ' engagement in extracurricular physical activity at school was positively related to the availability of organized activities at school . for girls , significant interactions with perceived benefits were found , with stronger correlations between organized activities and engagement in extracurricular activities among girls reporting more benefits . in contrast to our results , the organization of school sports was defined as unrelated to adolescents ' physical activity levels in a recent review . on the other hand and in line with our findings , environmental intervention studies already showed that offering extra physical activities at school appeared to be an effective strategy for increasing physical activity engagement at school ( e.g. , ) . results from the present study furthermore revealed that the provision of supervision was also positively related to participation in extracurricular activities . again , significant interactions with perceived benefits were found , with stronger correlations between supervision and engagement in extracurricular activities among boys reporting more benefits . the importance of supervision was also exposed in a study among american adolescents showing that boys were more active when supervision was provided . in contrast to the study among norwegian adolescents [ 12 , 13 ] , the present study found that access to accommodation and sports materials were both unrelated to participation in extracurricular activities at school . a second purpose of the present study was to investigate correlates of participation in leisure time sports in a sample of flemish adolescents . for adolescents , engagement in sport activities can be considered as an important leisure time activity , making it highly relevant to look at environmental correlates for this specific behavior in this age group . for engagement in leisure time sports , the neighborhood environment is particularly important to consider . in the present study only , one neighborhood environmental factor , namely , the perceived convenience of facilities for adolescents was included . in the american or canadian context , availability of facilities for adolescents was found to be unrelated adolescents ' engagement in physical activities . on the other hand , studies in adults found positive associations between perceived convenience of facilities and leisure time physical activity . the results of the present study , however , revealed that perceived convenience of facilities was unrelated to leisure time sports among adolescents . however , among girls an interaction with barriers for physical activity was found , with stronger correlations between convenience of facilities and leisure time sports among girls perceiving more barriers . clearly , future research should include more extensive measurements of those neighborhood factors ( i.e. , access to facilities , influence of parks , etc . ) a recently published study of canadian adolescents showed that beside the number of available recreation facilities , access to facilities might also be important to consider . additionally , some studies have recently shown that the objectively measured availability and accessibility of physical activity facilities [ 3133 ] might be more important when compared to perceived environmental factors . future studies should , therefore , include objective measurements of the actual neighborhood environment such as the application of geographic information systems ( giss ) data in a european adolescent population . the availability of equipment ( e.g. , playstation ) that facilitates sedentary behavior might discourage adolescents from going outside and engaging in sports activities . results showed that the availability of sedentary equipment was indeed negatively related to participation in leisure time sports , but only among boys . negative correlations between sedentary equipment and leisure time sports were only found among boys perceiving fewer barriers to be physically active . studies have shown that boys more frequently engage in screen time compared to girls , and playing computer games is considered as a rather masculine activity . therefore , it is plausible that boys more often use sedentary equipment when it is available ; whereas the availability of sedentary equipment might be less attractive to girls . future research should elaborate on these findings by adding questions on time spent using sedentary equipment or rules regarding the use of sedentary equipment at home . studies among adults revealed positive relationships between the availability of physical activity equipment in the home environment and activity levels among men and women . in the present sample of adolescents , the availability of physical activity equipment was also positively related to engagement in leisure time sports among girls . among boys , interactions with self - efficacy were found , with stronger correlations between equipment and leisure time sports among boys reporting higher levels of self - efficacy . however , in adult studies , the argument is raised that people who engage in sport activities more regularly might be more likely to purchase physical activity equipment . therefore , also among adolescents , engagement in leisure time sports might more likely be an antecedent rather than a consequence of availability of sports equipment in the home environment . in addition , for adolescents , the availability of sports equipment might also be closely related to the activity levels of other family members such as parents who are known to influence adolescents ' activity levels . participation in mvpa is essential for improved health . in line with findings from jago et al . only the interaction between self - efficacy and supervision was significant , with stronger correlations between supervision and mvpa among those reporting higher levels of self - efficacy . however , research that further investigates the relationships between environmental factors and objectively measured mvpa in larger samples is needed to be conclusive . according to ecological models of behavior change , the environment does not influence behavior separate from individual determinants [ 3 , 4 ] . previous intervention studies have shown favorable changes in physical activity levels as a result of the combination of environmental strategies with personal interventions [ 27 , 28 , 39 ] . the results of the present study revealing several significant interactions between personal and environmental variables are supportive of such multicomponent intervention designs . some limitations of the present study need to be addressed . first , the questionnaire to measure school level variables was not validated yet ; therefore , conclusions concerning school environmental factors should be treated with considerable caution . the development of a comprehensive valid and reliable questionnaire to measure school environmental factors by use of self - reports is a priority for future research . although it is the strength of the present study that physical activity was also measured more objectively with accelerometers , the subsample in which these measurements occurred was very small . hence , most of the conclusions in the present study were based on self - reported physical activity measures and although validated questionnaires were used , this is a limitation . although the questionnaire on psychosocial determinants was already extensive , no questions on social norms were included . especially among adolescents , apart from social support , social norms ( i.e. , influence of peers ) might be important to consider . furthermore , given the cross - sectional design of the present study , no causal conclusions can be drawn . although cross - sectional studies are necessary to understand the relationships between environmental factors and physical activity levels , prospective studies that further explore the causal relationships between the change in personal , intrapersonal , and environmental factors and the change in physical activity levels are needed to formulate recommendations for physical activity promotion . this explorative study provided initial insight into the relationship between school environmental factors and participation in extracurricular physical activity in a sample of european adolescents . the findings showed that increased availability of extracurricular activities and supervision are related to greater participation in extracurricular activities among adolescent boys and girls . the findings showed that perceived convenience of neighborhood facilities was unrelated , whereas the availability of sedentary and physical activity equipment at home was related to time spent in leisure time sports . overall , findings were supportive of ecological theories stating that behaviors are influenced by personal and environmental factors that are constantly interacting .

the present study aimed at investigating the influence of home , neighbourhood and school environmental factors on adolescents ' engagement in self - reported extracurricular physical activity and leisure time sports and on mvpa objectively measured by accelerometers . environmental factors were assessed using questionnaires . gender specific hierarchical regression analyses were conducted , with demographic variables entered in the first block , and environmental , psychosocial factors and interactions terms entered in the second block . participation in extracurricular activities at school was positively related to the number of organized activities and the provision of supervision . perceived accessibility of neighborhood facilities was not related to engagement in leisure time sports , whereas the availability of sedentary and physical activity equipment was . findings were generally supportive of ecological theories stating that behaviors are influenced by personal and environmental factors that are constantly interacting .

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

in order to effectively promote physical activity among adolescents , it is necessary to understand factors that influence adolescents ' physical activity levels . ecological models such as the social ecological theory and the social cognitive theory recognize the importance of simultaneously investigating demographic , psychosocial , and environmental factors to understand physical activity behaviors . in addition , it has been demonstrated that relationships between personal or environmental factors and physical activity levels differ according to the type of physical activity behavior and the context in which it takes place . although a recent review showed that participation in extracurricular activities at school considerably contributes to adolescents overall engagement in physical activities of moderate - to - vigorous intensity , few studies looked at this specific type of behavior typically related to the school context [ 2 , 8 ] . at the same time , schools are considered as preferred environments for promoting physical activity among adolescents [ 9 , 10 ] . , physical education classes , extracurricular physical activities , and recess periods ) and adolescents spend large amounts of time at school . although it is recommended to promote physical activity during physical education classes , physical education time is limited and even the best school physical education programs do not provide enough physical activity to meet health - related recommendations . therefore , it has been suggested that the organization of physical activities at school outside physical education classes , such as during the lunch breaks , during recesses , and during after school hours , might be important to reach the recommended daily physical activity time of one hour . one study in american middle schools found that schools that organize extracurricular activities might increase physical activity levels among boys ( grades 68 ) . the same study pointed out that the objectively measured school environment ( area type , area size , supervision , equipment , improvements ) explains the largest amount of variance in the number of boys and girls engaging in mvpa at school . boys were most active on outdoor courts with high levels of supervision or when both equipment and supervision were provided . , basketball hoops , volleyball nets , tennis courts ) and high levels of supervision . two studies among norwegian adolescents showed that self - reported activity levels during recess were positively related to the number of available outdoor spaces [ 12 , 13 ] and the availability of playground equipment . however , the authors argued that more in - depth research simultaneously addressing the impact of individual , sociocultural , environmental factors , and the interaction between them is needed to get a better understanding of activity levels at school . therefore , the first purpose of this explorative study was to investigate the relationship between environmental features of the school environment such as accommodation and supervision and participation in extracurricular physical activities at school , while simultaneously including psychosocial determinants . next to school - based activities , also leisure time sports take a significant place in the activity culture of many adolescents . for engagement in leisure time sports , the home and neighborhood environment might be important contexts to consider . , access to facilities ) and participation in leisure time physical activity [ 1517 ] . the results of a recent review showed that among adolescents , only four neighborhood environmental factors ( access to facilities , availability of facilities , availability of equipment , crime incidence ) were examined in more than three samples . the same review furthermore revealed that only one adolescent study looked at the influence of distance to destinations and residential density , suggesting that studies including these variables are needed . with regard to the home environment , most prior studies explored sociocultural ( i.e. , family structure , modelling and support from family and friends ) correlates of physical activity among adolescents . only seven studies investigated the effects of availability of physical activity equipment and most of these studies were conducted in the us and canada . availability of exercise equipment was found to be unrelated to adolescents ' physical activity levels . additionally , none of these studies included a measure of availability of sedentary equipment ( e.g. therefore , a second purpose of this explorative study was to investigate the relationship between adolescents ' engagement in leisure time sports and neighborhood and home environmental features , while simultaneously including psychosocial determinants and the interactions between the two . finally , as health - related guidelines specifically address engagement in physical activity of moderate - to - vigorous intensity ( mvpa ) , the final purpose of this study was to investigate psychosocial and environmental correlates of objectively measured mvpa by means of accelerometers . participants were recruited from four randomly selected middle schools offering technical - vocational education in west - flanders ( belgium ) . parents of 634 ( 95% ) of those adolescents , 523 completed all questionnaires ; missing data were due to absence on the day of measurements or questionnaires filled out inaccurately , yielding in a final response rate of 78% . from each of the four schools , one class of seventh graders was randomly selected for more in - depth physical activity measurements with accelerometers , which resulted in a subsample of 62 adolescents . data on demographics like gender , birth date , and occupation of father and mother were collected in the first part of the questionnaire . participation in extracurricular activities at school and leisure time sports was determined in a second part of the questionnaire using a selection of questions from the flemish physical activity questionnaire ( fpaq , see supplementary material available online at doi:10.1155/2009/320372 ) . one question addressed time spent in extracurricular physical activities at school ( question 4 , see supplementary material ) . three questions asked for frequency and duration of time spent in leisure time sports ( question 7 , see supplementary material ) . validity and reliability of a computerized version of the questionnaire used in the present study was investigated in a separate study with different participants aged 1218 . for all types of physical activity test - retest , icc 's exceeded 0.70 . pearson correlations were significant and ranged between 0.43 ( total activity levels ) and 0.78 ( minutes spent in vigorous activities ) , indicating acceptable validity of the instrument used in the present study . ecological models recognize the importance of the interactions between personal - level factors and the environment . to investigate these interactions , measures of students ' general - affective attitudes , that is , social support , self - efficacy , perceived benefits , and barriers , were assessed by 29 items with a 5-point scale . general - affective attitudes ( 4 items ) toward physical activity were assessed using bipolar adjectives . participants were asked whether sports and physical activity are not pleasant - pleasant , bad - good , healthy - unhealthy , , and dangerous - safe ( cronbach 's = 0.79 ) . social support ( 4 items ) was assessed by asking respondents how frequently their parents , brothers and sisters , friends , and teachers encouraged them to be physically active ( cronbach 's = 0.79 ) . self - efficacy ( 2 items ) was measured by asking how easy or difficult it is to be active at their school or at their home ( cronbach 's = 0.38 ) . perceived benefits and barriers with regard to physical activity were investigated by asking respondents to rate their agreement with possible effects of sports and physical activity ( 8 items : weight and physical appearance , health and fitness , social interaction , pleasure , competition , stress and depression , admiration of others , relaxation from ( school ) work , cronbach 's = 0.85 ) , and the frequency with which barriers prevented them from exercising ( 11 items : lack of time , lack of discipline , lack of interest , health problems , personal problems , not skilled enough , too expensive , no transportation , not liking to sweat , fear of being laughed at , lack of facilities at school , cronbach 's = 0.88 ) . for measures of psychosocial determinants physical activity levels were also assessed using accelerometers ( model 7164 , computer science application , inc . accelerometers are considered as valid and reliable tools for assessment of physical activity among adolescents . accelerometer measurements could only be conducted in a subsample of one class per school ( n = 62 adolescents ) because given the expense of these instruments , only a limited number of instruments were available . independent sample t - tests with self - reported physical activity levels as dependent variables indicated that there were no significant differences between those students with and without accelerometer data ( all t 0.9 ) . accelerometer data were used to determine engagement in physical activity of moderate to vigorous intensity ( mvpa ) . to measure potential environmental correlates of physical activity among adolescents , a modified version of a questionnaire validated in adults was used . the scale composition , scale items , response categories , reliability , and validity data are all represented in table 1 . to measure neighborhood residential density adolescents rated on a three - point scale ( none - some - much ) , how many detached single - family residences , row houses , and apartments there were in their neighborhood . the number of tv 's , computers , and playstations was questioned to get a measure of the availability of sedentary equipment at home . finally , home availability of physical activity equipment ( 13 items ) was questioned ( see table 1 ) . a questionnaire for measuring the school environment related to physical activity accessibility of sports facilities and sports materials availability of supervision and extracurricular activities ( during breaks and after school hours ) was measured using a three - point answering scale ( daily - weekly - never ) . multiple linear hierarchical regression analyses with extracurricular physical activity , leisure time sports , and mvpa as dependent variables were conducted to investigate the relationship between personal , environmental factors , and these physical activity measurements . all analyses were controlled for age and ses by entering these factors into the first block . in the first series of regression analyses ( model 1 ) , neighborhood ( convenience of facilities ) , home ( sedentary and sports equipment ) , and school ( availability of physical activities , accessibility of sports accommodation and sports materials , supervision ) environmental variables were entered into the second block . for analyses on time spent in extracurricular physical activity only neighborhood and home environmental factors were entered into the second block . for mvpa school , in the second series of regression analyses ( model 2 ) psychosocial determinants ( attitude , self - efficacy , social support , perceived benefits , and perceived barriers ) were entered into the second block . in the third series of regression analyses ( model 3 ) , interactions between environmental and psychosocial determinants were entered into the second block . in a first phase , hierarchical regression analyses with one environmental correlate , and the interaction terms between that specific correlate and the psychosocial determinants were conducted , to test which variables needed to be included in the final models . in a second phase , only significant correlates were included in the regression analyses ( see tables 37 ) . in all of the models , variation inflation factors were below 10 , indicating that there were no problems of multicollinearity . previous studies showed gender differences in levels of physical activity [ 1 , 26 ] or intervention effects [ 2729 ] . preliminary analyses furthermore revealed differences in physical activity levels and correlates of physical activity for boys and girls of the present sample . therefore , all analyses were conducted in boys and girls separately . however , due to the small sample size ( n = 62 ) , analyses on mvpa were conducted in boys and girls together . hierarchical regression analyses on participation in extracurricular physical activities are presented in table 3 for boys and table 4 for girls . among boys , the total model explained 28% of the variance in participation in extracurricular physical activity . availability of extra physical activities was the only significant correlate within the second block . in the second model , the psychosocial determinants explained 19% of the variance . self - efficacy was the only significant correlate within the second block . in the third model , the interaction terms explained 10% of the variance . among girls , the total model explained 17% of the variance in participation in extracurricular physical activity . supervision was the only significant correlate within the second block . in the second model , psychosocial determinants explained 7% of the variance . self - efficacy and perceived barriers were significant correlates of extracurricular physical activity . in the third model , the interactions terms explained 4.0% of the variance . hierarchical regression analyses on self - reported leisure time sports levels are presented in table 5 for boys and table 6 for girls . among boys , the entire model explained 32% of the variance in leisure time sports . perceived benefits were significantly positively related to leisure time sports among boys . in the third block , the interactions terms between sedentary equipment and social support and sedentary equipment and barriers were significant . among girls , the entire model explained 27% of the variance in leisure time sports . availability of pa equipment was the only significant environmental correlate . in the second model , psychosocial determinants explained 21% of the variance . attitude and self - efficacy were significantly positively related to leisure time sports among girls . in the third block , the entire model explained 51% of the variance in mvpa . in the first model , environmental factors explained 1% of the variance in mvpa . in the second model , the psychosocial determinants explained 18% of the variance . perceived benefits were positively related to mvpa , whereas the attitude was negatively correlated to mvpa . in the third model , the first purpose of this explorative study was to investigate the relationship between environmental features of the school environment and participation in extracurricular activities at school . the time spent in extracurricular activities at school contributes considerably to overall physical activity levels . findings of the present study showed that adolescents ' engagement in extracurricular physical activity at school was positively related to the availability of organized activities at school . for girls , significant interactions with perceived benefits were found , with stronger correlations between organized activities and engagement in extracurricular activities among girls reporting more benefits . in contrast to our results , the organization of school sports was defined as unrelated to adolescents ' physical activity levels in a recent review . on the other hand and in line with our findings , environmental intervention studies already showed that offering extra physical activities at school appeared to be an effective strategy for increasing physical activity engagement at school ( e.g. results from the present study furthermore revealed that the provision of supervision was also positively related to participation in extracurricular activities . again , significant interactions with perceived benefits were found , with stronger correlations between supervision and engagement in extracurricular activities among boys reporting more benefits . the importance of supervision was also exposed in a study among american adolescents showing that boys were more active when supervision was provided . in contrast to the study among norwegian adolescents [ 12 , 13 ] , the present study found that access to accommodation and sports materials were both unrelated to participation in extracurricular activities at school . a second purpose of the present study was to investigate correlates of participation in leisure time sports in a sample of flemish adolescents . for adolescents , engagement in sport activities can be considered as an important leisure time activity , making it highly relevant to look at environmental correlates for this specific behavior in this age group . for engagement in leisure time sports , the neighborhood environment is particularly important to consider . in the present study only , one neighborhood environmental factor , namely , the perceived convenience of facilities for adolescents was included . in the american or canadian context , availability of facilities for adolescents was found to be unrelated adolescents ' engagement in physical activities . on the other hand , studies in adults found positive associations between perceived convenience of facilities and leisure time physical activity . the results of the present study , however , revealed that perceived convenience of facilities was unrelated to leisure time sports among adolescents . however , among girls an interaction with barriers for physical activity was found , with stronger correlations between convenience of facilities and leisure time sports among girls perceiving more barriers . , access to facilities , influence of parks , etc . ) a recently published study of canadian adolescents showed that beside the number of available recreation facilities , access to facilities might also be important to consider . additionally , some studies have recently shown that the objectively measured availability and accessibility of physical activity facilities [ 3133 ] might be more important when compared to perceived environmental factors . the availability of equipment ( e.g. results showed that the availability of sedentary equipment was indeed negatively related to participation in leisure time sports , but only among boys . negative correlations between sedentary equipment and leisure time sports were only found among boys perceiving fewer barriers to be physically active . studies have shown that boys more frequently engage in screen time compared to girls , and playing computer games is considered as a rather masculine activity . therefore , it is plausible that boys more often use sedentary equipment when it is available ; whereas the availability of sedentary equipment might be less attractive to girls . future research should elaborate on these findings by adding questions on time spent using sedentary equipment or rules regarding the use of sedentary equipment at home . studies among adults revealed positive relationships between the availability of physical activity equipment in the home environment and activity levels among men and women . in the present sample of adolescents , the availability of physical activity equipment was also positively related to engagement in leisure time sports among girls . among boys , interactions with self - efficacy were found , with stronger correlations between equipment and leisure time sports among boys reporting higher levels of self - efficacy . however , in adult studies , the argument is raised that people who engage in sport activities more regularly might be more likely to purchase physical activity equipment . therefore , also among adolescents , engagement in leisure time sports might more likely be an antecedent rather than a consequence of availability of sports equipment in the home environment . in addition , for adolescents , the availability of sports equipment might also be closely related to the activity levels of other family members such as parents who are known to influence adolescents ' activity levels . participation in mvpa is essential for improved health . only the interaction between self - efficacy and supervision was significant , with stronger correlations between supervision and mvpa among those reporting higher levels of self - efficacy . however , research that further investigates the relationships between environmental factors and objectively measured mvpa in larger samples is needed to be conclusive . previous intervention studies have shown favorable changes in physical activity levels as a result of the combination of environmental strategies with personal interventions [ 27 , 28 , 39 ] . the results of the present study revealing several significant interactions between personal and environmental variables are supportive of such multicomponent intervention designs . some limitations of the present study need to be addressed . first , the questionnaire to measure school level variables was not validated yet ; therefore , conclusions concerning school environmental factors should be treated with considerable caution . the development of a comprehensive valid and reliable questionnaire to measure school environmental factors by use of self - reports is a priority for future research . although it is the strength of the present study that physical activity was also measured more objectively with accelerometers , the subsample in which these measurements occurred was very small . hence , most of the conclusions in the present study were based on self - reported physical activity measures and although validated questionnaires were used , this is a limitation . , influence of peers ) might be important to consider . furthermore , given the cross - sectional design of the present study , no causal conclusions can be drawn . although cross - sectional studies are necessary to understand the relationships between environmental factors and physical activity levels , prospective studies that further explore the causal relationships between the change in personal , intrapersonal , and environmental factors and the change in physical activity levels are needed to formulate recommendations for physical activity promotion . this explorative study provided initial insight into the relationship between school environmental factors and participation in extracurricular physical activity in a sample of european adolescents . the findings showed that increased availability of extracurricular activities and supervision are related to greater participation in extracurricular activities among adolescent boys and girls . the findings showed that perceived convenience of neighborhood facilities was unrelated , whereas the availability of sedentary and physical activity equipment at home was related to time spent in leisure time sports . overall , findings were supportive of ecological theories stating that behaviors are influenced by personal and environmental factors that are constantly interacting .

seventy - six college students ( mean age = 21.3 years old ; range = 19 27 years old ; male = 8 ; female = 68 ) completed the immediate and delayed tests in partial fulfillment of a psychology course requirement . at the beginning , ninety - one college students were invited to participate in the present study . data of fifteen participants were discarded because nine of them were over 27 years - old ( to keep the age range within 10 ) , two of them did not show up for the delayed free - recall test , and four of them did not follow instruction to provide complete data . the procedures met all american psychological association ( apa ) ethical principles for use of human subjects ( apa , 2002 ) , and participants were provided informed consent in accordance with guidelines set by the institutional review board of the university . forty stimulus words were taken from the words used by craik and tulving ( 1975 , experiment 9 , see table 1 ) . from the mrc psycholinguistic database ( wilson , 1998 ) , the average number of letters was 4.75 ( sd = .74 ) , the average number of syllables was 1.23 ( sd = .42 ) , the average printed kucera - francis word frequency was 16.3 per million ( sd = 14.45 ) , the average concreteness rating was 571.91 ( sd = 40.7 ) , and the average familiarity rating was 507.73 ( sd = 54.06 ) . a 2 2 2 mixed anovas was used with two between - subject factors of test trial ( single test , repeated test ) and processing level ( shallow , deep ) , and one within - subject factor of final recall ( immediate , delayed ) . they were then randomly assigned again to the shallow processing level and the deep processing level . therefore , there were 38 participants in each test trial ( single test and repeated test ) and each processing level ( shallow and deep ) . the design for the test trial was based on that in roediger and karpicke ( 2006a ) and wheeler and roediger ( 1992 ) . in the single test trial , participants studied the stimulus words three times and took one free - recall test in each cycle . in the repeated test trial , participants studied the stimulus words once and took three consecutive free - recall tests in each cycle . there were three cycles of study / test trials ( either ssst or sttt ) for 12 trials total . there were nine study and three test trials in the single test trial , and there were three study and nine test trials in the repeated test trial . in the shallow processing level , participants were asked whether each stimulus word was presented in capital letter or in small letter . in the deep processing level , participants were asked whether each stimulus word belonged to a particular category ( see table 1 ) . the final immediate free - recall test was administered five minutes after the 12 study and test trials , whereas the delayed free - recall test was administered one week later . they were told to study and recall a list of words , and answer some questions to help them remember the words . the task was programmed by e - prime experimental software ( version 1.1 ; schneider , eschman , & zuccolotto , 2002 ) . before the word list was presented , participants were given a practice list of two words to familiarize themselves with the task and the presentation rate , and a practice recall test to familiarize themselves with the testing procedure . the learning phase consisted of 12 study and test trials and took about 30 minutes . at the beginning of each study trial , participants were asked to rest their hands on a key labeled yes and the other on a key labeled no on the computer keyboard . prompt was shown on the computer screen for 1 s. the typescript question or category question was then shown for 1 s , and participants were asked to answer the question by pressing the appropriate key . the typescript question was asked in the form , is the word in capital letter ? or is the word in small letter ? the category question was asked in the form , is the word ( a category ) ? both typescript and category questions were counterbalanced , so that half of the answers to the questions was yes and half was no . the purpose of the question was to induce the participant to process the word at a relatively shallow level ( typescript questions ) or at a relatively deep level ( category questions ) . no matter if participants answered the typescript or category questions , stimuli words were presented on a computer at 2 s per word and the screen proceeded to the next word after 2 s. to present 40 stimuli words , the total time for one study trial was 80 s. participants who were not able to answer the questions correctly over 80% were discarded from the analysis . the beginning of each test trial was indicated by a tone ( presented over headphones for 0.5 s ) and a recall prompt that remained on the computer screen throughout the test . during each test trial , participants were given 80 s to write down as many of the words as possible , in any order , on a response booklet . therefore , the time of exposure to materials on study trials and test trials was equated ( both are 80 s ) . the transition from one test trial to another ( in the repeated test condition ) was indicated by a tone as well as a change in the background color on the computer screen : the background was blue during the first test , green during the second test , and red during the third test . at the end of each test trial , participants were instructed to turn to the next page on their response booklets and not to look back at any of their previous responses at any time during the learning phase . after the learning phase of three cycles of 12 study and test trials , participants proceeded to the testing phase and were asked to complete mazes for five minutes . participants were then given an immediate free - recall test to write down as many of the words as they could recall in 10 minutes , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . this procedure ensured that participants had exhausted their knowledge by the end of the 10 minutes recall test and allowed the researcher to measure the number of words recalled . all participants , except two , returned for the delayed free - recall test one week later . they were given 10 minutes to write down as many of the words as they could recall , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . finally , participants were asked whether they expected to be given a test in the second session and whether they consciously rehearsed the test items after the first session . at the end of the delayed free - recall test seventy - six college students ( mean age = 21.3 years old ; range = 19 27 years old ; male = 8 ; female = 68 ) completed the immediate and delayed tests in partial fulfillment of a psychology course requirement . at the beginning , ninety - one college students were invited to participate in the present study . data of fifteen participants were discarded because nine of them were over 27 years - old ( to keep the age range within 10 ) , two of them did not show up for the delayed free - recall test , and four of them did not follow instruction to provide complete data . the procedures met all american psychological association ( apa ) ethical principles for use of human subjects ( apa , 2002 ) , and participants were provided informed consent in accordance with guidelines set by the institutional review board of the university . forty stimulus words were taken from the words used by craik and tulving ( 1975 , experiment 9 , see table 1 ) . from the mrc psycholinguistic database ( wilson , 1998 ) , the average number of letters was 4.75 ( sd = .74 ) , the average number of syllables was 1.23 ( sd = .42 ) , the average printed kucera - francis word frequency was 16.3 per million ( sd = 14.45 ) , the average concreteness rating was 571.91 ( sd = 40.7 ) , and the average familiarity rating was 507.73 ( sd = 54.06 ) . a 2 2 2 mixed anovas was used with two between - subject factors of test trial ( single test , repeated test ) and processing level ( shallow , deep ) , and one within - subject factor of final recall ( immediate , delayed ) . they were then randomly assigned again to the shallow processing level and the deep processing level . therefore , there were 38 participants in each test trial ( single test and repeated test ) and each processing level ( shallow and deep ) . the design for the test trial was based on that in roediger and karpicke ( 2006a ) and wheeler and roediger ( 1992 ) . in the single test trial , participants studied the stimulus words three times and took one free - recall test in each cycle . in the repeated test trial , participants studied the stimulus words once and took three consecutive free - recall tests in each cycle . there were three cycles of study / test trials ( either ssst or sttt ) for 12 trials total . there were nine study and three test trials in the single test trial , and there were three study and nine test trials in the repeated test trial . in the shallow processing level , participants were asked whether each stimulus word was presented in capital letter or in small letter . in the deep processing level , participants were asked whether each stimulus word belonged to a particular category ( see table 1 ) . the final immediate free - recall test was administered five minutes after the 12 study and test trials , whereas the delayed free - recall test was administered one week later . participants were tested in groups of five or fewer . they were told to study and recall a list of words , and answer some questions to help them remember the words . the task was programmed by e - prime experimental software ( version 1.1 ; schneider , eschman , & zuccolotto , 2002 ) . before the word list was presented , participants were given a practice list of two words to familiarize themselves with the task and the presentation rate , and a practice recall test to familiarize themselves with the testing procedure . the learning phase consisted of 12 study and test trials and took about 30 minutes . at the beginning of each study trial , participants were asked to rest their hands on a key labeled yes and the other on a key labeled no on the computer keyboard . prompt was shown on the computer screen for 1 s. the typescript question or category question was then shown for 1 s , and participants were asked to answer the question by pressing the appropriate key . the typescript question was asked in the form , is the word in capital letter ? or is the word in small letter ? the category question was asked in the form , is the word ( a category ) ? both typescript and category questions were counterbalanced , so that half of the answers to the questions was yes and half was no . the purpose of the question was to induce the participant to process the word at a relatively shallow level ( typescript questions ) or at a relatively deep level ( category questions ) . no matter if participants answered the typescript or category questions , stimuli words were presented on a computer at 2 s per word and the screen proceeded to the next word after 2 s. to present 40 stimuli words , the total time for one study trial was 80 s. participants who were not able to answer the questions correctly over 80% were discarded from the analysis . the beginning of each test trial was indicated by a tone ( presented over headphones for 0.5 s ) and a recall prompt that remained on the computer screen throughout the test . during each test trial , participants were given 80 s to write down as many of the words as possible , in any order , on a response booklet . therefore , the time of exposure to materials on study trials and test trials was equated ( both are 80 s ) . the transition from one test trial to another ( in the repeated test condition ) was indicated by a tone as well as a change in the background color on the computer screen : the background was blue during the first test , green during the second test , and red during the third test . at the end of each test trial , participants were instructed to turn to the next page on their response booklets and not to look back at any of their previous responses at any time during the learning phase . after the learning phase of three cycles of 12 study and test trials , participants proceeded to the testing phase and were asked to complete mazes for five minutes . participants were then given an immediate free - recall test to write down as many of the words as they could recall in 10 minutes , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . this procedure ensured that participants had exhausted their knowledge by the end of the 10 minutes recall test and allowed the researcher to measure the number of words recalled . all participants , except two , returned for the delayed free - recall test one week later . they were given 10 minutes to write down as many of the words as they could recall , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . finally , participants were asked whether they expected to be given a test in the second session and whether they consciously rehearsed the test items after the first session . at the end of the delayed free - recall test the mean number of correct words recalled out of 40 words on the immediate and delayed free - recall tests is presented in figure 1 , as a function of test trial ( single test , repeated test ) and processing level ( shallow , deep ) . a significance level of .05 is used for all analyses in this study . a 2 test trial ( single test vs. repeated test ) 2 processing levels ( shallow vs. deep ) 2 final recall ( immediate vs. delay ) mixed analysis of variance ( anova ) revealed a main effect of final recall , f(1 , 72 ) = 400.446 , p < .001 , partial = .848 . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in five minutes ( 23.868 ) was significantly higher than words recalled in one week ( 14.395 ) , p < .001 . means and standard error of the number of words ( total = 40 ) recalled in immediate and delayed final recall by test trial and processing level ( n = 76 ) . results showed a main effect of test trial , f(1 , 72 ) = 13.7 , p < .001 , partial = .160 . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in single test trial ( 21.737 ) was significantly higher than those recalled in repeated test trial ( 16.526 ) , p < .001 . there was also a main effect of processing level , f(1 , 72 ) = 34.676 , p < .001 , partial = .325 . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in deep processing level ( 23.276 ) was significantly higher than those recalled in shallow processing level ( 14.987 ) , p < .001 . there was an interaction between final recall and test trial , f(1 , 72 ) = 7.119 , p = .009 , partial = .09 . there was an interaction between final recall and processing level , f(1 , 72 ) = 39.454 , p < .001 , partial = .354 . no interaction was found between test trial and processing level , f(1 , 72 ) = 1.762 , p = .189 , partial = .024 . there was an interaction between final recall , test trial and processing level , f(1 , 72 ) = 9.347 , p = .003 , partial = .115 . simple main effects analysis was then conducted to investigate the interaction between final recall and test trial ( table 2 ) . in immediate recall , the number of words recalled at the single test trial ( m = 27.11 , sd = 8.611 ) was significantly higher than those recalled in repeated test trial [ m = 20.63 , sd = 8.274 ; f(1 , 72 ) = 20.027 , p < .001 ] . in delayed recall , the number of words recalled at the single test trial ( m = 16.37 , sd = 7.134 ) was significantly higher than those recalled in repeated test trial [ m = 12.42 , sd = 7.417 ; f(1 , 72 ) = 6.719 , p = .012 ] . in single test trial , the number of words in immediate recall ( m = 27.11 , sd = 8.611 ) was significantly higher than those in delayed recall [ m = 16.37 , sd = 7.134 ; f(1 , 72 ) = 257.176 , p the number of words in immediate recall ( m = 20.63 , sd = 8.274 ) was significantly higher than those in delayed recall [ m = 12.42 , sd = 7.417 ; f(1 , 72 ) = 150.39 , p < .001 ] . simple main effects analysis was also conducted to investigate the interaction between final recall and processing level ( table 3 ) . in immediate recall , the number of words recalled at deep processing ( m = 29.5 , sd = 6.185 ) was significantly higher than those recalled in shallow processing [ m = 18.24 , sd = 7.793 ; f(1 , 72 ) = 60.621 , p < .001 ] . in delayed recall , the number of words recalled at deep processing ( m = 17.05 , sd = 6.363 ) was significantly higher than those recalled in shallow processing [ m = 11.74 , sd = 7.675 ; f(1 , 72 ) = 12.186 , p = .001 ] . in shallow processing , the number of words in immediate recall ( m = 18.24 , sd = 7.793 ) was significantly higher than those in delayed recall [ m = 11.74 , sd = 7.675 ; f(1 , 72 ) = 94.255 , p < .001 ] . in deep processing , the number of words in immediate recall ( m = 29.5 , sd = 6.185 ) was significantly higher than those in delayed recall [ m = 17.05 , sd = 6.363 ; f(1 , 72 ) = 345.646 , p < .001 ] . another simple main effects analysis was conducted to investigate the interaction among final recall , test trial and processing level ( table 4 ) . in shallow processing and immediate recall , the number of words recalled at the single test trial ( m = 21.684 , sd = 8.226 ) was significantly higher than those recalled in repeated test trial [ m = 14.789 , sd = 5.663 ; f(1 , 72 ) = 11.358 , p = .001 ] . in shallow processing and delayed recall , the number of words recalled at the single test trial ( m = 15.37 , sd = 8.348 ) was significantly higher than those recalled in repeated test trial [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 11.374 , p = .001 ] . in deep processing and immediate recall , the number of words recalled at the single test trial ( m = 32.526 , sd = 4.765 ) was significantly higher than those recalled in repeated test trial [ m = 26.474 , sd = 6.05 ; f(1 , 72 ) = 8.753 , p = .004 ] . in deep processing and delayed recall , no difference was found between the number of words recalled at the single test trial ( m = 17.37 , sd = 5.727 ) and those recalled in repeated test trial [ m = 16.74 , sd = 7.086 ; f(1 , 72 ) = .086 , p = .77 ] . in single test trial and immediate recall , the number of words in deep processing ( m = 32.526 , sd = 4.765 ) was significantly higher than those in shallow processing [ m = 21.684 , sd = 8.226 ; f(1 , 72 ) = 28.087 , p < .001 ] . in single test trial and delayed recall , no difference was found between the number of words in deep processing ( m = 17.37 , sd = 5.727 ) and those in shallow processing [ m = 15.37 , sd = 8.348 ; f(1 , 72 ) = .862 , p = .356 ] . in repeated test trial and immediate recall , the number of words in deep processing ( m = 26.474 , sd = 6.05 ) was significantly higher than those in shallow processing [ m = 14.789 , sd = 5.663 ; f(1 , 72 ) = 32.619 , p < delayed recall , the number of words in deep processing ( m = 16.74 , sd = 7.086 ) was significantly higher than those in shallow processing [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 16.064 , p < .001 ] . in single test trial and shallow processing , the number of words in immediate recall ( m = 21.684 , sd = 8.226 ) was significantly higher than those in delayed recall [ m = 15.37 , sd = 8.348 ; f(1 , 72 ) = 44.494 , p < .001 ] . in single test trial and deep processing , the number of words in immediate recall ( m = 32.526 , sd = 4.765 ) was significantly higher than those in delayed recall [ m = 17.37 , sd = 5.727 ; f(1 , 72 ) = 256.286 , p < .001 ] . in repeated test trial and shallow processing , the number of words in immediate recall ( m = 14.789 , sd = 5.663 ) was significantly higher than those in delayed recall [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 49.836 , p < .001 ] . in repeated test trial and deep processing , the number of words in immediate recall ( m = 26.474 , sd = 6.05 ) was significantly higher than those in delayed recall [ m = 16.74 , sd = 7.086 ; f(1 , 72 ) = 105.751 , p < .001 ] . the free - recall tests in the learning phase were also analyzed to see if results were similar to the free - recall tests in the testing phase ( immediate and delay ) . another 2 test trial ( single test vs. repeated test ) 2 processing levels ( shallow vs. deep ) 3 recall ( recall 1 vs. recall 2 vs. recall 3 ) mixed analysis of variance ( anova ) was conducted . similar results were found with main effects in test trial , f(1 , 72 ) = 28.704 , p < .001 , partial = .285 ; processing level , f(1 , 72 ) = 32.054 , p < .001 , partial = .308 ; and recall , f(2 , 144 ) = 124.216 , p < .001 , partial = .633 . table 5 shows that the mean number of words recalled in single test trial ( 15.588 ) was significantly higher than those recalled in repeated test trial ( 10.64 ) ; those in deep processing level ( 15.728 ) was significantly higher than those in shallow processing level ( 10.50 ) , those in the third recall test ( 16.618 ) was significantly higher than those in the second ( 13.697 ) and first recall tests ( 9.026 ) in the learning phase . however , no interactions were found between recall and test trial , f(2 , 144 ) = .362 , p = .697 , partial = .005 , recall and processing level , f(2 , 144 ) = 1.593 , p = .207 , partial = .022 ; test trial and processing level , f(1 , 72 ) = .159 , p = .691 , partial 2 = .002 ; and among recall , test trial and processing level , f(2 , 144 ) = .356 , p = .701 , partial 2 = .005 . the present study investigated the effects of test trial and processing level on immediate and delayed retention . there was an interaction between final recall and test trial ; between final recall and processing level ; and among final recall , test trial and processing level . the finding that participants in single test trial recalled more words than repeated test trial in immediate final free - recall test was consistent with previous studies that single test trial produced more short - term benefits than repeated test trial ( roediger & karpicke , 2006a ; wheeler et al . , 2003 ) . however , the dominance of single test trial over repeated test trial in delayed retention was different from previous studies that repeated test trial produced more long - term benefits than single test trial ( roediger & karpicke , 2006a ; wheeler et al . , 2003 ) . participants in the single test trial were exposed to the words in 9 study trials and those in the repeated test trial were exposed to the words in 3 study trials . the additional exposure to the words in single test trial may lead to overlearning of the words and better retention on immediate and delayed test . on the other hand , participants in the repeated test trial took 9 test trials and those in the single test trial took 3 test trials . the additional test trials were supposed to give participants in the repeated test trial more retrieval practice . bjork ( 1975 ) stated that the retrieval process increased the elaboration of memory trace and enhanced the testing effect . however , duchastel ( 1981 ) noted that the free - recall test contained no cues to assist participants in answering the test and might therefore result in recall of only part of the contents , or a lesser testing effect . with less exposure time to the words in the study trials and no cues to assist free recall in the test trials , participants in the repeated test trial failed to produce greater benefits on the delayed recall test . the finding that participants in deep processing performed better than those in shallow processing in both immediate and delayed retention was consistent with previous studies that deeper encodings led to higher levels of performance on subsequent retention test ( craik & tulving , 1975 ; jacoby et al . , 2005 ) . the effort participants put forth to differentiate if each stimulus word belonged to a particular category promoted a deep processing of the words whereas the effort to differentiate if the words were presented in capital letters encouraged a shallow processing . craik and tulving explained that memory performance depended on the elaborateness of the encoding , and retention was enhanced when the encoding context was more fully descriptive . even though no interaction was found between test trial and processing level , there was an interaction among test trial , processing level and final recall . no matter whether it was in shallow or deep processing , participants in single test trial performed better than those in repeated test trial in immediate retention . the advantage of single test trial over repeated test trial carried over to delayed retention in shallow processing , but not in deep processing . similarly , no matter whether it was in immediate or delayed retention , participants in deep processing performed better than shallow processing in repeated test trial . the advantage of deep processing over shallow processing carried over to single test trial in immediate retention , but not delayed retention . the most interesting finding was the delayed retention of participants in the deep processing and single test trial . the current study showed a dominance of single test trial over repeated test trial and deep processing over shallow processing in retention . however , the dominance of single test trial and deep processing did not happen in delayed retention . even though participants in the repeated test trial were only exposed to the words in 3 study trials , their delayed retention was the same as those in the single test trial who were exposed to the words in 9 study trials . when participants studied the words in deep processing , the number of study and test trials did not matter . no testing effect was found because the repeated test trial still did not outperform the single test trial in delayed retention . kang , mcdermott , and roediger ( 2007 ) pointed that testing could be of little help when very few items were successfully retrieved on test trials . a further look at the recall performance in the learning phase found that participants in the repeated test trial did not recall more items than those in the single test trial . with a disadvantage of the fewer items retrieved in the learning phase , the repeated test trials managed to perform the same as the single test trial in delayed retention , but did not perform well enough to bring the testing effect . the advantage of deep processing over shallow processing prevailed when participants only studied the words 3 times in repeated study trial . however , when participants studied the words 9 times in single test trial , the deep processing advantage disappeared in delayed retention . when participants studied more times , the depth of processing did not mediate the delayed retention . craik ( 2002 ) stated that initial encoding determined the potential for later retrieval , while retrieval environment determined the degree to which that potential will be realized . deep processing has the potential for assisting later performance but the retrieval environment makes the potential possible . even though shallow processing does not have the potential for greater retrieval , the number of study trials may have increased the odds of the environment for greater retrieval . the present study found the level of processing effect or the superiority of deep processing over shallow processing on subsequent retention tests , but did not find any testing effect or the superiority of repeated testing over simple testing on subsequent retention tests . even though testing effect was not found in delayed retention , the depth of processing did mediate the delayed retention . in deep processing , participants managed to perform the same no matter whether they were in single or repeated test trial . it showed that the number of study and test trials did not affect the delayed retention when participants studied the words in deep processing . once participants established a connection of the word to the category in 3 study trials , the additional 6 study trials did not further enhance retention . in single test trial when participants studied the words 9 times , they performed the same no matter whether they processed the words in shallow or deep encoding . it showed that the number of study and test trials affected the level of processing effect . even when participants processed the words in shallow encoding , they could perform as well as those in deep processing when both studied the words 9 times . in conclusion , additional study trials did not further enhance the delayed retention of words encoded in deep processing , but did enhance the delayed retention of words encoded in shallow processing .

the purpose of the present study was to investigate the effects of test trial and processing level on immediate and delayed retention . a 2 2 2 mixed anovas was used with two between - subject factors of test trial ( single test , repeated test ) and processing level ( shallow , deep ) , and one within - subject factor of final recall ( immediate , delayed ) . seventy - six college students were randomly assigned first to the single test ( studied the stimulus words three times and took one free - recall test ) and the repeated test trials ( studied the stimulus words once and took three consecutive free - recall tests ) , and then to the shallow processing level ( asked whether each stimulus word was presented in capital letter or in small letter ) and the deep processing level ( whether each stimulus word belonged to a particular category ) to study forty stimulus words . the immediate test was administered five minutes after the trials , whereas the delayed test was administered one week later . results showed that single test trial recalled more words than repeated test trial in immediate final free - recall test , participants in deep processing performed better than those in shallow processing in both immediate and delayed retention . however , the dominance of single test trial and deep processing did not happen in delayed retention . additional study trials did not further enhance the delayed retention of words encoded in deep processing , but did enhance the delayed retention of words encoded in shallow processing .

Method Participants Materials Design Procedure Results Discussion Conclusion

seventy - six college students ( mean age = 21.3 years old ; range = 19 27 years old ; male = 8 ; female = 68 ) completed the immediate and delayed tests in partial fulfillment of a psychology course requirement . data of fifteen participants were discarded because nine of them were over 27 years - old ( to keep the age range within 10 ) , two of them did not show up for the delayed free - recall test , and four of them did not follow instruction to provide complete data . a 2 2 2 mixed anovas was used with two between - subject factors of test trial ( single test , repeated test ) and processing level ( shallow , deep ) , and one within - subject factor of final recall ( immediate , delayed ) . they were then randomly assigned again to the shallow processing level and the deep processing level . therefore , there were 38 participants in each test trial ( single test and repeated test ) and each processing level ( shallow and deep ) . in the single test trial , participants studied the stimulus words three times and took one free - recall test in each cycle . in the repeated test trial , participants studied the stimulus words once and took three consecutive free - recall tests in each cycle . there were nine study and three test trials in the single test trial , and there were three study and nine test trials in the repeated test trial . in the shallow processing level , participants were asked whether each stimulus word was presented in capital letter or in small letter . in the deep processing level , participants were asked whether each stimulus word belonged to a particular category ( see table 1 ) . the final immediate free - recall test was administered five minutes after the 12 study and test trials , whereas the delayed free - recall test was administered one week later . before the word list was presented , participants were given a practice list of two words to familiarize themselves with the task and the presentation rate , and a practice recall test to familiarize themselves with the testing procedure . the transition from one test trial to another ( in the repeated test condition ) was indicated by a tone as well as a change in the background color on the computer screen : the background was blue during the first test , green during the second test , and red during the third test . after the learning phase of three cycles of 12 study and test trials , participants proceeded to the testing phase and were asked to complete mazes for five minutes . participants were then given an immediate free - recall test to write down as many of the words as they could recall in 10 minutes , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . all participants , except two , returned for the delayed free - recall test one week later . at the end of the delayed free - recall test seventy - six college students ( mean age = 21.3 years old ; range = 19 27 years old ; male = 8 ; female = 68 ) completed the immediate and delayed tests in partial fulfillment of a psychology course requirement . data of fifteen participants were discarded because nine of them were over 27 years - old ( to keep the age range within 10 ) , two of them did not show up for the delayed free - recall test , and four of them did not follow instruction to provide complete data . a 2 2 2 mixed anovas was used with two between - subject factors of test trial ( single test , repeated test ) and processing level ( shallow , deep ) , and one within - subject factor of final recall ( immediate , delayed ) . they were then randomly assigned again to the shallow processing level and the deep processing level . therefore , there were 38 participants in each test trial ( single test and repeated test ) and each processing level ( shallow and deep ) . in the single test trial , participants studied the stimulus words three times and took one free - recall test in each cycle . in the repeated test trial , participants studied the stimulus words once and took three consecutive free - recall tests in each cycle . there were nine study and three test trials in the single test trial , and there were three study and nine test trials in the repeated test trial . in the shallow processing level , participants were asked whether each stimulus word was presented in capital letter or in small letter . in the deep processing level , participants were asked whether each stimulus word belonged to a particular category ( see table 1 ) . the final immediate free - recall test was administered five minutes after the 12 study and test trials , whereas the delayed free - recall test was administered one week later . the purpose of the question was to induce the participant to process the word at a relatively shallow level ( typescript questions ) or at a relatively deep level ( category questions ) . the transition from one test trial to another ( in the repeated test condition ) was indicated by a tone as well as a change in the background color on the computer screen : the background was blue during the first test , green during the second test , and red during the third test . after the learning phase of three cycles of 12 study and test trials , participants proceeded to the testing phase and were asked to complete mazes for five minutes . participants were then given an immediate free - recall test to write down as many of the words as they could recall in 10 minutes , and were instructed to draw a line on their recall sheet to mark their progress at one minute intervals . all participants , except two , returned for the delayed free - recall test one week later . at the end of the delayed free - recall test the mean number of correct words recalled out of 40 words on the immediate and delayed free - recall tests is presented in figure 1 , as a function of test trial ( single test , repeated test ) and processing level ( shallow , deep ) . a 2 test trial ( single test vs. repeated test ) 2 processing levels ( shallow vs. deep ) 2 final recall ( immediate vs. delay ) mixed analysis of variance ( anova ) revealed a main effect of final recall , f(1 , 72 ) = 400.446 , p < .001 , partial = .848 . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in five minutes ( 23.868 ) was significantly higher than words recalled in one week ( 14.395 ) , p < .001 . means and standard error of the number of words ( total = 40 ) recalled in immediate and delayed final recall by test trial and processing level ( n = 76 ) . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in single test trial ( 21.737 ) was significantly higher than those recalled in repeated test trial ( 16.526 ) , p < .001 . further pairwise comparisons using a bonferroni correction showed that the mean number of words recalled in deep processing level ( 23.276 ) was significantly higher than those recalled in shallow processing level ( 14.987 ) , p < .001 . simple main effects analysis was then conducted to investigate the interaction between final recall and test trial ( table 2 ) . in immediate recall , the number of words recalled at the single test trial ( m = 27.11 , sd = 8.611 ) was significantly higher than those recalled in repeated test trial [ m = 20.63 , sd = 8.274 ; f(1 , 72 ) = 20.027 , p < .001 ] . in delayed recall , the number of words recalled at the single test trial ( m = 16.37 , sd = 7.134 ) was significantly higher than those recalled in repeated test trial [ m = 12.42 , sd = 7.417 ; f(1 , 72 ) = 6.719 , p = .012 ] . in single test trial , the number of words in immediate recall ( m = 27.11 , sd = 8.611 ) was significantly higher than those in delayed recall [ m = 16.37 , sd = 7.134 ; f(1 , 72 ) = 257.176 , p the number of words in immediate recall ( m = 20.63 , sd = 8.274 ) was significantly higher than those in delayed recall [ m = 12.42 , sd = 7.417 ; f(1 , 72 ) = 150.39 , p < .001 ] . simple main effects analysis was also conducted to investigate the interaction between final recall and processing level ( table 3 ) . in immediate recall , the number of words recalled at deep processing ( m = 29.5 , sd = 6.185 ) was significantly higher than those recalled in shallow processing [ m = 18.24 , sd = 7.793 ; f(1 , 72 ) = 60.621 , p < .001 ] . in delayed recall , the number of words recalled at deep processing ( m = 17.05 , sd = 6.363 ) was significantly higher than those recalled in shallow processing [ m = 11.74 , sd = 7.675 ; f(1 , 72 ) = 12.186 , p = .001 ] . in shallow processing , the number of words in immediate recall ( m = 18.24 , sd = 7.793 ) was significantly higher than those in delayed recall [ m = 11.74 , sd = 7.675 ; f(1 , 72 ) = 94.255 , p < .001 ] . in deep processing , the number of words in immediate recall ( m = 29.5 , sd = 6.185 ) was significantly higher than those in delayed recall [ m = 17.05 , sd = 6.363 ; f(1 , 72 ) = 345.646 , p < .001 ] . another simple main effects analysis was conducted to investigate the interaction among final recall , test trial and processing level ( table 4 ) . in shallow processing and immediate recall , the number of words recalled at the single test trial ( m = 21.684 , sd = 8.226 ) was significantly higher than those recalled in repeated test trial [ m = 14.789 , sd = 5.663 ; f(1 , 72 ) = 11.358 , p = .001 ] . in shallow processing and delayed recall , the number of words recalled at the single test trial ( m = 15.37 , sd = 8.348 ) was significantly higher than those recalled in repeated test trial [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 11.374 , p = .001 ] . in deep processing and immediate recall , the number of words recalled at the single test trial ( m = 32.526 , sd = 4.765 ) was significantly higher than those recalled in repeated test trial [ m = 26.474 , sd = 6.05 ; f(1 , 72 ) = 8.753 , p = .004 ] . in deep processing and delayed recall , no difference was found between the number of words recalled at the single test trial ( m = 17.37 , sd = 5.727 ) and those recalled in repeated test trial [ m = 16.74 , sd = 7.086 ; f(1 , 72 ) = .086 , p = .77 ] . in single test trial and immediate recall , the number of words in deep processing ( m = 32.526 , sd = 4.765 ) was significantly higher than those in shallow processing [ m = 21.684 , sd = 8.226 ; f(1 , 72 ) = 28.087 , p < .001 ] . in single test trial and delayed recall , no difference was found between the number of words in deep processing ( m = 17.37 , sd = 5.727 ) and those in shallow processing [ m = 15.37 , sd = 8.348 ; f(1 , 72 ) = .862 , p = .356 ] . in repeated test trial and immediate recall , the number of words in deep processing ( m = 26.474 , sd = 6.05 ) was significantly higher than those in shallow processing [ m = 14.789 , sd = 5.663 ; f(1 , 72 ) = 32.619 , p < delayed recall , the number of words in deep processing ( m = 16.74 , sd = 7.086 ) was significantly higher than those in shallow processing [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 16.064 , p < .001 ] . in single test trial and shallow processing , the number of words in immediate recall ( m = 21.684 , sd = 8.226 ) was significantly higher than those in delayed recall [ m = 15.37 , sd = 8.348 ; f(1 , 72 ) = 44.494 , p < .001 ] . in single test trial and deep processing , the number of words in immediate recall ( m = 32.526 , sd = 4.765 ) was significantly higher than those in delayed recall [ m = 17.37 , sd = 5.727 ; f(1 , 72 ) = 256.286 , p < .001 ] . in repeated test trial and shallow processing , the number of words in immediate recall ( m = 14.789 , sd = 5.663 ) was significantly higher than those in delayed recall [ m = 8.11 , sd = 4.852 ; f(1 , 72 ) = 49.836 , p < .001 ] . in repeated test trial and deep processing , the number of words in immediate recall ( m = 26.474 , sd = 6.05 ) was significantly higher than those in delayed recall [ m = 16.74 , sd = 7.086 ; f(1 , 72 ) = 105.751 , p < .001 ] . the free - recall tests in the learning phase were also analyzed to see if results were similar to the free - recall tests in the testing phase ( immediate and delay ) . another 2 test trial ( single test vs. repeated test ) 2 processing levels ( shallow vs. deep ) 3 recall ( recall 1 vs. recall 2 vs. recall 3 ) mixed analysis of variance ( anova ) was conducted . table 5 shows that the mean number of words recalled in single test trial ( 15.588 ) was significantly higher than those recalled in repeated test trial ( 10.64 ) ; those in deep processing level ( 15.728 ) was significantly higher than those in shallow processing level ( 10.50 ) , those in the third recall test ( 16.618 ) was significantly higher than those in the second ( 13.697 ) and first recall tests ( 9.026 ) in the learning phase . however , no interactions were found between recall and test trial , f(2 , 144 ) = .362 , p = .697 , partial = .005 , recall and processing level , f(2 , 144 ) = 1.593 , p = .207 , partial = .022 ; test trial and processing level , f(1 , 72 ) = .159 , p = .691 , partial 2 = .002 ; and among recall , test trial and processing level , f(2 , 144 ) = .356 , p = .701 , partial 2 = .005 . the present study investigated the effects of test trial and processing level on immediate and delayed retention . the finding that participants in single test trial recalled more words than repeated test trial in immediate final free - recall test was consistent with previous studies that single test trial produced more short - term benefits than repeated test trial ( roediger & karpicke , 2006a ; wheeler et al . however , the dominance of single test trial over repeated test trial in delayed retention was different from previous studies that repeated test trial produced more long - term benefits than single test trial ( roediger & karpicke , 2006a ; wheeler et al . participants in the single test trial were exposed to the words in 9 study trials and those in the repeated test trial were exposed to the words in 3 study trials . the additional exposure to the words in single test trial may lead to overlearning of the words and better retention on immediate and delayed test . on the other hand , participants in the repeated test trial took 9 test trials and those in the single test trial took 3 test trials . however , duchastel ( 1981 ) noted that the free - recall test contained no cues to assist participants in answering the test and might therefore result in recall of only part of the contents , or a lesser testing effect . with less exposure time to the words in the study trials and no cues to assist free recall in the test trials , participants in the repeated test trial failed to produce greater benefits on the delayed recall test . the finding that participants in deep processing performed better than those in shallow processing in both immediate and delayed retention was consistent with previous studies that deeper encodings led to higher levels of performance on subsequent retention test ( craik & tulving , 1975 ; jacoby et al . the effort participants put forth to differentiate if each stimulus word belonged to a particular category promoted a deep processing of the words whereas the effort to differentiate if the words were presented in capital letters encouraged a shallow processing . no matter whether it was in shallow or deep processing , participants in single test trial performed better than those in repeated test trial in immediate retention . the advantage of single test trial over repeated test trial carried over to delayed retention in shallow processing , but not in deep processing . similarly , no matter whether it was in immediate or delayed retention , participants in deep processing performed better than shallow processing in repeated test trial . the advantage of deep processing over shallow processing carried over to single test trial in immediate retention , but not delayed retention . the most interesting finding was the delayed retention of participants in the deep processing and single test trial . the current study showed a dominance of single test trial over repeated test trial and deep processing over shallow processing in retention . however , the dominance of single test trial and deep processing did not happen in delayed retention . even though participants in the repeated test trial were only exposed to the words in 3 study trials , their delayed retention was the same as those in the single test trial who were exposed to the words in 9 study trials . when participants studied the words in deep processing , the number of study and test trials did not matter . no testing effect was found because the repeated test trial still did not outperform the single test trial in delayed retention . a further look at the recall performance in the learning phase found that participants in the repeated test trial did not recall more items than those in the single test trial . with a disadvantage of the fewer items retrieved in the learning phase , the repeated test trials managed to perform the same as the single test trial in delayed retention , but did not perform well enough to bring the testing effect . however , when participants studied the words 9 times in single test trial , the deep processing advantage disappeared in delayed retention . when participants studied more times , the depth of processing did not mediate the delayed retention . the present study found the level of processing effect or the superiority of deep processing over shallow processing on subsequent retention tests , but did not find any testing effect or the superiority of repeated testing over simple testing on subsequent retention tests . even though testing effect was not found in delayed retention , the depth of processing did mediate the delayed retention . in deep processing , participants managed to perform the same no matter whether they were in single or repeated test trial . it showed that the number of study and test trials did not affect the delayed retention when participants studied the words in deep processing . once participants established a connection of the word to the category in 3 study trials , the additional 6 study trials did not further enhance retention . even when participants processed the words in shallow encoding , they could perform as well as those in deep processing when both studied the words 9 times . in conclusion , additional study trials did not further enhance the delayed retention of words encoded in deep processing , but did enhance the delayed retention of words encoded in shallow processing .

bacterial artificial chromosomes ( bacs ) are convenient vectors for many experiments because of their capability in propagating large dna payloads . however , their size and sequence diversity largely eliminates conventional molecular biology methods as an option for manipulating these constructs . to address this issue , techniques such as recombineering and cre / lox methods have been used to manipulate bac constructs . recombineering is the regulated use of dna recombination / repair machinery to stimulate homologous recombination between two constructs . bacterial strains used to propagate large constructs must , in general , have a reduced capacity for dna recombination and repair in order to maintain the stability of the construct they bear . recombination machinery therefore needs to be reintroduced to stimulate homologous recombination between the bac construct and targeting vector . frequently , these proteins are expressed from a donor plasmid or in a regulated manner from the host genome [ 3 , 4 ] . in both cases , expression must be extinguished after the targeting event to maintain stability of the resultant construct . cre / lox techniques have become key tools used alongside recombineering because of their simple and specific behavior . cre recombinase is a type i topoisomerase that binds a 34-bp target site defined as the lox site . the lox site is composed of two 13-bp inverted repeats separated by an 8-bp spacer . after cre binds as a dimer to a target lox site , strand exchange can occur with another cre - bound lox site . the product of the reaction is determined by the orientation of the spacers within the lox site . direct repeats result in the circularization and excision of the intervening dna leaving a single lox site . if the spacer pairs are inverted repeats , then the sequence between the two sites will be inverted in the presence of cre . cre - mediated recombination does not require energy other than the temperature necessary for enzymatic activity and requires no cofactors other than magnesium ions . recombineering in combination with cre / lox technology has been indispensable in a wide range of genetic and genomic studies . many steps in generating constructs for genetic manipulations rely heavily upon these techniques , such as placement of a lox site in a conditional targeted mutagenesis construct , the capture of homology arms to create a targeting vector , and placement and removal of selection cassettes . these manipulations generally employ only a single type of lox site and leave a functional lox site after every cre - mediated excision . this effectively limits their complexity and presents a significant hurdle in additional manipulations because the left - over site will compete with any subsequent cre / lox reactions that are attempted . lr lox variants produce a lox72 site after recombination that has reduced function as a lox site . utilizing these lr elements might allow serial modification of bacs , recycling selection cassettes after each modification , and permitting the production of bacs with sophisticated iterative modifications . while this technique has been applied to generate a double knockout in the genome of the bacteria , lactobacillus plantarum , it has yet to be demonstrated for multiple bac modifications in e. coli . to investigate the efficacy of making serial manipulations through this approach , we explored the use of several lox site variants in combination with a bac containing multiple genes of the mouse hoxb cluster to insert fluorescent reporter proteins into four adjacent hoxb genes . we utilized the spacer variants loxp , lox5171 , lox2722 , loxm2 , the inverted repeat variants lox71 ( l ) and lox66 ( r ) , and also the combinations of the spacer and inverted repeat variants [ 7 , 9 ] . our experiments demonstrate that combinations of these variants can be used to effectively achieve a series of sophisticated manipulations in bacs , which has important implications for expanding the potential of experimental approaches using bacs and recombineering . 100 g of pcrb - loxx - amp - loxx variant plasmids were digested with hindiii and spei to linearize the template . digests were precipitated in isopropanol , washed in 70% ethanol , dried , and resuspended in tris - edta buffer ph 8 . 5 g of purified template was then incubated in cre reaction buffer with 20 u of cre recombinase at 37c replicated five times . samples were removed at time point 0 , 1 m , 2 m , 5 m , 10 m , 15 m , 30 m , 1 hr , and 2 hr combined with loading dye including proteinase k and frozen . prior to gel electrophoresis , samples were treated with proteinase k for 2 hr at 37c . after electrophoresis gel bands were recorded on a typhoon imaging system and quantitated with imagequant tl v2003.02 software . mmp5 is a bac containing a region of the mouse hoxb complex ( beginning sequence of mmp-5 aagcttcacatcagccacggtaattctccatctctttttt end sequence of mmp-5 atgggctggtggctccaaagggcccccagaaagctt . ucsc genome brower coordinates ( as of 10/1/10 ) : chromosome 11 : 96165471 - 96309155 ) isolated from a library prepared by partial hind iii digest of genomic dna cloned into the pbelo backbone . dna encoding the different h2b fluorescent proteins was inserted into the respective hoxb gene at the position of amino acid 35 in the predicted hox protein sequence . in all cases , this would create a fusion protein containing the first 35 aa of the hox protein and the h2b - fp domain . h2b - mcherry was inserted in hoxb1 , h2b - yfp ( venus ) into hoxb2 , h2b - cfp ( cerulean ) into hoxb3 , and h2b - egfp into hoxb4 . primers common to all of the fluorochrome bearing selection cassettes were used for amplification . for targeting 50 bp of targeting homology targeting fragments were generated by pcr using oligos ( 50 bp homology + amplification primer ( primer list 2.12 ) ) and platinum pfx polymerase pcr system ( invitrogen ) . cycling parameters were 95c 10 min and then 30 cycles of 95c 30 sec , 6468c 30 sec , and 68c for 4 min followed by a final step of 68c for 10 min . after amplification pcr fragments were digested with dpni enzyme for 1 hr at 37c and then either used directly or gel purified using fermentas dna extraction kit as per the manufacturer 's directions . bac harboring recombineering strains of bacteria ( el350 or sw106 ) [ 4 , 10 ] were grown overnight in 3 ml of lb broth at 32c shaking at 200 rpm250 rpm . one ml of this culture was used to inoculate 50 ml of lb and grown ( 32c shaking 200250 rpm ) 23 hr until mid - log phase growth ( abs @600 nm of 0.40.6 ) . bacteria were then pelleted by centrifugation for 6 min at 5500 g , washed 2 times with 100 ml ice cold dh20 , washed a final time in 2 ml cold dh20 , and resuspended in 550 l of cold dh20 . 0.52 l of purified targeting fragment was electroporated into 50 l of electrocompetent bacteria ( 1.8 kv , 25 f , 200 ohm ) . electroporated bacteria were allowed to recover with 24 hr of growth in soc medium at 32c shaking 200250 rpm . colonies were picked , stab cultured , and inoculated into a pcr reaction containing a primer inside and outside the targeting fragment . cycling parameters were 95c 10 min and then 30 cycles of 95c 30 sec , 55c 30 sec , and 72c for 40 sec followed by a final step of 72c for 10 min . stab cultures of clones producing appropriate pcr bands were used to inoculate 5 ml lb overnight cultures . from these cultures , 800 l was used to produce glycerol stocks of each clone , and 4 ml was used for bac isolation via alkaline lysis . isolated bac pellets were dissolved in 19 l of digestion buffer and incubated at 55c for 1 hr . one l of nhei enzyme was then added to each isolate and digested for 14 hrs . digests were then loaded on a 25 cm 0.8%1% agarose gel and run at 4050 v overnight . banding patterns were then recorded by gel documentation systems or on a typhoon imaging system . streak plates made from glycerol stocks of recombined bac clones were incubated overnight at 32c . individual colonies were grown in lb + 0.1% l - arabinose for 1 hr and plated with chloramphenicol only selection . the resultant clones were analyzed by digestion analysis ( section 2.5 ) , and positive clones were then subjected to targeted recombination . lox flanked ampicillin cassettes generated in section 2.1 were transferred into the pbelo bac backbone in the el350 strain by recombineering . three clones were selected for each transfer and pbelo + cassette was purified from each clone and sequenced . clones were then grown overnight at 32c in lb + 0.1% l - arabinose and plated . resultant clones were analyzed by colony pcr ( section 2.4 ) with multiplex pcr capable of detecting all reactants and products . after two sequential rounds of targeted recombination using pcr generated targeting fragments containing two different antibiotic selection cassettes ( completed as described in section 2.2 ) , resultant clones were subjected to simultaneous selection cassette removal ( completed as described in section 2.6 ) . labeled fragments used for fcs experiments were generated by pcr using plasmids bearing the respective loxx t7 primer and a ( ) strand primer at the 5 end ampicillin ( mep-047 ) were used to produce a 360 bp amplicon which was subsequently reduced to 142 bp by digestion with nhei restriction enzyme . after column purification , fluorescence correlation spectroscopy ( fcs ) was performed with a carl zeiss lsm 510 meta microscope incorporating a confocor 3 fcs attachment and a 40x c - apochromat water objective . for all experiments the 488 nm laser power was 0.05% within the confocor software , corresponding to approximately 6 w at the sample , and the pinhole was set at one airy unit ( 70 m ) . the temperature within the microscope chamber was maintained at 37 1c . samples were pipetted directly onto # 1.5 coverslips which were placed directly on the objective with a small amount of water . intensity trajectories were collected in either 5 or 20 sec segments for a total acquisition time of 120 sec . raw data was converted into photons per 10 s time period and correlated via a fast fourier transform using custom software written in java . diffusion times were obtained by fitting the data using levenberg - marquardt nonlinear least squares using the following formula : ( 1)g()=g(0)(1+/d)1+(02/z02)(/d ) , where d is the diffusion time , 0 is the radial waist of the confocal focus , and z0 is the axial waist of the focus . the z0/0 ratio was fixed to a value of 5 for this analysis according to the recommendations of hess and webb . diffusion times were converted into hydrodynamic volumes using the following relationships : ( 2)d=024d , r = kt6d , v=43r3 , where d is the diffusion coefficient , k is boltzmann 's constant , t is the temperature in kelvin 's , is the viscosity , r is the hydrodynamic radius , and v is the hydrodynamic volume . the hydrodynamic volume scales with the molecular weight of the diffusing complex as long as there are no shape changes in the complex . this may not be the case for the cre - oligonucleotide complex . nevertheless , our analysis only requires that the bound complex has a different hydrodynamic volume than the dna alone . bac dna was purified from 500 ml of overnight culture by either quiagen or nucleobond maxi purification kits with the following modifications . alkaline lysis volumes were increased from 10 ml to 50 ml , and elution from the column was done with elution buffer heated to 70c . transgenic mice carrying modified bac dna constructs with the fluorescent proteins were generated using well - established methods [ 1416 ] . the dna was resuspended at 1 ng/l and used for pronuclear injection into fertilized eggs from a cross of c57b6/jxcba - f1 mice . injected embryos were cultured overnight to the 2-cell stage and then transferred into the uterine horns bilaterally ( 1020 embryos per side ) of anesthetized pseudopregnant recipient hosts . embryos were harvested from the hosts at 9.5 days postcoitum ( dpc ) , and transgenic founder embryos ( f0 ) were identified by genotyping for gfp . to determine whether the bac manipulations generated the expected fluorescent proteins with appropriate activity in transgenic embryos , f0 transgenic embryos were examined using fluorescent microscopy and established methods for multispectral detection [ 1722 ] . we successfully detected the four fluorescent proteins , h2b - egfp , h2b - cfp , h2b - yfp , and h2b - mcherry using a leica uv fluo 206 stereoscope with appropriate filters . global expression patterns of the reporters were scored in whole embryos , and hindbrain tissues were also dissected from the embryos and placed in flat - mount preparations to examine the specific restricted patterns of reporter expression in hindbrain segments . in both cases , the patterns of reporter activity observed correlated with previously described expression of the endogenous hoxb genes . initially to assess the possibility of utilizing multiple lox sequences simultaneously , we evaluated the reaction rates of a series of individual lox variants to verify that they were similar . figure 1 illustrates the loxp , lox5171 , lox2722 , and loxm2 spacer variants and the lox71 ( l ) , lox66 ( r ) , and lox72 inverted repeat variants [ 7 , 9 ] which we evaluated . a linear template containing various lox site pairs of was subjected to cre - mediated recombination which produced a shorter linear fragment and an excised circle ( figure 2(a ) ) . the progress of the reaction was measured by determining the ratio of the linear product / template as measured by gel electrophoresis and band quantitation . samples were taken at various time points from t = 0 to t = 120 min . loxp ( wt ) sites showed measurable recombination even at 1 min incubation with cre at 37c ( figure 2(b ) ) . this proceeded over the 120 min time course to produce a ratio of 1.5 . lox5171 sites exhibited an even higher initial reaction rate ( figure 2(b ) ) ; however , at the end time - point the lox5171 ratio was only 50.7% of the loxp ratio ( table 1 ) . lox2722 and loxm2 lagged behind loxp and lox5171 in the accumulation of cre products ( figure 2(b ) ) however ; at t = 120 min . their product ratios were 41 and 34% of the loxp ratio ( table 1 ) . cre reactions for each of the lox spacer variants produced substantial amounts of products over the time course , opening the opportunity for the use of multiple sites to generate products produced by each lox spacer variant . while the functionality of individual spacer variants has been previously demonstrated , the functionality and reaction rates of lox spacer variants combined with the inverted repeat variants lox71/66 ( lr ) have not been analyzed . we therefore generated linear dna templates containing the spacer variants combined with lr - lox variants to test the functionality and reaction rates of these lox site combinations . the relative order of reaction rates compared with the spacer variants alone remained the same , lr - loxp > lr - lox5171 > lr - lox2722 > lr - loxm2 ( figure 2(c ) ) . the lox5171 spacer again had an initial reaction rate that was higher than loxp but was not sustained . while lr - loxp and lr - lox5171 produced similar amounts of reaction products at t = 120 to loxp without lr inverted repeat mutations , the disparity between loxp and lox2722/loxm2 rates was enhanced when combined with lr lox variants . lr - lox2722 and lr - loxm2 reaction progress ratios at t = 120 were only 5.1 and 2.3% of the loxp ratio at the same time point ( table 2 ) . in fact , inclusion of lr variations of the lox site reduced the reaction rates for all of the spacer variants ( figures 2(c ) and 2(d ) ) . lr - lox5171 was the least affected and the rates of lox2722 and loxm2 were reduced almost 10 folds compared to the respective lox spacer variant alone ( table 2 ) . it has been shown that cre recombinase binds as a dimer to each lox site cooperatively with an effective affinity of 7.4 10 m and induces an asymmetric bend in lox site containing dna [ 24 , 25 ] . if cre had an altered affinity for the different lox sites or was less effective at bending the dna , this might result in altered reaction rates for lox site variants . to investigate this possibility , we generated dna fragments containing a single lox site labeled at one end with alexa fluor 488 ( figure 3(a ) ) . fluorescence correlation spectroscopy ( fcs ) was used to measure the diffusion coefficients for lox site bearing fragments in the presence or absence of cre recombinase ( figure 3(b ) ) . labeled dna fragments alone had a diffusion rate of 360 sec . dna fragments with lox spacer variants loxp , 5171 , 2722 , and m2 all had similar diffusion rates ranging between 453488 sec in the presence of cre . in contrast , all lox site variants that included the l - lox inverted repeat variant had an increased diffusion rate , suggesting a reduced binding of cre and/or inhibition of cre - mediated bending ( figure 3(b ) ) . examination of the concentration dependence of this effect on loxp with or without the l - lox inverted repeat variation showed a concentration dependence of the l - lox effect on the diffusion rate in the presence of cre ( figure 3(c ) ) . this indicates that the binding of cre to l - lox is impaired rather than the ability of cre to induce a bend in the lox containing dna , since bending should independent of cre concentration . exploiting multiple lox variants opens the possibility of multiple manipulations of the same bac or the simultaneous manipulation of multiple loci . to test the feasibility of serial modifications , we have used a mouse bac , mmp5 , which contains a portion of the mouse hoxb cluster as a substrate ( figure 4(a ) ) . the objective was to sequentially insert each of the four different fluorescent protein reporters ( h2b - egfp ( gfp ) , h2b - cerulean ( cfp ) , h2b - venus ( yfp ) , and h2b - mcherry ) into four adjacent hoxb genes and assay for predicted functional activity in transgenic mice to evaluate success of the approach ( figure 4(a ) ) . we have used a pcr strategy to generate the series of targeting fragments that encode four different fluorochrome reporter proteins . figure 4(b ) illustrates the strategy for generating and applying a fragment with specific homology arms to hoxb4 and containing green fluorescent protein ( egfp ) . in a similar manner , specific homology arms were used for targeting h2b - mcherry to hoxb1 , h2b - yfp to hoxb2 , and h2b - cfp to hoxb3 . each insertion was designed to create a fusion protein with the first 35 amino acids of the respective hox protein whose expression would be under control of the endogenous promoter . each fluorochrome was inserted into a locus by homologous recombination stimulated by temperature regulated recombination machinery in the recombineering strains el350 or sw106 [ 4 , 10 ] . positive clones from each step were selected for using kanamycin resistance conferred by the selection cassette at the 3 end of each fluorochrome targeting fragment ( figure 4(b ) ) . correct targeting events were identified by colony pcr , and potential positives were subjected to a secondary validation via a nhei digest of purified bac dna . figure 4(c ) shows the results for targeting h2b - venus into the hoxb2 gene and removal of the selection cassette , in a version of mmp5 which already contained h2b - mcherry in the hoxb1 locus . by this method , the presence of a single , correctly targeted insertion could be verified , and the integrity of the remainder of the bac could be confirmed by comparing the predicted banding patterns of the product bac with that of the initial bac used for recombination . utilizing this approach , we were able to successfully generate a hoxb bac containing the four different fluorescent protein reporters at specific sites by serial rounds of targeted homologous recombination , each followed by cre - mediated excision of the selection cassette ( figure 4 ) . the intermediate stages and final bac were verified by sequencing across homology regions and coding regions , as occasionally errors occur due to error rates in the generation of long primers or as a result of the induction of recombination machinery . we next tested the functional activity of the four targeted fluorescent protein reporters by generating transgenic mice carrying the modified mmp5 bac . a 9.5 dpc f0 mouse founder embryo carrying the mmp5 bac transgene is shown in figure 4(a ) . the fluorescent illuminations display of the same embryos shows nested and spatially restricted patterns of expression . this functionally demonstrates that each of the serially targeted reporter fragments inserted into the proper location and displayed an expression pattern appropriate for its site in the hoxb cluster . furthermore , flat - mount preparations of the dissected hindbrain from this embryo display reporter activity in rhombomeric segments that also correlate with those expected for the endogenous genes ( figure 4(a ) ) . in the serial targeting experiments , while we exploited the expected preference against utilization of lox72 ( the result of l - lox r - lox recombination ) sites in recombination , such reactions did occur . during the fourth round of cre - mediated selection cassette excision in which the attempted reaction ( l - loxp r - loxp ) was challenged with two additional lox72 sites and a lox72 - 5171 site present within the bac , many of the bac products resulting from this reaction exhibited recombination between lox72 and the l - loxp or r - loxp sites . two of 17 clones displayed the desired banding pattern while 10 of 17 had nhei digest patterns consistent with utilization of the lox72 site in the recombination ( data not shown ) . in order for multiple lox variants to be used simultaneously , cre - mediated recombination must be highly specific for recombination between homologous spacer variants . the specificity of lox5171 and lox2722 has been partially compared in vitro however , a detailed comparison of the four sites against each other has not been done . therefore , we induced cre expression in vivo in a single - copy template system challenging variants against each other in the presence of a wt loxp site ( figure 5(a ) ) . these experiments were done in a single - copy format because cre reactions done in vitro or in multicopy plasmids resulted in complex inter - molecular reaction products . as each template is challenged with cre three products are possible and multiplex pcr with four primers distinguishes among these events ( figure 5(b ) ) . after overnight l - arabinose cre induction , clones were plated and colony pcr was performed to identify the products present in each clone ( figure 5(c ) ) . while some promiscuity was observed between lox72 and lox66/71 inverted repeat lox site variants , cre - mediated recombination between nonhomologous spacers was rare . all possible combinations of lox spacer variants were tested in this manner ( table 2 ) . one instance of a loxp loxm2 recombination , however , was recorded ( table 2 ) . given that all spacer variant pairs tested against each other were done in the presence of a wt loxp site , the resultant rate of loxp loxm2 hetero - specific reactions was rare at a frequency of 1/566 . given the selectivity of lox spacer variants for homo - specific cre - mediated recombination , if multiple selection cassettes are available , simultaneous cre excision of such cassettes should be possible allowing more rapid generation of multiply targeted constructs . to verify the feasibility of this technique , we inserted two different pcr generated fluorochrome bearing targeting cassettes to hoxb1 and hoxb2 in the bac mmp5 ( figure 6(a ) ) . a h2b - egfp fusion protein was targeted into the hoxb1 locus using an l - loxp kanamycin r - loxp , and h2b - dsred was targeted into the hoxb2 locus using a l - lox-5171 ampicillin r - lox-5171 resistance cassette . subsequently , selection cassette removal was achieved by l - arabinose induced cre expression , and resultant clones were analyzed by their nhei banding pattern . of the 28 clones analyzed , 23 had recombined in the expected manner while 5 remained in an unrecombined state ( figure 6(b ) ) . in this study , we described the functional characterization of four lox spacer variants and two inverted repeat variants for use in bac modifications . while all of the spacer - inverted repeat variant combinations were functional , clearly the l - lox2722 r - lox2722 and the l - loxm2 r - loxm2 combinations had reaction rates that were between 1 - 2 orders of magnitude less than the wt loxp rate . while the rate might be increased by raising the concentration of cre in the reaction , this is not always feasible , prohibiting their use at standard concentrations of cre recombinase . by using lox spacer variants in combination with inverted repeat variants , we were able to generate a hoxb bac ( mmp5 ) with four specific , independent , and functional targeting events . we observed that lox72 sites , which are the result of a l - lox r - lox cre - mediated reaction , retain some function , although at a reduced rate . the reduced affinity of inverted repeat variants for cre , as shown by fcs , could be overcome by increased cre concentration . the amount of cre production stimulated by l - arabinose induction of recombineering bacterial strains in these experiments was unknown . it is possible that the levels of cre in these experiments exceeded the concentration range in which lox72 would exhibit reduced activity . perhaps l - arabinose concentrations could be titrated in these recombineering strains to decrease the possibility of lox72 site use in cre recombination reactions . diffusion rates of lox sites measured by fcs in the presence of cre showed that l - lox had increased diffusion compared to the wt lox site , independent of spacer sequence but related to a reduced affinity of cre for the l inverted repeat variant . while these sites had reduced binding of cre , they were still fully functional for recombination , as shown by their misdirection of selection cassette removal in the final step of a serial bac modification . fcs measurements of cre binding to inverted repeat variants demonstrated that at higher cre concentrations these variants were bound just as frequently as the wt lox site . such behavior may explain the use of cryptic lox sites ( sites of cre / lox recombination that are divergent from the known lox variants ) that are inevitably present in large genomes . while the function of inverted variants was dependent upon the concentration of cre , lox spacer variants demonstrated a high degree of fidelity in the selection of only homologous sites for recombination . if the selectivity of the cre / lox reaction is dependent primarily upon the spacer sequence , identification of cryptic lox sites by the sequence of the spacer may be a more informative method than scanning the genome with the entire lox sequence . if the functionality of the inverted repeat is limited to its affinity for cre , functional variants of the inverted repeats should be easily determined by measuring affinity of a specific sequence for cre recombinase . this could lead to the discovery of lr pairs that are even less favored than the lox72 site and are truly irreversible after recombination . these lr - lox site pairs may be useful in systems where cre concentration can be monitored and controlled however , for the large number of experiments that are done in vivo , this is unlikely to be the case , reducing the possibilities for using lr lox site pairs for multistep serial bac modifications . while lox72 showed promiscuous reactivity with other lox sites , the spacer variants showed high selectivity for reactions only between homologous sites . for four or less serial bac modifications , utilization of loxp , 5171 , 2722 , and m2 spacer variants should produce very efficient and specific reaction products . if greater numbers of modifications are required , use of inverted repeat - spacer variant combinations could expand the number of modifications possible or perhaps additional spacer variants could be identified to increase the number of specific modifications feasible . the similar rates of reaction for the lox spacer variants and their high selectivity open the possibility for their simultaneous use in vivo . with four antibiotic resistance cassettes , four serial recombinations could be achieved and the selection cassettes subsequently removed in a single step . often , when a gene is deleted in mice similar / paralogous genes can compensate for its loss and mask phenotypes . using this system , at least four floxed genes could be deleted simultaneously avoiding many complexities caused by compensation and allowing for temporal and spatial precision through regulation of cre expression .

bacterial artificial chromosomes ( bacs ) are vital tools in mouse genomic analyses because of their ability to propagate large inserts . the size of these constructs , however , prevents the use of conventional molecular biology techniques for modification and manipulation . techniques such as recombineering and cre / lox methodologies have thus become heavily relied upon for such purposes . in this work , we investigate the applicability of lox variant sites for serial and/or simultaneous manipulations of bacs . we show that lox spacer mutants are very specific , and inverted repeat variants reduce lox reaction rates through reducing the affinity of cre for the site , while retaining some functionality . employing these methods , we produced serial modifications encompassing four independent changes which generated a mouse hoxb bac with fluorescent reporter proteins inserted into four adjacent hox genes . we also generated specific , simultaneous deletions using combinations of spacer variants and inverted repeat variants . these techniques will facilitate bac manipulations and open a new repertoire of methods for bac and genome manipulation .

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

bacterial artificial chromosomes ( bacs ) are convenient vectors for many experiments because of their capability in propagating large dna payloads . however , their size and sequence diversity largely eliminates conventional molecular biology methods as an option for manipulating these constructs . to address this issue , techniques such as recombineering and cre / lox methods have been used to manipulate bac constructs . recombineering is the regulated use of dna recombination / repair machinery to stimulate homologous recombination between two constructs . bacterial strains used to propagate large constructs must , in general , have a reduced capacity for dna recombination and repair in order to maintain the stability of the construct they bear . cre / lox techniques have become key tools used alongside recombineering because of their simple and specific behavior . after cre binds as a dimer to a target lox site , strand exchange can occur with another cre - bound lox site . if the spacer pairs are inverted repeats , then the sequence between the two sites will be inverted in the presence of cre . recombineering in combination with cre / lox technology has been indispensable in a wide range of genetic and genomic studies . many steps in generating constructs for genetic manipulations rely heavily upon these techniques , such as placement of a lox site in a conditional targeted mutagenesis construct , the capture of homology arms to create a targeting vector , and placement and removal of selection cassettes . these manipulations generally employ only a single type of lox site and leave a functional lox site after every cre - mediated excision . this effectively limits their complexity and presents a significant hurdle in additional manipulations because the left - over site will compete with any subsequent cre / lox reactions that are attempted . utilizing these lr elements might allow serial modification of bacs , recycling selection cassettes after each modification , and permitting the production of bacs with sophisticated iterative modifications . to investigate the efficacy of making serial manipulations through this approach , we explored the use of several lox site variants in combination with a bac containing multiple genes of the mouse hoxb cluster to insert fluorescent reporter proteins into four adjacent hoxb genes . we utilized the spacer variants loxp , lox5171 , lox2722 , loxm2 , the inverted repeat variants lox71 ( l ) and lox66 ( r ) , and also the combinations of the spacer and inverted repeat variants [ 7 , 9 ] . our experiments demonstrate that combinations of these variants can be used to effectively achieve a series of sophisticated manipulations in bacs , which has important implications for expanding the potential of experimental approaches using bacs and recombineering . digests were precipitated in isopropanol , washed in 70% ethanol , dried , and resuspended in tris - edta buffer ph 8 . 5 g of purified template was then incubated in cre reaction buffer with 20 u of cre recombinase at 37c replicated five times . samples were removed at time point 0 , 1 m , 2 m , 5 m , 10 m , 15 m , 30 m , 1 hr , and 2 hr combined with loading dye including proteinase k and frozen . mmp5 is a bac containing a region of the mouse hoxb complex ( beginning sequence of mmp-5 aagcttcacatcagccacggtaattctccatctctttttt end sequence of mmp-5 atgggctggtggctccaaagggcccccagaaagctt . dna encoding the different h2b fluorescent proteins was inserted into the respective hoxb gene at the position of amino acid 35 in the predicted hox protein sequence . h2b - mcherry was inserted in hoxb1 , h2b - yfp ( venus ) into hoxb2 , h2b - cfp ( cerulean ) into hoxb3 , and h2b - egfp into hoxb4 . bacteria were then pelleted by centrifugation for 6 min at 5500 g , washed 2 times with 100 ml ice cold dh20 , washed a final time in 2 ml cold dh20 , and resuspended in 550 l of cold dh20 . colonies were picked , stab cultured , and inoculated into a pcr reaction containing a primer inside and outside the targeting fragment . from these cultures , 800 l was used to produce glycerol stocks of each clone , and 4 ml was used for bac isolation via alkaline lysis . the resultant clones were analyzed by digestion analysis ( section 2.5 ) , and positive clones were then subjected to targeted recombination . for all experiments the 488 nm laser power was 0.05% within the confocor software , corresponding to approximately 6 w at the sample , and the pinhole was set at one airy unit ( 70 m ) . diffusion times were obtained by fitting the data using levenberg - marquardt nonlinear least squares using the following formula : ( 1)g()=g(0)(1+/d)1+(02/z02)(/d ) , where d is the diffusion time , 0 is the radial waist of the confocal focus , and z0 is the axial waist of the focus . diffusion times were converted into hydrodynamic volumes using the following relationships : ( 2)d=024d , r = kt6d , v=43r3 , where d is the diffusion coefficient , k is boltzmann 's constant , t is the temperature in kelvin 's , is the viscosity , r is the hydrodynamic radius , and v is the hydrodynamic volume . this may not be the case for the cre - oligonucleotide complex . alkaline lysis volumes were increased from 10 ml to 50 ml , and elution from the column was done with elution buffer heated to 70c . embryos were harvested from the hosts at 9.5 days postcoitum ( dpc ) , and transgenic founder embryos ( f0 ) were identified by genotyping for gfp . to determine whether the bac manipulations generated the expected fluorescent proteins with appropriate activity in transgenic embryos , f0 transgenic embryos were examined using fluorescent microscopy and established methods for multispectral detection [ 1722 ] . we successfully detected the four fluorescent proteins , h2b - egfp , h2b - cfp , h2b - yfp , and h2b - mcherry using a leica uv fluo 206 stereoscope with appropriate filters . global expression patterns of the reporters were scored in whole embryos , and hindbrain tissues were also dissected from the embryos and placed in flat - mount preparations to examine the specific restricted patterns of reporter expression in hindbrain segments . initially to assess the possibility of utilizing multiple lox sequences simultaneously , we evaluated the reaction rates of a series of individual lox variants to verify that they were similar . figure 1 illustrates the loxp , lox5171 , lox2722 , and loxm2 spacer variants and the lox71 ( l ) , lox66 ( r ) , and lox72 inverted repeat variants [ 7 , 9 ] which we evaluated . lox5171 sites exhibited an even higher initial reaction rate ( figure 2(b ) ) ; however , at the end time - point the lox5171 ratio was only 50.7% of the loxp ratio ( table 1 ) . lox2722 and loxm2 lagged behind loxp and lox5171 in the accumulation of cre products ( figure 2(b ) ) however ; at t = 120 min . cre reactions for each of the lox spacer variants produced substantial amounts of products over the time course , opening the opportunity for the use of multiple sites to generate products produced by each lox spacer variant . while the functionality of individual spacer variants has been previously demonstrated , the functionality and reaction rates of lox spacer variants combined with the inverted repeat variants lox71/66 ( lr ) have not been analyzed . we therefore generated linear dna templates containing the spacer variants combined with lr - lox variants to test the functionality and reaction rates of these lox site combinations . the relative order of reaction rates compared with the spacer variants alone remained the same , lr - loxp > lr - lox5171 > lr - lox2722 > lr - loxm2 ( figure 2(c ) ) . while lr - loxp and lr - lox5171 produced similar amounts of reaction products at t = 120 to loxp without lr inverted repeat mutations , the disparity between loxp and lox2722/loxm2 rates was enhanced when combined with lr lox variants . in fact , inclusion of lr variations of the lox site reduced the reaction rates for all of the spacer variants ( figures 2(c ) and 2(d ) ) . lr - lox5171 was the least affected and the rates of lox2722 and loxm2 were reduced almost 10 folds compared to the respective lox spacer variant alone ( table 2 ) . it has been shown that cre recombinase binds as a dimer to each lox site cooperatively with an effective affinity of 7.4 10 m and induces an asymmetric bend in lox site containing dna [ 24 , 25 ] . if cre had an altered affinity for the different lox sites or was less effective at bending the dna , this might result in altered reaction rates for lox site variants . to investigate this possibility , we generated dna fragments containing a single lox site labeled at one end with alexa fluor 488 ( figure 3(a ) ) . fluorescence correlation spectroscopy ( fcs ) was used to measure the diffusion coefficients for lox site bearing fragments in the presence or absence of cre recombinase ( figure 3(b ) ) . dna fragments with lox spacer variants loxp , 5171 , 2722 , and m2 all had similar diffusion rates ranging between 453488 sec in the presence of cre . in contrast , all lox site variants that included the l - lox inverted repeat variant had an increased diffusion rate , suggesting a reduced binding of cre and/or inhibition of cre - mediated bending ( figure 3(b ) ) . examination of the concentration dependence of this effect on loxp with or without the l - lox inverted repeat variation showed a concentration dependence of the l - lox effect on the diffusion rate in the presence of cre ( figure 3(c ) ) . this indicates that the binding of cre to l - lox is impaired rather than the ability of cre to induce a bend in the lox containing dna , since bending should independent of cre concentration . exploiting multiple lox variants opens the possibility of multiple manipulations of the same bac or the simultaneous manipulation of multiple loci . to test the feasibility of serial modifications , we have used a mouse bac , mmp5 , which contains a portion of the mouse hoxb cluster as a substrate ( figure 4(a ) ) . the objective was to sequentially insert each of the four different fluorescent protein reporters ( h2b - egfp ( gfp ) , h2b - cerulean ( cfp ) , h2b - venus ( yfp ) , and h2b - mcherry ) into four adjacent hoxb genes and assay for predicted functional activity in transgenic mice to evaluate success of the approach ( figure 4(a ) ) . we have used a pcr strategy to generate the series of targeting fragments that encode four different fluorochrome reporter proteins . in a similar manner , specific homology arms were used for targeting h2b - mcherry to hoxb1 , h2b - yfp to hoxb2 , and h2b - cfp to hoxb3 . each fluorochrome was inserted into a locus by homologous recombination stimulated by temperature regulated recombination machinery in the recombineering strains el350 or sw106 [ 4 , 10 ] . correct targeting events were identified by colony pcr , and potential positives were subjected to a secondary validation via a nhei digest of purified bac dna . by this method , the presence of a single , correctly targeted insertion could be verified , and the integrity of the remainder of the bac could be confirmed by comparing the predicted banding patterns of the product bac with that of the initial bac used for recombination . utilizing this approach , we were able to successfully generate a hoxb bac containing the four different fluorescent protein reporters at specific sites by serial rounds of targeted homologous recombination , each followed by cre - mediated excision of the selection cassette ( figure 4 ) . this functionally demonstrates that each of the serially targeted reporter fragments inserted into the proper location and displayed an expression pattern appropriate for its site in the hoxb cluster . furthermore , flat - mount preparations of the dissected hindbrain from this embryo display reporter activity in rhombomeric segments that also correlate with those expected for the endogenous genes ( figure 4(a ) ) . during the fourth round of cre - mediated selection cassette excision in which the attempted reaction ( l - loxp r - loxp ) was challenged with two additional lox72 sites and a lox72 - 5171 site present within the bac , many of the bac products resulting from this reaction exhibited recombination between lox72 and the l - loxp or r - loxp sites . in order for multiple lox variants to be used simultaneously , cre - mediated recombination must be highly specific for recombination between homologous spacer variants . the specificity of lox5171 and lox2722 has been partially compared in vitro however , a detailed comparison of the four sites against each other has not been done . therefore , we induced cre expression in vivo in a single - copy template system challenging variants against each other in the presence of a wt loxp site ( figure 5(a ) ) . while some promiscuity was observed between lox72 and lox66/71 inverted repeat lox site variants , cre - mediated recombination between nonhomologous spacers was rare . all possible combinations of lox spacer variants were tested in this manner ( table 2 ) . one instance of a loxp loxm2 recombination , however , was recorded ( table 2 ) . given that all spacer variant pairs tested against each other were done in the presence of a wt loxp site , the resultant rate of loxp loxm2 hetero - specific reactions was rare at a frequency of 1/566 . given the selectivity of lox spacer variants for homo - specific cre - mediated recombination , if multiple selection cassettes are available , simultaneous cre excision of such cassettes should be possible allowing more rapid generation of multiply targeted constructs . to verify the feasibility of this technique , we inserted two different pcr generated fluorochrome bearing targeting cassettes to hoxb1 and hoxb2 in the bac mmp5 ( figure 6(a ) ) . a h2b - egfp fusion protein was targeted into the hoxb1 locus using an l - loxp kanamycin r - loxp , and h2b - dsred was targeted into the hoxb2 locus using a l - lox-5171 ampicillin r - lox-5171 resistance cassette . subsequently , selection cassette removal was achieved by l - arabinose induced cre expression , and resultant clones were analyzed by their nhei banding pattern . in this study , we described the functional characterization of four lox spacer variants and two inverted repeat variants for use in bac modifications . while all of the spacer - inverted repeat variant combinations were functional , clearly the l - lox2722 r - lox2722 and the l - loxm2 r - loxm2 combinations had reaction rates that were between 1 - 2 orders of magnitude less than the wt loxp rate . while the rate might be increased by raising the concentration of cre in the reaction , this is not always feasible , prohibiting their use at standard concentrations of cre recombinase . by using lox spacer variants in combination with inverted repeat variants , we were able to generate a hoxb bac ( mmp5 ) with four specific , independent , and functional targeting events . the reduced affinity of inverted repeat variants for cre , as shown by fcs , could be overcome by increased cre concentration . the amount of cre production stimulated by l - arabinose induction of recombineering bacterial strains in these experiments was unknown . it is possible that the levels of cre in these experiments exceeded the concentration range in which lox72 would exhibit reduced activity . diffusion rates of lox sites measured by fcs in the presence of cre showed that l - lox had increased diffusion compared to the wt lox site , independent of spacer sequence but related to a reduced affinity of cre for the l inverted repeat variant . while these sites had reduced binding of cre , they were still fully functional for recombination , as shown by their misdirection of selection cassette removal in the final step of a serial bac modification . fcs measurements of cre binding to inverted repeat variants demonstrated that at higher cre concentrations these variants were bound just as frequently as the wt lox site . such behavior may explain the use of cryptic lox sites ( sites of cre / lox recombination that are divergent from the known lox variants ) that are inevitably present in large genomes . while the function of inverted variants was dependent upon the concentration of cre , lox spacer variants demonstrated a high degree of fidelity in the selection of only homologous sites for recombination . if the selectivity of the cre / lox reaction is dependent primarily upon the spacer sequence , identification of cryptic lox sites by the sequence of the spacer may be a more informative method than scanning the genome with the entire lox sequence . if the functionality of the inverted repeat is limited to its affinity for cre , functional variants of the inverted repeats should be easily determined by measuring affinity of a specific sequence for cre recombinase . these lr - lox site pairs may be useful in systems where cre concentration can be monitored and controlled however , for the large number of experiments that are done in vivo , this is unlikely to be the case , reducing the possibilities for using lr lox site pairs for multistep serial bac modifications . while lox72 showed promiscuous reactivity with other lox sites , the spacer variants showed high selectivity for reactions only between homologous sites . for four or less serial bac modifications , utilization of loxp , 5171 , 2722 , and m2 spacer variants should produce very efficient and specific reaction products . if greater numbers of modifications are required , use of inverted repeat - spacer variant combinations could expand the number of modifications possible or perhaps additional spacer variants could be identified to increase the number of specific modifications feasible . the similar rates of reaction for the lox spacer variants and their high selectivity open the possibility for their simultaneous use in vivo . using this system , at least four floxed genes could be deleted simultaneously avoiding many complexities caused by compensation and allowing for temporal and spatial precision through regulation of cre expression .

data were collected prospectively in 76 full - heritage pima indians from the gila river indian community . since 1965 , all residents of the community age 5 years or older have been invited to participate in a longitudinal study of health that included a biennial health exam as well as a 75-g oral glucose tolerance test ( ogtt ) . for children under the age of 18 years , written informed consent was provided by a parent or guardian and the child assented . for this subset analysis , we selected the earliest exam in those participants who were evaluated between their 5th and 16th birthdays , with all measures recorded for bmi , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , cholesterol , and fasting and 2-h plasma glucose and insulin levels during the ogtt ( n = 1,435 ) and excluded those children with type 2 diabetes , as determined by the 2003 american diabetes association ( ada ) criteria ( 11 ) ( n = 27 ) . of the remaining 1,408 individuals , 198 participated as nonsmoking healthy adults in our study investigating metabolic determinants of type 2 diabetes ( 12 ) with complete data for m and air measurements . for the purposes of ensuring adequate separation between childhood and adult measures , only adults 21 years or older were included in this analysis . as adults , we excluded those individuals with impaired glucose regulation ( n = 117 ) and those with in utero exposure to diabetes ( n = 5 ) , a factor known to dramatically increase the risk of developing diabetes by the third decade ( 13 ) and to reduce acute insulin release in pima indians ( 14 ) . in utero exposure to diabetes was defined by a confirmed date of diabetes diagnosis in the mother that preceded the child 's birth date or an ogtt during pregnancy with a 2-h value of 11 mmol / l . in the final subset of 76 subjects ( 38% of eligible adult subjects ) , the childhood evaluations spanned the years 19791994 , and the adult visits occurred over the years 19872004 . both study protocols were approved by the institutional review board of the national institute of diabetes and digestive and kidney diseases . plasma glucose was measured by an autoanalyzer using the potassium ferricyanide method ( technicon instruments , tarrytown , ny ) . serum insulin concentrations were measured by radioimmunoassay using the herbert modification of the yalow and berson method ( 15 ) or an automated analyzer ( concept 4 ; icn radiochemicals , costa mesa , ca ) . all insulin values were converted using regression equations to the earlier radioimmunoassay values for comparability . height was measured using a stadiometer and weight using calibrated scales by a set of trained observers . ogtts ( 75 g ) were used to exclude people with diabetes before enrollment and to classify glucose tolerance . normal glucose regulation was determined using 2003 ada criteria , a fasting glucose < 5.6 mmol / l , and a 2hg < 7.8 mmol / l ( 11 ) . all plasma glucose concentrations were determined by the glucose oxidase method ( beckman instruments , fullerton , ca ) . upon admission to the clinical research unit , participants were given a weight - maintaining diet ( 50% carbohydrate , 30% fat , and 20% protein ) and asked to abstain from strenuous exercise . body composition was estimated by underwater weighing with determination of residual lung volume by helium dilution ( 20 participants ) or by total - body dual - energy x - ray absorptiometry ( dpx - l ; lunar radiation , madison , wi ) ( 56 participants ) . percent body fat measurements from the dual - energy x - ray absorptiometry scan were converted to underwater values using a conversion equation ( 16 ) . a 25-g glucose bolus was given over 3 min , and blood was drawn every minute until the fifth minute for measurement of insulin levels . the air was calculated as the average increase in plasma insulin from baseline during this time period . plasma insulin concentrations were determined by radioimmunoassay via an automated analyzer , either the concept 4 analyzer described previously or an access analyzer ( beckman instruments ) . all insulin measurements were normalized to the earliest radioimmunoassay using regression equations for comparability with childhood values . after an overnight fast , a primed continuous insulin infusion was administered through an antecubital vein at 40 mu / m per minute for 100 min . the plasma glucose concentrations were measured every 5 min after the start of the insulin infusion , and a variable infusion of 20% dextrose was used to maintain plasma glucose at 5.6 the rate of glucose required to maintain euglycemia , as a measure of insulin sensitivity ( m ) , was calculated for the last 40 min of the insulin infusion and corrected for both steady - state plasma insulin levels and endogenous glucose production . endogenous glucose production was determined at baseline ( 120 min ) and during the insulin infusion using a primed continuous infusion ( 0.3 ci / min ) of [ 3-h]glucose . m values were normalized to estimated metabolic body size , or fat - free mass + 17.7 kg ( 17 ) . subject characteristics are presented as means sd if normally distributed and as median with 25th and 75th percentiles if skewed . m , air , and insulin levels were log10 transformed to meet the assumptions of linear regression . linear regression models were used to determine subsets of childhood predictors of adult m and air using backward selection , i.e. , all factors were initially placed in the model , and the weakest factors were sequentially rejected until only those with a p < 0.1 remained . adult and childhood age , sex , and percent body fat were not made available to the stepwise procedure and thus , by default , were included in the final model . because of concerns about the small sample size relative to the number of predictor variables , the models were also run using forward selection , i.e. , the variables most strongly related to the outcome are entered into the model one at a time until none of the remaining variables improve the model , with identical results . the validity of the selected models was assessed using bootstrapping ( 18 ) with 1,000 replicates , each with a sample size of 76 , created by sampling with replacement from the original dataset . bootstrapping provides an estimate of what might be observed with multiple repetitions of a study . the backward selection procedure was applied to each of the 1,000 replicates to verify the stability of the chosen predictors in the final models . the final model for air was also applied to adult 30-min insulin levels during the ogtt , a frequently used surrogate for air . a 75-g ogtt was performed . fasting and 2-h blood samples were drawn for measurement of glucose and insulin concentrations . plasma glucose was measured by an autoanalyzer using the potassium ferricyanide method ( technicon instruments , tarrytown , ny ) . serum insulin concentrations were measured by radioimmunoassay using the herbert modification of the yalow and berson method ( 15 ) or an automated analyzer ( concept 4 ; icn radiochemicals , costa mesa , ca ) . all insulin values were converted using regression equations to the earlier radioimmunoassay values for comparability . height was measured using a stadiometer and weight using calibrated scales by a set of trained observers . ogtts ( 75 g ) were used to exclude people with diabetes before enrollment and to classify glucose tolerance . normal glucose regulation was determined using 2003 ada criteria , a fasting glucose < 5.6 mmol / l , and a 2hg < 7.8 mmol / l ( 11 ) . all plasma glucose concentrations were determined by the glucose oxidase method ( beckman instruments , fullerton , ca ) . upon admission to the clinical research unit , participants were given a weight - maintaining diet ( 50% carbohydrate , 30% fat , and 20% protein ) and asked to abstain from strenuous exercise . body composition was estimated by underwater weighing with determination of residual lung volume by helium dilution ( 20 participants ) or by total - body dual - energy x - ray absorptiometry ( dpx - l ; lunar radiation , madison , wi ) ( 56 participants ) . percent body fat measurements from the dual - energy x - ray absorptiometry scan were converted to underwater values using a conversion equation ( 16 ) . a 25-g glucose bolus was given over 3 min , and blood was drawn every minute until the fifth minute for measurement of insulin levels . the air was calculated as the average increase in plasma insulin from baseline during this time period . plasma insulin concentrations were determined by radioimmunoassay via an automated analyzer , either the concept 4 analyzer described previously or an access analyzer ( beckman instruments ) . all insulin measurements were normalized to the earliest radioimmunoassay using regression equations for comparability with childhood values . after an overnight fast , a primed continuous insulin infusion was administered through an antecubital vein at 40 mu / m per minute for 100 min . the plasma glucose concentrations were measured every 5 min after the start of the insulin infusion , and a variable infusion of 20% dextrose was used to maintain plasma glucose at 5.6 the rate of glucose required to maintain euglycemia , as a measure of insulin sensitivity ( m ) , was calculated for the last 40 min of the insulin infusion and corrected for both steady - state plasma insulin levels and endogenous glucose production . endogenous glucose production was determined at baseline ( 120 min ) and during the insulin infusion using a primed continuous infusion ( 0.3 ci / min ) of [ 3-h]glucose . m values were normalized to estimated metabolic body size , or fat - free mass + 17.7 kg ( 17 ) . subject characteristics are presented as means sd if normally distributed and as median with 25th and 75th percentiles if skewed . m , air , and insulin levels were log10 transformed to meet the assumptions of linear regression . linear regression models were used to determine subsets of childhood predictors of adult m and air using backward selection , i.e. , all factors were initially placed in the model , and the weakest factors were sequentially rejected until only those with a p < 0.1 remained . adult and childhood age , sex , and percent body fat were not made available to the stepwise procedure and thus , by default , were included in the final model . because of concerns about the small sample size relative to the number of predictor variables , the models were also run using forward selection , i.e. , the variables most strongly related to the outcome are entered into the model one at a time until none of the remaining variables improve the model , with identical results . the validity of the selected models was assessed using bootstrapping ( 18 ) with 1,000 replicates , each with a sample size of 76 , created by sampling with replacement from the original dataset . bootstrapping provides an estimate of what might be observed with multiple repetitions of a study . the backward selection procedure was applied to each of the 1,000 replicates to verify the stability of the chosen predictors in the final models . the final model for air was also applied to adult 30-min insulin levels during the ogtt , a frequently used surrogate for air . subject characteristics for the 76 individuals ( 63% male ) included in this study during both childhood and adulthood are presented in table 1 . the median duration of follow - up time between the childhood and adult evaluations was 13.4 years ( range 6.521.0 ) . a total of 55% had at least one parent who developed type 2 diabetes before the age of 45 years . however , parental diabetes before age 45 years , if included in either model , was not a significant predictor of m or air in this subset . at the childhood visit , the individuals included in this analysis compared with those 1,332 children not included were of a similar age but had significantly lower average bmi , cholesterol , sbp , fasting insulin , and 2hins levels . the differences in sbp and 2hins levels were attributable to the bmi differences . on average , the individuals in this analysis , as adults , were relatively young and obese with an average bmi of 34.2 kg / m and body fat of 32.3% . this is not substantially different from the complete group of full heritage adult pima indians who have participated in our metabolic studies . by design , as expected , there was a high degree of correlation between the childhood factors that were evaluated as predictors of m and air ( table 2 ) . the highest correlations were between bmi and insulin levels as well as glucose and insulin levels , with no difference between sexes . subject characteristics during childhood and adulthood data are means sd or medians ( 25th to 75th percentile ) . the median duration of follow - up between evaluations was 13.4 years ( range 6.521.0 ) . pearson correlations between childhood metabolic factors evaluated as predictors of adult insulin action and air data are pearson correlation coefficient ( p value ) . insulin action , air , and insulin levels were log10-transformed to approximate a normal distribution . the final regression model included childhood sbp ( p = 0.007 ) and childhood bmi ( p = 0.01 ) as negative predictors of insulin action after controlling for sex , adult percentage body fat , and child and adult age ( f = 9.62 , r = 0.46 , p < 0.0001 ) ( fig . 1 ) . these two childhood measures explained an additional 14% of the variance in adult m over the reduced model . because insulin action was log transformed , the clinical meaning of the relationship between m and the childhood factors is reported in terms of percent change in m. for every 5 kg / m increase in childhood bmi , adult m decreased by 7.4% ( 95% ci 12.7 to 1.8% ) . for every 10 mmhg increase in childhood sbp , adult m decreased by 5.0% ( 95% ci 8.4 to 1.4% ) . bootstrapping confirmed that childhood bmi and sbp were the two strongest childhood predictors of m ( chosen in 812 and 768 of the 1,000 replicates ) . simple linear regression plots between childhood predictors and adult measures ; r and p values are indicated on the graphs . a : negative association between childhood bmi and log10 adult insulin action ( m ) . b : negative association between childhood sbp and log10 adult insulin action ( m ) . c : positive relationship between log10 of childhood 2-h insulin levels during an ogtt and log10 adult air . all measures of insulin in this study were log10-transformed to meet the assumptions of linear regression . we did not have enough individuals or pubertal information to be able to separate children into pre- and postpubertal developmental stages ; however , in the small group of children with data before the age of 12 years ( n = 37 ) , the relationships between childhood factors and adult measurements maintain the same directionality as those in the larger group , but only the association between childhood bmi and adult m was significant ( data not shown ) . both childhood 2hg ( p = 0.086 ) and 2hins ( p = 0.014 ) were chosen for the final model of air , but only 2hins was statistically significant ( f = 2.68 , r = 0.23 , p = 0.017 ) ( fig . 1 ) . in a reduced model without 2hins , neither the model ( p = 0.09 ) nor 2hg ( p = 0.85 ) were statistically significant . additionally , without 2hg in the model , 2hins was no longer significant ( p = 0.08 ) , indicating that 2hg is a suppresser confounder for 2hins , i.e. , a variable associated with both the risk factor and the outcome , which when adjusted for , allows the relationship between the factor and the outcome to become apparent ( 22 ) . the insulin sensitivity index combining glucose and insulin measurements into a single factor was not significant in the model and did not improve the fit of the model . childhood 2hins explained an additional 8.6% of variance in adult air over the reduced model . adjusting for childhood bmi in the final model for every 25% increase in childhood 2hins , adult air increased 7.3% ( 95% ci 1.513.5% ) . bootstrapping also confirmed that childhood 2hins was the strongest childhood predictor of adult air ( 810 of 1,000 replicates ) . notably , childhood fasting insulin was only chosen in 177 out of the 1,000 replicates . to further corroborate these findings , the final model was applied to adult 30-min insulin levels from the ogtt , a commonly used surrogate for air . childhood 2hins was significant in the model ( = 0.7 , 95% ci 0.21.2 ) with ( p = 0.007 ) and without ( p = 0.04 ) 2hg , but the association was stronger when 2hg was included . subject characteristics for the 76 individuals ( 63% male ) included in this study during both childhood and adulthood are presented in table 1 . the median duration of follow - up time between the childhood and adult evaluations was 13.4 years ( range 6.521.0 ) . a total of 55% had at least one parent who developed type 2 diabetes before the age of 45 years . however , parental diabetes before age 45 years , if included in either model , was not a significant predictor of m or air in this subset . at the childhood visit , the individuals included in this analysis compared with those 1,332 children not included were of a similar age but had significantly lower average bmi , cholesterol , sbp , fasting insulin , and 2hins levels . the differences in sbp and 2hins levels were attributable to the bmi differences . on average , the individuals in this analysis , as adults , were relatively young and obese with an average bmi of 34.2 kg / m and body fat of 32.3% . this is not substantially different from the complete group of full heritage adult pima indians who have participated in our metabolic studies . by design , as expected , there was a high degree of correlation between the childhood factors that were evaluated as predictors of m and air ( table 2 ) . the highest correlations were between bmi and insulin levels as well as glucose and insulin levels , with no difference between sexes . subject characteristics during childhood and adulthood data are means sd or medians ( 25th to 75th percentile ) . the median duration of follow - up between evaluations was 13.4 years ( range 6.521.0 ) . pearson correlations between childhood metabolic factors evaluated as predictors of adult insulin action and air data are pearson correlation coefficient ( p value ) . insulin action , air , and insulin levels were log10-transformed to approximate a normal distribution . the final regression model included childhood sbp ( p = 0.007 ) and childhood bmi ( p = 0.01 ) as negative predictors of insulin action after controlling for sex , adult percentage body fat , and child and adult age ( f = 9.62 , r = 0.46 , p < 0.0001 ) ( fig . 1 ) . these two childhood measures explained an additional 14% of the variance in adult m over the reduced model . because insulin action was log transformed , the clinical meaning of the relationship between m and the childhood factors is reported in terms of percent change in m. for every 5 kg / m increase in childhood bmi , adult m decreased by 7.4% ( 95% ci 12.7 to 1.8% ) . for every 10 mmhg increase in childhood sbp , adult m decreased by 5.0% ( 95% ci 8.4 to 1.4% ) . bootstrapping confirmed that childhood bmi and sbp were the two strongest childhood predictors of m ( chosen in 812 and 768 of the 1,000 replicates ) . simple linear regression plots between childhood predictors and adult measures ; r and p values are indicated on the graphs . a : negative association between childhood bmi and log10 adult insulin action ( m ) . b : negative association between childhood sbp and log10 adult insulin action ( m ) . c : positive relationship between log10 of childhood 2-h insulin levels during an ogtt and log10 adult air . all measures of insulin in this study were log10-transformed to meet the assumptions of linear regression . we did not have enough individuals or pubertal information to be able to separate children into pre- and postpubertal developmental stages ; however , in the small group of children with data before the age of 12 years ( n = 37 ) , the relationships between childhood factors and adult measurements maintain the same directionality as those in the larger group , but only the association between childhood bmi and adult m was significant ( data not shown ) . both childhood 2hg ( p = 0.086 ) and 2hins ( p = 0.014 ) were chosen for the final model of air , but only 2hins was statistically significant ( f = 2.68 , r = 0.23 , p = 0.017 ) ( fig . 1 ) . in a reduced model without 2hins , neither the model ( p = 0.09 ) nor 2hg ( p = 0.85 ) were statistically significant . additionally , without 2hg in the model , 2hins was no longer significant ( p = 0.08 ) , indicating that 2hg is a suppresser confounder for 2hins , i.e. , a variable associated with both the risk factor and the outcome , which when adjusted for , allows the relationship between the factor and the outcome to become apparent ( 22 ) . the insulin sensitivity index combining glucose and insulin measurements into a single factor was not significant in the model and did not improve the fit of the model . childhood 2hins explained an additional 8.6% of variance in adult air over the reduced model . adjusting for childhood bmi in the final model for every 25% increase in childhood 2hins , adult air increased 7.3% ( 95% ci 1.513.5% ) . bootstrapping also confirmed that childhood 2hins was the strongest childhood predictor of adult air ( 810 of 1,000 replicates ) . notably , childhood fasting insulin was only chosen in 177 out of the 1,000 replicates . to further corroborate these findings , the final model was applied to adult 30-min insulin levels from the ogtt , a commonly used surrogate for air . childhood 2hins was significant in the model ( = 0.7 , 95% ci 0.21.2 ) with ( p = 0.007 ) and without ( p = 0.04 ) 2hg , but the association was stronger when 2hg was included . in this longitudinal observational study , childhood factors predicted adult insulin action and secretion in adults with normal glucose regulation , i.e. , before clinical evidence of glucose dysregulation . the main findings are first that childhood bmi and sbp are negatively associated with adult m and , second , that 2hins during a childhood ogtt is positively associated with adult air after adjustment for childhood 2hg . both m and air predict type 2 diabetes in the pima population ( 12 ) , even in individuals with normal glucose regulation ( 4 ) . in the development of type 2 diabetes , both insulin secretion and insulin action these prior studies indicate that preventing diabetes should begin at an early stage and target both insulin resistance and insulin secretion . our work expands on this idea by identifying early life measures that are associated with these pre - diabetic elements . we have previously shown that childhood metabolic factors predict development of type 2 diabetes at a young age in pima indians ( 5 ) . this study found that 2hg , waist circumference , and bmi were the strongest predictors , whereas sbp and dbp , although predictors , were relatively weak . an earlier study which did include 2hins measurements found that in pima indians , parental diabetes , weight relative to height , and 2hg predicted adult risk of type 2 diabetes ( 10 ) . in this study , weight relative to height and fasting and 2hg and insulin levels were associated with parental diabetes . we did not find parental diabetes to be an important contributor to either m or air in our study , but this may be due to use of m and air as outcomes in the present analysis and diabetes in the previous articles ( 5,10 ) . our analysis separates the physiologic components of diabetes to determine individual predictors in this population based dataset of longitudinal data . we show that these childhood factors are predictive even across life stages , with a minimum separation of 6.5 years . we purposefully only looked at children between the ages of 5 and 16 years before the completion of growth and development and reevaluated these individuals after the age of 21 years when development was complete . our data indicate that changes contributing to later risk of diabetes may begin during childhood . childhood sbp and bmi , both measures related to adiposity ( 23 ) , independently predict adult insulin action . while lifelong obesity may be a causative factor as childhood obesity is strongly associated with adult obesity ( 6,7 ) , the relationships in this study were independent of adult percent body fat . this implies that excess adiposity during development may have adverse effects independent of later body size . the association of childhood sbp with adult m may reflect common underlying mechanisms that begin early in life and may be as simple as a sedentary lifestyle or more complex such as sympathetic hyperfunction or increased inflammation . others have found that sbp in childhood , but not dbp , predicts metabolic syndrome , a component of which is impaired fasting glucose , with or without hypertension later in life ( 8) . our study adds to this previous study by separating the effects of insulin action from insulin release . in the bogalusa heart study , the corollary was found where people who developed hypertension as adults were more likely to have had insulin resistance during childhood ( 19 ) . also , in the bogalusa heart study , development of insulin resistance syndrome as an adult was strongly predicted by childhood bmi ( 20 ) . in another study , childhood bmi and sbp were related to adult insulin sensitivity ; however , these associations were attenuated when adult waist circumference and maximal aerobic capacity were included as confounders ( 21 ) . we also found that childhood insulin response during ogtt is a predictor of adult air independent of childhood bmi and adult percent body fat . the explanation for 2hg as a suppressor may be that a less robust insulin response is required to maintain a relatively low glucose level in an insulin - sensitive child . the association between 2hins and future air implies that -cell capacity may be set early in life . whether this capacity is determined by genetic factors , in utero exposures ( 14 ) , or other undiscovered reasons it has been shown that insulin release in pima indians is similar to african americans and exaggerated when compared with caucasians ( 23 ) . although the prevalence of diabetes is much higher in the pima indian population , in general , diabetes in this population has been prototypic of type 2 diabetes in other populations . overall , our population had lower childhood bmis and lower average sbp and 2hins levels than the general pima indian population with childhood measures ; the lower sbp and insulin levels were attributable to the lower bmi levels . this is likely due to a survivor effect given our selection of adults with normal glucose regulation , who may have had relatively lower childhood bmis than those excluded for impaired glucose regulation or diabetes . nevertheless , as m and air are predictors of type 2 diabetes , even in adults with normal glucose regulation , our results are still relevant . another limitation of this study is the small number of participants who met our inclusion criteria for this analysis . this may have resulted in overfitting of our model given the number of childhood factors evaluated relative to our sample size . however , we did rerun the models using forward selection , which is better suited for dealing with small sample sizes ( 22 ) with identical results . the results from our analysis are also consistent with our bootstrapped analysis and with the literature previously mentioned . nevertheless , given the small sample size , the findings should be interpreted cautiously pending replication in other studies . in conclusion , abnormalities contributing to adult insulin resistance and release and , thus , risk of diabetes appear to begin in childhood or earlier . while it is important to implement preventive lifestyle measures once impaired glucose regulation develops , identifying childhood factors that contribute to or associate with pathological declines in m and air may uncover earlier preventive targets in advance of clinical evidence of dysfunction . in particular , our study implies that prevention of future declines in insulin action may be possible through modification of childhood body weight , but insulin secretion may not be modifiable through lifestyle interventions .

objectivebecause declines in acute insulin response ( air ) and insulin action ( m ) predict development of type 2 diabetes , we sought to determine childhood factors that predict insulin action and air using longitudinal data from young pima indian adults with normal glucose regulation.research design and methodspredictors of adult m , measured by the euglycemic - hyperinsulinemic clamp , and air , measured after a 25-g glucose bolus , were assessed in 76 individuals from a set of childhood data ( bmi , systolic blood pressure [ sbp ] and diastolic blood pressure , cholesterol , fasting and 2-h insulin , and glucose levels during an oral glucose tolerance test).resultsafter adjustment for sex , adult percent body fat , adult and childhood age , childhood bmi , and sbp were negative and independent predictors of adult m. a 5 kg / m2 increase in childhood bmi was associated with a 7.4% decrease in adult insulin action ( 95% ci 12.7 to 1.8% , p = 0.01 ) and a 10-mmhg increase in childhood sbp with a 5.0% decrease in adult m ( 95% ci 8.4 to 1.4% , p = 0.007 ) . after a similar adjustment with m as an additional covariate , childhood 2-h insulin was a positive predictor of adult air such that a 25% increase predicted a 7.3% increase in adult air ( 95% ci 1.513.5% , p = 0.014).conclusionschildhood insulin response during an oral glucose challenge predicts adult air , indicating that -cell capacity may be set early in life . childhood measures related to adiposity predict adult insulin action , which may reflect common underlying mechanisms that may be amenable to modification through programs targeting prevention or treatment of childhood obesity .

RESEARCH DESIGN AND METHODS Childhood measurements Adult measurements Statistical analysis RESULTS Subject characteristics Childhood predictors of adult insulin action Childhood predictors of adult acute insulin secretion CONCLUSIONS

since 1965 , all residents of the community age 5 years or older have been invited to participate in a longitudinal study of health that included a biennial health exam as well as a 75-g oral glucose tolerance test ( ogtt ) . for this subset analysis , we selected the earliest exam in those participants who were evaluated between their 5th and 16th birthdays , with all measures recorded for bmi , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , cholesterol , and fasting and 2-h plasma glucose and insulin levels during the ogtt ( n = 1,435 ) and excluded those children with type 2 diabetes , as determined by the 2003 american diabetes association ( ada ) criteria ( 11 ) ( n = 27 ) . of the remaining 1,408 individuals , 198 participated as nonsmoking healthy adults in our study investigating metabolic determinants of type 2 diabetes ( 12 ) with complete data for m and air measurements . as adults , we excluded those individuals with impaired glucose regulation ( n = 117 ) and those with in utero exposure to diabetes ( n = 5 ) , a factor known to dramatically increase the risk of developing diabetes by the third decade ( 13 ) and to reduce acute insulin release in pima indians ( 14 ) . in the final subset of 76 subjects ( 38% of eligible adult subjects ) , the childhood evaluations spanned the years 19791994 , and the adult visits occurred over the years 19872004 . plasma glucose was measured by an autoanalyzer using the potassium ferricyanide method ( technicon instruments , tarrytown , ny ) . serum insulin concentrations were measured by radioimmunoassay using the herbert modification of the yalow and berson method ( 15 ) or an automated analyzer ( concept 4 ; icn radiochemicals , costa mesa , ca ) . height was measured using a stadiometer and weight using calibrated scales by a set of trained observers . normal glucose regulation was determined using 2003 ada criteria , a fasting glucose < 5.6 mmol / l , and a 2hg < 7.8 mmol / l ( 11 ) . upon admission to the clinical research unit , participants were given a weight - maintaining diet ( 50% carbohydrate , 30% fat , and 20% protein ) and asked to abstain from strenuous exercise . percent body fat measurements from the dual - energy x - ray absorptiometry scan were converted to underwater values using a conversion equation ( 16 ) . a 25-g glucose bolus was given over 3 min , and blood was drawn every minute until the fifth minute for measurement of insulin levels . the plasma glucose concentrations were measured every 5 min after the start of the insulin infusion , and a variable infusion of 20% dextrose was used to maintain plasma glucose at 5.6 the rate of glucose required to maintain euglycemia , as a measure of insulin sensitivity ( m ) , was calculated for the last 40 min of the insulin infusion and corrected for both steady - state plasma insulin levels and endogenous glucose production . m , air , and insulin levels were log10 transformed to meet the assumptions of linear regression . linear regression models were used to determine subsets of childhood predictors of adult m and air using backward selection , i.e. , all factors were initially placed in the model , and the weakest factors were sequentially rejected until only those with a p < 0.1 remained . adult and childhood age , sex , and percent body fat were not made available to the stepwise procedure and thus , by default , were included in the final model . the final model for air was also applied to adult 30-min insulin levels during the ogtt , a frequently used surrogate for air . fasting and 2-h blood samples were drawn for measurement of glucose and insulin concentrations . plasma glucose was measured by an autoanalyzer using the potassium ferricyanide method ( technicon instruments , tarrytown , ny ) . height was measured using a stadiometer and weight using calibrated scales by a set of trained observers . ogtts ( 75 g ) were used to exclude people with diabetes before enrollment and to classify glucose tolerance . normal glucose regulation was determined using 2003 ada criteria , a fasting glucose < 5.6 mmol / l , and a 2hg < 7.8 mmol / l ( 11 ) . upon admission to the clinical research unit , participants were given a weight - maintaining diet ( 50% carbohydrate , 30% fat , and 20% protein ) and asked to abstain from strenuous exercise . percent body fat measurements from the dual - energy x - ray absorptiometry scan were converted to underwater values using a conversion equation ( 16 ) . a 25-g glucose bolus was given over 3 min , and blood was drawn every minute until the fifth minute for measurement of insulin levels . the plasma glucose concentrations were measured every 5 min after the start of the insulin infusion , and a variable infusion of 20% dextrose was used to maintain plasma glucose at 5.6 the rate of glucose required to maintain euglycemia , as a measure of insulin sensitivity ( m ) , was calculated for the last 40 min of the insulin infusion and corrected for both steady - state plasma insulin levels and endogenous glucose production . m , air , and insulin levels were log10 transformed to meet the assumptions of linear regression . linear regression models were used to determine subsets of childhood predictors of adult m and air using backward selection , i.e. , all factors were initially placed in the model , and the weakest factors were sequentially rejected until only those with a p < 0.1 remained . adult and childhood age , sex , and percent body fat were not made available to the stepwise procedure and thus , by default , were included in the final model . , the variables most strongly related to the outcome are entered into the model one at a time until none of the remaining variables improve the model , with identical results . the final model for air was also applied to adult 30-min insulin levels during the ogtt , a frequently used surrogate for air . subject characteristics for the 76 individuals ( 63% male ) included in this study during both childhood and adulthood are presented in table 1 . a total of 55% had at least one parent who developed type 2 diabetes before the age of 45 years . at the childhood visit , the individuals included in this analysis compared with those 1,332 children not included were of a similar age but had significantly lower average bmi , cholesterol , sbp , fasting insulin , and 2hins levels . on average , the individuals in this analysis , as adults , were relatively young and obese with an average bmi of 34.2 kg / m and body fat of 32.3% . by design , as expected , there was a high degree of correlation between the childhood factors that were evaluated as predictors of m and air ( table 2 ) . pearson correlations between childhood metabolic factors evaluated as predictors of adult insulin action and air data are pearson correlation coefficient ( p value ) . insulin action , air , and insulin levels were log10-transformed to approximate a normal distribution . the final regression model included childhood sbp ( p = 0.007 ) and childhood bmi ( p = 0.01 ) as negative predictors of insulin action after controlling for sex , adult percentage body fat , and child and adult age ( f = 9.62 , r = 0.46 , p < 0.0001 ) ( fig . these two childhood measures explained an additional 14% of the variance in adult m over the reduced model . because insulin action was log transformed , the clinical meaning of the relationship between m and the childhood factors is reported in terms of percent change in m. for every 5 kg / m increase in childhood bmi , adult m decreased by 7.4% ( 95% ci 12.7 to 1.8% ) . for every 10 mmhg increase in childhood sbp , adult m decreased by 5.0% ( 95% ci 8.4 to 1.4% ) . bootstrapping confirmed that childhood bmi and sbp were the two strongest childhood predictors of m ( chosen in 812 and 768 of the 1,000 replicates ) . a : negative association between childhood bmi and log10 adult insulin action ( m ) . b : negative association between childhood sbp and log10 adult insulin action ( m ) . c : positive relationship between log10 of childhood 2-h insulin levels during an ogtt and log10 adult air . we did not have enough individuals or pubertal information to be able to separate children into pre- and postpubertal developmental stages ; however , in the small group of children with data before the age of 12 years ( n = 37 ) , the relationships between childhood factors and adult measurements maintain the same directionality as those in the larger group , but only the association between childhood bmi and adult m was significant ( data not shown ) . both childhood 2hg ( p = 0.086 ) and 2hins ( p = 0.014 ) were chosen for the final model of air , but only 2hins was statistically significant ( f = 2.68 , r = 0.23 , p = 0.017 ) ( fig . in a reduced model without 2hins , neither the model ( p = 0.09 ) nor 2hg ( p = 0.85 ) were statistically significant . additionally , without 2hg in the model , 2hins was no longer significant ( p = 0.08 ) , indicating that 2hg is a suppresser confounder for 2hins , i.e. , a variable associated with both the risk factor and the outcome , which when adjusted for , allows the relationship between the factor and the outcome to become apparent ( 22 ) . childhood 2hins explained an additional 8.6% of variance in adult air over the reduced model . adjusting for childhood bmi in the final model for every 25% increase in childhood 2hins , adult air increased 7.3% ( 95% ci 1.513.5% ) . bootstrapping also confirmed that childhood 2hins was the strongest childhood predictor of adult air ( 810 of 1,000 replicates ) . notably , childhood fasting insulin was only chosen in 177 out of the 1,000 replicates . childhood 2hins was significant in the model ( = 0.7 , 95% ci 0.21.2 ) with ( p = 0.007 ) and without ( p = 0.04 ) 2hg , but the association was stronger when 2hg was included . a total of 55% had at least one parent who developed type 2 diabetes before the age of 45 years . at the childhood visit , the individuals included in this analysis compared with those 1,332 children not included were of a similar age but had significantly lower average bmi , cholesterol , sbp , fasting insulin , and 2hins levels . on average , the individuals in this analysis , as adults , were relatively young and obese with an average bmi of 34.2 kg / m and body fat of 32.3% . by design , as expected , there was a high degree of correlation between the childhood factors that were evaluated as predictors of m and air ( table 2 ) . pearson correlations between childhood metabolic factors evaluated as predictors of adult insulin action and air data are pearson correlation coefficient ( p value ) . insulin action , air , and insulin levels were log10-transformed to approximate a normal distribution . the final regression model included childhood sbp ( p = 0.007 ) and childhood bmi ( p = 0.01 ) as negative predictors of insulin action after controlling for sex , adult percentage body fat , and child and adult age ( f = 9.62 , r = 0.46 , p < 0.0001 ) ( fig . these two childhood measures explained an additional 14% of the variance in adult m over the reduced model . because insulin action was log transformed , the clinical meaning of the relationship between m and the childhood factors is reported in terms of percent change in m. for every 5 kg / m increase in childhood bmi , adult m decreased by 7.4% ( 95% ci 12.7 to 1.8% ) . for every 10 mmhg increase in childhood sbp , adult m decreased by 5.0% ( 95% ci 8.4 to 1.4% ) . bootstrapping confirmed that childhood bmi and sbp were the two strongest childhood predictors of m ( chosen in 812 and 768 of the 1,000 replicates ) . a : negative association between childhood bmi and log10 adult insulin action ( m ) . b : negative association between childhood sbp and log10 adult insulin action ( m ) . c : positive relationship between log10 of childhood 2-h insulin levels during an ogtt and log10 adult air . we did not have enough individuals or pubertal information to be able to separate children into pre- and postpubertal developmental stages ; however , in the small group of children with data before the age of 12 years ( n = 37 ) , the relationships between childhood factors and adult measurements maintain the same directionality as those in the larger group , but only the association between childhood bmi and adult m was significant ( data not shown ) . both childhood 2hg ( p = 0.086 ) and 2hins ( p = 0.014 ) were chosen for the final model of air , but only 2hins was statistically significant ( f = 2.68 , r = 0.23 , p = 0.017 ) ( fig . in a reduced model without 2hins , neither the model ( p = 0.09 ) nor 2hg ( p = 0.85 ) were statistically significant . additionally , without 2hg in the model , 2hins was no longer significant ( p = 0.08 ) , indicating that 2hg is a suppresser confounder for 2hins , i.e. , a variable associated with both the risk factor and the outcome , which when adjusted for , allows the relationship between the factor and the outcome to become apparent ( 22 ) . the insulin sensitivity index combining glucose and insulin measurements into a single factor was not significant in the model and did not improve the fit of the model . childhood 2hins explained an additional 8.6% of variance in adult air over the reduced model . adjusting for childhood bmi in the final model for every 25% increase in childhood 2hins , adult air increased 7.3% ( 95% ci 1.513.5% ) . bootstrapping also confirmed that childhood 2hins was the strongest childhood predictor of adult air ( 810 of 1,000 replicates ) . notably , childhood fasting insulin was only chosen in 177 out of the 1,000 replicates . childhood 2hins was significant in the model ( = 0.7 , 95% ci 0.21.2 ) with ( p = 0.007 ) and without ( p = 0.04 ) 2hg , but the association was stronger when 2hg was included . in this longitudinal observational study , childhood factors predicted adult insulin action and secretion in adults with normal glucose regulation , i.e. the main findings are first that childhood bmi and sbp are negatively associated with adult m and , second , that 2hins during a childhood ogtt is positively associated with adult air after adjustment for childhood 2hg . both m and air predict type 2 diabetes in the pima population ( 12 ) , even in individuals with normal glucose regulation ( 4 ) . in the development of type 2 diabetes , both insulin secretion and insulin action these prior studies indicate that preventing diabetes should begin at an early stage and target both insulin resistance and insulin secretion . we have previously shown that childhood metabolic factors predict development of type 2 diabetes at a young age in pima indians ( 5 ) . this study found that 2hg , waist circumference , and bmi were the strongest predictors , whereas sbp and dbp , although predictors , were relatively weak . an earlier study which did include 2hins measurements found that in pima indians , parental diabetes , weight relative to height , and 2hg predicted adult risk of type 2 diabetes ( 10 ) . in this study , weight relative to height and fasting and 2hg and insulin levels were associated with parental diabetes . we did not find parental diabetes to be an important contributor to either m or air in our study , but this may be due to use of m and air as outcomes in the present analysis and diabetes in the previous articles ( 5,10 ) . our analysis separates the physiologic components of diabetes to determine individual predictors in this population based dataset of longitudinal data . we show that these childhood factors are predictive even across life stages , with a minimum separation of 6.5 years . childhood sbp and bmi , both measures related to adiposity ( 23 ) , independently predict adult insulin action . while lifelong obesity may be a causative factor as childhood obesity is strongly associated with adult obesity ( 6,7 ) , the relationships in this study were independent of adult percent body fat . the association of childhood sbp with adult m may reflect common underlying mechanisms that begin early in life and may be as simple as a sedentary lifestyle or more complex such as sympathetic hyperfunction or increased inflammation . others have found that sbp in childhood , but not dbp , predicts metabolic syndrome , a component of which is impaired fasting glucose , with or without hypertension later in life ( 8) . also , in the bogalusa heart study , development of insulin resistance syndrome as an adult was strongly predicted by childhood bmi ( 20 ) . in another study , childhood bmi and sbp were related to adult insulin sensitivity ; however , these associations were attenuated when adult waist circumference and maximal aerobic capacity were included as confounders ( 21 ) . we also found that childhood insulin response during ogtt is a predictor of adult air independent of childhood bmi and adult percent body fat . the explanation for 2hg as a suppressor may be that a less robust insulin response is required to maintain a relatively low glucose level in an insulin - sensitive child . the association between 2hins and future air implies that -cell capacity may be set early in life . although the prevalence of diabetes is much higher in the pima indian population , in general , diabetes in this population has been prototypic of type 2 diabetes in other populations . overall , our population had lower childhood bmis and lower average sbp and 2hins levels than the general pima indian population with childhood measures ; the lower sbp and insulin levels were attributable to the lower bmi levels . this is likely due to a survivor effect given our selection of adults with normal glucose regulation , who may have had relatively lower childhood bmis than those excluded for impaired glucose regulation or diabetes . nevertheless , as m and air are predictors of type 2 diabetes , even in adults with normal glucose regulation , our results are still relevant . this may have resulted in overfitting of our model given the number of childhood factors evaluated relative to our sample size . however , we did rerun the models using forward selection , which is better suited for dealing with small sample sizes ( 22 ) with identical results . in conclusion , abnormalities contributing to adult insulin resistance and release and , thus , risk of diabetes appear to begin in childhood or earlier . while it is important to implement preventive lifestyle measures once impaired glucose regulation develops , identifying childhood factors that contribute to or associate with pathological declines in m and air may uncover earlier preventive targets in advance of clinical evidence of dysfunction . in particular , our study implies that prevention of future declines in insulin action may be possible through modification of childhood body weight , but insulin secretion may not be modifiable through lifestyle interventions .

quantum memories are the essential building blocks in a number of quantum information processing protocols . examples include building a quantum network connecting space - separated atomic memories via optical fibers , quantum computation , quantum communication , entanglement and multiple photon generation on demand . a number of different implementations of quantum memory have been realized so far including electromagnetically induced transparency , raman scattering , atomic frequency comb , gradient echo memory and coherent population oscillations . among the most popular media for storage of the quantum information are cold alkaline atoms [ 1417 ] , room temperature atomic vapors in cells [ 9,1821 ] and in hollow - core fibers , molecular gases , nv - centers and optical phonons in diamonds , and rare - earth - doped solids . in this work , we focus on probably the most widespread and renowned implementation based on warm alkali atom vapor ( rubidium-87 ) contained in a glass cell together with noble buffer gas ( krypton ) to reduce the effects of thermal motion . photons are interfaced with atomic collective excitations , spin waves , via off - resonant raman transitions . we operate on memory in spatially multimode regime using large diameter driving beams . in general , multimode operation is natural and readily achievable in a number of spontaneous and parametric processes [ 3133 ] such as spontaneous parametric down - conversion in nonlinear crystals and instantaneous four - wave mixing in warm atomic vapors . in our application , spatially multimode operational mode can be applied to store transversely varying information , such as images , in quantum memories . in our setup , typically over one hundred distinguishable spatial modes of raman light are coupled with independent spin - wave modes . spontaneous raman scattering in warm and high optical depth ensembles is most efficient at the detuning of the order of 1 ghz and requires significant drive laser energy . thus , filtering out of the driving beams requires very large attenuation , around 1011 . in our experiments , first we use crystal polarizers , as the raman photons are polarized orthogonally to the driving light . next , a rubidium-85 absorption filter of magnetically broadened absorption line width is used to stop any remaining leakage . the second type of noise is an omnidirectional spontaneous fluorescence directly from the excited state , observed in a number of experiments and discussed theoretically [ 4346 ] . our experiments also indicate the presence of broadband and omnidirectional noise light of an intensity proportional both to the concentration of rubidium and the buffer gas , likely due to the scattering of laser light by the long - lived rb - buffer gas molecules . to filter out this light , a narrow bandpass filter transmitting the raman light only is necessary . however , it is unsuitable for our purposes , because it can not transmit hundreds of spatial modes at the same frequency . therefore , we use a faraday filter with rubidium-87 vapor rotator as a second stage . by properly adjusting the temperature and magnetic field in both filters , we are able to attenuate the driving beams by a factor of 1011 and reach a transmission of 65% for the stokes and 45% for the anti - stokes light . we demonstrate the immense effect of combined filtering on the statistical properties of the registered light . however , only due to the application of the second filter intensity correlations between the stokes and anti - stokes scattered light can be seen down to a single - photon level . the approach we demonstrate is a robust alternative to filtering with the use of interference cavities . it is insensitive to direction and precise alignment although its tuning is possible just through adjusting the magnetic field and temperature in both filters . in our experiments , we start with an ensemble of 87rb atoms pumped to f=1 state . we write to the memory by driving spontaneous stokes transition , which produces pairs of collective excitations to the f=2 state , i.e. spin waves and scattered stokes photons . after adjustable storage time , the spin wave can be converted to anti - stokes photons by sending read laser pulse . both drive lasers work near d1-line transitions . the detunings of write and read lasers from resonances f=1f=1 and f=2f=2 equal w=1 ghz and r=800 mhz , respectively , as indicated in figure 1 . write and read lasers are virtually resonant with doppler- and magnetically broadened 85rb isotope transitions in absorption filter as opposed to scattered photons . ( the colour version of this figure is included in the online version of the journal . ) the simplified version of the experimental setup is depicted in figure 2 . we utilize three external cavity diode lasers for optical pumping and raman scattering in the 87rb cell . the raman scattered light has a polarization orthogonal to the driving lasers , so we use wollaston prisms to pre - filter the raman scattered light . due to the finite extinction of the wollaston prisms ( 105:1 ) , a small portion of laser light leaks through together with the raman scattered light . usage of the 85rb leads to a high absorption coefficient of the driving lasers , whereas transmission of raman scattered light remains at the level of 80% . properties of the atomic absorption filter are independent of the transmitting light properties , e.g. direction , spatial mode profile or polarization , in contrast to the properties of the faraday or voigt filters . finally , the broadband light coming from the resonant fluorescence is filtered out with the use of the faraday filter . experimental setup of quantum memory and filters system . from top - left corner : 87rb atomic memory cell inside magnetic shielding , magnetically broadened 85rb absorption filter , faraday filter and a low - noise , single - photon - sensitive intensified scmos camera ( i - scmos ) as a detector . the blue squares stand for polarizing beam splitter cubes or wollaston polarizers ( woll ) . ( the colour version of this figure is included in the online version of the journal . ) the photons are detected with a high - resolution scmos camera with an image intensifier ( i - scmos ) [ 5154 ] . this enables us to work in a single - photon - level regime and to measure photon statistics in a similar manner as with multiplexed on off detectors [ 51,5355 ] . further details of the experimental setup and the full operational scheme of our quantum memory protocol are described in . absorption and transmission spectra of the three rubidium cells used in the experiment are presented in figure 3 along with the positions of the laser beams frequencies , as well as stokes and anti - stokes scattered light . detuning of about 1 ghz for both write - in and readout beams is a compromise between the high efficiency of raman scattering and the low absorption coefficient inside the filtering cells . ( b ) 85rb absorption filter at t=100c and ( c ) 87rb faraday filter for the magnetic field amplitude b=10 - 3 t and temperature t=102c . the frequency of the laser beams and scattered stokes and anti - stokes light are marked by detunings measured from f=2f=2 transition on d1-line in 87rb . ( the colour version of this figure is included in the online version of the journal . ) the 30-cm long rubidium 85rb cell in the transverse static magnetic field , drawn in figure 4 , is used to stop the strong laser beams and transmit the raman scattered single photons which are very narrowband but their frequencies lie out of the wings region of the spectrum . the transverse magnetic field inside the filter is emitted by the ferrite magnets located on both sides of the rubidium cell . by changing the distance between the magnets , we effectively change the magnetic field inside the cell and thence the line broadening . the width of the absorption spectrum controlled by the magnetic field ranges from 5.5 ghz up to 8.4 ghz , depending on the distance between the magnets , as depicted in figure 5 . for our purposes , we set the magnetic field to 10 - 2 t for a 9-cm distance between magnets , to move 85rb lines close enough to 87rb where driving lasers operate . the opaqueness and to some extent the width of the filter lines are tuned by changing the temperature of the filter . the filter is heated in a range of 20-140c to vary the optical density of atomic vapor that grows exponentially with temperature . in practice , the operational range is limited , as for temperatures below 90c the absorption of drive lasers is too low , while above 120c the transmission of the raman scattered light drops abruptly . the whole filter is inserted inside the two - layer soft steel magnetic shielding that confines the magnetic field inside the filter suppressing the stray external fields down to bext10 - 4 t. we heat the vapors in the rubidium cell using the high - power resistor mounted under the cell which dissipates around p80 w power . filter is embedded in a two - layer magnetic shielding protecting main quantum memory cell from the corrupting stray magnetic field . ( the colour version of this figure is included in the online version of the journal . ) large broadening of the lines of absorption filter due to transverse magnetic field , tuned by changing distance between ferrite magnets . for visualization purposes presented transmission spectra were measured below operation temperature , at t=60c . for our operation , we selected a distance of 9 cm corresponding to 10 - 2 t inside the cell with 85rb . ( the colour version of this figure is included in the online version of the journal . ) 85rb filter transmission spectra for a range of temperatures . absorption lines are significantly broadened due to transverse magnetic field of approximately 10 - 2 t. the detuning is measured from f=2f=2 transition on the d1-line of 87rb . ( the colour version of this figure is included in the online version of the journal . ) measured transmission spectra for different temperatures in the frequency range of both lasers and the scattered light are shown in figure 6 . for large positive detunings , the wing of the spectrum transmission is smaller than for negative detunings due to the residual content of 87rb in the filter cell . for optimal temperature tabs=100c , the suppression of the drive lasers is 50 db , while the transmission of the raman scattered light is at the level of 80% . the frequencies of stokes and anti - stokes scattered light in figure 6 correspond to detunings s=-2.3 and as=7.8 ghz , respectively . to filter out the broadband fluorescence and four - wave mixing accompanying write - in and readout from the memory , we utilize a faraday filter which consists of two orthogonal polarizers and a faraday rotator in between . in this arrangement , we use the standard configuration of the filter , but for temperature stabilization purposes , we apply water cooling to compensate the relatively high power emitted by coils producing magnetic field . the rotation of the polarization occurs in a 30-cm long cell containing the 87rb isotope heated up to 100c for us to obtain appropriate optical density of vapors . the magnetic field inside the filter is about b=10 - 2 t and directed along the beam . the average magnetic field does not depend on the transverse position inside the cell , i.e. it is near perfectly uniform in each plane perpendicular to the laser beam direction . the imperfections of the magnetic field in the transverse directions are smaller than 10 - 5 t. the cell is placed in a pvc tube and heated with a current flowing through the coils wound around the 87rb glass cell . the filter is inside a soft steel pipe with lids to contain the magnetic field inside the filter . the whole system is additionally sealed in another , outer pipe , where circulating water transfers out the heat emitted by the coils . the transmission through the system with two orthogonal polarizers and the rotator between reads t = trotsin2 , where trot is the transmission through the rotator and is the rotation angle . to elucidate the above expression , consider the linearly polarized light entering the rotator decomposed in a left ( l ) and right ( r ) circular polarization basis . each of them has a refractive index nl , r and an absorption coefficient l , r . traveling through the rotator , each circular polarization acquires a phase exp(ikl , rz ) , where kl , r = nl , r/c+il , rz is the complex wavevector . the resulting rotation angle due to the difference between refractive indices equals =(nl - nr)/2c . for the sake of simplicity , we assume equal absorption coefficients for both circular polarization components lr= , yielding the expression for the total transmission trot = exp(-z ) . the transmittance spectrum of the faraday filter with two orthogonal polarizers is presented in figure 7 for the whole spectrum of rubidium-87 d1-line . the transmission spectrum is measured for different values of the external magnetic field at the temperature of t=68c . for very weak magnetic fields , there is almost no transmission while in strong magnetic fields numerous separate high - transmission bands emerge . figure 8 presents analogous spectra but measured in a detailed frequency scale at a higher temperature t=110c , separately for the two different regions of the d1-line of rubidium : near the f=1f=2 and f=2f=2 transitions , respectively . figure 9 compares two relative orientations of polarizers parallel and perpendicular to each other at t=90c . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter with a hot cell at t=110c for different values of magnetic field zoomed to the vicinity of the 87rb resonances . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter at t=90 c with ( a ) perpendicular and ( b ) parallel polarizers for reference . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of both filters for different magnetic fields inside the faraday filter . temperatures of the filters were tabs=100c and tfarad=102c , respectively , for the absorption and faraday filer . ( the colour version of this figure is included in the online version of the journal . ) for the perpendicular polarizers , the transmission t=trotsin2 reaches its maximum for a rotation angle =+2k provided the transmission through rotator trot is nonzero . although in the vicinity of the resonance the rotation may be high , the rotator becomes opaque . on the other hand , further away from the resonances the net polarization rotation is very small , since both circular polarization components are subject to virtually the same retardation . practically , the filter transmits frequencies detuned by 13 ghz from any of the four resonances of 87rb d1-line , depicted in figure3(a ) . the transmission through both filters for the d1-line rubidium spectrum is depicted in figure 10 . both filters transmit light at the frequencies detuned to the red from the f=2f=2 resonant transition . the frequency corresponding to the maximum transmission can be adjusted by proper settings of the faraday filter s temperature and the internal magnetic field . for positive detunings , we observe a sharp edge of absorption region resulting from the presence of the absorption filter . we found the optimal conditions for the operation of the filtering system to be : tabs=100c and tfarad=102 c , with the magnetic field amplitude inside the faraday filter b=10 - 2 t. for such settings , the combined transmission through absorption and faraday filters is depicted in figure 3 . remarkably for raman scattered light detuned from the f=2f=2 resonant transition by s=-2.3 ghz and as=7.8 ghz , we achieved a high transmission of 65 and 40% for stokes and anti - stokes light , respectively . the 30-cm long rubidium 85rb cell in the transverse static magnetic field , drawn in figure 4 , is used to stop the strong laser beams and transmit the raman scattered single photons which are very narrowband but their frequencies lie out of the wings region of the spectrum . the transverse magnetic field inside the filter is emitted by the ferrite magnets located on both sides of the rubidium cell . by changing the distance between the magnets , we effectively change the magnetic field inside the cell and thence the line broadening . the width of the absorption spectrum controlled by the magnetic field ranges from 5.5 ghz up to 8.4 ghz , depending on the distance between the magnets , as depicted in figure 5 . for our purposes , we set the magnetic field to 10 - 2 t for a 9-cm distance between magnets , to move 85rb lines close enough to 87rb where driving lasers operate . the opaqueness and to some extent the width of the filter lines are tuned by changing the temperature of the filter . the filter is heated in a range of 20-140c to vary the optical density of atomic vapor that grows exponentially with temperature . in practice , the operational range is limited , as for temperatures below 90c the absorption of drive lasers is too low , while above 120c the transmission of the raman scattered light drops abruptly . the whole filter is inserted inside the two - layer soft steel magnetic shielding that confines the magnetic field inside the filter suppressing the stray external fields down to bext10 - 4 t. we heat the vapors in the rubidium cell using the high - power resistor mounted under the cell which dissipates around p80 w power . filter is embedded in a two - layer magnetic shielding protecting main quantum memory cell from the corrupting stray magnetic field . ( the colour version of this figure is included in the online version of the journal . ) large broadening of the lines of absorption filter due to transverse magnetic field , tuned by changing distance between ferrite magnets . for visualization purposes presented transmission spectra were measured below operation temperature , at t=60c . for our operation , we selected a distance of 9 cm corresponding to 10 - 2 t inside the cell with 85rb . ( the colour version of this figure is included in the online version of the journal . ) 85rb filter transmission spectra for a range of temperatures . absorption lines are significantly broadened due to transverse magnetic field of approximately 10 - 2 t. the detuning is measured from f=2f=2 transition on the d1-line of 87rb . ( the colour version of this figure is included in the online version of the journal . ) measured transmission spectra for different temperatures in the frequency range of both lasers and the scattered light are shown in figure 6 . for large positive detunings , the wing of the spectrum transmission is smaller than for negative detunings due to the residual content of 87rb in the filter cell . for optimal temperature tabs=100c , the suppression of the drive lasers is 50 db , while the transmission of the raman scattered light is at the level of 80% . the frequencies of stokes and anti - stokes scattered light in figure 6 correspond to detunings s=-2.3 and as=7.8 ghz , respectively . to filter out the broadband fluorescence and four - wave mixing accompanying write - in and readout from the memory , we utilize a faraday filter which consists of two orthogonal polarizers and a faraday rotator in between . in this arrangement , we use the standard configuration of the filter , but for temperature stabilization purposes , we apply water cooling to compensate the relatively high power emitted by coils producing magnetic field . the rotation of the polarization occurs in a 30-cm long cell containing the 87rb isotope heated up to 100c for us to obtain appropriate optical density of vapors . the magnetic field inside the filter is about b=10 - 2 t and directed along the beam . the average magnetic field does not depend on the transverse position inside the cell , i.e. it is near perfectly uniform in each plane perpendicular to the laser beam direction . the imperfections of the magnetic field in the transverse directions are smaller than 10 - 5 t. the cell is placed in a pvc tube and heated with a current flowing through the coils wound around the 87rb glass cell . the filter is inside a soft steel pipe with lids to contain the magnetic field inside the filter . the whole system is additionally sealed in another , outer pipe , where circulating water transfers out the heat emitted by the coils . the transmission through the system with two orthogonal polarizers and the rotator between reads t = trotsin2 , where trot is the transmission through the rotator and is the rotation angle . to elucidate the above expression , consider the linearly polarized light entering the rotator decomposed in a left ( l ) and right ( r ) circular polarization basis . each of them has a refractive index nl , r and an absorption coefficient l , r . traveling through the rotator , each circular polarization acquires a phase exp(ikl , rz ) , where kl , r = nl , r/c+il , rz is the complex wavevector . the resulting rotation angle due to the difference between refractive indices equals =(nl - nr)/2c . for the sake of simplicity , we assume equal absorption coefficients for both circular polarization components lr= , yielding the expression for the total transmission trot = exp(-z ) . the transmittance spectrum of the faraday filter with two orthogonal polarizers is presented in figure 7 for the whole spectrum of rubidium-87 d1-line . the transmission spectrum is measured for different values of the external magnetic field at the temperature of t=68c . for very weak magnetic fields , there is almost no transmission while in strong magnetic fields numerous separate high - transmission bands emerge . figure 8 presents analogous spectra but measured in a detailed frequency scale at a higher temperature t=110c , separately for the two different regions of the d1-line of rubidium : near the f=1f=2 and f=2f=2 transitions , respectively . figure 9 compares two relative orientations of polarizers parallel and perpendicular to each other at t=90c . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter with a hot cell at t=110c for different values of magnetic field zoomed to the vicinity of the 87rb resonances . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter at t=90 c with ( a ) perpendicular and ( b ) parallel polarizers for reference . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of both filters for different magnetic fields inside the faraday filter . the detuning is measured from f=2f=2 transition on the d1-line of 87rb . temperatures of the filters were tabs=100c and tfarad=102c , respectively , for the absorption and faraday filer . ( the colour version of this figure is included in the online version of the journal . ) for the perpendicular polarizers , the transmission t=trotsin2 reaches its maximum for a rotation angle =+2k provided the transmission through rotator trot is nonzero . although in the vicinity of the resonance the rotation may be high , the rotator becomes opaque . on the other hand , further away from the resonances the net polarization rotation is very small , since both circular polarization components are subject to virtually the same retardation . practically , the filter transmits frequencies detuned by 13 ghz from any of the four resonances of 87rb d1-line , depicted in figure3(a ) . the transmission through both filters for the d1-line rubidium spectrum is depicted in figure 10 . both filters transmit light at the frequencies detuned to the red from the f=2f=2 resonant transition . the frequency corresponding to the maximum transmission can be adjusted by proper settings of the faraday filter s temperature and the internal magnetic field . for positive detunings , we observe a sharp edge of absorption region resulting from the presence of the absorption filter . we found the optimal conditions for the operation of the filtering system to be : tabs=100c and tfarad=102 c , with the magnetic field amplitude inside the faraday filter b=10 - 2 t. for such settings , the combined transmission through absorption and faraday filters is depicted in figure 3 . remarkably for raman scattered light detuned from the f=2f=2 resonant transition by s=-2.3 ghz and as=7.8 ghz , we achieved a high transmission of 65 and 40% for stokes and anti - stokes light , respectively . to qualitatively assess the performance of the filtering system , we measured stokes photons along with subsequently retrieved anti - stokes photons in a similar manner as , but in a low - gain regime exciting only a few photons per spatial mode . we observe an increase in the signal - to - noise ratio owing to the filtering system by measuring average intensities and correlations between the number of photons generated in the write - in and readout processes . the average intensities from 2105 frames registered on the i - scmos camera are depicted in figure 11 . the first line presents the angularly broad stokes and anti - stokes light intensities without the faraday filter , while the second one includes faraday filter operation where the isotropic noise background is greatly suppressed . for comparison , the residual laser beams ( angularly narrow ) leakage is let through the filters in the system by with cold 85rb filter , as depicted in the third line . normalized average light intensities of angularly broad stokes ( left ) and anti - stokes ( right ) scattering registered by the i - scmos camera along the x axis . raman scattering is compared with angularly narrow laser beam leakage which dominates without absorption filter . ( the colour version of this figure is included in the online version of the journal . ) comparison of measured joint photon statistics ( a b ) and spatial correlations between the stokes and anti - stokes photons ( c d ) with ( left panel ) and without ( right panel ) faraday filter . the joint count statistics of centrally emitted stokes and anti - stokes ( top row ) p(ns , nas ) and maps of intensity correlations between different directions ( bottom row ) . ( the colour version of this figure is included in the online version of the journal . ) the joint statistics p(ns , nas ) of counts between stokes and anti - stokes photons from the write - in and readout processes are depicted in figure 12(a ) and ( b ) , with and without the faraday filter , respectively . we count the number of stokes photons ns scattered on the camera region around the write - in laser beam ( 6 mrad in diameter ) and the number of anti - stokes photons nas around the readout beam . comparing the maps of the joint statistics , one can see that application of the faraday filter enables observation of a correlation between the amount of stokes and anti - stokes photons scattered in each realization of the experiment . the ratio of the size of the diagonal and anti - diagonal joint probability distribution is a measure of correlation coefficient and thus the signal - to - noise ratio in the registered data . without the faraday filter , the number of anti - stokes scattered photons is almost independent of the number of stokes scattered photons . this is due to spurious incoherent light which is intense enough to cover the raman scattering signal . to demonstrate the multimode capacity of our memory and filter system , we conduct direction - resolved measurements of correlations . to this end , we divide the camera region along the horizontal line to the left and right around the write and read beams into small circular regions ai and aj , each of the area of 0.02 mrad2 and with 0.70.8 single photon on average . we calculated the correlation coefficient cij=ns(ai)nas(aj)/(ns(ai))2(nas(aj))2 between the number of stokes ns(ai ) and anti - stokes nas(aj ) photons scattered in the circular regions ai , aj around the write and read beams , respectively . the maps of correlation coefficient between the number of stokes and anti - stokes photons scattered into each pair of circular regions are depicted in figure 12(c ) and ( d ) . using faraday filter , then , we compare the number of coincidences of stokes and anti - stokes photons from each of these small regions . including the faraday filter , we are able to observe the elliptic structure which is evidence for the generation of correlated photon pairs with a correlation coefficient of 0.38 which is smaller than unity because of the fluorescence signal from the quantum memory cell . without the narrowband faraday filter , we do not observe any correlations on the illumination level of n=30 photons per camera frame ( from which nb=1.5 is the light - independent equivalent background illumination noise of the image intensifier ) . the correlation coefficient between the stokes and anti - stokes photons scattered in the opposite directions extracted from the measured data is as little as ca . 0.03 on average which is negligible as compared to figure 12(c ) with faraday filter . moreover , in figure 12(d ) , there is no visible structure , which is due to the large amount of fluorescence light comparable to the raman scattered photons which are correlated . in conclusion , we built and characterized a two - step filtering setup which enables observation of the correlations between stokes and anti - stokes raman scattering at a single - photon level . polarization pre - filtering and a rubidium absorption filter are used to eliminate the laser beams leakage by attenuating them by more than 109 , while an additional combination with a faraday filter yields a total attenuation of 1011 . moreover , the faraday filter blocks the fluorescence and the four - wave mixing noise coming from the atomic memory medium . the filter transmits only the narrowband spectrum of frequencies corresponding to the raman scattered light in 87rb . in the case of our filter construction , it corresponds to the frequencies detuned approximately by s=-2 ghz and as=7 ghz from the f=2f=2 resonant transition for stokes and anti - stokes light , respectively . noticeably , regardless of such small detunings , we achieve a transmission for the raman scattered light as high as 5060% , depending on the faraday filter settings . what is more , the frequencies with high transmission can be tuned by several hundreds of mhz by changing the magnetic field amplitude inside the faraday filter , whereas absorption edges of 85rb filter can be moved magnetically as widely as up to 8.4 ghz . eventually , we can obtain the optimal , high transmission on the raman scattered light frequencies while blocking frequencies corresponding to the broadband fluorescence , laser beams and four - wave mixing . the performance of the system in the regime of a few atomic collective excitations is evaluated in statistical measurements of the scattered photons . the obtained correlation maps are strong evidence for the directional coincidences between raman scattered light produced in the write - in and readout processes of quantum memory . however , the output signal is dominated by noise and no correlation appears above the background if no filtering is applied . application of the filtering system we present can enhance the fidelity of retrieved photons and extend other teams operational regime into the multimode quantum memory . undoubtedly such system will be essential to measure nonclassical g(2 ) cross - correlation function between stored and retrieved photons , which is prerequisite for further applications in quantum information processing . importantly , presented filtering solution can find applications far beyond quantum memories including raman spectroscopy and biological applications .

warm atomic vapor quantum memories are simple and robust , yet suffer from a number of parasitic processes which produce excess noise . for operating in a single - photon regime precise filtering of the output light is essential . here , we report a combination of magnetically tuned absorption and faraday filters , both light direction insensitive , which stop the driving lasers and attenuate spurious fluorescence and four - wave mixing while transmitting narrowband stokes and anti - stokes photons generated in write - in and readout processes . we characterize both filters with respect to adjustable working parameters . we demonstrate a significant increase in the signal - to - noise ratio upon applying the filters seen qualitatively in measurements of correlation between the raman scattered photons .

Introduction Memory setup Filtering system details Absorption filter Faraday filter Transmission of combined filters Performance filtering photons from the memory Conclusions

quantum memories are the essential building blocks in a number of quantum information processing protocols . a number of different implementations of quantum memory have been realized so far including electromagnetically induced transparency , raman scattering , atomic frequency comb , gradient echo memory and coherent population oscillations . in this work , we focus on probably the most widespread and renowned implementation based on warm alkali atom vapor ( rubidium-87 ) contained in a glass cell together with noble buffer gas ( krypton ) to reduce the effects of thermal motion . in general , multimode operation is natural and readily achievable in a number of spontaneous and parametric processes [ 3133 ] such as spontaneous parametric down - conversion in nonlinear crystals and instantaneous four - wave mixing in warm atomic vapors . in our setup , typically over one hundred distinguishable spatial modes of raman light are coupled with independent spin - wave modes . thus , filtering out of the driving beams requires very large attenuation , around 1011 . in our experiments , first we use crystal polarizers , as the raman photons are polarized orthogonally to the driving light . next , a rubidium-85 absorption filter of magnetically broadened absorption line width is used to stop any remaining leakage . the second type of noise is an omnidirectional spontaneous fluorescence directly from the excited state , observed in a number of experiments and discussed theoretically [ 4346 ] . therefore , we use a faraday filter with rubidium-87 vapor rotator as a second stage . by properly adjusting the temperature and magnetic field in both filters , we are able to attenuate the driving beams by a factor of 1011 and reach a transmission of 65% for the stokes and 45% for the anti - stokes light . we demonstrate the immense effect of combined filtering on the statistical properties of the registered light . however , only due to the application of the second filter intensity correlations between the stokes and anti - stokes scattered light can be seen down to a single - photon level . after adjustable storage time , the spin wave can be converted to anti - stokes photons by sending read laser pulse . ( the colour version of this figure is included in the online version of the journal . ) the raman scattered light has a polarization orthogonal to the driving lasers , so we use wollaston prisms to pre - filter the raman scattered light . due to the finite extinction of the wollaston prisms ( 105:1 ) , a small portion of laser light leaks through together with the raman scattered light . usage of the 85rb leads to a high absorption coefficient of the driving lasers , whereas transmission of raman scattered light remains at the level of 80% . direction , spatial mode profile or polarization , in contrast to the properties of the faraday or voigt filters . from top - left corner : 87rb atomic memory cell inside magnetic shielding , magnetically broadened 85rb absorption filter , faraday filter and a low - noise , single - photon - sensitive intensified scmos camera ( i - scmos ) as a detector . ( the colour version of this figure is included in the online version of the journal . ) this enables us to work in a single - photon - level regime and to measure photon statistics in a similar manner as with multiplexed on off detectors [ 51,5355 ] . absorption and transmission spectra of the three rubidium cells used in the experiment are presented in figure 3 along with the positions of the laser beams frequencies , as well as stokes and anti - stokes scattered light . detuning of about 1 ghz for both write - in and readout beams is a compromise between the high efficiency of raman scattering and the low absorption coefficient inside the filtering cells . the frequency of the laser beams and scattered stokes and anti - stokes light are marked by detunings measured from f=2f=2 transition on d1-line in 87rb . ( the colour version of this figure is included in the online version of the journal . ) the 30-cm long rubidium 85rb cell in the transverse static magnetic field , drawn in figure 4 , is used to stop the strong laser beams and transmit the raman scattered single photons which are very narrowband but their frequencies lie out of the wings region of the spectrum . the transverse magnetic field inside the filter is emitted by the ferrite magnets located on both sides of the rubidium cell . by changing the distance between the magnets , we effectively change the magnetic field inside the cell and thence the line broadening . the width of the absorption spectrum controlled by the magnetic field ranges from 5.5 ghz up to 8.4 ghz , depending on the distance between the magnets , as depicted in figure 5 . for our purposes , we set the magnetic field to 10 - 2 t for a 9-cm distance between magnets , to move 85rb lines close enough to 87rb where driving lasers operate . the filter is heated in a range of 20-140c to vary the optical density of atomic vapor that grows exponentially with temperature . in practice , the operational range is limited , as for temperatures below 90c the absorption of drive lasers is too low , while above 120c the transmission of the raman scattered light drops abruptly . the whole filter is inserted inside the two - layer soft steel magnetic shielding that confines the magnetic field inside the filter suppressing the stray external fields down to bext10 - 4 t. we heat the vapors in the rubidium cell using the high - power resistor mounted under the cell which dissipates around p80 w power . ( the colour version of this figure is included in the online version of the journal . ) ( the colour version of this figure is included in the online version of the journal . ) ( the colour version of this figure is included in the online version of the journal . ) measured transmission spectra for different temperatures in the frequency range of both lasers and the scattered light are shown in figure 6 . for large positive detunings , the wing of the spectrum transmission is smaller than for negative detunings due to the residual content of 87rb in the filter cell . for optimal temperature tabs=100c , the suppression of the drive lasers is 50 db , while the transmission of the raman scattered light is at the level of 80% . the frequencies of stokes and anti - stokes scattered light in figure 6 correspond to detunings s=-2.3 and as=7.8 ghz , respectively . to filter out the broadband fluorescence and four - wave mixing accompanying write - in and readout from the memory , we utilize a faraday filter which consists of two orthogonal polarizers and a faraday rotator in between . in this arrangement , we use the standard configuration of the filter , but for temperature stabilization purposes , we apply water cooling to compensate the relatively high power emitted by coils producing magnetic field . the rotation of the polarization occurs in a 30-cm long cell containing the 87rb isotope heated up to 100c for us to obtain appropriate optical density of vapors . the imperfections of the magnetic field in the transverse directions are smaller than 10 - 5 t. the cell is placed in a pvc tube and heated with a current flowing through the coils wound around the 87rb glass cell . figure 8 presents analogous spectra but measured in a detailed frequency scale at a higher temperature t=110c , separately for the two different regions of the d1-line of rubidium : near the f=1f=2 and f=2f=2 transitions , respectively . ( the colour version of this figure is included in the online version of the journal . ) ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter at t=90 c with ( a ) perpendicular and ( b ) parallel polarizers for reference . ( the colour version of this figure is included in the online version of the journal . ) temperatures of the filters were tabs=100c and tfarad=102c , respectively , for the absorption and faraday filer . ( the colour version of this figure is included in the online version of the journal . ) although in the vicinity of the resonance the rotation may be high , the rotator becomes opaque . practically , the filter transmits frequencies detuned by 13 ghz from any of the four resonances of 87rb d1-line , depicted in figure3(a ) . the transmission through both filters for the d1-line rubidium spectrum is depicted in figure 10 . for positive detunings , we observe a sharp edge of absorption region resulting from the presence of the absorption filter . we found the optimal conditions for the operation of the filtering system to be : tabs=100c and tfarad=102 c , with the magnetic field amplitude inside the faraday filter b=10 - 2 t. for such settings , the combined transmission through absorption and faraday filters is depicted in figure 3 . remarkably for raman scattered light detuned from the f=2f=2 resonant transition by s=-2.3 ghz and as=7.8 ghz , we achieved a high transmission of 65 and 40% for stokes and anti - stokes light , respectively . the 30-cm long rubidium 85rb cell in the transverse static magnetic field , drawn in figure 4 , is used to stop the strong laser beams and transmit the raman scattered single photons which are very narrowband but their frequencies lie out of the wings region of the spectrum . by changing the distance between the magnets , we effectively change the magnetic field inside the cell and thence the line broadening . the width of the absorption spectrum controlled by the magnetic field ranges from 5.5 ghz up to 8.4 ghz , depending on the distance between the magnets , as depicted in figure 5 . for our purposes , we set the magnetic field to 10 - 2 t for a 9-cm distance between magnets , to move 85rb lines close enough to 87rb where driving lasers operate . the filter is heated in a range of 20-140c to vary the optical density of atomic vapor that grows exponentially with temperature . in practice , the operational range is limited , as for temperatures below 90c the absorption of drive lasers is too low , while above 120c the transmission of the raman scattered light drops abruptly . ( the colour version of this figure is included in the online version of the journal . ) for our operation , we selected a distance of 9 cm corresponding to 10 - 2 t inside the cell with 85rb . ( the colour version of this figure is included in the online version of the journal . ) ( the colour version of this figure is included in the online version of the journal . ) measured transmission spectra for different temperatures in the frequency range of both lasers and the scattered light are shown in figure 6 . for large positive detunings , the wing of the spectrum transmission is smaller than for negative detunings due to the residual content of 87rb in the filter cell . for optimal temperature tabs=100c , the suppression of the drive lasers is 50 db , while the transmission of the raman scattered light is at the level of 80% . the frequencies of stokes and anti - stokes scattered light in figure 6 correspond to detunings s=-2.3 and as=7.8 ghz , respectively . to filter out the broadband fluorescence and four - wave mixing accompanying write - in and readout from the memory , we utilize a faraday filter which consists of two orthogonal polarizers and a faraday rotator in between . in this arrangement , we use the standard configuration of the filter , but for temperature stabilization purposes , we apply water cooling to compensate the relatively high power emitted by coils producing magnetic field . the rotation of the polarization occurs in a 30-cm long cell containing the 87rb isotope heated up to 100c for us to obtain appropriate optical density of vapors . the imperfections of the magnetic field in the transverse directions are smaller than 10 - 5 t. the cell is placed in a pvc tube and heated with a current flowing through the coils wound around the 87rb glass cell . figure 8 presents analogous spectra but measured in a detailed frequency scale at a higher temperature t=110c , separately for the two different regions of the d1-line of rubidium : near the f=1f=2 and f=2f=2 transitions , respectively . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter with a hot cell at t=110c for different values of magnetic field zoomed to the vicinity of the 87rb resonances . ( the colour version of this figure is included in the online version of the journal . ) transmission spectra of the faraday filter at t=90 c with ( a ) perpendicular and ( b ) parallel polarizers for reference . ( the colour version of this figure is included in the online version of the journal . ) temperatures of the filters were tabs=100c and tfarad=102c , respectively , for the absorption and faraday filer . ( the colour version of this figure is included in the online version of the journal . ) although in the vicinity of the resonance the rotation may be high , the rotator becomes opaque . the frequency corresponding to the maximum transmission can be adjusted by proper settings of the faraday filter s temperature and the internal magnetic field . for positive detunings , we observe a sharp edge of absorption region resulting from the presence of the absorption filter . we found the optimal conditions for the operation of the filtering system to be : tabs=100c and tfarad=102 c , with the magnetic field amplitude inside the faraday filter b=10 - 2 t. for such settings , the combined transmission through absorption and faraday filters is depicted in figure 3 . remarkably for raman scattered light detuned from the f=2f=2 resonant transition by s=-2.3 ghz and as=7.8 ghz , we achieved a high transmission of 65 and 40% for stokes and anti - stokes light , respectively . to qualitatively assess the performance of the filtering system , we measured stokes photons along with subsequently retrieved anti - stokes photons in a similar manner as , but in a low - gain regime exciting only a few photons per spatial mode . we observe an increase in the signal - to - noise ratio owing to the filtering system by measuring average intensities and correlations between the number of photons generated in the write - in and readout processes . the first line presents the angularly broad stokes and anti - stokes light intensities without the faraday filter , while the second one includes faraday filter operation where the isotropic noise background is greatly suppressed . for comparison , the residual laser beams ( angularly narrow ) leakage is let through the filters in the system by with cold 85rb filter , as depicted in the third line . normalized average light intensities of angularly broad stokes ( left ) and anti - stokes ( right ) scattering registered by the i - scmos camera along the x axis . ( the colour version of this figure is included in the online version of the journal . ) comparison of measured joint photon statistics ( a b ) and spatial correlations between the stokes and anti - stokes photons ( c d ) with ( left panel ) and without ( right panel ) faraday filter . the joint count statistics of centrally emitted stokes and anti - stokes ( top row ) p(ns , nas ) and maps of intensity correlations between different directions ( bottom row ) . ( the colour version of this figure is included in the online version of the journal . ) the joint statistics p(ns , nas ) of counts between stokes and anti - stokes photons from the write - in and readout processes are depicted in figure 12(a ) and ( b ) , with and without the faraday filter , respectively . we count the number of stokes photons ns scattered on the camera region around the write - in laser beam ( 6 mrad in diameter ) and the number of anti - stokes photons nas around the readout beam . comparing the maps of the joint statistics , one can see that application of the faraday filter enables observation of a correlation between the amount of stokes and anti - stokes photons scattered in each realization of the experiment . the ratio of the size of the diagonal and anti - diagonal joint probability distribution is a measure of correlation coefficient and thus the signal - to - noise ratio in the registered data . without the faraday filter , the number of anti - stokes scattered photons is almost independent of the number of stokes scattered photons . to demonstrate the multimode capacity of our memory and filter system , we conduct direction - resolved measurements of correlations . to this end , we divide the camera region along the horizontal line to the left and right around the write and read beams into small circular regions ai and aj , each of the area of 0.02 mrad2 and with 0.70.8 single photon on average . we calculated the correlation coefficient cij=ns(ai)nas(aj)/(ns(ai))2(nas(aj))2 between the number of stokes ns(ai ) and anti - stokes nas(aj ) photons scattered in the circular regions ai , aj around the write and read beams , respectively . the maps of correlation coefficient between the number of stokes and anti - stokes photons scattered into each pair of circular regions are depicted in figure 12(c ) and ( d ) . using faraday filter , then , we compare the number of coincidences of stokes and anti - stokes photons from each of these small regions . including the faraday filter , we are able to observe the elliptic structure which is evidence for the generation of correlated photon pairs with a correlation coefficient of 0.38 which is smaller than unity because of the fluorescence signal from the quantum memory cell . without the narrowband faraday filter , we do not observe any correlations on the illumination level of n=30 photons per camera frame ( from which nb=1.5 is the light - independent equivalent background illumination noise of the image intensifier ) . the correlation coefficient between the stokes and anti - stokes photons scattered in the opposite directions extracted from the measured data is as little as ca . moreover , in figure 12(d ) , there is no visible structure , which is due to the large amount of fluorescence light comparable to the raman scattered photons which are correlated . in conclusion , we built and characterized a two - step filtering setup which enables observation of the correlations between stokes and anti - stokes raman scattering at a single - photon level . moreover , the faraday filter blocks the fluorescence and the four - wave mixing noise coming from the atomic memory medium . the filter transmits only the narrowband spectrum of frequencies corresponding to the raman scattered light in 87rb . in the case of our filter construction , it corresponds to the frequencies detuned approximately by s=-2 ghz and as=7 ghz from the f=2f=2 resonant transition for stokes and anti - stokes light , respectively . noticeably , regardless of such small detunings , we achieve a transmission for the raman scattered light as high as 5060% , depending on the faraday filter settings . eventually , we can obtain the optimal , high transmission on the raman scattered light frequencies while blocking frequencies corresponding to the broadband fluorescence , laser beams and four - wave mixing . the performance of the system in the regime of a few atomic collective excitations is evaluated in statistical measurements of the scattered photons . the obtained correlation maps are strong evidence for the directional coincidences between raman scattered light produced in the write - in and readout processes of quantum memory .

early reports of an increased risk of cancer in a particular exposed group have led to the identification of several agents as human carcinogens ( 3 ) , but for many other agents the suspicion of a carcinogenic effect has not been confirmed in subsequent studies . because cancer epidemiology has great societal impact , and avoidance of false - positive reports in this field deserves particular attention , we have selected one example from environmental epidemiology and one example of a reported occupational cancer association . obviously , considerations addressed in these examples are applicable to other areas of epidemiological research as well . the first example is of an apparently strong positive result for a pollutant in the general environment that was initially suspected to give rise to cancer but for which such an association has not been confirmed in subsequent studies . in 1993 , a report of a case control study nested in a new york city based cohort ( 10 ) showed an association between breast cancer risk and serum levels of 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene ( dde ) , the major metabolite of 1,1,1,-trichloro-2,2-bis(p - chlorophenyl)ethane ( ddt ) , an organochlorine pesticide that was used extensively until the early 1970s . the study involved 58 women who had been diagnosed with breast cancer within 6 months of entry in the prospective cohort study and 171 control subjects from the same cohort , and it reported a relative risk of 3.7 ( 95% confidence interval [ ci ] = 1.0 to 13.5 ) for the highest vs lowest 20% of the dde distribution in serum . this study was prompted by earlier results of small ( < 50 case patients ) case control studies ( 1114 ) that had suggested a modest association between breast cancer and exposure to dde and polychlorinated biphenyls . an editorial accompanying the report of the new york city study ( 15 ) stated that these findings may have extraordinary implications for the prevention of breast cancer and that this study should serve as a wake - up call for further urgent research . accordingly , a series of studies were subsequently launched , including a large case control study of breast cancer in long island , ny ( 16 ) , but they failed to replicate the earlier findings . the results on serum dde level from the prospective studies , including an expanded analysis of women who had been included in the original study from new york city and a combined analysis of five prospective studies , were published between 1994 and 2001 ( 1728 ) ( table 1 ) . we performed a cumulative meta - analysis of the initial study and seven subsequent studies published in 19942000 ( figure 1 ) . the final pooled estimate of the relative risk of breast cancer for the highest vs lowest dde category was 0.95 ( 95% ci = 0.7 to 1.3 ) . results of prospective studies on serum 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene level and risk of breast cancer * dde = 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene ; rr = relative risk ; ci = confidence interval ; hmo = health maintenance organization ; na = not available . dde serum levels are either not lipid adjusted ( expressed as parts per billion [ ppb ] , equivalent to milligrams per gram serum ) or lipid adjusted ( expressed as nanograms per gram lipid ) . mean level , control subjects : 1260 ng / g ; case patients : 1230 ng / g . cumulative meta - analysis of cohort studies of breast cancer and serum level of 1,1-dichloro-2,2-bis(p - chlorophenyl ) ethylene ( highest vs lowest category in each study ) . estimated relative risks ( rrs ) are shown with 95% confidence intervals ( cis ) ( error bars ) by year of publication of subsequent reports . in parentheses are references of studies included in the cumulative meta - analyses ( see table 1 for details ) . the reason for the positive findings in the original new york city study is unknown . the short time that elapsed between blood collection and diagnosis of breast cancer may be another explanation because organochlorine compounds are stored in fat tissue , whose metabolism may be affected by the neoplastic process itself ( 29 ) . overinterpretation and an apparent lack of skepticism had major roles in the wide acceptance of the result . although the relative risk estimate of breast cancer among women in the highest quintile of serum dde compared with the lowest was almost 4.0 , the width of the confidence interval and the marginal level of statistical significance should have tempered the enthusiasm for the finding . similarly , a recently reported relative risk estimate of 5.0 ( 95% ci = 1.7 to 14.8 ) for serum levels of p , p-ddt and breast cancer risk , based on subgroup analyses in a nested case what is of ongoing concern is that given the history of studies of this exposure disease issue , the results were still interpreted with few reservations and little if any caution . a second example relates to the results of occupational cohort studies of lung cancer risk among workers who were exposed to acrylonitrile . acrylonitrile is a chemical used in the manufacture of acrylic fibers , resins , and nitrile rubber , as an intermediate in the chemical industry and , in the past , as a fumigant . the first study of lung cancer mortality among workers who were exposed to acrylonitrile in a textile factory in the united states , which was reported in 1978 ( 31 ) , showed a fourfold increased risk , based on six deaths from lung cancer . an international agency for research on cancer ( iarc ) working group cited this study in its review of acrylonitrile in 1979 and concluded that while confirmatory evidence in experimental animals and humans is desirable , acrylonitrile should be regarded as if it were carcinogenic to humans ( 32 ) . following the initial report , a total of 16 partially overlapping occupational cohort studies have provided results on lung cancer risk among acrylonitrile - exposed workers , including an expanded analysis of the first study , which identified two additional observed and 2.9 additional expected lung cancer deaths ( 33 ) ( table 2 ) . results of cohort studies on lung cancer risk among workers who were exposed to acrylonitrile * n = number of observed lung cancer deaths ; rr = relative risk ; ci = confidence interval . for studies overlapping with previous reports , only the additional observed lung cancers are reported ( and the relative risk is based on these additional cancers only ) . when we performed a cumulative meta - analysis of the initial 1978 findings and of 15 subsequent studies of acrylonitrile and lung cancer that were published in 19801998 , we found a steady decrease over time in the reported overall relative risk estimate of lung cancer in acrylonitrile workers ( figure 2 ) . the final pooled estimate of the relative risk of lung cancer among the workers was 1.1 ( 95% ci = 0.9 to 1.4 ) . this approach admittedly represents an overly simplified review because , for the sake of comparability , we restricted the meta - analysis to historical cohort studies and did not consider case control studies [ eg , sclo et al . ( 48 ) ] that may provide additional relevant information , we did not consider the results of internal ( eg , dose response ) analyses available for several cohorts , and we did not consider the evidence for or against bias and confounding in each of the studies . however , the declining trend in the summary relative risk estimate as further data accumulated provides evidence that the initial finding was a false - positive result . in an updated review of the evidence in 1999 , an iarc working group modified the classification for acrylonitrile from cumulative meta - analysis of cohort studies of lung cancer and occupational exposure to acrylonitrile . estimated relative risks ( rrs ) are shown with 95% confidence intervals ( cis ) ( error bars ) by year of publication of subsequent reports . in parentheses are references of studies included in the cumulative meta - analyses ( see table 2 for details ) . similar examples of false - positive results are relevant to the evidence linking exposure to established occupational carcinogens with cancer of organs that are not the primary target of their carcinogenic effect . a detailed analysis of results on other suspected occupational carcinogens is beyond the scope of this commentary , but for several agents it would show the same pattern seen for lung cancer among workers who were exposed to acrylonitrile , that is , early positive results were not confirmed , or were shown to be less strong , in subsequent studies . the issue of multiplicity of exposures and outcomes , and consequent multiple comparisons and subgroup analyses leading to many statistically significant ( yet false ) findings arising by chance , is likely the major contributor to false - positive findings in epidemiology . epidemiological studies , whether cross - sectional , case control , or cohort in design , tend to obtain information on multiple exposures and disease outcomes , so the possibility often exists for examining hundreds or thousands of exposure disease combinations . a similar situation arises in emerging genetic epidemiological studies in which even larger numbers of traits , up to tens or hundreds of thousands of variants , can be examined . in a quantitative approach to this problem , ( 8) reviewed 36 genetic disease associations and found that for 25 of them , the first report gave a stronger estimate of genetic association than did subsequent studies ; in 10 of these associations , the difference between the first and the subsequent results was statistically significant . the same authors subsequently considered the results of 55 meta - analyses ( 50 ) , including 579 study comparisons of genetic associations , and found that for each association the largest study generally yielded more conservative results than the meta - analysis , that in 26% of the meta - analyses the association was statistically significantly stronger in the first study , and that in only 16% was the genetic association found in the first study replicated without evidence of heterogeneity and bias . statistical techniques aimed at reducing the likelihood of false - positive associations , such as false discovery rate ( 51,52 ) , false - positive report probability ( 53 ) , and bayesian false discovery probability ( 54 ) , are now included in the analysis of databases with large numbers of genetic variants [ eg , wellcome trust case control consortium ( 55 ) ] . it seems appropriate that similar approaches be applied in other areas of cancer epidemiology that are prone to reporting false - positive results , including studies of potential occupational and environmental carcinogens . there are multiple reasons besides chance why positive results , particularly initial results , in epidemiological studies may be false ( 2,56 ) . to formally address the determinants of false - positive results in occupational cancer , swaen et al . ( 57 ) scored the main characteristics of studies reporting a positive association with established target ( true positive ) and nontarget ( false positive ) organs of 19 occupational carcinogens . the main determinants of false - positive findings were the absence of a specific a priori hypothesis , a small magnitude of the association , the absence of a dose effect relationship , and the lack of adjustment for tobacco smoking . although this analysis can be criticized because of subjective aspects in the definition of target organs and in quality scoring , it highlights the important roles of confounding and bias , together with chance , in the generation of false - positive results . although chance is a major cause of false - positive results , it is less appreciated that false leads may also be generated by bias . bias consists of a systematic alteration of the research findings due to factors related to study design , data acquisition , analysis , or reporting of results . there is a fundamental distinction between false - positive results that are generated by chance and those caused by bias the former will rarely be replicated in subsequent investigations , whereas bias may operate in a similar fashion in different settings and populations and thus will provide a consistent pattern of independently generated results . even if only a relatively low proportion of results are generated by bias information bias , or recall bias , is a likely source of false - positive findings . early studies examining a possible association between induced abortion and breast cancer risk that were based on retrospectively ascertained information generated evidence indicating that women who had undergone an induced abortion were at increased risk of breast cancer ( 58 ) . a collaborative reanalysis of data from 53 epidemiological studies comparing women with prospectively ascertained records of one or more induced abortions vs women with no such record ( 59 ) reported a relative risk of breast cancer equal to 0.93 ( 95% ci = 0.89 to 0.96 ) . it is now apparent that information bias had a major role in generating the early false - positive results . control women were less likely to report their previous induced abortions than were women with breast cancer ( 58,60 ) . in general , patients have greater motivation ( eg , to explain their disease ) and thus are more likely to remember and/or report certain past exposures than are healthy individuals who are selected as control subjects . a likely example of selection bias is the 1981 report of increased pancreatic cancer risk associated with coffee consumption in one of the earliest epidemiological studies of this disease ( 61 ) . many investigators aimed to replicate these findings , but the results of subsequent studies were in general null and by the end of the 1980s a causal association between coffee drinking and pancreatic cancer was considered unlikely ( 62,63 ) . yet for several years the suspicion that coffee drinking was associated with a highly lethal cancer was widespread in the medical community and the general public . this false - positive result was generated , at least in part , by exclusions from the control patient population , but not from the case patients , of individuals with a history of diseases related to cigarette smoking and alcohol consumption ; because these exposures were highly correlated with coffee consumption , the exclusions likely led to a deficit of coffee consumers in the control group . another source of bias and false - positive findings in observational studies is confounding resulting either from incomplete statistical adjustment for measured variables or from the inability to adjust for unmeasured distorting variables . for example , results of early epidemiological studies showed an increased risk of cervical cancer among women who were infected with agents such as herpes simplex virus 2 and chlamydia . when sensitive and specific markers of infection with human papilloma virus became available , however , it was clear that the early positive results for the other sexually transmitted agents were due to confounding by human papilloma virus ( 64 ) . although the importance of residual confounding and unmeasured confounders as a source of bias in epidemiological studies has been downplayed by many ( 65 ) , a recent statistical simulation study ( 66 ) showed that with plausible assumptions , effect sizes on the order of 1.52.0 , which is a magnitude frequently reported in epidemiology studies , can be generated by residual and/or unmeasured confounding . the real effect of unmeasured confounders is of course unknown but may explain some of the notorious differences between well - known cohort studies and subsequent randomized trials . the randomized controlled trial is often presented as the gold standard of epidemiological studies , with the random assignment of treatments mitigating against the potential influences of bias and confounding . systematic reviews have found evidence of higher relative risks in observational studies as compared with randomized controlled trials addressing the same question ( 67,68 ) . however , even randomized controlled studies are not exempt from reporting of false - positive results that arise by chance . in a review of 39 highly cited ( defined as studies cited 1000 or more times in the literature ) randomized controlled trials that reported an original claim of an effect ( 69 ) , the results of 19 were replicated by subsequent studies , whereas for nine trials subsequent results either contradicted the findings of the original report or provided evidence of a weaker effect ( the remaining 11 trials did not have subsequent attempts of replication ) . premature conclusions can be avoided by cautious interpretation of initial trials , especially when they are small , and by trial registration , which is a requirement of the international committee of medical journal editors ( 70 ) . moreover , there is an unfortunate tendency to highlight positive or statistically significant findings in the abstracts of both observational studies and randomized trials , even when the results are doubtful or open to criticism ( 71 ) . another cause of false - positive reporting in cancer epidemiology is publication or reporting bias . it originates from the tendency of authors and journal reviewers and editors to report and publish positive or statistically significant results over null or non statistically significant results , particularly if the findings appear to confirm a previously reported association ( ie , the bandwagon effect ) . as with other forms of bias , statistical tests have been proposed to assess the presence of publication bias ( 7275 ) ; unfortunately , these tests tend to have low power and require a large number of independent studies to provide evidence of bias . there are , however , suspected occupational and environmental carcinogens for which a sufficiently large number of studies are available to allow testing for publication bias . one group of suspected environmental carcinogens is the polychlorinated dibenzo - para - dioxins , which are by - products of the manufacture of chlorophenols and chlorophenoxy herbicides ; they are also produced during incineration of organic material and in other thermal processes , such as metal processing and paper pulp bleaching . the most toxic is 2,3,7,8-tetrachlorodibenzo - p - dioxin ( 2,3,7,8-tcdd ) , which has been found to cause tumors in rodents under certain conditions ( 62 ) . concern about the carcinogenicity of dioxins in general , and 2,3,7,8-tcdd in particular , has led to epidemiological studies that are both occupationally based ( eg , workers in the chemical industry , pesticide applicators ) and community based ( eg , residents in contaminated areas , the general population ) . non - hodgkin lymphoma ( nhl ) has been among the suspected targets of dioxin carcinogenicity in humans . at the time of the most recent iarc monograph evaluation ( 76 ) , results on nhl risk were available from 16 studies of 2,3,7,8-tcdd exposed populations eight cohort studies and eight case control studies ( 76 ) . although a meta - analysis suggests an increased risk of nhl in these populations ( meta - relative risk = 1.6 , 95% ci = 1.2 to 2.1 ) , there is evidence of publication bias [ figure 3 ; p = .02 , begg test ( 75 ) ] . in the absence of publication bias , the results of individual studies plotted against their precision , as in figure 3 , should be distributed in a triangular ( funnel ) pattern , with less precise studies ( on the right side of the graph ) randomly dispersed around the mean . publication bias is suggested by the absence of results in one of the parts of the funnel ( eg , lower right corner in figure 3 , where small null studies would be plotted ) . formal testing of publication bias is helpful , but keeping the bandwagon effect concept in mind would go a long way to instilling skepticism when assessing the strength of evidence provided by a new report on a popular hypothesis . funnel plot of results of studies on dioxin exposure and risk of non - hodgkin lymphoma . the examples presented in this commentary suggest that false - positive results are a common problem in cancer and other types of epidemiological studies . what can be done within the practice of epidemiology to reduce the problem ? one of the simplest yet potentially most effective remedies involves increasing emphasis on skepticism when assessing study results , particularly when they are new . put another way , epidemiologists should practice some epistemological modesty when interpreting and presenting their findings . epidemiologists by training are most often aware of the methodological limitations of observational studies , particularly those done by others , yet when it comes to practice , and especially the interpretation of their own study results , methodological vigilance is often absent . this absence of skepticism in reporting results in published papers increases the likelihood that a positive finding will receive unwarranted attention in the media and by the public . the tendency to emphasize and overinterpret what appear to be new findings is commonplace , perhaps in part because of a belief that the findings provide information that may ultimately improve public health . although the results may turn out to be wrong , some authors feel it is better to err on the side of overreporting or overstating potentially false - positive results than to miss the identification of a potential new hazard or an opportunity for career advancement . this tendency to hype new findings increases the likelihood of downplaying inconsistencies within the data or any lack of concordance with other sources of evidence . furthermore , the clear acknowledgement that the statistically significant findings may arise from multiple comparisons or subgroup analyses is often missing ; results from a single study are often dispersed across multiple publications , sometimes without reference to each other , hindering the detection of the multiple comparisons or subgroup reporting . strict adherence to the highest epidemiological standards in the design , analysis , reporting , and interpretation of studies would help reduce the likelihood and impact of false - positive results . these standards include provisions to reduce the opportunity for bias and confounding in study design , adequate statistical power , avoidance ( or at least cautious interpretation ) of data - driven subgroup analyses , and accounting for multiple comparisons . the strategy for reporting study results should be specified before the results are known , and selective reporting or emphasis of statistically significant results based on ex post facto subgroup analyses should be discouraged . the interpretation of positive results , especially if they are not supported by additional evidence ( eg , other epidemiological or experimental studies ) , should be careful and cautious . they are also in line with many of the recommendations in the strengthening the reporting of observational studies in epidemiology ( strobe ) statement , which is aimed at strengthening the reporting of epidemiological studies ( 77,78 ) . we propose that the policies of some journals , which require the explicit acknowledgement of study limitations up front in the abstract or in a note box , become standard practice . caution should be applied in the communication of results to the media and the general public , because positive findings tend to attract the media and public attention , whereas findings that do not confirm a previously reported association or do not indicate a new association often receive no attention . consequently , false - positive results tend to survive longer and have larger long - term consequences in the general public than in the scientific community . likewise , users of epidemiological results outside the scientific community ( eg , regulatory agencies , stakeholders , media , advocacy groups , trial lawyers , the general public ) should be aware of the fact that statistically significant or positive results are often false , in particular when they are not supported by related studies or other lines of evidence . the examples we have presented in this commentary were restricted largely to the field of cancer epidemiology . however , we believe that similar problems affect other areas of epidemiological research , in particular those involving studies with large sets of determinant and outcome variables . a careful analysis of factors enhancing the likelihood of false - positive reporting should be part of the training of epidemiologists , and these issues should be addressed in the discussion section of every epidemiological paper . in general , epidemiologists should recognize the reporting of false - positive results as a major challenge to the scientific credibility of their discipline and institutionalize a greater level of epistemological modesty in this regard ( 4,79 ) .

false - positive results are inherent in the scientific process of testing hypotheses concerning the determinants of cancer and other human illnesses . although much of what is known about the etiology of human cancers has arisen from well - conducted epidemiological studies , epidemiology has been increasingly criticized for producing findings that are often sensationalized in the media and fail to be upheld in subsequent studies . herein we describe examples from cancer epidemiology of likely false - positive findings and discuss conditions under which such results may occur . we suggest general guidelines or principles , including the endorsement of editorial policies requiring the prominent listing of study caveats , which may help reduce the reporting of misleading results . increased epistemological humility regarding findings in epidemiology would go a long way to diminishing the detrimental effects of false - positive results on the allocation of limited research resources , on the advancement of knowledge of the causes and prevention of cancer , and on the scientific reputation of epidemiology and would help to prevent oversimplified interpretations of results by the media and the public .

Examples of False-Positive Results in Environmental and Occupational Cancer Epidemiology Sources of False-Positive Results Publication Bias Caveats and Conclusions

early reports of an increased risk of cancer in a particular exposed group have led to the identification of several agents as human carcinogens ( 3 ) , but for many other agents the suspicion of a carcinogenic effect has not been confirmed in subsequent studies . because cancer epidemiology has great societal impact , and avoidance of false - positive reports in this field deserves particular attention , we have selected one example from environmental epidemiology and one example of a reported occupational cancer association . the first example is of an apparently strong positive result for a pollutant in the general environment that was initially suspected to give rise to cancer but for which such an association has not been confirmed in subsequent studies . the study involved 58 women who had been diagnosed with breast cancer within 6 months of entry in the prospective cohort study and 171 control subjects from the same cohort , and it reported a relative risk of 3.7 ( 95% confidence interval [ ci ] = 1.0 to 13.5 ) for the highest vs lowest 20% of the dde distribution in serum . this study was prompted by earlier results of small ( < 50 case patients ) case control studies ( 1114 ) that had suggested a modest association between breast cancer and exposure to dde and polychlorinated biphenyls . an editorial accompanying the report of the new york city study ( 15 ) stated that these findings may have extraordinary implications for the prevention of breast cancer and that this study should serve as a wake - up call for further urgent research . accordingly , a series of studies were subsequently launched , including a large case control study of breast cancer in long island , ny ( 16 ) , but they failed to replicate the earlier findings . the results on serum dde level from the prospective studies , including an expanded analysis of women who had been included in the original study from new york city and a combined analysis of five prospective studies , were published between 1994 and 2001 ( 1728 ) ( table 1 ) . we performed a cumulative meta - analysis of the initial study and seven subsequent studies published in 19942000 ( figure 1 ) . the final pooled estimate of the relative risk of breast cancer for the highest vs lowest dde category was 0.95 ( 95% ci = 0.7 to 1.3 ) . results of prospective studies on serum 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene level and risk of breast cancer * dde = 1,1-dichloro-2,2-bis(p - chlorophenyl)ethylene ; rr = relative risk ; ci = confidence interval ; hmo = health maintenance organization ; na = not available . cumulative meta - analysis of cohort studies of breast cancer and serum level of 1,1-dichloro-2,2-bis(p - chlorophenyl ) ethylene ( highest vs lowest category in each study ) . in parentheses are references of studies included in the cumulative meta - analyses ( see table 1 for details ) . the reason for the positive findings in the original new york city study is unknown . the short time that elapsed between blood collection and diagnosis of breast cancer may be another explanation because organochlorine compounds are stored in fat tissue , whose metabolism may be affected by the neoplastic process itself ( 29 ) . overinterpretation and an apparent lack of skepticism had major roles in the wide acceptance of the result . although the relative risk estimate of breast cancer among women in the highest quintile of serum dde compared with the lowest was almost 4.0 , the width of the confidence interval and the marginal level of statistical significance should have tempered the enthusiasm for the finding . similarly , a recently reported relative risk estimate of 5.0 ( 95% ci = 1.7 to 14.8 ) for serum levels of p , p-ddt and breast cancer risk , based on subgroup analyses in a nested case what is of ongoing concern is that given the history of studies of this exposure disease issue , the results were still interpreted with few reservations and little if any caution . acrylonitrile is a chemical used in the manufacture of acrylic fibers , resins , and nitrile rubber , as an intermediate in the chemical industry and , in the past , as a fumigant . the first study of lung cancer mortality among workers who were exposed to acrylonitrile in a textile factory in the united states , which was reported in 1978 ( 31 ) , showed a fourfold increased risk , based on six deaths from lung cancer . following the initial report , a total of 16 partially overlapping occupational cohort studies have provided results on lung cancer risk among acrylonitrile - exposed workers , including an expanded analysis of the first study , which identified two additional observed and 2.9 additional expected lung cancer deaths ( 33 ) ( table 2 ) . for studies overlapping with previous reports , only the additional observed lung cancers are reported ( and the relative risk is based on these additional cancers only ) . when we performed a cumulative meta - analysis of the initial 1978 findings and of 15 subsequent studies of acrylonitrile and lung cancer that were published in 19801998 , we found a steady decrease over time in the reported overall relative risk estimate of lung cancer in acrylonitrile workers ( figure 2 ) . the final pooled estimate of the relative risk of lung cancer among the workers was 1.1 ( 95% ci = 0.9 to 1.4 ) . ( 48 ) ] that may provide additional relevant information , we did not consider the results of internal ( eg , dose response ) analyses available for several cohorts , and we did not consider the evidence for or against bias and confounding in each of the studies . however , the declining trend in the summary relative risk estimate as further data accumulated provides evidence that the initial finding was a false - positive result . in an updated review of the evidence in 1999 , an iarc working group modified the classification for acrylonitrile from cumulative meta - analysis of cohort studies of lung cancer and occupational exposure to acrylonitrile . in parentheses are references of studies included in the cumulative meta - analyses ( see table 2 for details ) . similar examples of false - positive results are relevant to the evidence linking exposure to established occupational carcinogens with cancer of organs that are not the primary target of their carcinogenic effect . a detailed analysis of results on other suspected occupational carcinogens is beyond the scope of this commentary , but for several agents it would show the same pattern seen for lung cancer among workers who were exposed to acrylonitrile , that is , early positive results were not confirmed , or were shown to be less strong , in subsequent studies . the issue of multiplicity of exposures and outcomes , and consequent multiple comparisons and subgroup analyses leading to many statistically significant ( yet false ) findings arising by chance , is likely the major contributor to false - positive findings in epidemiology . epidemiological studies , whether cross - sectional , case control , or cohort in design , tend to obtain information on multiple exposures and disease outcomes , so the possibility often exists for examining hundreds or thousands of exposure disease combinations . a similar situation arises in emerging genetic epidemiological studies in which even larger numbers of traits , up to tens or hundreds of thousands of variants , can be examined . in a quantitative approach to this problem , ( 8) reviewed 36 genetic disease associations and found that for 25 of them , the first report gave a stronger estimate of genetic association than did subsequent studies ; in 10 of these associations , the difference between the first and the subsequent results was statistically significant . the same authors subsequently considered the results of 55 meta - analyses ( 50 ) , including 579 study comparisons of genetic associations , and found that for each association the largest study generally yielded more conservative results than the meta - analysis , that in 26% of the meta - analyses the association was statistically significantly stronger in the first study , and that in only 16% was the genetic association found in the first study replicated without evidence of heterogeneity and bias . statistical techniques aimed at reducing the likelihood of false - positive associations , such as false discovery rate ( 51,52 ) , false - positive report probability ( 53 ) , and bayesian false discovery probability ( 54 ) , are now included in the analysis of databases with large numbers of genetic variants [ eg , wellcome trust case control consortium ( 55 ) ] . it seems appropriate that similar approaches be applied in other areas of cancer epidemiology that are prone to reporting false - positive results , including studies of potential occupational and environmental carcinogens . there are multiple reasons besides chance why positive results , particularly initial results , in epidemiological studies may be false ( 2,56 ) . to formally address the determinants of false - positive results in occupational cancer , swaen et al . ( 57 ) scored the main characteristics of studies reporting a positive association with established target ( true positive ) and nontarget ( false positive ) organs of 19 occupational carcinogens . the main determinants of false - positive findings were the absence of a specific a priori hypothesis , a small magnitude of the association , the absence of a dose effect relationship , and the lack of adjustment for tobacco smoking . although this analysis can be criticized because of subjective aspects in the definition of target organs and in quality scoring , it highlights the important roles of confounding and bias , together with chance , in the generation of false - positive results . although chance is a major cause of false - positive results , it is less appreciated that false leads may also be generated by bias . bias consists of a systematic alteration of the research findings due to factors related to study design , data acquisition , analysis , or reporting of results . there is a fundamental distinction between false - positive results that are generated by chance and those caused by bias the former will rarely be replicated in subsequent investigations , whereas bias may operate in a similar fashion in different settings and populations and thus will provide a consistent pattern of independently generated results . even if only a relatively low proportion of results are generated by bias information bias , or recall bias , is a likely source of false - positive findings . a collaborative reanalysis of data from 53 epidemiological studies comparing women with prospectively ascertained records of one or more induced abortions vs women with no such record ( 59 ) reported a relative risk of breast cancer equal to 0.93 ( 95% ci = 0.89 to 0.96 ) . it is now apparent that information bias had a major role in generating the early false - positive results . a likely example of selection bias is the 1981 report of increased pancreatic cancer risk associated with coffee consumption in one of the earliest epidemiological studies of this disease ( 61 ) . many investigators aimed to replicate these findings , but the results of subsequent studies were in general null and by the end of the 1980s a causal association between coffee drinking and pancreatic cancer was considered unlikely ( 62,63 ) . yet for several years the suspicion that coffee drinking was associated with a highly lethal cancer was widespread in the medical community and the general public . this false - positive result was generated , at least in part , by exclusions from the control patient population , but not from the case patients , of individuals with a history of diseases related to cigarette smoking and alcohol consumption ; because these exposures were highly correlated with coffee consumption , the exclusions likely led to a deficit of coffee consumers in the control group . another source of bias and false - positive findings in observational studies is confounding resulting either from incomplete statistical adjustment for measured variables or from the inability to adjust for unmeasured distorting variables . for example , results of early epidemiological studies showed an increased risk of cervical cancer among women who were infected with agents such as herpes simplex virus 2 and chlamydia . when sensitive and specific markers of infection with human papilloma virus became available , however , it was clear that the early positive results for the other sexually transmitted agents were due to confounding by human papilloma virus ( 64 ) . although the importance of residual confounding and unmeasured confounders as a source of bias in epidemiological studies has been downplayed by many ( 65 ) , a recent statistical simulation study ( 66 ) showed that with plausible assumptions , effect sizes on the order of 1.52.0 , which is a magnitude frequently reported in epidemiology studies , can be generated by residual and/or unmeasured confounding . the real effect of unmeasured confounders is of course unknown but may explain some of the notorious differences between well - known cohort studies and subsequent randomized trials . the randomized controlled trial is often presented as the gold standard of epidemiological studies , with the random assignment of treatments mitigating against the potential influences of bias and confounding . however , even randomized controlled studies are not exempt from reporting of false - positive results that arise by chance . in a review of 39 highly cited ( defined as studies cited 1000 or more times in the literature ) randomized controlled trials that reported an original claim of an effect ( 69 ) , the results of 19 were replicated by subsequent studies , whereas for nine trials subsequent results either contradicted the findings of the original report or provided evidence of a weaker effect ( the remaining 11 trials did not have subsequent attempts of replication ) . premature conclusions can be avoided by cautious interpretation of initial trials , especially when they are small , and by trial registration , which is a requirement of the international committee of medical journal editors ( 70 ) . moreover , there is an unfortunate tendency to highlight positive or statistically significant findings in the abstracts of both observational studies and randomized trials , even when the results are doubtful or open to criticism ( 71 ) . another cause of false - positive reporting in cancer epidemiology is publication or reporting bias . as with other forms of bias , statistical tests have been proposed to assess the presence of publication bias ( 7275 ) ; unfortunately , these tests tend to have low power and require a large number of independent studies to provide evidence of bias . one group of suspected environmental carcinogens is the polychlorinated dibenzo - para - dioxins , which are by - products of the manufacture of chlorophenols and chlorophenoxy herbicides ; they are also produced during incineration of organic material and in other thermal processes , such as metal processing and paper pulp bleaching . the most toxic is 2,3,7,8-tetrachlorodibenzo - p - dioxin ( 2,3,7,8-tcdd ) , which has been found to cause tumors in rodents under certain conditions ( 62 ) . concern about the carcinogenicity of dioxins in general , and 2,3,7,8-tcdd in particular , has led to epidemiological studies that are both occupationally based ( eg , workers in the chemical industry , pesticide applicators ) and community based ( eg , residents in contaminated areas , the general population ) . non - hodgkin lymphoma ( nhl ) has been among the suspected targets of dioxin carcinogenicity in humans . at the time of the most recent iarc monograph evaluation ( 76 ) , results on nhl risk were available from 16 studies of 2,3,7,8-tcdd exposed populations eight cohort studies and eight case control studies ( 76 ) . although a meta - analysis suggests an increased risk of nhl in these populations ( meta - relative risk = 1.6 , 95% ci = 1.2 to 2.1 ) , there is evidence of publication bias [ figure 3 ; p = .02 , begg test ( 75 ) ] . in the absence of publication bias , the results of individual studies plotted against their precision , as in figure 3 , should be distributed in a triangular ( funnel ) pattern , with less precise studies ( on the right side of the graph ) randomly dispersed around the mean . publication bias is suggested by the absence of results in one of the parts of the funnel ( eg , lower right corner in figure 3 , where small null studies would be plotted ) . formal testing of publication bias is helpful , but keeping the bandwagon effect concept in mind would go a long way to instilling skepticism when assessing the strength of evidence provided by a new report on a popular hypothesis . funnel plot of results of studies on dioxin exposure and risk of non - hodgkin lymphoma . the examples presented in this commentary suggest that false - positive results are a common problem in cancer and other types of epidemiological studies . what can be done within the practice of epidemiology to reduce the problem ? one of the simplest yet potentially most effective remedies involves increasing emphasis on skepticism when assessing study results , particularly when they are new . epidemiologists by training are most often aware of the methodological limitations of observational studies , particularly those done by others , yet when it comes to practice , and especially the interpretation of their own study results , methodological vigilance is often absent . this absence of skepticism in reporting results in published papers increases the likelihood that a positive finding will receive unwarranted attention in the media and by the public . the tendency to emphasize and overinterpret what appear to be new findings is commonplace , perhaps in part because of a belief that the findings provide information that may ultimately improve public health . although the results may turn out to be wrong , some authors feel it is better to err on the side of overreporting or overstating potentially false - positive results than to miss the identification of a potential new hazard or an opportunity for career advancement . furthermore , the clear acknowledgement that the statistically significant findings may arise from multiple comparisons or subgroup analyses is often missing ; results from a single study are often dispersed across multiple publications , sometimes without reference to each other , hindering the detection of the multiple comparisons or subgroup reporting . strict adherence to the highest epidemiological standards in the design , analysis , reporting , and interpretation of studies would help reduce the likelihood and impact of false - positive results . these standards include provisions to reduce the opportunity for bias and confounding in study design , adequate statistical power , avoidance ( or at least cautious interpretation ) of data - driven subgroup analyses , and accounting for multiple comparisons . the strategy for reporting study results should be specified before the results are known , and selective reporting or emphasis of statistically significant results based on ex post facto subgroup analyses should be discouraged . the interpretation of positive results , especially if they are not supported by additional evidence ( eg , other epidemiological or experimental studies ) , should be careful and cautious . they are also in line with many of the recommendations in the strengthening the reporting of observational studies in epidemiology ( strobe ) statement , which is aimed at strengthening the reporting of epidemiological studies ( 77,78 ) . we propose that the policies of some journals , which require the explicit acknowledgement of study limitations up front in the abstract or in a note box , become standard practice . caution should be applied in the communication of results to the media and the general public , because positive findings tend to attract the media and public attention , whereas findings that do not confirm a previously reported association or do not indicate a new association often receive no attention . consequently , false - positive results tend to survive longer and have larger long - term consequences in the general public than in the scientific community . likewise , users of epidemiological results outside the scientific community ( eg , regulatory agencies , stakeholders , media , advocacy groups , trial lawyers , the general public ) should be aware of the fact that statistically significant or positive results are often false , in particular when they are not supported by related studies or other lines of evidence . the examples we have presented in this commentary were restricted largely to the field of cancer epidemiology . a careful analysis of factors enhancing the likelihood of false - positive reporting should be part of the training of epidemiologists , and these issues should be addressed in the discussion section of every epidemiological paper . in general , epidemiologists should recognize the reporting of false - positive results as a major challenge to the scientific credibility of their discipline and institutionalize a greater level of epistemological modesty in this regard ( 4,79 ) .

according to european hernia society guidelines , patients with symptomatic inguinal hernias should be operated on electively to reduce complaints and to prevent complications such as incarceration or strangulation . it is believed that inguinal hernia occurs in 5% of adult men , and 515% of patients with inguinal hernia will need emergency surgery due to incarceration . polypropylene meshes can be safely used in inguinal hernia surgery , even when intestine resection is needed [ 46 ] . emphasized surgical treatment of inguinal hernia in 6.65% of infertile men as a main cause of infertility , and semen quality was markedly reduced in comparison with that of fertile men . the connection between decreased testicular arterial flow and immunosensitization has been demonstrated in animal models . unilateral testicular ischemia in the rat results in morphological damage in the contralateral testis due to increase in serum cytotoxic antisperm antibodies . it has also been proven in animal models that mesh , besides direct compression on the spermatic cord , additionally narrows the lumen of the deferent duct by its contraction , and also reduces the arterial supply of the testis , consequently affecting spermatogenesis . support the hypothesis that mesh techniques in inguinal hernia repair significantly change the doppler parameters in the early postoperative period , but do not have a significant effect on sperm concentration or rate of progressive motility . in contrast , the study by ramadan et al . showed that mesh placement does not adversely affect ipsilateral testicular flow . the measurement of doppler ultrasound parameters at the level of the testicular artery has been used to assess testicular flow in various studies , and pinggera et al . hillelsohn et al . confirmed those findings and supported intratesticular ri use as an independent indicator of testicular function . they also proposed further investigations of doppler changes occurring after surgical therapy and their association with spermatogenesis . measured the flow in testicular , capsular , and intratesticular arteries following mesh hernia repair in elective patients ; they found only transitory changes in testicular perfusion , without any long - lasting adverse effects on microcirculation . immune infertility. the incidence of sperm autoimmunity in infertile couples is about 20% ; asa assessment is considered as an essential part of infertility management . it is not clear how the mechanism of asa causes male infertility , but recent studies showed significant negative effects of asa on sperm concentration and sperm motility . testicles are paired organs ; it has been proven that the unilateral damage to the deferent duct may affect fertility by production of asa , which can be determined in serum before an evident histological unilateral or bilateral damage , as shown in a rat model . also , it can be affected by free radicals discharged after ischemia in gastrointestinal organs . this is an autoimmune reaction which is responsible for damaging the function of the contralateral testis . it was hypothesized that increased testicular tissue ischemia and hypoxia damage to the basement membrane of endothelial and sertoli cells in the seminiferous tubules may lead to the destruction of the blood - testis barrier , activating autoimmune reaction to produce asa and result in infertility by direct interaction with sperm or indirect change to the local microenvironment . in their study , haoxu et al . compared the effects of different types of hernia mesh on the reproductive function of adult male rats following open tension - free hernioplasty . in comparison with polytetrafluoroethylene groups , the concentration of asa the influence of elective inguinal hernia mesh repair on sperm autoimmunity and testicular flow was investigated by tula et al . , and there was no effect on testicular flow change on the blood - testicle barrier that could lead to a significant immune reaction . the aim of this study was to determine the influence of incarcerated inguinal hernia mesh repair on testicular circulation and to investigate an autoimmunological reaction as a possible reason for infertility . the prospective study was performed over a 3-year period ( january 2012 to december 2014 ) in 25 male patients with incarcerated indirect inguinal hernia who underwent urgent surgical procedure at the department of surgery , university hospital split . exclusion criteria were previous history of fertility problems , testicular trauma , operation or clinical detectable testicular disease , previous hernia repair , genitourinary infection , or immunodeficiency . also , patients with direct inguinal hernias were not included in the study because of anatomical features of direct hernia and consequently less expected negative impact on testicular circulation . the control group consisted of 25 male patients hospitalized for a planned elective surgical treatment of indirect inguinal hernias who had given written consent to participate in the survey and have no exclusionary factors . the approval of the ethics committee of split university hospital was obtained for this study . the preoperative preparation and postoperative care for patients who were treated electively was uniform . also , preoperative preparation for patients who were treated from incarcerated inguinal hernia was also equable . low - molecular weight heparin prophylaxis was administered to all patients before surgery , whereas a single dose of antibiotic prophylaxis ( cefazolin 1 g ) was administered to patients treated for incarcerated hernia . general anesthesia was administered to all patients , and all surgical procedures were performed by the same surgeon , using only anterior tension - free polypropylene mesh repair ( lichtenstein technique ) . there were no signs of ischemic necrosis of content as a result of strangulation , and was no bowel resections were performed , so further procedures were identical in both groups . the mesh was surgically shaped with a vertical cut cranial to the inguinal ligament and a round aperture was positioned around the spermatic cord and fixed with polyglactin 910 interrupted sutures ( vycril , ethicon , norderstedt , germany ) . color doppler ultrasound evaluation of the testicular blood flow was performed in all patients before the operation , in the early postoperative period ( between 24 and 48 h after the operation ) , and in the late postoperative period ( 5 months after operation ) ( figure 1 ) . testicular , capsular , and intratesticular arterial flow dynamics were measured by a single experienced radiologist . the patients were scanned in supine position using an ultrasound unit ( acuson x500 , siemens medical systems , erlangen , germany ) with a 10 mhz linear array probe placed directly on the scrotal skin . realtime scans were obtained in standard longitudinal and transverse plans to identify testicular blood flow on the hernia side . after that , point spectral analysis was performed over the testicular artery 1 cm superior to the pole of the testicle , over the capsular artery , and over the largest intratesticular vessel identified , in the middle and on both poles of the testicle . after testicular blood flow had been identified , the following parameters were evaluated : peak systolic velocity ( psv ) , end - diastolic velocity ( edv ) , pulsative index ( pi ) , and resistive index ( ri ) . the serum samples were immediately frozen and kept at a temperature of 70c until they were analyzed . the antisperm antibodies were analyzed by enzyme - linked immunoadsorbent assay ( elisa , iason , germany ) . the data were analyzed using nonparametric tests ( friedman , wilcoxon , mann - whitney , test ; statistica for windows release 7.0 , statsoft , tulsa , ok ) . there were no signs of ischemic necrosis of content as a result of strangulation , and was no bowel resections were performed , so further procedures were identical in both groups . the mesh was surgically shaped with a vertical cut cranial to the inguinal ligament and a round aperture was positioned around the spermatic cord and fixed with polyglactin 910 interrupted sutures ( vycril , ethicon , norderstedt , germany ) . color doppler ultrasound evaluation of the testicular blood flow was performed in all patients before the operation , in the early postoperative period ( between 24 and 48 h after the operation ) , and in the late postoperative period ( 5 months after operation ) ( figure 1 ) . testicular , capsular , and intratesticular arterial flow dynamics were measured by a single experienced radiologist . the patients were scanned in supine position using an ultrasound unit ( acuson x500 , siemens medical systems , erlangen , germany ) with a 10 mhz linear array probe placed directly on the scrotal skin . realtime scans were obtained in standard longitudinal and transverse plans to identify testicular blood flow on the hernia side . after that , point spectral analysis was performed over the testicular artery 1 cm superior to the pole of the testicle , over the capsular artery , and over the largest intratesticular vessel identified , in the middle and on both poles of the testicle . after testicular blood flow had been identified , the following parameters were evaluated : peak systolic velocity ( psv ) , end - diastolic velocity ( edv ) , pulsative index ( pi ) , and resistive index ( ri ) . the serum samples were immediately frozen and kept at a temperature of 70c until they were analyzed . the antisperm antibodies were analyzed by enzyme - linked immunoadsorbent assay ( elisa , iason , germany ) . the data were analyzed using nonparametric tests ( friedman , wilcoxon , mann - whitney , test ; statistica for windows release 7.0 , statsoft , tulsa , ok ) . fifty male patients underwent indirect inguinal hernia open repair using tension - free polypropylene mesh technique between january 2012 and december 2014 . twenty - five of them underwent elective open repair ( group i ) , and the other twenty - five had incarcerated hernia and underwent urgent open repair ( group ii ) . the median age of patients in group i was 57 years ( range 4081 years ) and in group ii 64 years ( range 2880 years ) . no significant differences between the 2 groups were found regarding age ( p=0.123 ) ( table 1 ) . color doppler ultrasound evaluation of the testicular blood flow ( resistive index - ri , pulsative index - pi , peak systolic velocity - psv , and end - diastolic velocity - edv ) was performed in all patients preoperatively , 2448 hours after the operation ( early postoperative ) , and in 49 patients 5 months after the operation ( late postoperative ) at testicular , capsular , and intratesticular arteries levels . no significant differences between the 2 groups were found in terms of doppler ultrasound parameters for the preoperative period ( mann - whitney test ; p>0.05 ) . friedman analysis showed significant difference of ri only in group ii at testicular artery level ( =32.4 ; p<0.001 ) , capsular artery level ( =23.6 ; p<0.001 ) , and intratesticular arteries level ( =20.9 ; p < 0.001 ) over observed time . in the late postoperative period median value of ri decreased by 0.17 at testicular artery level , 0.14 at capsular artery level , and 0.09 at intratesticular arteries level in comparison to the preoperative values ( table 2 ) . friedman analysis showed significant difference of pi only in group ii at testicular artery level ( =11.2 ; p=0.004 ) , capsular artery level ( =7.9 ; p=0.019 ) , and intratesticular arteries level ( =10.3 ; p=0.006 ) over observed time . in the late postoperative period the median value of pi decreased by 0.41 at testicular artery level , by 0.1 at capsular artery level , and by 0.1 at intratesticular arteries level in comparison to the preoperative values ( table 3 ) . in the group i median psv value rose for 0.7 cm / s at testicular artery level ( =10.2 ; p=0.006 ) and for 0.6 cm / s at intratesticular arteries level ( =6.2 ; p=0.044 ) in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring ( table 4 ) . psv also changed in group ii during observed time but only at the intratesticular arteries level ( =12.2 ; p=0.002 ) . median psv value rose by 0.8 cm / s in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring . in group i , median edv value rose from 0.25 cm / s at the intratesticular artery level ( =6.33 ; p=0.042 ) in the early postoperative period in comparison to the preoperative value , and returned to its preoperative value in the late postoperative measuring . in group ii , median edv value rose from 0.8 cm / s at testicular arteries level ( =10 ; p=0.007 ) and from 0.4 cm / s at intratesticular arteries level ( =12.4 ; p=0.002 ) in the early postoperative period in comparison to the preoperative value and remained at that level in the late postoperative measurement ( table 5 ) . both preoperative and late postoperative antisperm antibodies ( asa ) values there were no statistically significant differences in basal values ( z=0.736 , p=0.220 , mann - whitney u test ) amongst the elective and urgent patient groups . with the elective patient group , median asa rose for 4.5 u / ml after surgery in comparison to the basal value ( z=3.36 ; p=0.001 ) . with the urgent patient group , median asa rose from 8.3 u / ml after the surgery in comparison to the preoperative value ( z=3.7 ; p<0.001 ) . the impact of inguinal hernia mesh repair on fertility of men remains a subject of scientific debate . the results of recent prospective studies in men do not support the hypothesis that inguinal hernia repair with alloplastic mesh prosthesis causes male infertility at a significantly greater rate than those operated on without mesh . a number of clinical studies indicate that potential adverse effects of mesh repairs do not seem to have a clinical impact on male fertility . the numbers of included patients were mainly limited , especially with bilateral hernias , and new observational cohort studies are being designed and in progress to assess the clinical relevance of the effects of inguinal hernia and hernia surgery on male fertility . despite the results stated in these studies , there is still a major ineradicable stance amongst surgeons that precautions are necessary when applying mesh on younger men who are planning to have children . besides the direct damage to testis circulation , a cause of infertility can also be damage to the vas deferens as well as an autoimmune reaction with patients treated with mesh . animal models show substantial effects of mesh hernia repair on structures in the spermatic cord . inguinal vessel obstruction related to inguinal herniorrhaphy in humans is an uncommon and frequently unrecognized cause of male infertility . infertility due to vas deferens dysfunction can be related to direct iatrogenic injury or delayed obstruction caused by scar tissue created as a result of inflammatory tissue response to a foreign body . shin et al . evaluated 14 men with infertility who underwent hernia repairs with polypropylene mesh . surgical exploration in the inguinal region at the previous hernia site revealed a dense fibro - plastic response encompassing the polypropylene mesh , with either trapped or obliterated vas deferens , and obstruction was confirmed by intraoperative vasograms . measured postoperative spermiogram after mesh hernia repair and did not find statistically significant differences to preoperative values . also , lee et al . reported that the presence of serum asa was highly accurate in predicting obstructive azoospermia . vas deferens injury , testicle ischemia , and inflammatory reaction are considered to be the main causes of intolerance to sperm antigens and asa production . asa can be detected prior to obvious testicle damage , and an antispermal immunological reaction can lead to dysfunction of the contralateral testis in infertile men who underwent unilateral mesh hernia repair . there are only a few studies that have investigated asa serum levels after hernia repair . matsuda et al . and friberg et al . found a significant increase of asa in infertile patients treated by non - mesh repair . on the other hand , kapral et al . investigated asa levels in 2 groups of patients and found higher asa values in the group treated by the anterior open free - tension method compared with the group treated by tapp , but the difference in asa value was not clinically important . a possible reason was greater tissue damage and consecutive inflammatory response , which is expected with the anterior approach . also , there was no significant correlation between the change in testicular flow and the increased asa value . the present study assessed asa values in patients treated for incarcerated hernia with mesh ; we expected to find a more pronounced sensitization and therefore higher asa values due to changes in testicular flow and consequently greater impact on blood - testicle barrier , in comparison to patients treated electively . time of postoperative asa measurement was based on 2 facts : asa production is not evident until 1530 days after the blood - testicle barrier is broken , and the possibility of extrinsic compression leaving a scar ends within 3 months when the wound healing process after repair is over . obviously , the short interval between incarceration and surgery was the explanation of immune response absence and preoperatively we did not found significant differences in asa values between the 2 groups ( p=0.220 ) . we found a statistically significant increase of antisperm antibodies ( asa ) in the late postoperative period compared to preoperative values in both groups of patients ( table 6 ) . the functional blood - testicle barrier is the result of cd8 + suppressor t lymphocytes domination . a possible reason for an increase in postoperative asa values is inflammatory reaction with increased prevalence of b - cells on immunosuppressor t cells after the mesh application and the consequent lack of autoimmune response inhibition . the asa values were within the reference range ( 060 iu / ml ) , except in 3 patients with incarcerated hernia who had postoperative values slightly above the upper limit ( 6063 iu / ml ) . extensive manipulation with spermatic cord structures during incarcerated hernia surgery may be the cause of some higher postoperative asa values in those patients . nevertheless , the 2 groups of patients did not show statistically significant differences in postoperatively measured asa values ( p=0.826 ) . until now there have been only a few studies of testicular flow and immunological response to sperm antigens that could be related to male infertility in patients treated by mesh hernia repair . koksal et al . compared testicular perfusion in 2 groups of patients : those treated by totally extraperitoneal mesh repair , and those treated by lichtenstein hernia repair . resistive index ( ri ) results in both groups were statistically insignificant in all preoperative , early , and late postoperative periods . in their ultrasound study , brisinda et al . measured testicular blood flow and testicular volume after tension - free inguinal hernia repair , and investigated testicular circulation using a linear array probe of 10 mhz which had a better resolution than a linear array probe of lower frequency used in other studies . in their study , measurements were made at 3 levels : testicular , capsular , and intratesticular . the study showed only transitory change in testicular perfusion without long - term adverse effects on microcirculation . hillelsohn et al . suggested that intratesticular ri can be a reliable indicator in routine clinical use to establish diagnostic criteria for normal and pathological sperm counts and to identify sub - fertile men . they noticed that ri values show greater testicular vascular resistance in patients with pathological sperm counts than in those with normal sperm counts . until now there has been no doppler study published which researched testicular flows in patients treated for hernia incarceration . our hypothesis was that patients treated for hernia incarceration by urgent surgical procedures with inguinal mesh placement would have significant changes in testicular flow in the late postoperative period in comparison to the control group . therefore , in our study , flow was measured on 3 levels : the testicular , capsular , and intratesticular artery level . this was mandatory due to the fact that increased vascular resistance inside testicular tissue is related to spermatogenesis disturbance . although resistance index ( ri ) and pulsative index ( pi ) values were of most importance , peak - systolic velocity ( psv ) and end - diastolic velocity ( edv ) values were also measured in our study as they are strongly correlated with testicular blood flow . in our study , preoperative ri was higher in patients with incarcerated hernia ( group ii ) compared to the control group at all levels , but we did not prove a significant difference . it was to be expected that incarcerated hernia caused greater testicular flow changes before surgical intervention ( table 2 ) . with patients who underwent elective surgery , ri values did not change significantly in the early and late postoperative periods , so we can conclude that the surgical procedure , with all its consequential tissue changes , does not lead to temporary or permanent changes which could reflect in changes of ri values , regardless of the level of measurement . the decompression effect of the surgical procedure and reactive hyperemia as a part of higher inflammatory response is a possible reason for the significant decrease of ri in the early postoperative period in group ii . it is known that testicular or funicular inflammatory processes can lead to significant ri decline . a surprising finding was the significant decline in ri measurements at all levels in the late postoperative period in group ii , compared to group i which had ultimately higher ri values in the late postoperative period . the development of collateral circulation as an adjustment due to chronic pressure on the narrow hernial neck is a possible explanation for lower ri values in the late postoperative period in group ii . preoperative values of pi were not different in the 2 groups of patients , which suggests that incarceration does not necessarily reduce testicular blood flow . in the electively operated group , pi increased in the early postoperative period and returned to baseline levels in the late postoperative period , and value differences were not statistically significant ( table 3 ) . on the other hand , measurements in emergency patients showed decline of pi values at all levels in the early postoperative period , with a statistically significant decrease in the late postoperative period . we found significantly lower values of pulsatile index at all levels in the late postoperative period in group ii . the dynamics of pi changes could be compared to ri changes in all patients , and both parameters show a good correlation with the testicular blood flow . end - diastolic velocity ( edv ) increased with all patients in the early postoperative period ( table 5 ) . the increase in value with group i was temporary and the values returned to their basal values in the late postoperative period , whereas with urgent patients ( group ii ) edv value remained at the same level as at previous measurements . although the dynamics of changes was similar at all levels of measuring , the difference was statistically significant at the testicular artery level in group i , and at intratesticular arteries level in patients from both groups . the dynamics of these transitory changes of edv can be explained by postoperative tissue changes which are reversible in elective patients , and possibly permanent in patients with incarcerated hernia . however , it should be noted that there were no statistically significant differences in the measured values of edv in the late postoperative period at the level of intratesticular arteries in both groups ( z=1.47 ; p=0.141 ) . peak systolic velocity ( psv ) showed a significant increase in the early postoperative period in both groups at the intratesticular artery level and in group i at the testicular artery level ( table 4 ) . the reason for the transient increase of psv may be an increase in vascular resistance or inflammatory reaction . in the late postoperative period , analyses of blood flow velocity results reveal that both groups of patients had similar changes in the early postoperative period , especially at intratesticular arteries level . the increased psv and edv values in both groups may be due to tissue reaction to surgical procedures . as postoperative hyperemia and edemas recede , psv returned to its initial values in the late postoperative period in both groups of patients , whereas edv had a similar dynamic only in elective patients . a possible explanation of retention of higher edv values in the late postoperative period with patients who underwent surgery due to hernia incarceration is the existence of a prominent testicular collateral irrigation , which explains the decrease in ri value , and a fall in peripheral resistance accelerates the diastolic flow . finally , ri values in the late postoperative period were within the reference ranges in both groups and the clinical significance of these changes is questionable . in our patients , there was no significant correlation between change in testicular flow and in the increase in asa value . although we measured an increase in asa in both groups of patients , we conclude that mesh hernia repair does not cause clinically significant immunological reactions , even in patients with incarcerated hernia . to the best of our knowledge , this is the first study to investigate both testicular circulation and antisperm antibodies in incarcerated hernia mesh repair . color doppler ultrasound evaluation of the testicular blood flow ( resistive index - ri , pulsative index - pi , peak systolic velocity - psv , and end - diastolic velocity - edv ) was performed in all patients preoperatively , 2448 hours after the operation ( early postoperative ) , and in 49 patients 5 months after the operation ( late postoperative ) at testicular , capsular , and intratesticular arteries levels . no significant differences between the 2 groups were found in terms of doppler ultrasound parameters for the preoperative period ( mann - whitney test ; p>0.05 ) . friedman analysis showed significant difference of ri only in group ii at testicular artery level ( =32.4 ; p<0.001 ) , capsular artery level ( =23.6 ; p<0.001 ) , and intratesticular arteries level ( =20.9 ; p < 0.001 ) over observed time . in the late postoperative period median value of ri decreased by 0.17 at testicular artery level , 0.14 at capsular artery level , and 0.09 at intratesticular arteries level in comparison to the preoperative values ( table 2 ) . friedman analysis showed significant difference of pi only in group ii at testicular artery level ( =11.2 ; p=0.004 ) , capsular artery level ( =7.9 ; p=0.019 ) , and intratesticular arteries level ( =10.3 ; p=0.006 ) over observed time . in the late postoperative period the median value of pi decreased by 0.41 at testicular artery level , by 0.1 at capsular artery level , and by 0.1 at intratesticular arteries level in comparison to the preoperative values ( table 3 ) . in the group i median psv value rose for 0.7 cm / s at testicular artery level ( =10.2 ; p=0.006 ) and for 0.6 cm / s at intratesticular arteries level ( =6.2 ; p=0.044 ) in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring ( table 4 ) . psv also changed in group ii during observed time but only at the intratesticular arteries level ( =12.2 ; p=0.002 ) . median psv value rose by 0.8 cm / s in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring . in group i , median edv value rose from 0.25 cm / s at the intratesticular artery level ( =6.33 ; p=0.042 ) in the early postoperative period in comparison to the preoperative value , and returned to its preoperative value in the late postoperative measuring . in group ii , median edv value rose from 0.8 cm / s at testicular arteries level ( =10 ; p=0.007 ) and from 0.4 cm / s at intratesticular arteries level ( =12.4 ; p=0.002 ) in the early postoperative period in comparison to the preoperative value and remained at that level in the late postoperative measurement ( table 5 ) . both preoperative and late postoperative antisperm antibodies ( asa ) values were measured in 46 patients ( table 6 ) . there were no statistically significant differences in basal values ( z=0.736 , p=0.220 , mann - whitney u test ) amongst the elective and urgent patient groups . with the elective patient group , median asa rose for 4.5 u / ml after surgery in comparison to the basal value ( z=3.36 ; p=0.001 ) . with the urgent patient group , median asa rose from 8.3 u / ml after the surgery in comparison to the preoperative value ( z=3.7 ; p<0.001 ) . the impact of inguinal hernia mesh repair on fertility of men remains a subject of scientific debate . the results of recent prospective studies in men do not support the hypothesis that inguinal hernia repair with alloplastic mesh prosthesis causes male infertility at a significantly greater rate than those operated on without mesh . a number of clinical studies indicate that potential adverse effects of mesh repairs do not seem to have a clinical impact on male fertility . the numbers of included patients were mainly limited , especially with bilateral hernias , and new observational cohort studies are being designed and in progress to assess the clinical relevance of the effects of inguinal hernia and hernia surgery on male fertility . despite the results stated in these studies , there is still a major ineradicable stance amongst surgeons that precautions are necessary when applying mesh on younger men who are planning to have children . besides the direct damage to testis circulation , a cause of infertility can also be damage to the vas deferens as well as an autoimmune reaction with patients treated with mesh . animal models show substantial effects of mesh hernia repair on structures in the spermatic cord . inguinal vessel obstruction related to inguinal herniorrhaphy in humans is an uncommon and frequently unrecognized cause of male infertility . infertility due to vas deferens dysfunction can be related to direct iatrogenic injury or delayed obstruction caused by scar tissue created as a result of inflammatory tissue response to a foreign body . shin et al . evaluated 14 men with infertility who underwent hernia repairs with polypropylene mesh . surgical exploration in the inguinal region at the previous hernia site revealed a dense fibro - plastic response encompassing the polypropylene mesh , with either trapped or obliterated vas deferens , and obstruction was confirmed by intraoperative vasograms . measured postoperative spermiogram after mesh hernia repair and did not find statistically significant differences to preoperative values . also , lee et al . reported that the presence of serum asa was highly accurate in predicting obstructive azoospermia . vas deferens injury , testicle ischemia , and inflammatory reaction are considered to be the main causes of intolerance to sperm antigens and asa production . asa can be detected prior to obvious testicle damage , and an antispermal immunological reaction can lead to dysfunction of the contralateral testis in infertile men who underwent unilateral mesh hernia repair . there are only a few studies that have investigated asa serum levels after hernia repair . matsuda et al . and friberg et al . found a significant increase of asa in infertile patients treated by non - mesh repair . on the other hand , kapral et al . tula et al . investigated asa levels in 2 groups of patients and found higher asa values in the group treated by the anterior open free - tension method compared with the group treated by tapp , but the difference in asa value was not clinically important . a possible reason was greater tissue damage and consecutive inflammatory response , which is expected with the anterior approach . also , there was no significant correlation between the change in testicular flow and the increased asa value . the present study assessed asa values in patients treated for incarcerated hernia with mesh ; we expected to find a more pronounced sensitization and therefore higher asa values due to changes in testicular flow and consequently greater impact on blood - testicle barrier , in comparison to patients treated electively . time of postoperative asa measurement was based on 2 facts : asa production is not evident until 1530 days after the blood - testicle barrier is broken , and the possibility of extrinsic compression leaving a scar ends within 3 months when the wound healing process after repair is over . obviously , the short interval between incarceration and surgery was the explanation of immune response absence and preoperatively we did not found significant differences in asa values between the 2 groups ( p=0.220 ) . we found a statistically significant increase of antisperm antibodies ( asa ) in the late postoperative period compared to preoperative values in both groups of patients ( table 6 ) . the functional blood - testicle barrier is the result of cd8 + suppressor t lymphocytes domination . a possible reason for an increase in postoperative asa values is inflammatory reaction with increased prevalence of b - cells on immunosuppressor t cells after the mesh application and the consequent lack of autoimmune response inhibition . the asa values were within the reference range ( 060 iu / ml ) , except in 3 patients with incarcerated hernia who had postoperative values slightly above the upper limit ( 6063 iu / ml ) . extensive manipulation with spermatic cord structures during incarcerated hernia surgery may be the cause of some higher postoperative asa values in those patients . nevertheless , the 2 groups of patients did not show statistically significant differences in postoperatively measured asa values ( p=0.826 ) . until now there have been only a few studies of testicular flow and immunological response to sperm antigens that could be related to male infertility in patients treated by mesh hernia repair . koksal et al . compared testicular perfusion in 2 groups of patients : those treated by totally extraperitoneal mesh repair , and those treated by lichtenstein hernia repair . resistive index ( ri ) results in both groups were statistically insignificant in all preoperative , early , and late postoperative periods . in their ultrasound study , brisinda et al . measured testicular blood flow and testicular volume after tension - free inguinal hernia repair , and no statistically significant differences investigated testicular circulation using a linear array probe of 10 mhz which had a better resolution than a linear array probe of lower frequency used in other studies . in their study , measurements were made at 3 levels : testicular , capsular , and intratesticular . the study showed only transitory change in testicular perfusion without long - term adverse effects on microcirculation . hillelsohn et al . suggested that intratesticular ri can be a reliable indicator in routine clinical use to establish diagnostic criteria for normal and pathological sperm counts and to identify sub - fertile men . they noticed that ri values show greater testicular vascular resistance in patients with pathological sperm counts than in those with normal sperm counts . until now there has been no doppler study published which researched testicular flows in patients treated for hernia incarceration . our hypothesis was that patients treated for hernia incarceration by urgent surgical procedures with inguinal mesh placement would have significant changes in testicular flow in the late postoperative period in comparison to the control group . therefore , in our study , flow was measured on 3 levels : the testicular , capsular , and intratesticular artery level . this was mandatory due to the fact that increased vascular resistance inside testicular tissue is related to spermatogenesis disturbance . although resistance index ( ri ) and pulsative index ( pi ) values were of most importance , peak - systolic velocity ( psv ) and end - diastolic velocity ( edv ) values were also measured in our study as they are strongly correlated with testicular blood flow . in our study , preoperative ri was higher in patients with incarcerated hernia ( group ii ) compared to the control group at all levels , but we did not prove a significant difference . it was to be expected that incarcerated hernia caused greater testicular flow changes before surgical intervention ( table 2 ) . with patients who underwent elective surgery , ri values did not change significantly in the early and late postoperative periods , so we can conclude that the surgical procedure , with all its consequential tissue changes , does not lead to temporary or permanent changes which could reflect in changes of ri values , regardless of the level of measurement . the decompression effect of the surgical procedure and reactive hyperemia as a part of higher inflammatory response is a possible reason for the significant decrease of ri in the early postoperative period in group ii . it is known that testicular or funicular inflammatory processes can lead to significant ri decline . a surprising finding was the significant decline in ri measurements at all levels in the late postoperative period in group ii , compared to group i which had ultimately higher ri values in the late postoperative period . the development of collateral circulation as an adjustment due to chronic pressure on the narrow hernial neck is a possible explanation for lower ri values in the late postoperative period in group ii . preoperative values of pi were not different in the 2 groups of patients , which suggests that incarceration does not necessarily reduce testicular blood flow . in the electively operated group , pi increased in the early postoperative period and returned to baseline levels in the late postoperative period , and value differences were not statistically significant ( table 3 ) . on the other hand , measurements in emergency patients showed decline of pi values at all levels in the early postoperative period , with a statistically significant decrease in the late postoperative period . we found significantly lower values of pulsatile index at all levels in the late postoperative period in group ii . the dynamics of pi changes could be compared to ri changes in all patients , and both parameters show a good correlation with the testicular blood flow . end - diastolic velocity ( edv ) increased with all patients in the early postoperative period ( table 5 ) . the increase in value with group i was temporary and the values returned to their basal values in the late postoperative period , whereas with urgent patients ( group ii ) edv value remained at the same level as at previous measurements . although the dynamics of changes was similar at all levels of measuring , the difference was statistically significant at the testicular artery level in group i , and at intratesticular arteries level in patients from both groups . the dynamics of these transitory changes of edv can be explained by postoperative tissue changes which are reversible in elective patients , and possibly permanent in patients with incarcerated hernia . however , it should be noted that there were no statistically significant differences in the measured values of edv in the late postoperative period at the level of intratesticular arteries in both groups ( z=1.47 ; p=0.141 ) . peak systolic velocity ( psv ) showed a significant increase in the early postoperative period in both groups at the intratesticular artery level and in group i at the testicular artery level ( table 4 ) . the reason for the transient increase of psv may be an increase in vascular resistance or inflammatory reaction . in the late postoperative period , psv values returned to their preoperative values in both groups of patients . analyses of blood flow velocity results reveal that both groups of patients had similar changes in the early postoperative period , especially at intratesticular arteries level . the increased psv and edv values in both groups may be due to tissue reaction to surgical procedures . as postoperative hyperemia and edemas recede , psv returned to its initial values in the late postoperative period in both groups of patients , whereas edv had a similar dynamic only in elective patients . a possible explanation of retention of higher edv values in the late postoperative period with patients who underwent surgery due to hernia incarceration is the existence of a prominent testicular collateral irrigation , which explains the decrease in ri value , and a fall in peripheral resistance accelerates the diastolic flow . finally , ri values in the late postoperative period were within the reference ranges in both groups and the clinical significance of these changes is questionable . in our patients , there was no significant correlation between change in testicular flow and in the increase in asa value . although we measured an increase in asa in both groups of patients , we conclude that mesh hernia repair does not cause clinically significant immunological reactions , even in patients with incarcerated hernia . to the best of our knowledge , this is the first study to investigate both testicular circulation and antisperm antibodies in incarcerated hernia mesh repair . transitory changes in testicular flow and autoimmune reaction due to changes on the blood - testicle barrier were comparable patients treated for incarcerated and those treated for non - complicated indirect inguinal hernia . further investigations are needed to make firm conclusions regarding the effect of incarcerated inguinal hernia mesh repair on immunologic sensitization and , perhaps , male fertility .

backgroundthe aim of our study was to determine an influence of incarcerated inguinal hernia mesh repair on testicular circulation and to investigate consequent sperm autoimmunity as a possible reason for infertility.material/methodsthis prospective study was performed over a 3-year period , and 50 male patients were included ; 25 of these patients underwent elective open mesh hernia repair ( group i ) . group ii consisted of 25 patients who had surgery for incarcerated inguinal hernia . doppler ultrasound evaluation of the testicular blood flow and blood samplings for antisperm antibodies ( asa ) was performed in all patients before the surgery , on the second day , and 5 months after . main outcome ultrasound measures were resistive index ( ri ) and pulsative index ( pi ) , as their values are inversely proportional to testicular blood flow.resultsin group i , ri , and pi temporarily increased after surgery and then returned to basal values in the late postoperative period . friedman analysis showed a significant difference in ri and pi for all measurements in group ii ( p<0.05 ) , with a significant decrease between the preoperative , early , and late postoperative periods . all final values were within reference range , including asa , despite significant increase of asa in the late postoperative period.conclusionsalthough statistically significant differences in values of testicular flow parameters and immunologic sensitization in observed time , final values remained within the reference ranges in all patients . our results suggest that the polypropylene mesh probably does not cause any clinically significant effect on testicular flow and immunologic response in both groups of patients .

Background Material and Methods Operative technique Color Doppler ultrasound Antisperm antibodies (ASA) Statistical analysis Results Testicular blood flow measurements Antisperm antibodies measurements Discussion Conclusions

support the hypothesis that mesh techniques in inguinal hernia repair significantly change the doppler parameters in the early postoperative period , but do not have a significant effect on sperm concentration or rate of progressive motility . in comparison with polytetrafluoroethylene groups , the concentration of asa the influence of elective inguinal hernia mesh repair on sperm autoimmunity and testicular flow was investigated by tula et al . , and there was no effect on testicular flow change on the blood - testicle barrier that could lead to a significant immune reaction . the aim of this study was to determine the influence of incarcerated inguinal hernia mesh repair on testicular circulation and to investigate an autoimmunological reaction as a possible reason for infertility . the prospective study was performed over a 3-year period ( january 2012 to december 2014 ) in 25 male patients with incarcerated indirect inguinal hernia who underwent urgent surgical procedure at the department of surgery , university hospital split . the control group consisted of 25 male patients hospitalized for a planned elective surgical treatment of indirect inguinal hernias who had given written consent to participate in the survey and have no exclusionary factors . color doppler ultrasound evaluation of the testicular blood flow was performed in all patients before the operation , in the early postoperative period ( between 24 and 48 h after the operation ) , and in the late postoperative period ( 5 months after operation ) ( figure 1 ) . after that , point spectral analysis was performed over the testicular artery 1 cm superior to the pole of the testicle , over the capsular artery , and over the largest intratesticular vessel identified , in the middle and on both poles of the testicle . after testicular blood flow had been identified , the following parameters were evaluated : peak systolic velocity ( psv ) , end - diastolic velocity ( edv ) , pulsative index ( pi ) , and resistive index ( ri ) . color doppler ultrasound evaluation of the testicular blood flow was performed in all patients before the operation , in the early postoperative period ( between 24 and 48 h after the operation ) , and in the late postoperative period ( 5 months after operation ) ( figure 1 ) . after that , point spectral analysis was performed over the testicular artery 1 cm superior to the pole of the testicle , over the capsular artery , and over the largest intratesticular vessel identified , in the middle and on both poles of the testicle . after testicular blood flow had been identified , the following parameters were evaluated : peak systolic velocity ( psv ) , end - diastolic velocity ( edv ) , pulsative index ( pi ) , and resistive index ( ri ) . twenty - five of them underwent elective open repair ( group i ) , and the other twenty - five had incarcerated hernia and underwent urgent open repair ( group ii ) . color doppler ultrasound evaluation of the testicular blood flow ( resistive index - ri , pulsative index - pi , peak systolic velocity - psv , and end - diastolic velocity - edv ) was performed in all patients preoperatively , 2448 hours after the operation ( early postoperative ) , and in 49 patients 5 months after the operation ( late postoperative ) at testicular , capsular , and intratesticular arteries levels . friedman analysis showed significant difference of ri only in group ii at testicular artery level ( =32.4 ; p<0.001 ) , capsular artery level ( =23.6 ; p<0.001 ) , and intratesticular arteries level ( =20.9 ; p < 0.001 ) over observed time . in the late postoperative period median value of ri decreased by 0.17 at testicular artery level , 0.14 at capsular artery level , and 0.09 at intratesticular arteries level in comparison to the preoperative values ( table 2 ) . friedman analysis showed significant difference of pi only in group ii at testicular artery level ( =11.2 ; p=0.004 ) , capsular artery level ( =7.9 ; p=0.019 ) , and intratesticular arteries level ( =10.3 ; p=0.006 ) over observed time . in the late postoperative period the median value of pi decreased by 0.41 at testicular artery level , by 0.1 at capsular artery level , and by 0.1 at intratesticular arteries level in comparison to the preoperative values ( table 3 ) . in the group i median psv value rose for 0.7 cm / s at testicular artery level ( =10.2 ; p=0.006 ) and for 0.6 cm / s at intratesticular arteries level ( =6.2 ; p=0.044 ) in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring ( table 4 ) . median psv value rose by 0.8 cm / s in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring . in group i , median edv value rose from 0.25 cm / s at the intratesticular artery level ( =6.33 ; p=0.042 ) in the early postoperative period in comparison to the preoperative value , and returned to its preoperative value in the late postoperative measuring . in group ii , median edv value rose from 0.8 cm / s at testicular arteries level ( =10 ; p=0.007 ) and from 0.4 cm / s at intratesticular arteries level ( =12.4 ; p=0.002 ) in the early postoperative period in comparison to the preoperative value and remained at that level in the late postoperative measurement ( table 5 ) . both preoperative and late postoperative antisperm antibodies ( asa ) values there were no statistically significant differences in basal values ( z=0.736 , p=0.220 , mann - whitney u test ) amongst the elective and urgent patient groups . found a significant increase of asa in infertile patients treated by non - mesh repair . we found a statistically significant increase of antisperm antibodies ( asa ) in the late postoperative period compared to preoperative values in both groups of patients ( table 6 ) . the asa values were within the reference range ( 060 iu / ml ) , except in 3 patients with incarcerated hernia who had postoperative values slightly above the upper limit ( 6063 iu / ml ) . until now there have been only a few studies of testicular flow and immunological response to sperm antigens that could be related to male infertility in patients treated by mesh hernia repair . compared testicular perfusion in 2 groups of patients : those treated by totally extraperitoneal mesh repair , and those treated by lichtenstein hernia repair . resistive index ( ri ) results in both groups were statistically insignificant in all preoperative , early , and late postoperative periods . measured testicular blood flow and testicular volume after tension - free inguinal hernia repair , and investigated testicular circulation using a linear array probe of 10 mhz which had a better resolution than a linear array probe of lower frequency used in other studies . although resistance index ( ri ) and pulsative index ( pi ) values were of most importance , peak - systolic velocity ( psv ) and end - diastolic velocity ( edv ) values were also measured in our study as they are strongly correlated with testicular blood flow . with patients who underwent elective surgery , ri values did not change significantly in the early and late postoperative periods , so we can conclude that the surgical procedure , with all its consequential tissue changes , does not lead to temporary or permanent changes which could reflect in changes of ri values , regardless of the level of measurement . the decompression effect of the surgical procedure and reactive hyperemia as a part of higher inflammatory response is a possible reason for the significant decrease of ri in the early postoperative period in group ii . a surprising finding was the significant decline in ri measurements at all levels in the late postoperative period in group ii , compared to group i which had ultimately higher ri values in the late postoperative period . the development of collateral circulation as an adjustment due to chronic pressure on the narrow hernial neck is a possible explanation for lower ri values in the late postoperative period in group ii . preoperative values of pi were not different in the 2 groups of patients , which suggests that incarceration does not necessarily reduce testicular blood flow . in the electively operated group , pi increased in the early postoperative period and returned to baseline levels in the late postoperative period , and value differences were not statistically significant ( table 3 ) . on the other hand , measurements in emergency patients showed decline of pi values at all levels in the early postoperative period , with a statistically significant decrease in the late postoperative period . we found significantly lower values of pulsatile index at all levels in the late postoperative period in group ii . the dynamics of pi changes could be compared to ri changes in all patients , and both parameters show a good correlation with the testicular blood flow . the increase in value with group i was temporary and the values returned to their basal values in the late postoperative period , whereas with urgent patients ( group ii ) edv value remained at the same level as at previous measurements . although the dynamics of changes was similar at all levels of measuring , the difference was statistically significant at the testicular artery level in group i , and at intratesticular arteries level in patients from both groups . however , it should be noted that there were no statistically significant differences in the measured values of edv in the late postoperative period at the level of intratesticular arteries in both groups ( z=1.47 ; p=0.141 ) . peak systolic velocity ( psv ) showed a significant increase in the early postoperative period in both groups at the intratesticular artery level and in group i at the testicular artery level ( table 4 ) . in the late postoperative period , analyses of blood flow velocity results reveal that both groups of patients had similar changes in the early postoperative period , especially at intratesticular arteries level . as postoperative hyperemia and edemas recede , psv returned to its initial values in the late postoperative period in both groups of patients , whereas edv had a similar dynamic only in elective patients . a possible explanation of retention of higher edv values in the late postoperative period with patients who underwent surgery due to hernia incarceration is the existence of a prominent testicular collateral irrigation , which explains the decrease in ri value , and a fall in peripheral resistance accelerates the diastolic flow . finally , ri values in the late postoperative period were within the reference ranges in both groups and the clinical significance of these changes is questionable . although we measured an increase in asa in both groups of patients , we conclude that mesh hernia repair does not cause clinically significant immunological reactions , even in patients with incarcerated hernia . to the best of our knowledge , this is the first study to investigate both testicular circulation and antisperm antibodies in incarcerated hernia mesh repair . color doppler ultrasound evaluation of the testicular blood flow ( resistive index - ri , pulsative index - pi , peak systolic velocity - psv , and end - diastolic velocity - edv ) was performed in all patients preoperatively , 2448 hours after the operation ( early postoperative ) , and in 49 patients 5 months after the operation ( late postoperative ) at testicular , capsular , and intratesticular arteries levels . friedman analysis showed significant difference of ri only in group ii at testicular artery level ( =32.4 ; p<0.001 ) , capsular artery level ( =23.6 ; p<0.001 ) , and intratesticular arteries level ( =20.9 ; p < 0.001 ) over observed time . in the late postoperative period median value of ri decreased by 0.17 at testicular artery level , 0.14 at capsular artery level , and 0.09 at intratesticular arteries level in comparison to the preoperative values ( table 2 ) . friedman analysis showed significant difference of pi only in group ii at testicular artery level ( =11.2 ; p=0.004 ) , capsular artery level ( =7.9 ; p=0.019 ) , and intratesticular arteries level ( =10.3 ; p=0.006 ) over observed time . in the late postoperative period the median value of pi decreased by 0.41 at testicular artery level , by 0.1 at capsular artery level , and by 0.1 at intratesticular arteries level in comparison to the preoperative values ( table 3 ) . in the group i median psv value rose for 0.7 cm / s at testicular artery level ( =10.2 ; p=0.006 ) and for 0.6 cm / s at intratesticular arteries level ( =6.2 ; p=0.044 ) in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring ( table 4 ) . median psv value rose by 0.8 cm / s in the early postoperative period in comparison to the preoperative value and returned to its preoperative value in the late postoperative measuring . in group i , median edv value rose from 0.25 cm / s at the intratesticular artery level ( =6.33 ; p=0.042 ) in the early postoperative period in comparison to the preoperative value , and returned to its preoperative value in the late postoperative measuring . in group ii , median edv value rose from 0.8 cm / s at testicular arteries level ( =10 ; p=0.007 ) and from 0.4 cm / s at intratesticular arteries level ( =12.4 ; p=0.002 ) in the early postoperative period in comparison to the preoperative value and remained at that level in the late postoperative measurement ( table 5 ) . both preoperative and late postoperative antisperm antibodies ( asa ) values were measured in 46 patients ( table 6 ) . found a significant increase of asa in infertile patients treated by non - mesh repair . we found a statistically significant increase of antisperm antibodies ( asa ) in the late postoperative period compared to preoperative values in both groups of patients ( table 6 ) . the asa values were within the reference range ( 060 iu / ml ) , except in 3 patients with incarcerated hernia who had postoperative values slightly above the upper limit ( 6063 iu / ml ) . resistive index ( ri ) results in both groups were statistically insignificant in all preoperative , early , and late postoperative periods . measured testicular blood flow and testicular volume after tension - free inguinal hernia repair , and no statistically significant differences investigated testicular circulation using a linear array probe of 10 mhz which had a better resolution than a linear array probe of lower frequency used in other studies . although resistance index ( ri ) and pulsative index ( pi ) values were of most importance , peak - systolic velocity ( psv ) and end - diastolic velocity ( edv ) values were also measured in our study as they are strongly correlated with testicular blood flow . with patients who underwent elective surgery , ri values did not change significantly in the early and late postoperative periods , so we can conclude that the surgical procedure , with all its consequential tissue changes , does not lead to temporary or permanent changes which could reflect in changes of ri values , regardless of the level of measurement . the decompression effect of the surgical procedure and reactive hyperemia as a part of higher inflammatory response is a possible reason for the significant decrease of ri in the early postoperative period in group ii . a surprising finding was the significant decline in ri measurements at all levels in the late postoperative period in group ii , compared to group i which had ultimately higher ri values in the late postoperative period . the development of collateral circulation as an adjustment due to chronic pressure on the narrow hernial neck is a possible explanation for lower ri values in the late postoperative period in group ii . preoperative values of pi were not different in the 2 groups of patients , which suggests that incarceration does not necessarily reduce testicular blood flow . in the electively operated group , pi increased in the early postoperative period and returned to baseline levels in the late postoperative period , and value differences were not statistically significant ( table 3 ) . on the other hand , measurements in emergency patients showed decline of pi values at all levels in the early postoperative period , with a statistically significant decrease in the late postoperative period . we found significantly lower values of pulsatile index at all levels in the late postoperative period in group ii . the dynamics of pi changes could be compared to ri changes in all patients , and both parameters show a good correlation with the testicular blood flow . the increase in value with group i was temporary and the values returned to their basal values in the late postoperative period , whereas with urgent patients ( group ii ) edv value remained at the same level as at previous measurements . although the dynamics of changes was similar at all levels of measuring , the difference was statistically significant at the testicular artery level in group i , and at intratesticular arteries level in patients from both groups . however , it should be noted that there were no statistically significant differences in the measured values of edv in the late postoperative period at the level of intratesticular arteries in both groups ( z=1.47 ; p=0.141 ) . peak systolic velocity ( psv ) showed a significant increase in the early postoperative period in both groups at the intratesticular artery level and in group i at the testicular artery level ( table 4 ) . in the late postoperative period , psv values returned to their preoperative values in both groups of patients . analyses of blood flow velocity results reveal that both groups of patients had similar changes in the early postoperative period , especially at intratesticular arteries level . as postoperative hyperemia and edemas recede , psv returned to its initial values in the late postoperative period in both groups of patients , whereas edv had a similar dynamic only in elective patients . a possible explanation of retention of higher edv values in the late postoperative period with patients who underwent surgery due to hernia incarceration is the existence of a prominent testicular collateral irrigation , which explains the decrease in ri value , and a fall in peripheral resistance accelerates the diastolic flow . finally , ri values in the late postoperative period were within the reference ranges in both groups and the clinical significance of these changes is questionable . although we measured an increase in asa in both groups of patients , we conclude that mesh hernia repair does not cause clinically significant immunological reactions , even in patients with incarcerated hernia . to the best of our knowledge , this is the first study to investigate both testicular circulation and antisperm antibodies in incarcerated hernia mesh repair . further investigations are needed to make firm conclusions regarding the effect of incarcerated inguinal hernia mesh repair on immunologic sensitization and , perhaps , male fertility .

the majority of such fractures occur at the thoracolumbar junction ( t11l2 ) , largely due to regional biomechanics in which the rigid , kyphotic thoracic spine is juxtaposed with the more mobile , lordotic lumbar spine.1 burst fractures account for 30 to 64% of the thoracolumbar spinal fractures.1 2 historically , burst fractures have been defined as involving the anterior and middle spinal columns based on the denis three - column spinal model.3 however , the more current classification of burst fractures utilizes the load - sharing classification.4 under the magerl fracture classification system , burst fractures are designated as type a3 compression fractures.2 the etiology is typically related to powerful axial compression forces , such as in falls from heights ( 34 to 54%),1 5 motor vehicle accidents ( 51 to 65%),1 5 or high - intensity sports ( 9%).5 6 7 8 9 despite an overwhelming amount of descriptive and clinical studies addressing traumatic thoracolumbar burst fractures , a significant controversy surrounds the optimum management strategies . typically , the management can be either operative or conservative : the former involves a combination of spinal fixation and fusion that may include interbody devices , with or without decompression,1 10 11 12 13 14 15 16 17 and the latter incorporates the use of braces / casts , bed rest , analgesics , or observation with no bed rest or casts.12 13 17 18 19 20 21 22 23 24 by extension , the algorithms to guide management decisions are limited , with one example being the thoracolumbar injury classification and severity score.25 with ambiguity regarding the best management , compiling and analyzing the levels of evidence ( loes ) for treatment practices can reform the decision - making process . the practice of evidence - based health care has gradually become more prevalent in modern medicine , as the pay - for - performance interventions with high loe lead to greater reimbursements.26 27 28 29 30 31 systematic reviews ( srs ) and meta - analyses provide the means to rapidly analyze a study 's quality , ultimately facilitating the introduction of recommendation grades and providing a foundation for establishing practice guidelines.32 to the author 's best knowledge , there are currently seven srs involving various aspects of traumatic thoracolumbar burst fractures.13 16 33 34 35 36 37 however , no review has yet evaluated the operative management of traumatic thoracolumbar burst fractures with particular attention placed on the loe from an established methodology . therefore , the objective of the present study was to characterize the literature evidence for traumatic thoracolumbar burst fractures , specifically by detailing the studies involving the operative management , with an loe of 2 or greater . a systematic literature review was conducted according to the cochrane guidelines to capture all studies pertaining to traumatic thoracolumbar burst fractures over the past 20 years.38 this time frame was arbitrarily predetermined , as the surgical management of burst fractures has greatly evolved during this period . an electronic search of pubmed ( medline ) was done on april 17 , 2014 with permutations of the following terms : ( spine and burst ) or burst fracture or ( burst and fracture ) or ( a3 and fracture and spine ) . the inclusion criteria included : ( 1 ) burst fractures resulting from traumatic mechanism and ( 2 ) fractures of the thoracic or lumbar spine . the exclusion criteria were : ( 1 ) osteoporotic burst fractures , ( 2 ) pathologic burst fractures , ( 3 ) cervical fractures , ( 4 ) biomechanical studies or those involving cadavers , and ( 5 ) computer - based studies such as finite element analyses . when inclusion or exclusion was unclear , full texts were retrieved ; if doubt remained , a decision was made through discussion and addition of a third reviewer if needed . following the search , only those involving operative management were selected to be reviewed in the present study . loes were assigned using criteria from the journal of clinical orthopaedics and related research,30 which is adapted from the oxford centre of evidence based medicine loes.39 40 the loe scale ranges from 1 being the highest to 5 the lowest . studies with level 1 or 2 loe were classified as high loe . ambiguities regarding loe were resolved through discussion and addition of a third reviewer if needed . once all the studies were assigned a loe , only those rated as level 1 and 2 were included . all data were stored in microsoft excel 2013 ( microsoft corp . , redmond , washington , united states ) . the search yielded a total of 1,138 abstracts , of which 272 met the criteria , involved operative management , as well as were published between january 1 , 1992 , and april 17 , 2014 . twenty - three of the studies ( 8.5% ) met level 1 ( n = 4 , 1.5% ) or 2 ( n = 19 , 7.0% ) criteria ( table 1 ) . fifteen of the studies ( 5.5% ) investigated the differences between operative versus nonoperative treatment . of these , seven qualified as high loe ( two level 1 , five level 2 ; table 2 ) . all others were classified as level 3 ( n = 6 ) and level 4 ( n = 2 ) . abbreviations : ais , american spinal injury association impairment scale ; asia , american spinal injury association ; ffbh - r , hannover functional questionnaire ; lbos , low back outcome score ; ls , long - segment ; lsc , load - sharing classification ; mpq , mcgill pain questionnaire ; mspi , monosegmental pedicle instrumentation ; na , not applicable ; odi , oswestry disability index ; rct , randomized controlled trial ; rmdq , roland - morris disability questionnaire ; sf-36 , short form-36 ; sspi , short - segment pedicle instrumentation ; tlif , transforaminal lumbar interbody fusion ; vas , visual analog scale . two studies one sr and one randomized controlled trial ( rct)met level 1 criteria . abudou et al reported an sr through the cochrane collaboration in which they searched for rcts comparing operative and nonoperative treatment in patients without neurologic deficit.41 due to strict inclusion and exclusion criteria , only two studies with contrasting results were identified ( siebenga et al and wood et al , discussed below),14 42 leading the authors to conclude that there is insufficient evidence to conclude whether surgical or conservative treatment yields superior functional outcomes.41 the second study was an rct reported by siebenga et al that compared the radiographic and functional outcomes in neurologically intact patients with burst fractures . the operative cohort underwent short - segment posterior pedicle screw fixation above and below the fracture level with an autologous fusion ; the nonoperative treatment involved rehabilitation and bed rest for 5 days following the injury . although both the patient groups had similar baseline local and regional sagittal angles and visual analog scale scores , the surgery resulted in significantly lower angles and less pain at the final follow - up . further , the authors reported that the conservative treatment led to higher roland morris disability questionnaire-24 ( rmdq-24 ) scores at the final follow - up , with fewer patients capable of returning to work.42 five studies met the level 2 criteria . in 1992 , dr . gertzbein and the scoliosis research society prospectively analyzed spinal fractures in 1,019 patients across 12 countries . the results revealed a statistically significant , albeit weak , relationship between the initial canal compromise and the neurologic deficit in burst fractures . however , there were no differences in the rates of the neurologic improvement between the treatment groups . of the operative patients , the neurologic outcomes were equivalent for anterior versus posterior surgery . among patients with preoperative neurologic deterioration , surgical management , particularly anterior surgery , led to greater improvement . at 2-year follow - up , a higher proportion of patients managed conservatively complained of severe / moderate pain compared with none / mild pain.1 hitchon et al performed a prospective study to outline the indications , benefits , and costs of bed rest compared with surgical stabilization with pedicle screws , rods , and plates ; luque or harington rods with sublaminar wiring ; or an anterior spinal device . although both cohorts improved neurologically , a progressive angular deformity was present , with rates twice as high in the surgery group.11 shen et al conducted an rct to examine nonoperative treatment ( a hyperextension brace for 3 months without fracture reduction ) versus short - segment posterior fixation . although the surgical group had better pain and clinical outcomes in the early postoperative period ( 3 to 6 months ) , these differences were not observed at final follow - up ( 2 years ) . regarding kyphosis correction , the operative group had an initial correction of 17 degrees , which was lost over time , and the nonoperative group had a loss of 4 degrees . also , hospital charges were 4 times greater with surgery.12 wood et al reported another rct in which nonoperative patients underwent a fracture reduction supplemented by a body cast or a thoracolumbar orthosis for 3 months . the operative patients underwent either a posterolateral spinal arthrodesis with pedicle screw and hook instrumentation and autologous bone grafting or an anterior two - level fibular and rib - strut construct arthrodesis with local autologous bone grafting and instrumentation . both groups were similar at final follow - up with respect to the radiographic and clinical outcomes.14 a level 2 meta - analysis reported by gnanenthiran et al pooled the results from the four aforementioned trials . the surgical group , however , did show better correction radiographically at follow - up , albeit this came with higher complication rates and costs.13 two hundred fifty - seven studies ( 9.5% ) examined the operative treatment and technique of thoracolumbar burst fractures . there were 16 high loe studies ( two level 1 , 14 level 2 ; table 2 ) , and the rest were level 3 ( n = 32 ) , level 4 ( n = 208 ) , and level 5 studies ( n = 1 ) . dai et al investigated posterolateral fusion in combination with short - segment posterior pedicle screw instrumentation into the levels cephalad and caudad to the fractured vertebra . a total of 37 patients underwent fusion and36 did not , and no differences in the radiographic or clinical outcomes were seen.43 in the second study , two muscle - sparing surgical approaches the percutaneous and paraspinal approaches for pedicle screw and rod fixation were compared by jiang et al for efficacy and safety . the percutaneous approach led to lower intraoperative blood loss , shorter surgical time , and shorter hospitalization . in the short - term postoperative period , this approach also yielded less pain and greater improvements in the functional scores . in contrast , the paraspinal approach resulted in better correction of the kyphosis and restoration of the vertebral height . the long - term clinical outcomes were equivalent.44 the first level 2 study was published by gertzbein and colleagues , in which 25 patients were treated with the ao internal fixator before or after the first 4 days postinjury . earlier intervention led to greater improvement in canal encroachment.45 vornanen et al compared three methods of internal fixation ( harrington rods , ao internal fixators , posterior segmental fixators ) with respect to improvement in canal encroachment . all methods improved encroachment , with harrington rods providing the best reduction.46 next , alanay et al reported an rct comparing short - segment pedicle instrumentation with or without transpedicular grafting . no differences in the radiographic , operative , pain , or functional outcomes were seen.47 stanci et al compared anterior and posterior surgical approaches for overall clinical outcomes and operative efficacy . of the 25 patients enrolled , 13 received anterior neurodecompression and fixation and 12 underwent posterior fixation by hook and rod and pedicle screw fixation . neither the neurologic outcomes nor economic / functional outcomes proved to be different . however , the posterior group suffered less blood loss and had shorter operative times , better aesthetic outcomes , and lower costs , complications , and donor site pain.48 tezeren and kuru performed a prospective study in which patients were treated with short- or long - segment pedicle fixation.49 the short - segment fixation involved fixation one level above and below the fracture ; long - segment instrumentation involved hook fixation two or three levels above plus pedicle fixation two levels below the fracture . at final follow - up , patients in the long - segment group showed significantly better correction of the sagittal index and anterior body compression.49 wood and colleagues compared anterior reconstruction , fusion , and instrumentation to posterior fusion with instrumentation.57 although the posterior approach had significantly less blood loss , no differences in radiographic , clinical , and functional outcomes were observed . in a prospective randomized trial by korovessis et al , patients received a combined anterior and posterior stabilization technique or standard posterior short - segment transpedicular fixation . both resulted in radiographic improvement but the combined group faced greater blood loss , operative time , complications , and duration of hospital stay . furthermore , visual analog scale and sf-36 scores did not correlate with the anatomical correction in either treatment group.50 dai et al prospectively evaluated the outcomes following anterior - only stabilization with either structural autografts or titanium mesh cages . apart from the differing instrumentation , the surgical approaches were similar between the groups . in the autograft group , the operative times were longer but no other differences were observed . both the treatment groups improved postoperatively and there were no significant differences.51 in 2009 , pang et al prospectively compared the outcomes and surgical efficiency / safety between an approach via the paravertebral muscle or the traditional posterior approach . the paravertebral muscle approach led to significantly better outcomes in terms of blood loss , drainage , duration of recumbency , and clinical outcome scores.52 tezeren et al prospectively investigated long - segment instrumentation with or without fusion . the average operation time and blood loss were significantly lower in the nonfusion group , but the two groups were otherwise similar in terms of the radiologic and clinical assessment measures that were analyzed.53 wei and colleagues published a clinical trial in 2010 comparing the clinical and radiologic outcomes between monosegmental transpedicular fixation and short - segment pedicle instrumentation . patients were analyzed based on the operative data , low back outcome score ( lbos ) , oswestry disability index , load - sharing classification , sagittal index , and percentage of anterior body height compression . monosegmental fixation resulted in lower operative times and blood loss but did lead to higher lbos scores.54 the groups were also similar at final follow - up for the sagittal index , local kyphosis , and average correction loss . following this , in 2012 , jindal et al assessed the benefits of short - segment pedicle fixation with or without fusion in an rct . ultimately , the fusion did not improve the radiographic or functional outcomes but did increase the operative time and necessity for blood transfusion.55 schmid et al prospectively compared thoracoscopy or transforaminal lumbar interbody fusion ( tlif ) in the anterior reconstruction of burst fractures . the tlif group received a monocortical strut graft and the thoracoscopy included tricortical strut grafts or titanium vertebral body - replacing implants . satisfactory outcomes were achieved in both groups , and no differences were seen in the radiographic or clinical outcomes . surgical decompression , however , was higher with tlif.56 in 2013 , xu et al performed a meta - analysis of rcts , quasi - rcts , or controlled trials investigating anterior versus posterior approaches in attempt to identify the optimal approach.37 the authors analyzed seven studies comparing 179 and 152 patients in the anterior and posterior groups , respectively . no significant differences were found between the approaches for neurologic recovery , return to work , complications , and deformity correction . however , the anterior group was associated with longer operative time , greater blood loss , and a higher cost than the posterior approach . the search yielded a total of 1,138 abstracts , of which 272 met the criteria , involved operative management , as well as were published between january 1 , 1992 , and april 17 , 2014 . twenty - three of the studies ( 8.5% ) met level 1 ( n = 4 , 1.5% ) or 2 ( n = 19 , 7.0% ) criteria ( table 1 ) . fifteen of the studies ( 5.5% ) investigated the differences between operative versus nonoperative treatment . of these , seven qualified as high loe ( two level 1 , five level 2 ; table 2 ) . all others were classified as level 3 ( n = 6 ) and level 4 ( n = 2 ) . abbreviations : ais , american spinal injury association impairment scale ; asia , american spinal injury association ; ffbh - r , hannover functional questionnaire ; lbos , low back outcome score ; ls , long - segment ; lsc , load - sharing classification ; mpq , mcgill pain questionnaire ; mspi , monosegmental pedicle instrumentation ; na , not applicable ; odi , oswestry disability index ; rct , randomized controlled trial ; rmdq , roland - morris disability questionnaire ; sf-36 , short form-36 ; sspi , short - segment pedicle instrumentation ; tlif , transforaminal lumbar interbody fusion ; vas , visual analog scale . two studies one sr and one randomized controlled trial ( rct)met level 1 criteria . abudou et al reported an sr through the cochrane collaboration in which they searched for rcts comparing operative and nonoperative treatment in patients without neurologic deficit.41 due to strict inclusion and exclusion criteria , only two studies with contrasting results were identified ( siebenga et al and wood et al , discussed below),14 42 leading the authors to conclude that there is insufficient evidence to conclude whether surgical or conservative treatment yields superior functional outcomes.41 the second study was an rct reported by siebenga et al that compared the radiographic and functional outcomes in neurologically intact patients with burst fractures . the operative cohort underwent short - segment posterior pedicle screw fixation above and below the fracture level with an autologous fusion ; the nonoperative treatment involved rehabilitation and bed rest for 5 days following the injury . although both the patient groups had similar baseline local and regional sagittal angles and visual analog scale scores , the surgery resulted in significantly lower angles and less pain at the final follow - up . further , the authors reported that the conservative treatment led to higher roland morris disability questionnaire-24 ( rmdq-24 ) scores at the final follow - up , with fewer patients capable of returning to work.42 five studies met the level 2 criteria . in 1992 , dr . gertzbein and the scoliosis research society prospectively analyzed spinal fractures in 1,019 patients across 12 countries . operative intervention was administered to 545 patients and 96 received conservative therapy . the results revealed a statistically significant , albeit weak , relationship between the initial canal compromise and the neurologic deficit in burst fractures . however , there were no differences in the rates of the neurologic improvement between the treatment groups . of the operative patients , the neurologic outcomes were equivalent for anterior versus posterior surgery . among patients with preoperative neurologic deterioration , surgical management , particularly anterior surgery , led to greater improvement . at 2-year follow - up , a higher proportion of patients managed conservatively complained of severe / moderate pain compared with none / mild pain.1 hitchon et al performed a prospective study to outline the indications , benefits , and costs of bed rest compared with surgical stabilization with pedicle screws , rods , and plates ; luque or harington rods with sublaminar wiring ; or an anterior spinal device . all patients were immobilized in thoracolumbar orthoses for 3 to 5 months . although both cohorts improved neurologically , a progressive angular deformity was present , with rates twice as high in the surgery group.11 shen et al conducted an rct to examine nonoperative treatment ( a hyperextension brace for 3 months without fracture reduction ) versus short - segment posterior fixation . although the surgical group had better pain and clinical outcomes in the early postoperative period ( 3 to 6 months ) , these differences were not observed at final follow - up ( 2 years ) . regarding kyphosis correction , the operative group had an initial correction of 17 degrees , which was lost over time , and the nonoperative group had a loss of 4 degrees . also , hospital charges were 4 times greater with surgery.12 wood et al reported another rct in which nonoperative patients underwent a fracture reduction supplemented by a body cast or a thoracolumbar orthosis for 3 months . the operative patients underwent either a posterolateral spinal arthrodesis with pedicle screw and hook instrumentation and autologous bone grafting or an anterior two - level fibular and rib - strut construct arthrodesis with local autologous bone grafting and instrumentation . both groups were similar at final follow - up with respect to the radiographic and clinical outcomes.14 a level 2 meta - analysis reported by gnanenthiran et al pooled the results from the four aforementioned trials . the surgical group , however , did show better correction radiographically at follow - up , albeit this came with higher complication rates and costs.13 two hundred fifty - seven studies ( 9.5% ) examined the operative treatment and technique of thoracolumbar burst fractures . there were 16 high loe studies ( two level 1 , 14 level 2 ; table 2 ) , and the rest were level 3 ( n = 32 ) , level 4 ( n = 208 ) , and level 5 studies ( n = 1 ) . dai et al investigated posterolateral fusion in combination with short - segment posterior pedicle screw instrumentation into the levels cephalad and caudad to the fractured vertebra . a total of 37 patients underwent fusion and36 did not , and no differences in the radiographic or clinical outcomes were seen.43 in the second study , two muscle - sparing surgical approaches the percutaneous and paraspinal approaches for pedicle screw and rod fixation were compared by jiang et al for efficacy and safety . the percutaneous approach led to lower intraoperative blood loss , shorter surgical time , and shorter hospitalization . in the short - term postoperative period , this approach also yielded less pain and greater improvements in the functional scores . in contrast , the paraspinal approach resulted in better correction of the kyphosis and restoration of the vertebral height . the long - term clinical outcomes were equivalent.44 the first level 2 study was published by gertzbein and colleagues , in which 25 patients were treated with the ao internal fixator before or after the first 4 days postinjury . earlier intervention led to greater improvement in canal encroachment.45 vornanen et al compared three methods of internal fixation ( harrington rods , ao internal fixators , posterior segmental fixators ) with respect to improvement in canal encroachment . all methods improved encroachment , with harrington rods providing the best reduction.46 next , alanay et al reported an rct comparing short - segment pedicle instrumentation with or without transpedicular grafting . no differences in the radiographic , operative , pain , or functional outcomes were seen.47 stanci et al compared anterior and posterior surgical approaches for overall clinical outcomes and operative efficacy . of the 25 patients enrolled , 13 received anterior neurodecompression and fixation and 12 underwent posterior fixation by hook and rod and pedicle screw fixation . neither the neurologic outcomes nor economic / functional outcomes proved to be different . however , the posterior group suffered less blood loss and had shorter operative times , better aesthetic outcomes , and lower costs , complications , and donor site pain.48 tezeren and kuru performed a prospective study in which patients were treated with short- or long - segment pedicle fixation.49 the short - segment fixation involved fixation one level above and below the fracture ; long - segment instrumentation involved hook fixation two or three levels above plus pedicle fixation two levels below the fracture . at final follow - up , patients in the long - segment group showed significantly better correction of the sagittal index and anterior body compression.49 wood and colleagues compared anterior reconstruction , fusion , and instrumentation to posterior fusion with instrumentation.57 although the posterior approach had significantly less blood loss , no differences in radiographic , clinical , and functional outcomes were observed . in a prospective randomized trial by korovessis et al , patients received a combined anterior and posterior stabilization technique or standard posterior short - segment transpedicular fixation . both resulted in radiographic improvement but the combined group faced greater blood loss , operative time , complications , and duration of hospital stay . furthermore , visual analog scale and sf-36 scores did not correlate with the anatomical correction in either treatment group.50 dai et al prospectively evaluated the outcomes following anterior - only stabilization with either structural autografts or titanium mesh cages . apart from the differing instrumentation , the surgical approaches were similar between the groups . in the autograft group , both the treatment groups improved postoperatively and there were no significant differences.51 in 2009 , pang et al prospectively compared the outcomes and surgical efficiency / safety between an approach via the paravertebral muscle or the traditional posterior approach . the paravertebral muscle approach led to significantly better outcomes in terms of blood loss , drainage , duration of recumbency , and clinical outcome scores.52 tezeren et al prospectively investigated long - segment instrumentation with or without fusion . the average operation time and blood loss were significantly lower in the nonfusion group , but the two groups were otherwise similar in terms of the radiologic and clinical assessment measures that were analyzed.53 wei and colleagues published a clinical trial in 2010 comparing the clinical and radiologic outcomes between monosegmental transpedicular fixation and short - segment pedicle instrumentation . patients were analyzed based on the operative data , low back outcome score ( lbos ) , oswestry disability index , load - sharing classification , sagittal index , and percentage of anterior body height compression . monosegmental fixation resulted in lower operative times and blood loss but did lead to higher lbos scores.54 the groups were also similar at final follow - up for the sagittal index , local kyphosis , and average correction loss . following this , in 2012 , jindal et al assessed the benefits of short - segment pedicle fixation with or without fusion in an rct . ultimately , the fusion did not improve the radiographic or functional outcomes but did increase the operative time and necessity for blood transfusion.55 schmid et al prospectively compared thoracoscopy or transforaminal lumbar interbody fusion ( tlif ) in the anterior reconstruction of burst fractures . the tlif group received a monocortical strut graft and the thoracoscopy included tricortical strut grafts or titanium vertebral body - replacing implants . satisfactory outcomes were achieved in both groups , and no differences were seen in the radiographic or clinical outcomes . surgical decompression , however , was higher with tlif.56 in 2013 , xu et al performed a meta - analysis of rcts , quasi - rcts , or controlled trials investigating anterior versus posterior approaches in attempt to identify the optimal approach.37 the authors analyzed seven studies comparing 179 and 152 patients in the anterior and posterior groups , respectively . no significant differences were found between the approaches for neurologic recovery , return to work , complications , and deformity correction . however , the anterior group was associated with longer operative time , greater blood loss , and a higher cost than the posterior approach . traumatic thoracolumbar burst fractures remain a challenge to treat , and optimal operative and nonoperative techniques have not yet been established . the goals of treatment are to obtain early patient mobilization and a painless , balanced , stable vertebral column with maximum spine mobility and optimal neurologic function . to achieve these goals , numerous studies have investigated different diagnostic , prognostic , and management approaches , with a greater emphasis in the latter . the present study attempted to characterize the high loe studies pertaining to the operative management of traumatic thoracolumbar burst fractures and found that the overall literature contained 23 studies ( 8.5% ) in pubmed that met an loe of 1 or 2 . when treating neurologically intact patients with thoracolumbar burst fractures , there is no difference in the clinical outcomes when comparing a conservative or open operative approach . this is supported by five of the seven high - level studies investigating this cohort . although better correction of the spinal deformity can be achieved surgically , three level 2 clinical studies demonstrated higher rates of complications and costs with this approach . two high - level srs confirmed no difference in the functional outcomes when the approaches were compared . these studies utilized different operative and conservative approaches , follow - up times , and inclusion / exclusion criteria . it is worth noting that there are limited numbers of patients in these studies and thus their results are inherently limited . there is a high loe for short- or long - segment pedicle instrumentation without fusion . tezeren and kuru compared short- versus long - segment pedicle fixation in a level 2 study and concluded that better correction can be achieved with the long construct , and lbos outcomes were similar.49 longer constructs provide the necessary rigidity to decrease failure rates , but they also decrease patient mobility due to the more rigid fixation , which may impact quality - of - life parameters that were not assessed . four high - level studies questioned the necessity of additional fusion to posterior short - segment pedicle instrumentation . dai et al ( level 1 ) , alanay et al ( level 2 ) , and jindal et al ( level 2 ) demonstrated that bone grafting was not necessary in short - segment pedicle instrumentation in rcts.43 47 55 tezeren et al ( level 2 ) also demonstrated that fusion did not change the radiologic or clinical outcomes in long - segment instrumentation.53 the operative time would decrease by forgoing fusion , and because bone graft is not needed , this would collectively decrease the risk of infections . as mentioned previously with studies involving neurologically intact patients , the discussed rcts also have small sample sizes and the conclusions from these studies should be considered within the context of that limitation . open approaches to spine surgery are associated with higher morbidity , and there has been a recent trend toward preserving thoracolumbar muscle attachments . the percutaneous and paraspinal approaches to thoracolumbar burst fractures are supported by level 1 and 2 studies . pang et al ( level 2 ) demonstrated that the paravertebral approach results in better clinical outcome scores than the traditional posterior approach.52 jiang et al ( level 1 ) demonstrated that shorter hospitalization time and equal 3-year clinical outcomes can be achieved with the percutaneous versus paraspinal approach in pedicle screw and rod fixation.44 however , better deformity correction can be achieved with the paraspinal approach . when investigating an anterior , posterior , or combined approach in instrumentation , four level 2 studies reported similar radiographic , clinical , and functional outcomes.37 48 50 57 furthermore , when compared with the anterior or combined approach , the posterior approach correlated with fewer complications and operative risks . when considering anterior reconstruction , dai et al demonstrated no significant differences in the use of titanium or autologous grafting for fusion.51 furthermore , schmid et al demonstrated that transforaminal lumbar interbody fusion may provide further surgical decompression than thoracoscopy.56 and finally , xu et al performed a meta - analysis and found similar results with the exception that the anterior approach also had a higher cost associated with it.37 the choice of the operative approach can be decided by the preference of the surgeon and patient , while acknowledging that greater risks and possible higher costs are associated with the anterior approach . additional considerations in the operative management of traumatic thoracolumbar burst fractures include both the correction of a kyphotic deformity and the potential loss of a kyphotic correction over time . the results regarding whether operative management maintained the correction more successfully than nonoperative management are mixed.12 14 42 all studies show a significant improvement postoperatively ; however , in the study by siebenga et al ( level 1),42 the operative group maintained the correction and the kyphosis was significantly less than in the nonoperative group at final follow - up ( a mean of 4.3 years ) . on the other hand , shen et al ( level 2 ) and wood et al ( level 2 ) did not show any differences in the deformity correction between operative and nonoperative management at the final follow - up ( 2 years).12 14 the clinical implication of kyphotic correction is questionable , as wood et al ( level 2 ) and korovessis et al ( level 2 ) both did not find any correlation between the degree of kyphosis at the final follow - up and the degree of pain or disability.14 50 operative management does improve pain and stability.12 13 14 16 42 most studies had similar radiographic results between the various operative techniques.37 43 47 51 53 54 55 56 57 specifically , fusion does not appear to affect the maintenance of radiographic correction as demonstrated by four high loe studies.43 47 53 55 however , jiang and colleagues ( level 1 ) found better long - term local kyphosis correction with a paraspinal approach versus a percutaneous.44 similarly , tezeren and kuruet ( level 2 ) found better kyphotic correction at final follow - up for patients who underwent long - segment pedicle fixation compared with short - segment fixation.49 the heterogeneity nature of burst fractures due to the different mechanisms of injury results in a variety of clinical presentations , and makes it difficult to formulate a universal approach . other srs were limited to select studies by the strict inclusion / exclusion criteria and therefore did not include most of the literature covered in this report . this report was conducted in accordance with the cochrane collaboration handbook on srs ; it highlights the current evidence and provides further insight into future investigations needed . this review is limited by studies found only in the pubmed database ( medline ) . however , the medline database is well accepted as the comprehensive source of relevant literature . the literature contains a high loe to support the operative management of traumatic thoracolumbar burst fractures . the highest degree of controversy involves the decision whether to operate on neurologically intact patients . for patients who are neurologically intact , a high loe demonstrated similar functional outcomes and lower complication rates and costs with conservative management when compared with surgical management . if a patient is determined to require surgery , there is no general consensus on the appropriate technique to be used . there is a high loe for short- or long - segment pedicle instrumentation without fusion and less invasive ( percutaneous and paraspinal ) approaches . furthermore , posterior approaches are associated with fewer complications as opposed to anterior or combined approaches .

study design systematic literature review . objective the management of traumatic thoracolumbar burst fractures ( tlbf ) remains challenging , and analyzing the levels of evidence ( loes ) for treatment practices can reform the decision - making process . however , no review has yet evaluated the operative management of traumatic thoracolumbar burst fractures with particular attention placed on loe from an established methodology . the objective of the present study was to characterize the literature evidence for tlbf , specifically for operative management . methods a comprehensive search of the english literature over the past 20 years was conducted using pubmed ( medline ) . the inclusion criteria consisted of ( 1 ) traumatic burst fractures ( 2 ) in the thoracic or lumbar spine . exclusion criteria included ( 1 ) osteoporotic burst fractures , ( 2 ) pathologic burst fractures , ( 3 ) cervical fractures , ( 4 ) biomechanical studies or those involving cadavers , and ( 5 ) computer - based studies . studies were assigned an loe and those meeting level 1 or 2 were included . results from 1,138 abstracts , 272 studies met the criteria . twenty - three studies ( 8.5% ) met level 1 ( n = 4 , 1.5% ) or 2 ( n = 19 , 7.0% ) criteria . all 23 studies were reported . conclusions the literature contains a high loe to support the operative management of traumatic thoracolumbar burst fractures . for patients who are neurologically intact , a high loe demonstrated similar functional outcomes , lower complication rates , and less costs with conservative management when compared with surgical management . there is a high loe for short- or long - segment pedicle instrumentation without fusion and less invasive ( percutaneous and paraspinal ) approaches . furthermore , the posterior approaches are associated with lower complications as opposed to the anterior or combined approaches .

Introduction Methods Results Overall Composition of the Literature Operative versus Nonoperative Management Treatment and Technique Discussion Conclusion

the majority of such fractures occur at the thoracolumbar junction ( t11l2 ) , largely due to regional biomechanics in which the rigid , kyphotic thoracic spine is juxtaposed with the more mobile , lordotic lumbar spine.1 burst fractures account for 30 to 64% of the thoracolumbar spinal fractures.1 2 historically , burst fractures have been defined as involving the anterior and middle spinal columns based on the denis three - column spinal model.3 however , the more current classification of burst fractures utilizes the load - sharing classification.4 under the magerl fracture classification system , burst fractures are designated as type a3 compression fractures.2 the etiology is typically related to powerful axial compression forces , such as in falls from heights ( 34 to 54%),1 5 motor vehicle accidents ( 51 to 65%),1 5 or high - intensity sports ( 9%).5 6 7 8 9 despite an overwhelming amount of descriptive and clinical studies addressing traumatic thoracolumbar burst fractures , a significant controversy surrounds the optimum management strategies . typically , the management can be either operative or conservative : the former involves a combination of spinal fixation and fusion that may include interbody devices , with or without decompression,1 10 11 12 13 14 15 16 17 and the latter incorporates the use of braces / casts , bed rest , analgesics , or observation with no bed rest or casts.12 13 17 18 19 20 21 22 23 24 by extension , the algorithms to guide management decisions are limited , with one example being the thoracolumbar injury classification and severity score.25 with ambiguity regarding the best management , compiling and analyzing the levels of evidence ( loes ) for treatment practices can reform the decision - making process . the practice of evidence - based health care has gradually become more prevalent in modern medicine , as the pay - for - performance interventions with high loe lead to greater reimbursements.26 27 28 29 30 31 systematic reviews ( srs ) and meta - analyses provide the means to rapidly analyze a study 's quality , ultimately facilitating the introduction of recommendation grades and providing a foundation for establishing practice guidelines.32 to the author 's best knowledge , there are currently seven srs involving various aspects of traumatic thoracolumbar burst fractures.13 16 33 34 35 36 37 however , no review has yet evaluated the operative management of traumatic thoracolumbar burst fractures with particular attention placed on the loe from an established methodology . therefore , the objective of the present study was to characterize the literature evidence for traumatic thoracolumbar burst fractures , specifically by detailing the studies involving the operative management , with an loe of 2 or greater . a systematic literature review was conducted according to the cochrane guidelines to capture all studies pertaining to traumatic thoracolumbar burst fractures over the past 20 years.38 this time frame was arbitrarily predetermined , as the surgical management of burst fractures has greatly evolved during this period . an electronic search of pubmed ( medline ) was done on april 17 , 2014 with permutations of the following terms : ( spine and burst ) or burst fracture or ( burst and fracture ) or ( a3 and fracture and spine ) . the inclusion criteria included : ( 1 ) burst fractures resulting from traumatic mechanism and ( 2 ) fractures of the thoracic or lumbar spine . the exclusion criteria were : ( 1 ) osteoporotic burst fractures , ( 2 ) pathologic burst fractures , ( 3 ) cervical fractures , ( 4 ) biomechanical studies or those involving cadavers , and ( 5 ) computer - based studies such as finite element analyses . following the search , only those involving operative management were selected to be reviewed in the present study . studies with level 1 or 2 loe were classified as high loe . once all the studies were assigned a loe , only those rated as level 1 and 2 were included . the search yielded a total of 1,138 abstracts , of which 272 met the criteria , involved operative management , as well as were published between january 1 , 1992 , and april 17 , 2014 . twenty - three of the studies ( 8.5% ) met level 1 ( n = 4 , 1.5% ) or 2 ( n = 19 , 7.0% ) criteria ( table 1 ) . fifteen of the studies ( 5.5% ) investigated the differences between operative versus nonoperative treatment . abbreviations : ais , american spinal injury association impairment scale ; asia , american spinal injury association ; ffbh - r , hannover functional questionnaire ; lbos , low back outcome score ; ls , long - segment ; lsc , load - sharing classification ; mpq , mcgill pain questionnaire ; mspi , monosegmental pedicle instrumentation ; na , not applicable ; odi , oswestry disability index ; rct , randomized controlled trial ; rmdq , roland - morris disability questionnaire ; sf-36 , short form-36 ; sspi , short - segment pedicle instrumentation ; tlif , transforaminal lumbar interbody fusion ; vas , visual analog scale . abudou et al reported an sr through the cochrane collaboration in which they searched for rcts comparing operative and nonoperative treatment in patients without neurologic deficit.41 due to strict inclusion and exclusion criteria , only two studies with contrasting results were identified ( siebenga et al and wood et al , discussed below),14 42 leading the authors to conclude that there is insufficient evidence to conclude whether surgical or conservative treatment yields superior functional outcomes.41 the second study was an rct reported by siebenga et al that compared the radiographic and functional outcomes in neurologically intact patients with burst fractures . at 2-year follow - up , a higher proportion of patients managed conservatively complained of severe / moderate pain compared with none / mild pain.1 hitchon et al performed a prospective study to outline the indications , benefits , and costs of bed rest compared with surgical stabilization with pedicle screws , rods , and plates ; luque or harington rods with sublaminar wiring ; or an anterior spinal device . the surgical group , however , did show better correction radiographically at follow - up , albeit this came with higher complication rates and costs.13 two hundred fifty - seven studies ( 9.5% ) examined the operative treatment and technique of thoracolumbar burst fractures . there were 16 high loe studies ( two level 1 , 14 level 2 ; table 2 ) , and the rest were level 3 ( n = 32 ) , level 4 ( n = 208 ) , and level 5 studies ( n = 1 ) . however , the posterior group suffered less blood loss and had shorter operative times , better aesthetic outcomes , and lower costs , complications , and donor site pain.48 tezeren and kuru performed a prospective study in which patients were treated with short- or long - segment pedicle fixation.49 the short - segment fixation involved fixation one level above and below the fracture ; long - segment instrumentation involved hook fixation two or three levels above plus pedicle fixation two levels below the fracture . at final follow - up , patients in the long - segment group showed significantly better correction of the sagittal index and anterior body compression.49 wood and colleagues compared anterior reconstruction , fusion , and instrumentation to posterior fusion with instrumentation.57 although the posterior approach had significantly less blood loss , no differences in radiographic , clinical , and functional outcomes were observed . the paravertebral muscle approach led to significantly better outcomes in terms of blood loss , drainage , duration of recumbency , and clinical outcome scores.52 tezeren et al prospectively investigated long - segment instrumentation with or without fusion . the average operation time and blood loss were significantly lower in the nonfusion group , but the two groups were otherwise similar in terms of the radiologic and clinical assessment measures that were analyzed.53 wei and colleagues published a clinical trial in 2010 comparing the clinical and radiologic outcomes between monosegmental transpedicular fixation and short - segment pedicle instrumentation . ultimately , the fusion did not improve the radiographic or functional outcomes but did increase the operative time and necessity for blood transfusion.55 schmid et al prospectively compared thoracoscopy or transforaminal lumbar interbody fusion ( tlif ) in the anterior reconstruction of burst fractures . surgical decompression , however , was higher with tlif.56 in 2013 , xu et al performed a meta - analysis of rcts , quasi - rcts , or controlled trials investigating anterior versus posterior approaches in attempt to identify the optimal approach.37 the authors analyzed seven studies comparing 179 and 152 patients in the anterior and posterior groups , respectively . however , the anterior group was associated with longer operative time , greater blood loss , and a higher cost than the posterior approach . the search yielded a total of 1,138 abstracts , of which 272 met the criteria , involved operative management , as well as were published between january 1 , 1992 , and april 17 , 2014 . twenty - three of the studies ( 8.5% ) met level 1 ( n = 4 , 1.5% ) or 2 ( n = 19 , 7.0% ) criteria ( table 1 ) . fifteen of the studies ( 5.5% ) investigated the differences between operative versus nonoperative treatment . abbreviations : ais , american spinal injury association impairment scale ; asia , american spinal injury association ; ffbh - r , hannover functional questionnaire ; lbos , low back outcome score ; ls , long - segment ; lsc , load - sharing classification ; mpq , mcgill pain questionnaire ; mspi , monosegmental pedicle instrumentation ; na , not applicable ; odi , oswestry disability index ; rct , randomized controlled trial ; rmdq , roland - morris disability questionnaire ; sf-36 , short form-36 ; sspi , short - segment pedicle instrumentation ; tlif , transforaminal lumbar interbody fusion ; vas , visual analog scale . abudou et al reported an sr through the cochrane collaboration in which they searched for rcts comparing operative and nonoperative treatment in patients without neurologic deficit.41 due to strict inclusion and exclusion criteria , only two studies with contrasting results were identified ( siebenga et al and wood et al , discussed below),14 42 leading the authors to conclude that there is insufficient evidence to conclude whether surgical or conservative treatment yields superior functional outcomes.41 the second study was an rct reported by siebenga et al that compared the radiographic and functional outcomes in neurologically intact patients with burst fractures . the surgical group , however , did show better correction radiographically at follow - up , albeit this came with higher complication rates and costs.13 two hundred fifty - seven studies ( 9.5% ) examined the operative treatment and technique of thoracolumbar burst fractures . there were 16 high loe studies ( two level 1 , 14 level 2 ; table 2 ) , and the rest were level 3 ( n = 32 ) , level 4 ( n = 208 ) , and level 5 studies ( n = 1 ) . however , the posterior group suffered less blood loss and had shorter operative times , better aesthetic outcomes , and lower costs , complications , and donor site pain.48 tezeren and kuru performed a prospective study in which patients were treated with short- or long - segment pedicle fixation.49 the short - segment fixation involved fixation one level above and below the fracture ; long - segment instrumentation involved hook fixation two or three levels above plus pedicle fixation two levels below the fracture . at final follow - up , patients in the long - segment group showed significantly better correction of the sagittal index and anterior body compression.49 wood and colleagues compared anterior reconstruction , fusion , and instrumentation to posterior fusion with instrumentation.57 although the posterior approach had significantly less blood loss , no differences in radiographic , clinical , and functional outcomes were observed . the paravertebral muscle approach led to significantly better outcomes in terms of blood loss , drainage , duration of recumbency , and clinical outcome scores.52 tezeren et al prospectively investigated long - segment instrumentation with or without fusion . the average operation time and blood loss were significantly lower in the nonfusion group , but the two groups were otherwise similar in terms of the radiologic and clinical assessment measures that were analyzed.53 wei and colleagues published a clinical trial in 2010 comparing the clinical and radiologic outcomes between monosegmental transpedicular fixation and short - segment pedicle instrumentation . ultimately , the fusion did not improve the radiographic or functional outcomes but did increase the operative time and necessity for blood transfusion.55 schmid et al prospectively compared thoracoscopy or transforaminal lumbar interbody fusion ( tlif ) in the anterior reconstruction of burst fractures . surgical decompression , however , was higher with tlif.56 in 2013 , xu et al performed a meta - analysis of rcts , quasi - rcts , or controlled trials investigating anterior versus posterior approaches in attempt to identify the optimal approach.37 the authors analyzed seven studies comparing 179 and 152 patients in the anterior and posterior groups , respectively . however , the anterior group was associated with longer operative time , greater blood loss , and a higher cost than the posterior approach . traumatic thoracolumbar burst fractures remain a challenge to treat , and optimal operative and nonoperative techniques have not yet been established . the present study attempted to characterize the high loe studies pertaining to the operative management of traumatic thoracolumbar burst fractures and found that the overall literature contained 23 studies ( 8.5% ) in pubmed that met an loe of 1 or 2 . when treating neurologically intact patients with thoracolumbar burst fractures , there is no difference in the clinical outcomes when comparing a conservative or open operative approach . there is a high loe for short- or long - segment pedicle instrumentation without fusion . tezeren and kuru compared short- versus long - segment pedicle fixation in a level 2 study and concluded that better correction can be achieved with the long construct , and lbos outcomes were similar.49 longer constructs provide the necessary rigidity to decrease failure rates , but they also decrease patient mobility due to the more rigid fixation , which may impact quality - of - life parameters that were not assessed . dai et al ( level 1 ) , alanay et al ( level 2 ) , and jindal et al ( level 2 ) demonstrated that bone grafting was not necessary in short - segment pedicle instrumentation in rcts.43 47 55 tezeren et al ( level 2 ) also demonstrated that fusion did not change the radiologic or clinical outcomes in long - segment instrumentation.53 the operative time would decrease by forgoing fusion , and because bone graft is not needed , this would collectively decrease the risk of infections . the percutaneous and paraspinal approaches to thoracolumbar burst fractures are supported by level 1 and 2 studies . pang et al ( level 2 ) demonstrated that the paravertebral approach results in better clinical outcome scores than the traditional posterior approach.52 jiang et al ( level 1 ) demonstrated that shorter hospitalization time and equal 3-year clinical outcomes can be achieved with the percutaneous versus paraspinal approach in pedicle screw and rod fixation.44 however , better deformity correction can be achieved with the paraspinal approach . when investigating an anterior , posterior , or combined approach in instrumentation , four level 2 studies reported similar radiographic , clinical , and functional outcomes.37 48 50 57 furthermore , when compared with the anterior or combined approach , the posterior approach correlated with fewer complications and operative risks . when considering anterior reconstruction , dai et al demonstrated no significant differences in the use of titanium or autologous grafting for fusion.51 furthermore , schmid et al demonstrated that transforaminal lumbar interbody fusion may provide further surgical decompression than thoracoscopy.56 and finally , xu et al performed a meta - analysis and found similar results with the exception that the anterior approach also had a higher cost associated with it.37 the choice of the operative approach can be decided by the preference of the surgeon and patient , while acknowledging that greater risks and possible higher costs are associated with the anterior approach . additional considerations in the operative management of traumatic thoracolumbar burst fractures include both the correction of a kyphotic deformity and the potential loss of a kyphotic correction over time . the results regarding whether operative management maintained the correction more successfully than nonoperative management are mixed.12 14 42 all studies show a significant improvement postoperatively ; however , in the study by siebenga et al ( level 1),42 the operative group maintained the correction and the kyphosis was significantly less than in the nonoperative group at final follow - up ( a mean of 4.3 years ) . on the other hand , shen et al ( level 2 ) and wood et al ( level 2 ) did not show any differences in the deformity correction between operative and nonoperative management at the final follow - up ( 2 years).12 14 the clinical implication of kyphotic correction is questionable , as wood et al ( level 2 ) and korovessis et al ( level 2 ) both did not find any correlation between the degree of kyphosis at the final follow - up and the degree of pain or disability.14 50 operative management does improve pain and stability.12 13 14 16 42 most studies had similar radiographic results between the various operative techniques.37 43 47 51 53 54 55 56 57 specifically , fusion does not appear to affect the maintenance of radiographic correction as demonstrated by four high loe studies.43 47 53 55 however , jiang and colleagues ( level 1 ) found better long - term local kyphosis correction with a paraspinal approach versus a percutaneous.44 similarly , tezeren and kuruet ( level 2 ) found better kyphotic correction at final follow - up for patients who underwent long - segment pedicle fixation compared with short - segment fixation.49 the heterogeneity nature of burst fractures due to the different mechanisms of injury results in a variety of clinical presentations , and makes it difficult to formulate a universal approach . the literature contains a high loe to support the operative management of traumatic thoracolumbar burst fractures . for patients who are neurologically intact , a high loe demonstrated similar functional outcomes and lower complication rates and costs with conservative management when compared with surgical management . there is a high loe for short- or long - segment pedicle instrumentation without fusion and less invasive ( percutaneous and paraspinal ) approaches . furthermore , posterior approaches are associated with fewer complications as opposed to anterior or combined approaches .

cystic fibrosis ( cf ) is a recessively inherited genetic disease in which the cystic fibrosis transmembrane conductance regulator ( cftr ) gene is mutated , leading either to partial or complete loss - of - function . cftr encodes a chloride ion channel , and the loss of ion transport across epithelial cell membranes leads to osmotic imbalance , and consequently to the buildup of sticky mucus in the lower respiratory tract.1 cf lungs are highly prone to microbial infection , with chronic infection beginning in infancy for the overwhelming majority of cases and continuing throughout the patient s life . pseudomonas aeruginosa is an opportunistic gram - negative pathogen and the predominant infective species isolated from late - stage cf lung infections.2,3 once established in the lungs of cf patients , p. aeruginosa infections are extremely difficult to completely eradicate , and the aggravation and progressive tissue degradation associated with repeated infectious relapses lead to morbidity and eventually death.3 p. aeruginosa lung infections typically progress clonally from infection with a single environmentally acquired genotype4,5 and , over the course of long - term chronic cf infections , undergo extensive genetic and phenotypic adaptation to the lung environment.6,7 there are two main consequences of this adaptation : first , the progressive transition toward a persistent , low virulence state , and second , a related diversification into a number of different phenotypes.6,7 these include mucoid cells , which overproduce alginate and form distinctive slimy colonies,8 and small colony variants ( scvs , figure 1 ) , which are typically slow - growing isolates that show strong attachment to surfaces , autoaggregation , enhanced exopolysaccharide production , and biofilm formation.9,10 in recent years , several studies have established a causal link between p. aeruginosa scvs and persistence of infection in animal models,1113 supporting the hypothesis that the scv phenotype confers a fitness advantage under chronic infection conditions , and thus plays an important role in the pathogenesis of p. aeruginosa lung infections . p. aeruginosa scvs also emerge in other situations that favor chronic infections , including in mechanically ventilated patients or in those suffering from chronic obstructive pulmonary disease.14,15 these studies suggest that persistent p. aeruginosa morphotypes such as scv represent genetic adaptations to the hostile milieu in the patient , with characteristics including resistance to phagocytosis,12 antimicrobial resistance due to slow growth or increased persister cell populations,16,17 and reduced virulence18 potentially contributing to selection . in this review we will discuss the initial discovery and characterization of scvs in the sputum of cf patients , the phenotypic characteristics associated with the scv morphotype , and the clinical implications of lung colonization with scvs . finally , we will address the molecular basis of the scv phenotype and possible evolutionary routes to scv generation , with an emphasis on scv evolution in the lung environment . phenotypic variation between different p. aeruginosa isolates in cf sputum has been recognized for many years . while the alginate - overproducing mucoid phenotype8 has until recently garnered the most attention , it has long been recognized that scvs ( previously known as dwarf colonies ) also arise in the chronically infected respiratory tract of cf patients.19 for example , thomassen et al20 examined p. aeruginosa isolates from 286 cf patients over a 3-month period in 19761977 and determined the frequency and distribution of different morphotypes . in this study , dwarf colonies were identified in the sputum of 14% of patients , although these colonies were always found in conjunction with other morphotypes.20 the first clinical study to specifically focus on the distribution and phenotypic characteristics of p. aeruginosa scv isolates from cf patients took place in germany from 1996 to 1998 . hussler et al17 tested the sputum of 86 cf patients for p. aeruginosa scvs , identifying them in 33 patients . over the course of this study , around 3.0% of the total p. aeruginosa isolates collected were classed as scvs . these colonies were characterized as small ( 13 mm in diameter ) , slow growing , and more resistant to several different classes of antibiotics than subsequent planktonic revertants.17 hussler et al17 went on to characterize some of their scv isolates , reporting that these strains were hyperpiliated and autoaggregative . the scv morphs tested showed increased twitching motility and a marked ability to form biofilms and to attach to human pneumocytes.10 simultaneously with these clinical scv studies , the molecular biology of scv formation was being investigated by two independent groups . drenkard and ausubel21 showed that p. aeruginosa scv formation could be induced under laboratory conditions by the addition of kanamycin and linked this phenotype to the putative cyclic - di - gmp ( cdg ) phosphodiesterase gene pvrr . at the same time , dargenio et al22 identified the wspr diguanylate cyclase ( dgc ) and showed that it was responsible for scv formation in the laboratory strain pa01 . in agreement with the early clinical research , clinical and laboratory - derived scvs generally display increased resistance to a broad range of antibiotics.17,21,23 exposure to subinhibitory antibiotic concentrations has been shown to induce attachment and scv formation in vitro , possibly as part of a defense response.21,24 scvs are also usually nonmotile , with flagellar motility absent in most scvs characterized to date.12,23,25 hyperpiliation has been reported for some strains , with these isolates displaying increased twitching motility as a result,10 although the distribution of this phenotype is unknown . probably the most prominent phenotype associated with scv formation is the significantly enhanced production of one or more exopolysaccharide molecules.9,12,25 p. aeruginosa produces at least three exopolysaccharides . the most noticeable of these in clinical cf isolates is alginate , the overproduction of which produces the slimy , mucoid phenotype.8 the role of alginate in scv formation is unclear , although scv strains have been identified that overproduce this molecule.26 also produced are the glucose - rich exopolysaccharide pel , and psl , which contains glucose , mannose , and rhamnose.27 both pel and psl overproduction have been explicitly linked to scv formation , and deletion of both pel and psl operons reverts an scv to a smooth phenotype.12,25 many of the other phenotypes associated with scv , including their enhanced biofilm formation and surface attachment,10 small size ( stemming from autoaggregation),17 resistance to phagocytosis,12 and infectious persistence,1113 have been linked to exopolysaccharide overproduction in these morphs . a striking characteristic of p. aeruginosa in the cf lung is its extraordinary degree of phenotypic diversity . for example , based on a year - long study of sputum samples from ten cf patients infected with the same initial p. aeruginosa strain , mowat et al28 examined the phenotypic characteristics of 1,720 individual isolates . the p. aeruginosa populations in each individual sputum sample in this study showed extensive phenotypic diversity , with the majority of diversity occurring within sputum samples rather than between patients . in agreement with this , workentine et al29 examined a more extensive series of phenotypes for 169 clonal isolates from a single chronically colonized patient . once again interestingly , every isolate in this study presented a different phenotypic profile from the others , with very little correlation seen between the majority of tested phenotypes.29 the implications of these findings for scvs in the lung environment are that apart from the core phenotypic characteristics by which scvs are identified in the clinic , a high degree of variation might be expected for every other phenotype . for example , hussler et al17 reported reduced growth rates for their clinical scv isolates , an observation that has since been repeated for other scv genotypes growing in liquid media.23 however , work from other researchers has shown that this does not apply to all strains or under all growth conditions.30 similarly , siderophore production has been reported as being downregulated in some scvs,30 but as being upregulated in others.12,23,31 cytotoxicity / virulence is another potentially plastic phenotype in scvs . long - term lung colonization is generally associated with a loss of cytotoxicity , and downregulation of the type iii secretion system ( t3ss).32 reduced cytotoxicity is a recognized characteristic of rsma- strains,13 which form scvs under laboratory conditions.33 however , a transcriptional analysis of the well - studied strain scv20265 showed significant upregulation of t3ss genes compared to its clonal predecessor.31 this increased expression agreed with the results of an earlier proteomic analysis of the same strain34 and corresponded to significantly increased cytotoxicity in a murine infection model.31 given that scvs generally arise after significant periods of lung colonization , many of the phenotypes associated with long - term , chronic lung infection ( loss of virulence , reduced quorum sensing , hypermutability , etc18,23,3537 ) might be expected to correlate with scv . however , as the studies above indicate , many exceptions exist , and phenotypic variation between scv morphotypes is likely to be extensive.28,29 at least two independent reports have identified a strong relationship between the presence of scvs in the cf lung and poor clinical outcomes . the 1999 hussler et al17 study of scvs in cf sputum examined a total of 86 patients with chronic p. aeruginosa lung infections . while only 3% of all colonies recovered during the study were classed as scvs , those patients from whom scvs were isolated ( 33 out of 86 ) displayed poorer lung function than those who did not display scv colonization . the forced expiratory volume in the first second ( fev1 ) score for these patients was 56% , which was significantly lower than the fev1 of scv - negative patients ( 80% ) . more recently , schneider et al38 looked for p. aeruginosa and staphylococcus aureus scvs in sputum samples from 98 cf patients . over a 3-month period , 9.2% of patients in this study were colonized with p. aeruginosa scvs.38 in agreement with the findings of hussler et al,17 patients having scvs had poorer lung function , with lower blood oxygen levels and a significantly lower fev1 score ( 39% ) than the non - scv patients ( 65.5% ) . the body mass of scv - colonized patients was also significantly lower ( mean 14.0% underweight compared with 2.7% for non - scv patients).38 while it is difficult to confidently infer a causal relationship between the scv phenotype and poor clinical prognosis based on correlation studies alone , there is substantial evidence supporting a role for scvs in persistence in a clinical setting . three independent research groups , using different animal infection models and genetically distinct scv strains , have established causal relationships between the scv phenotype and persistence of infection.1113 furthermore , many of the phenotypes associated with scv have been directly linked to advantageous survival traits during chronic infection . for example , malone et al12 showed that extracellular polysaccharides ( eps ) overproduction rendered p. aeruginosa scvs highly resistant to macrophage phagocytosis . as mentioned previously , scvs generally show enhanced antibiotic resistance compared to wild - type p. aeruginosa.17,21,23 while a causal relationship again remains to be established , this antibiotic resistance may contribute to scv survival in the cf lung during antibiotic chemotherapy . fully defining the relationship between scvs and persistence in the cf lung is likely to remain a challenge for the foreseeable future . while correlations between clinical scv colonization and poor lung function are clear , and scvs contribute to infectious persistence in laboratory and animal studies , it is currently unknown whether scvs trigger poor clinical outcomes or just preferentially associate with deteriorating lungs . short of invasive or postmortem investigations of cf lungs , an interesting recent advance that may provide an answer is the ex vivo porcine lung model developed by harrison et al.39 this system has been successfully tested with p. aeruginosa quorum sensing mutants and promises to shed further light onto microbial colonization of the lung environment . following early indications from two independent research labs,21,22 strong evidence has accumulated for a causal link between the scv phenotype and the bacterial second messenger cdg.12,25,4042 cdg is a ubiquitous bacterial signaling molecule that controls a wide range of cellular processes involved in the transition between motile , virulent , and sessile biofilm forming lifestyles.43,44 the cyclic dinucleotide is produced from two molecules of gtp ( guanosine triphosphate ) by dgcs and degraded to pgpg ( 5-phosphoguanylyl-(35)-guanosineguanosine ) by phosphodiesterases;45 enzymes containing the conserved ggdef and eal / hd - gyp domains , respectively.4649 in general , cdg production is associated with community behavior phenotypes such as eps production and biofilm formation , while low cdg levels lead to enhanced motility , virulence , and a single - celled lifestyle43 ( figure 2 ) . cdg signal transduction is a highly complex process , with many bacterial species containing dozens of different cdg - signaling proteins.43 p. aeruginosa is no exception , with 33 predicted ggdef and 17 eal domain - containing proteins.50,51 these cdg metabolic enzymes control the intracellular level of cdg and hence regulate the expression of various phenotypic outputs . in p. aeruginosa , this includes exopolysaccharide production,5254 production and deployment of proteinaceous adhesins,55,56 siderophore production,12 rhamnolipid biosynthesis,57 and virulence and cytotoxicity systems,51,5860 as well as the assembly , function , and control of type iv pili61,62 and the bacterial flagellum.54,6365 cdg affects cell behavior by controlling phenotypic outputs at every regulatory level , from gene expression through to allosteric modulation of phenotypic outputs.43 these effects occur through binding to specific effector proteins , whose activity is altered upon interaction with cdg . consistent with the high complexity of cdg signal transduction , binding is highly promiscuous and occurs with a wide range of different protein folds.66,67 for example , the pilz domain is a dedicated cdg binding protein , with examples like alg44 in p. aeruginosa , which is a regulator of alginate biosynthesis.52 furthermore , not all ggdef , hd - gyp , and eal domain proteins have cdg metabolic activity . a substantial minority have degenerate active sites and instead function by binding cdg , via protein protein interactions or through other alternative mechanisms.66,67 for example , the p. aeruginosa eps synthase component peld binds to cdg , at a conserved site on its degenerate ggdef domain , and induces a conformational change in the pel synthase machinery that leads to activation and eps production.68 cdg also binds to a number of different transcriptional regulators . in p. aeruginosa , this includes the flagellar and eps - gene master regulator fleq , which controls pel and psl eps operon transcription.54,69 the metabolism of cdg is under extensive spatial and temporal control in p. aeruginosa . expression and translation of cdg genes is regulated by various cellular inputs , including sigma factors , cell cycle control , quorum sensing , and translational regulation.43,60,70 a further , significant level of control exists at the posttranslational level , with the majority of ggdef , eal , and hd - gyp domain proteins also containing diverse sensory inputs , including pas , gaf , and response - regulator receiver domains.43 the role of cdg in p. aeruginosa scv formation is now well - established . for example , a strong scv phenotype can be triggered under laboratory conditions by overexpressing a dgc gene in trans,44 a phenotype that can be reversed by disrupting the pel and psl eps synthase operons ( figure 3).12 the intracellular level of cdg has been shown to be elevated in several clinical and laboratory - derived scvs.12,25,42 furthermore , mutations in several p. aeruginosa cdg signaling pathways have been shown to induce scv phenotypes in vitro . in each case , elevated intracellular levels of cdg have been linked to overproduction of either exopolysaccharides9,25,41 or fimbrial adhesins,40,71 and consequently to scv formation . as well as cdg signaling pathways , other genetic loci have been implicated in persistence of infection and/or scv formation in p. aeruginosa . a prominent example is the gacas / rsmazy signaling system ( figure 4 ) , an important regulator of the switch between chronic and acute infectious lifestyles in many gram - negative bacterial species , including p. aeruginosa.70,72 rsma is a small , translational regulator that specifically recognizes and binds to gga sequences in the 5 leader region of target mrnas . rsma reciprocally controls the translation of mrnas associated with type iii / type vi secretion and psl exopolysaccharide synthesis,33,70 leading to the suppression of biofilm - associated phenotypes and promoting secondary metabolism , motility , and virulence . deletion of the rsma gene in p. aeruginosa pa01 induces an scv phenotype as a consequence of increased psl mrna translation,33 and has been shown to increase infectious persistence in a murine lung model.13 in pseudomonas spp . the activity of rsma is controlled by the gacas two - component signal transduction system . gacs is a transmembrane histidine protein kinase and activates its cognate response regulator gaca by phosphotransfer . phosphorylated gaca promotes transcription of the antagonistic srnas , rsmy and rsmz.73 rsmy / rsmz contain multiple gga trinucleotides in the exposed stem - loops of their predicted secondary structures and inhibit rsma activity by competing for rsma binding , thus titrating the translational regulator away from its target mrnas ( figure 4 ) . the gacas system is itself controlled by the two - component signal hybrid proteins lads and rets . rets is an antagonist of gacs activity , and thus suppresses rsmz / y levels . rather than operating via a conventional histidine protein kinase phosphotransfer mechanism , rets forms heterodimers with gacs , and thus blocks gacs autophosphorylation phosphotransfer to gaca.74 the lads protein positively controls rsmz / y expression , and thus works to suppress rsma activity . although the mechanism of lads function is currently uncharacterized , it may work by counteracting rets activity ( figure 4).72 signal transduction through the gac / rsm pathway is highly complex , and affects the production of dozens of different proteins.70 in addition to direct rsma translational regulation , the gac / rsm system indirectly affects numerous additional cellular behaviors via the regulation of signaling pathways , including both cdg metabolism and quorum sensing.60,70 similar to cdg , mutations in quorum sensing genes are strongly associated with persistence during chronic cf lung infections.6,75 having colonized the respiratory passage and lungs of a cf patient , in most cases , a p. aeruginosa infection persists throughout the patient s lifetime . patients are typically colonized by a single environmentally acquired genotype4,5 ( although some genotypes such as the liverpool epidemic strain are infectious and can be transmitted from patient to patient76 ) . p. aeruginosa cf lung infections generally alternate between periods of chronic , largely asymptomatic colonization and relapses into aggravated infection.1 over several years , the progressive tissue degradation that accompanies these repeated infectious relapses leads to lung failure and premature death.3 as stated above , p. aeruginosa strains undergo extensive adaptation to the lung environment throughout the period of chronic lung infection,6,7 with multiple distinct phenotypes including scvs arising in the lung population.7,10,77 despite generally originating from a single clonal genotype , significant genetic variation exists between individual sputum isolates sampled at any one time,28,29 providing an explanation for the long - observed phenotypic heterogeneity in cf p. aeruginosa isolates.19,20 perhaps unsurprisingly , the most commonly identified scv - inducing mutations are loss - of - function mutations in repressor proteins that control the activity of dgcs ( table 1 ) . a well - studied example is the wsp pathway,41 which contains a methyl - accepting chemotaxis receptor ( wspa ) and a dgc response regulator ( wspr ) . wspr overproduction / activation induces an scv phenotype,22,41 displaying strong attachment and increased expression of the pel and psl exopolysaccharide operons . under laboratory conditions , the principle route to scv evolution is via loss - of - function mutations in the methylesterase gene wspf.25,41 without wspf , wspa and hence wspr are constitutively activated , leading to cdg synthesis and scv formation through eps overproduction ( figure 5).41,78 there is evidence that wspf loss - of - function also represents a route to clinical scv evolution . in 2006 , smith et al6 carried out a longitudinal study of lung - adapted p. aeruginosa isolates and identified several different backgrounds in which the wspf gene was mutated . deletion of fleq induces an autoaggregative phenotype in pa01 , with many of the characteristics of scv colonies.54 while the morphologies of the lung isolates in the smith et al6 study were not characterized in detail , these data nonetheless implicate both wspf and fleq as potential mutagenic targets for scv generation in the cf lung ( figure 5 ) . blanka et al42 recently identified an interesting additional mechanism for scv evolution in the cf lung isolate scv20265 . comparison of the scv20265 genome with those of several laboratory - derived revertants identified a causal mutation for the scv20265 aggregative phenotype . this study showed that a point mutation in the 5 untranslated region of accbc , a gene cluster responsible for fatty acid biosynthesis , leads to mrna stabilization and a consequent increase in the proportion of short - chain fatty acids in the plasma membrane . in turn , this change in membrane composition triggered activation of the wsp system and cdg overproduction via wspr.42 intriguingly , the genome of scv20265 was also shown to contain a second , additive mutation in the methylesterase gene wspf , shown in previous studies to induce an scv phenotype.6,25 these findings suggest that both regulatory and genetic inputs combine to control cdg production , and hence scv evolution , in the cf lung.42 the yfibnr signaling operon12 represents a further , well - characterized genetic target for clinically - arising scvs . yfin is a membrane - bound dgc whose activity is normally allosterically repressed by the soluble periplasmic repressor yfir . release of yfir repression , either through a loss - of - function mutation in yfir or an escape mutation in yfin , leads to dgc activation , cdg overproduction , and scv formation under laboratory conditions.12,26 as with wspf and fleq , activating mutations arise in the yfi locus ( in both yfir and yfin ) during long - term cf lung infections , driving scv formation in vivo ( figure 5).26 interestingly , further examination of the cf isolates included in this study identified mutational these strains contained both activating and loss - of - function mutations in the yfin dgc gene , supporting the idea that yfin activity is under both positive and negative selection in vivo , with yfi - scvs acting as an environmental pool for the generation of new smooth morphotypes in the complex and heterogeneous fitness landscape of the cf lung.26 another class of mutations associated with the clinical emergence of the scv phenotype are those in muts and other mismatch repair genes . these hypermutatable strains arise with high frequency in the course of chronic cf lung infection.37,79 mena et al35 showed that a p. aeruginosa mismatch - repair mutation increased the long - term persistence of oropharyngeal colonization in cf mice , and that scv mutants readily emerged over time in this background . the phenomenon of scv morphotypes arising in hypermutatable p. aeruginosa backgrounds has been independently observed for another muts mutant strain,80 which was also shown to produce a large number of mutations in the lasr quorum sensing locus.36 the work of mena et al35 strongly suggests that mismatch repair mutants are selected in chronic p. aeruginosa lungs due to their increased persistence . as a possible explanation for this , hypermutatable strains have been linked to the rsma signaling network in erwinia carotovora,81 placing rsma downstream of muts and suggesting that the persistence of these strains may result ( at least in part ) from reduced levels of rsma ( figure 5 ) . another possibility is simply that the increased rate of mutation in these strains facilitates the emergence of mutations in the gac / rsm or cdg signaling pathways . genetic adaptation to the cf lung has been shown to be catalyzed by the initial acquisition of mismatch repair mutations , with subsequent mutations arising much more rapidly than in nonmutator lines.82 mutants in the cdg signaling genes fleq and wspf were identified in this study , although the isolate library used was the same as described in the earlier work of smith et al,6 where the wspf and fleq mutations were initially identified . large - scale dna inversions have also been associated with the generation of morphological diversity , and hence potentially scv generation , in the lung environment . in a study of cf lung isolates , rmling et al83 showed that 50% of tested p. aeruginosa genomes contained large chromosomal inversions . these inversions were exclusively detected in cf lung isolates , suggesting that they are selected during adaptation to chronic lung infection.83 chromosomal inversions have been linked to the mobile element is6100 and are proposed to represent a source of phenotypic variation,84 similar to that emerging from the loss of mismatch repair described above.80 furthermore , reversible genomic inversion has been shown to induce an scv phenotype in s. aureus.85 recently , the advent of next - generation sequencing has enabled the molecular basis of scv formation to be examined much more closely . the complete genome sequences of at least two clinically - evolved scvs have been published , allowing candidate causal mutations for generation of the scv phenotype to be investigated . for scv20265,86 the mutational routes to scv are known,42 while for the urethral catheter scv mh27,87 the underlying genetics of the scv phenotype has yet to be established . as sequencing technology continues to improve and becomes ever more accessible , it is likely that the coming years will see a much more complete examination of the mutational and regulatory routes to scv generation in the cf lung environment . the appearance of these distinctive , small , autoaggregative colonies strongly correlates with both deteriorating lung function and associated poor clinical prognosis . whether this connection is causal or simply correlative is not yet clear . nonetheless , the importance of the scv phenotype in persistence of p. aeruginosa infection is beyond doubt , with scvs conferring both increased antibiotic tolerance and resistance to immune phagocytosis . hopefully , an increased awareness of the importance of scvs in chronic cf infection , alongside recent improvements in both diagnostic and analytical tools , will allow us to make headway in the treatment of these unusual morphotypes .

pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis ( cf ) lung infections . over many years of chronic lung colonization , p. aeruginosa undergoes extensive adaptation to the lung environment , evolving both toward a persistent , low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs . these lung - adapted p. aeruginosa strains include the small colony variants ( scvs ) , small , autoaggregative isolates that show enhanced biofilm formation , strong attachment to surfaces , and increased production of exopolysaccharides . their appearance in the sputum of cf patients correlates with increased resistance to antibiotics , poor lung function , and prolonged persistence of infection , increasing their relevance as a subject for clinical investigation . the evolution of scvs in the cf lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic - di - gmp , with increased cyclic - di - gmp levels shown to be responsible for the scv phenotype in a number of different cf lung isolates . here , we review the current state of research in clinical p. aeruginosa scvs . we will discuss the phenotypic characteristics underpinning the scv morphotype , the clinical implications of lung colonization with scvs , and the molecular basis and clinical evolution of the scv phenotype in the cf lung environment .

Introduction Discovery and initial characterization of SCVs in the CF lung Phenotypic characteristics of the SCV morphotype The clinical implications of SCV lung infection The molecular bases of the SCV phenotype: cyclic-di-GMP and the GacAS/RsmAZY pathway Evolution of SCVs in the CF lung Summary

cystic fibrosis ( cf ) is a recessively inherited genetic disease in which the cystic fibrosis transmembrane conductance regulator ( cftr ) gene is mutated , leading either to partial or complete loss - of - function . cftr encodes a chloride ion channel , and the loss of ion transport across epithelial cell membranes leads to osmotic imbalance , and consequently to the buildup of sticky mucus in the lower respiratory tract.1 cf lungs are highly prone to microbial infection , with chronic infection beginning in infancy for the overwhelming majority of cases and continuing throughout the patient s life . pseudomonas aeruginosa is an opportunistic gram - negative pathogen and the predominant infective species isolated from late - stage cf lung infections.2,3 once established in the lungs of cf patients , p. aeruginosa infections are extremely difficult to completely eradicate , and the aggravation and progressive tissue degradation associated with repeated infectious relapses lead to morbidity and eventually death.3 p. aeruginosa lung infections typically progress clonally from infection with a single environmentally acquired genotype4,5 and , over the course of long - term chronic cf infections , undergo extensive genetic and phenotypic adaptation to the lung environment.6,7 there are two main consequences of this adaptation : first , the progressive transition toward a persistent , low virulence state , and second , a related diversification into a number of different phenotypes.6,7 these include mucoid cells , which overproduce alginate and form distinctive slimy colonies,8 and small colony variants ( scvs , figure 1 ) , which are typically slow - growing isolates that show strong attachment to surfaces , autoaggregation , enhanced exopolysaccharide production , and biofilm formation.9,10 in recent years , several studies have established a causal link between p. aeruginosa scvs and persistence of infection in animal models,1113 supporting the hypothesis that the scv phenotype confers a fitness advantage under chronic infection conditions , and thus plays an important role in the pathogenesis of p. aeruginosa lung infections . p. aeruginosa scvs also emerge in other situations that favor chronic infections , including in mechanically ventilated patients or in those suffering from chronic obstructive pulmonary disease.14,15 these studies suggest that persistent p. aeruginosa morphotypes such as scv represent genetic adaptations to the hostile milieu in the patient , with characteristics including resistance to phagocytosis,12 antimicrobial resistance due to slow growth or increased persister cell populations,16,17 and reduced virulence18 potentially contributing to selection . in this review we will discuss the initial discovery and characterization of scvs in the sputum of cf patients , the phenotypic characteristics associated with the scv morphotype , and the clinical implications of lung colonization with scvs . finally , we will address the molecular basis of the scv phenotype and possible evolutionary routes to scv generation , with an emphasis on scv evolution in the lung environment . while the alginate - overproducing mucoid phenotype8 has until recently garnered the most attention , it has long been recognized that scvs ( previously known as dwarf colonies ) also arise in the chronically infected respiratory tract of cf patients.19 for example , thomassen et al20 examined p. aeruginosa isolates from 286 cf patients over a 3-month period in 19761977 and determined the frequency and distribution of different morphotypes . in this study , dwarf colonies were identified in the sputum of 14% of patients , although these colonies were always found in conjunction with other morphotypes.20 the first clinical study to specifically focus on the distribution and phenotypic characteristics of p. aeruginosa scv isolates from cf patients took place in germany from 1996 to 1998 . hussler et al17 tested the sputum of 86 cf patients for p. aeruginosa scvs , identifying them in 33 patients . drenkard and ausubel21 showed that p. aeruginosa scv formation could be induced under laboratory conditions by the addition of kanamycin and linked this phenotype to the putative cyclic - di - gmp ( cdg ) phosphodiesterase gene pvrr . in agreement with the early clinical research , clinical and laboratory - derived scvs generally display increased resistance to a broad range of antibiotics.17,21,23 exposure to subinhibitory antibiotic concentrations has been shown to induce attachment and scv formation in vitro , possibly as part of a defense response.21,24 scvs are also usually nonmotile , with flagellar motility absent in most scvs characterized to date.12,23,25 hyperpiliation has been reported for some strains , with these isolates displaying increased twitching motility as a result,10 although the distribution of this phenotype is unknown . the most noticeable of these in clinical cf isolates is alginate , the overproduction of which produces the slimy , mucoid phenotype.8 the role of alginate in scv formation is unclear , although scv strains have been identified that overproduce this molecule.26 also produced are the glucose - rich exopolysaccharide pel , and psl , which contains glucose , mannose , and rhamnose.27 both pel and psl overproduction have been explicitly linked to scv formation , and deletion of both pel and psl operons reverts an scv to a smooth phenotype.12,25 many of the other phenotypes associated with scv , including their enhanced biofilm formation and surface attachment,10 small size ( stemming from autoaggregation),17 resistance to phagocytosis,12 and infectious persistence,1113 have been linked to exopolysaccharide overproduction in these morphs . a striking characteristic of p. aeruginosa in the cf lung is its extraordinary degree of phenotypic diversity . for example , based on a year - long study of sputum samples from ten cf patients infected with the same initial p. aeruginosa strain , mowat et al28 examined the phenotypic characteristics of 1,720 individual isolates . once again interestingly , every isolate in this study presented a different phenotypic profile from the others , with very little correlation seen between the majority of tested phenotypes.29 the implications of these findings for scvs in the lung environment are that apart from the core phenotypic characteristics by which scvs are identified in the clinic , a high degree of variation might be expected for every other phenotype . long - term lung colonization is generally associated with a loss of cytotoxicity , and downregulation of the type iii secretion system ( t3ss).32 reduced cytotoxicity is a recognized characteristic of rsma- strains,13 which form scvs under laboratory conditions.33 however , a transcriptional analysis of the well - studied strain scv20265 showed significant upregulation of t3ss genes compared to its clonal predecessor.31 this increased expression agreed with the results of an earlier proteomic analysis of the same strain34 and corresponded to significantly increased cytotoxicity in a murine infection model.31 given that scvs generally arise after significant periods of lung colonization , many of the phenotypes associated with long - term , chronic lung infection ( loss of virulence , reduced quorum sensing , hypermutability , etc18,23,3537 ) might be expected to correlate with scv . however , as the studies above indicate , many exceptions exist , and phenotypic variation between scv morphotypes is likely to be extensive.28,29 at least two independent reports have identified a strong relationship between the presence of scvs in the cf lung and poor clinical outcomes . the 1999 hussler et al17 study of scvs in cf sputum examined a total of 86 patients with chronic p. aeruginosa lung infections . over a 3-month period , 9.2% of patients in this study were colonized with p. aeruginosa scvs.38 in agreement with the findings of hussler et al,17 patients having scvs had poorer lung function , with lower blood oxygen levels and a significantly lower fev1 score ( 39% ) than the non - scv patients ( 65.5% ) . the body mass of scv - colonized patients was also significantly lower ( mean 14.0% underweight compared with 2.7% for non - scv patients).38 while it is difficult to confidently infer a causal relationship between the scv phenotype and poor clinical prognosis based on correlation studies alone , there is substantial evidence supporting a role for scvs in persistence in a clinical setting . three independent research groups , using different animal infection models and genetically distinct scv strains , have established causal relationships between the scv phenotype and persistence of infection.1113 furthermore , many of the phenotypes associated with scv have been directly linked to advantageous survival traits during chronic infection . as mentioned previously , scvs generally show enhanced antibiotic resistance compared to wild - type p. aeruginosa.17,21,23 while a causal relationship again remains to be established , this antibiotic resistance may contribute to scv survival in the cf lung during antibiotic chemotherapy . fully defining the relationship between scvs and persistence in the cf lung is likely to remain a challenge for the foreseeable future . short of invasive or postmortem investigations of cf lungs , an interesting recent advance that may provide an answer is the ex vivo porcine lung model developed by harrison et al.39 this system has been successfully tested with p. aeruginosa quorum sensing mutants and promises to shed further light onto microbial colonization of the lung environment . following early indications from two independent research labs,21,22 strong evidence has accumulated for a causal link between the scv phenotype and the bacterial second messenger cdg.12,25,4042 cdg is a ubiquitous bacterial signaling molecule that controls a wide range of cellular processes involved in the transition between motile , virulent , and sessile biofilm forming lifestyles.43,44 the cyclic dinucleotide is produced from two molecules of gtp ( guanosine triphosphate ) by dgcs and degraded to pgpg ( 5-phosphoguanylyl-(35)-guanosineguanosine ) by phosphodiesterases;45 enzymes containing the conserved ggdef and eal / hd - gyp domains , respectively.4649 in general , cdg production is associated with community behavior phenotypes such as eps production and biofilm formation , while low cdg levels lead to enhanced motility , virulence , and a single - celled lifestyle43 ( figure 2 ) . consistent with the high complexity of cdg signal transduction , binding is highly promiscuous and occurs with a wide range of different protein folds.66,67 for example , the pilz domain is a dedicated cdg binding protein , with examples like alg44 in p. aeruginosa , which is a regulator of alginate biosynthesis.52 furthermore , not all ggdef , hd - gyp , and eal domain proteins have cdg metabolic activity . a substantial minority have degenerate active sites and instead function by binding cdg , via protein protein interactions or through other alternative mechanisms.66,67 for example , the p. aeruginosa eps synthase component peld binds to cdg , at a conserved site on its degenerate ggdef domain , and induces a conformational change in the pel synthase machinery that leads to activation and eps production.68 cdg also binds to a number of different transcriptional regulators . for example , a strong scv phenotype can be triggered under laboratory conditions by overexpressing a dgc gene in trans,44 a phenotype that can be reversed by disrupting the pel and psl eps synthase operons ( figure 3).12 the intracellular level of cdg has been shown to be elevated in several clinical and laboratory - derived scvs.12,25,42 furthermore , mutations in several p. aeruginosa cdg signaling pathways have been shown to induce scv phenotypes in vitro . a prominent example is the gacas / rsmazy signaling system ( figure 4 ) , an important regulator of the switch between chronic and acute infectious lifestyles in many gram - negative bacterial species , including p. aeruginosa.70,72 rsma is a small , translational regulator that specifically recognizes and binds to gga sequences in the 5 leader region of target mrnas . deletion of the rsma gene in p. aeruginosa pa01 induces an scv phenotype as a consequence of increased psl mrna translation,33 and has been shown to increase infectious persistence in a murine lung model.13 in pseudomonas spp . although the mechanism of lads function is currently uncharacterized , it may work by counteracting rets activity ( figure 4).72 signal transduction through the gac / rsm pathway is highly complex , and affects the production of dozens of different proteins.70 in addition to direct rsma translational regulation , the gac / rsm system indirectly affects numerous additional cellular behaviors via the regulation of signaling pathways , including both cdg metabolism and quorum sensing.60,70 similar to cdg , mutations in quorum sensing genes are strongly associated with persistence during chronic cf lung infections.6,75 having colonized the respiratory passage and lungs of a cf patient , in most cases , a p. aeruginosa infection persists throughout the patient s lifetime . p. aeruginosa cf lung infections generally alternate between periods of chronic , largely asymptomatic colonization and relapses into aggravated infection.1 over several years , the progressive tissue degradation that accompanies these repeated infectious relapses leads to lung failure and premature death.3 as stated above , p. aeruginosa strains undergo extensive adaptation to the lung environment throughout the period of chronic lung infection,6,7 with multiple distinct phenotypes including scvs arising in the lung population.7,10,77 despite generally originating from a single clonal genotype , significant genetic variation exists between individual sputum isolates sampled at any one time,28,29 providing an explanation for the long - observed phenotypic heterogeneity in cf p. aeruginosa isolates.19,20 perhaps unsurprisingly , the most commonly identified scv - inducing mutations are loss - of - function mutations in repressor proteins that control the activity of dgcs ( table 1 ) . in 2006 , smith et al6 carried out a longitudinal study of lung - adapted p. aeruginosa isolates and identified several different backgrounds in which the wspf gene was mutated . deletion of fleq induces an autoaggregative phenotype in pa01 , with many of the characteristics of scv colonies.54 while the morphologies of the lung isolates in the smith et al6 study were not characterized in detail , these data nonetheless implicate both wspf and fleq as potential mutagenic targets for scv generation in the cf lung ( figure 5 ) . in turn , this change in membrane composition triggered activation of the wsp system and cdg overproduction via wspr.42 intriguingly , the genome of scv20265 was also shown to contain a second , additive mutation in the methylesterase gene wspf , shown in previous studies to induce an scv phenotype.6,25 these findings suggest that both regulatory and genetic inputs combine to control cdg production , and hence scv evolution , in the cf lung.42 the yfibnr signaling operon12 represents a further , well - characterized genetic target for clinically - arising scvs . release of yfir repression , either through a loss - of - function mutation in yfir or an escape mutation in yfin , leads to dgc activation , cdg overproduction , and scv formation under laboratory conditions.12,26 as with wspf and fleq , activating mutations arise in the yfi locus ( in both yfir and yfin ) during long - term cf lung infections , driving scv formation in vivo ( figure 5).26 interestingly , further examination of the cf isolates included in this study identified mutational these strains contained both activating and loss - of - function mutations in the yfin dgc gene , supporting the idea that yfin activity is under both positive and negative selection in vivo , with yfi - scvs acting as an environmental pool for the generation of new smooth morphotypes in the complex and heterogeneous fitness landscape of the cf lung.26 another class of mutations associated with the clinical emergence of the scv phenotype are those in muts and other mismatch repair genes . these hypermutatable strains arise with high frequency in the course of chronic cf lung infection.37,79 mena et al35 showed that a p. aeruginosa mismatch - repair mutation increased the long - term persistence of oropharyngeal colonization in cf mice , and that scv mutants readily emerged over time in this background . the phenomenon of scv morphotypes arising in hypermutatable p. aeruginosa backgrounds has been independently observed for another muts mutant strain,80 which was also shown to produce a large number of mutations in the lasr quorum sensing locus.36 the work of mena et al35 strongly suggests that mismatch repair mutants are selected in chronic p. aeruginosa lungs due to their increased persistence . genetic adaptation to the cf lung has been shown to be catalyzed by the initial acquisition of mismatch repair mutations , with subsequent mutations arising much more rapidly than in nonmutator lines.82 mutants in the cdg signaling genes fleq and wspf were identified in this study , although the isolate library used was the same as described in the earlier work of smith et al,6 where the wspf and fleq mutations were initially identified . large - scale dna inversions have also been associated with the generation of morphological diversity , and hence potentially scv generation , in the lung environment . in a study of cf lung isolates , rmling et al83 showed that 50% of tested p. aeruginosa genomes contained large chromosomal inversions . these inversions were exclusively detected in cf lung isolates , suggesting that they are selected during adaptation to chronic lung infection.83 chromosomal inversions have been linked to the mobile element is6100 and are proposed to represent a source of phenotypic variation,84 similar to that emerging from the loss of mismatch repair described above.80 furthermore , reversible genomic inversion has been shown to induce an scv phenotype in s. aureus.85 recently , the advent of next - generation sequencing has enabled the molecular basis of scv formation to be examined much more closely . the complete genome sequences of at least two clinically - evolved scvs have been published , allowing candidate causal mutations for generation of the scv phenotype to be investigated . for scv20265,86 the mutational routes to scv are known,42 while for the urethral catheter scv mh27,87 the underlying genetics of the scv phenotype has yet to be established . as sequencing technology continues to improve and becomes ever more accessible , it is likely that the coming years will see a much more complete examination of the mutational and regulatory routes to scv generation in the cf lung environment . the appearance of these distinctive , small , autoaggregative colonies strongly correlates with both deteriorating lung function and associated poor clinical prognosis . nonetheless , the importance of the scv phenotype in persistence of p. aeruginosa infection is beyond doubt , with scvs conferring both increased antibiotic tolerance and resistance to immune phagocytosis . hopefully , an increased awareness of the importance of scvs in chronic cf infection , alongside recent improvements in both diagnostic and analytical tools , will allow us to make headway in the treatment of these unusual morphotypes .

in mammals , hedgehog ( hh ) signaling plays a crucial role in embryonic development , adult tissue homeostasis and pathogenesis of human diseases . in normal situation , hh signaling is regulated by one of the three ligands : sonic hedgehog ( shh ) , indian hedgehog ( ihh ) , and desert hedgehog ( dhh ) . hh ligands activate signaling in target cells by binding to the 12-pass transmembrane receptor patched ( ptc ) . in the absence of these ligands , ptc prevents the 7-transmembrane protein , smoothened ( smo ) , from transducing signal to downstream gli transcription factors , and the pathway is in the off state . hedgehog binding to ptch leads to smo signaling to downstream effectors , leading to gli - induced target gene expression , and the pathway is turned on . primary cilium is a microtubule - based non - motile antenna - like structure that emanates from cell surface of virtually all mammalian cells . gli1 and gli2 are generally regarded as transcriptional activators whereas gli3 is often viewed as a repressor . activation of gli proteins via the hh - ptc - smo route is regarded as the canonical hh signaling pathway . in addition to the canonical pathway , the molecules can bypass the ligand - receptor signaling axis to activate gli , and these types of regulation are regarded as non - canonical hh signaling . ras signaling , tgf , pi3k and pkc are reported to regulate hh signaling via non - canonical pathways . non - canonical hh signaling is often observed in malignant diseases and have been summarized in another recent published review . pancreatic cancer is a devastating malignant disease with a very high mortality . despite its low incidence ( 2% of all cancer cases ) , pancreatic cancer is the fourth leading cause of cancer - related deaths in the us and expected to become the second cause of cancer - related deaths in a few years . after several decades of efforts , the 5-year survival rate of pancreatic cancer remains around 5% , without dramatic improvement . the high mortality rate and poor prognosis are largely due to its aggressive and metastatic nature . by the time of diagnosis , more than 80% of cases are locally advanced or distally metastasized , and are not eligible for surgical resection , which is the most effective treatment option . even in patients with resected pancreatic cancer , the outcomes are not as good as other resected solid tumors . for pancreatic cancer patients , the most common histologic type of pancreatic cancer is pancreatic ductal adenocarcinoma ( pdac ) , accounting for > 90% of pancreatic cancer cases . the exact cellular origin of pdac is still not completely known . by histological studies and clinical observation , it is postulated that before the final formation of invasive cancer , there is a stepwise progression of precursor lesions , including pancreatic intraepithelial neoplasia ( panin ) , intraductal papillary mucinous neoplasms ( ipmn ) and mucinous cystic neoplasms ( mcn ) . panin , the most common precursor lesion of pdac , is a type of microscopic precursor lesion . based on the degree of cytonuclear and architectural atypia , panins are divided in three grades : panin-1 ( subdivided into panin-1a and panin-1b ) , panin-2 and panin-3/in situ carcinoma , reflecting a progressive increase in histologic grade . recent genetic studies indicate a possibility that pdac arises from acinar cells instead of ductal cells . clinically , pancreatic cancer can be divided into four stages ( i , ii , iii , iv ) based on the tumor size , and appearance of lymph node or distal metastasis . although whether the tumor is resectable or not will require radiology data , stage - i and some stage - ii tumors are generally resectable or borderline resectable whereas all stage - iv and some stage - iii tumors are not resectable . the most common genetic event in pancreatic cancer is oncogenic kras mutation , which is almost universally present in pdac ( > 90% ) . since over 90% of low - grade panin ( panin-1 ) lesions also harbor oncogenic kras mutations , and mice conditionally expressing mutant kras develop panin , mutated kras is considered an early and initiating event in pdac development . this mutation alone , however , may not be sufficient to drive the progression of invasive cancer . molecular profiling studies revealed that during the panin - to - pdac progression , inactivating mutations of three tumor suppressor genes are commonly found : telomere shortening ( panin-1 ) p16/ cdkn2a ( some panin-1b and most panin-2 ) , tumor protein 53 ( tp53 , panin-3 ) , brca2 ( panin-3 ) and smad family member 4 ( smad4 , panin-3 ) ( fig . 1 ) . deficiency in the p16/cdkn2a axis is detectable in the early panin lesions ( 30% of panin-1b ) and in nearly all pdac , whereas inactivation of tp53 and smad4 is mainly found in panin-3 , and is associated with tumor progression ( fig . in addition to these four frequently mutated genes ( designated mountains in the genetic landscape of the pdac genomes ) , comprehensive genetic analysis has also uncovered alterations of numerous candidate cancer genes at low frequency ( designated hills ) , indicating the complexity and heterogeneity of pdac . development of pancreatic cancer is a multiple - step process , involving in formation of pancreatic intraepitheial lesions ( panin ) and carcinoma . in this process , the tumor compartment starts to have activated mutation of kras and loss of p16 in early stages whereas loss of smad4 and p53 are often found in later stages . accompanying the alterations in the tumor , accumulating changes in the stroma also occur , including expansion of fibroblasts , stellate cells , and an increase in tumor stromal fibers . these cellular changes are associated with elevated expression of many growth factors , cytokines and chemokines . shh is one of the factors secreted from the tumor compartment to affect the tumor microenvironment . another important feature of pancreatic cancer is the dense stroma , which is composed of fibroblasts , stellate cells , extracellular matrix and immune cells . the direct regulation of tgf signaling on pancreatic cancer desmoplasia has been reviewed elsewhere , and will not be repeated here . moreover , rooted from genetic alterations , many cytokines , growth factors , and their receptors as well as the associated signaling pathways are involved in the development and maintenance of pdac , reinforcing the heterogeneous features of this deadly disease . while genetic alterations during tumor development are well characterized , changes in pdac metastasis are not well studied . lack of typical symptoms at early stages , the complicated and heterogeneous genetic makeup of the tumor , the existence of extensive stroma and less well characterized metastatic tumors all increase the difficulty to make clinical advances . in the rest of the review , we will focus on the role of hh signaling in pancreatic cancer . activation of the hh pathway is necessary for early embryonic specification of the gastrointestinal tract , but downregulation of the hh pathway is critical for pancreatic development . ectopic expression of hh or aberrant activation of this pathway at the onset of pancreas organogenesis results in gain of tissues with duodenal properties and loss of pancreatic tissue , whereas the inactivation of hh pathway promotes the development of pancreatic tissue . in adult pancreas , the activity of the hh pathway is very limited and restricted to beta - cells of the endocrine pancreas in regulation of insulin production , but is also required for regeneration of the exocrine pancreas under circumstances such as injury or disease . the aberrant activation of the hh pathway in human pancreatic cancer was first reported by two independent studies . overexpression of shh is observed in both pre - invasive and invasive epithelium of 70% of human pancreatic cancer samples , and detectable as early as panin1 and throughout all disease progression , but is absent in normal pancreas . conversely , aberrant hh ligand expression has been identified in the majority of pancreatic cancer cell lines . this observation in human pdac was also confirmed in a genetically engineered mouse model . the aberrant expression of shh is directly associated with oncogenic kras expression in pdac . ectopic expression of oncogenic kras in normal human pancreatic ductal cells leads to increase of shh transcript , indicating that shh is a downstream effector of oncogenic kras in pancreatic cancer development . it was further shown that nf-b is constitutively active in pancreatic cancer , and shh is a target gene of nf-b . the human shh promoter region contains putative nf-b binding sites and activation of nf-b can promote the transcriptional activity of shh in cell - based and in vivo models . thus , it is possible that oncogenic kras promotes shh expression via nf-b signaling ( fig . shh expression is induced by kras and nfkb pathways . as a result , shh can either activate hh signaling in the cancer cell or the stromal cell , through canonical hh signaling . in addition , other signaling pathways , such as kras , can also induce gli transcriptional activity ( non - canonical hh signaling ) in the tumor compartment . as a result of hh signaling activation , cancer cells will be more proliferative , more invasive and more resistant to apoptosis . conversely , hh signaling activation in the stromal cells can feedback to stimulate cancer cell proliferation . despite the above promising data , recent studies indicate that the roles of hh pathway for pancreatic cancer may not be that simple . initially , it was thought that overexpressed shh by cancer or pre - cancer cells promotes pdac by activation of hh signaling in the stroma or in the tumor proper , and application of hh inhibitors will bring hope for patients with pancreatic cancer . however , gene knockout of smo in the pancreas has no effects on kras - mediated pancreatic cancer development , and removal of stromal hh signaling actually accelerates kras - mediated tumor development , a result opposite to the prediction . below we will discuss activation and function of hh signaling in the stroma and cancer cells of pdac ( fig . activation of the hh pathway is necessary for early embryonic specification of the gastrointestinal tract , but downregulation of the hh pathway is critical for pancreatic development . ectopic expression of hh or aberrant activation of this pathway at the onset of pancreas organogenesis results in gain of tissues with duodenal properties and loss of pancreatic tissue , whereas the inactivation of hh pathway promotes the development of pancreatic tissue . in adult pancreas , the activity of the hh pathway is very limited and restricted to beta - cells of the endocrine pancreas in regulation of insulin production , but is also required for regeneration of the exocrine pancreas under circumstances such as injury or disease . the aberrant activation of the hh pathway in human pancreatic cancer was first reported by two independent studies . overexpression of shh is observed in both pre - invasive and invasive epithelium of 70% of human pancreatic cancer samples , and detectable as early as panin1 and throughout all disease progression , but is absent in normal pancreas . conversely , aberrant hh ligand expression has been identified in the majority of pancreatic cancer cell lines . this observation in human pdac was also confirmed in a genetically engineered mouse model . the aberrant expression of shh is directly associated with oncogenic kras expression in pdac . ectopic expression of oncogenic kras in normal human pancreatic ductal cells leads to increase of shh transcript , indicating that shh is a downstream effector of oncogenic kras in pancreatic cancer development . it was further shown that nf-b is constitutively active in pancreatic cancer , and shh is a target gene of nf-b . the human shh promoter region contains putative nf-b binding sites and activation of nf-b can promote the transcriptional activity of shh in cell - based and in vivo models . thus , it is possible that oncogenic kras promotes shh expression via nf-b signaling ( fig . shh expression is induced by kras and nfkb pathways . as a result , shh can either activate hh signaling in the cancer cell or the stromal cell , through canonical hh signaling . in addition , other signaling pathways , such as kras , can also induce gli transcriptional activity ( non - canonical hh signaling ) in the tumor compartment . as a result of hh signaling activation , cancer cells will be more proliferative , more invasive and more resistant to apoptosis . conversely , hh signaling activation in the stromal cells can feedback to stimulate cancer cell proliferation . despite the above promising data , recent studies indicate that the roles of hh pathway for pancreatic cancer may not be that simple . initially , it was thought that overexpressed shh by cancer or pre - cancer cells promotes pdac by activation of hh signaling in the stroma or in the tumor proper , and application of hh inhibitors will bring hope for patients with pancreatic cancer . however , gene knockout of smo in the pancreas has no effects on kras - mediated pancreatic cancer development , and removal of stromal hh signaling actually accelerates kras - mediated tumor development , a result opposite to the prediction . below we will discuss activation and function of hh signaling in the stroma and cancer cells of pdac ( fig . one of the notorious features of pdac is desmoplasia , characterized by activation and proliferation of fibroblasts and production of collagens , laminin , and fibronectin by stromal cells . shh ligand secreted from cancerous epithelial cells can activate smo - dependent signaling in adjacent stromal cells according to the canonical hh pathway , leading to desmoplasia . fibroblasts in tumor microenvironment , also named cancer - associated fibroblasts ( cafs ) , are widely considered to promote cancer development , and this theory is also evidenced in pdac . co - culture of fibroblasts , isolated from resected pancreatic adenocarcinoma samples , increased proliferation , migration , invasion , and colony formation of cancer cells . fibroblasts also increases gemcitabine resistance in vitro and promoted tumor growth and metastasis in vivo . using mouse embryonic fibroblasts ( mefs ) as a substitute for cafs , the growth of tumor after co - injection with smo - deficient mefs was much slower compared with those from cancer cells plus wild type mefs . furthermore , orthotopic xenograft of pancreatic tumor cell line ectopically expressing shh induces primary tumor size and promotes metastasis . taken together , all these data demonstrate that epithelium - derived shh is a major regulator of fibrosis in pdac , and the activated stroma promotes tumor in pdac progression . in another word , cancer or pre - cancer cells pharmacologic blockade of the canonical hh pathway with smo antagonist , such as cyclopamine , hhantag and shh ligand - blocking antibody 5e1 has been reported to reduce the growth and distal metastases of human pancreatic tumors in immunodeficient mice , and also in one genetically engineered mouse ( gem ) model . the desmoplastic feature of pcac not only facilitates tumor growth but also protect them from chemotherapy . it is proposed that failure to treat this disease by chemotherapy is likely due to an inability of the drugs to penetrate the dense stroma to reach cancer cells . using a kpc mouse model ( pdx1-cre ; lsl - kras ; trp53 or pdx1-cre ; lsl - kras ; trp53 ) , olive and colleagues found that tumors contain an extensive stroma and poor vascular density and lead to the limitation of the chemotherapeutic agent delivery and reduction of the effectiveness of chemotherapy . smo inhibitor treatment can decrease the fibroblastic components and transiently increases blood perfusion in the tumor by increasing vasculature density . combination of smo antagonist and gemcitabine leads to increased gemcitabine accumulation in the tumor , leading to enhanced mouse survival . these studies further support that elimination of desmoplasia by shh inhibitors in pdac will result in effective delivery of chemotherapeutic agents to the tumor , and thus better clinical outcomes . clinical trials using smo inhibitors in pdac patients have shown little to no efficacy when combined with gemcitabine . another phase ii clinical trial of smo inhibitor ipi-926 in com bination with gemcitabine on pdac was suspended because patients receiving the combination had a worse outcome when compared to the placebo group ( infinity corp reports , 2012 ) . furthermore , recent published studies from two groups revealed that either genetic ablation of shh in kpc mice or prolonged exposure to hh inhibitors led to more frequent adm and panin lesions , less well - differentiated , more proliferative and metastatic tumors compared with the control litter - mates . these dogma - challenging studies indicate that the stromal cells may play a restraining role during pdac development by promoting differentiation and inhibiting aggressiveness of cancer cells , a mechanism also reported in bladder cancer . taken together , it is possible that the impact of stroma on pancreatic cancer is highly circumstantial , probably determined by temporal stage of cancer progression . further studies are definitely needed to delineate the biological function of stroma in pdac , and more importantly , smo inhibitor should be more carefully applied in cancer patients before better understanding of stromal functions for cancer development . earlier studies suggested that cancer cell - derived shh signals both via paracrine fashion to communicate with stromal cells and via autocrine signaling to support self - survival . the evidence for autocrine signaling is that in vivo hh signaling pathway components such as smo and ptc are also expressed in pdac and in pancreatic cancer cell lines . more recent studies revealed that hh signaling is restricted to the stromal compartment during pancreatic carcinogenesis and pdac cells do not respond to hh ligand . in the pdac gem mouse model based on oncogenic kras expression , conditional deletion of smo in the same cells has no effects on pancreas development or on the multistage development of pdac , indicating that the canonical hh signaling is indispensable for pdac progression . expression of smom2 , an oncogenic smoothened , using pdx1 promoter - driven cre recombinase does not result in hh signaling activation , and has no impact on krasg12v - induced tumor development . smom2 , however , is able to transduce hh signaling in several pancreatic cancer cell lines and orthotopic mouse models . additionally , in a subcutaneous xenograft model , yauch and colleagues showed that tumors from mouse xenografts displayed significant inhibition of tumor growth after treatment with a smo inhibitor , followed by decreased expression of mouse hh target genes without effects on human counterpart . taken all these data together , it seems that canonical ( ligand - dependent ) hh signaling is not activated in the tumor compartment of pdac . however , it is hard to rule out the possibility of non - canonical hh signaling in human pdac and a potential role for hh signaling in a minor subpopulation of epithelial tumor cells , such as cancer initiating cells . in our studies , we found that smo signaling inhibition in orthotopic xenografts of human pancreatic cancer almost completely suppresses hh signaling in the stromal cells but only reduces 50% of the hh signaling activity in cancer cells as indicated by hh target gene expression ( our unpublished data ) . these results indicate the coexistence of canonical and non - canonical hh signaling in pancreatic cancer cells . we also found that smo signaling inhibition in vitro reduces stem cell population , suggesting a role of ligand - dependent hh pathway in the maintenance of cancer stem cell population in pdac . recently , sharma and colleagues also demonstrated that nvp - bez-235 , another smoothened inhibitor , can also inhibit the self - renewal of pancreatic cancer stem cells ( cscs ) by suppressing the ligand dependent hh signaling pathway . thus , hh may play different roles in different cell types within the same tumor . although the involvement of the upstream part of the canonical hh signaling pathway in pancreatic cancer cells is controversial , gli proteins , the downstream transcription factors , do play a role in pancreatic cancer development . demonstrated that conditional deletion of smo does nt affect gli1 expression in cancer cells , indicating that gli transcription in cancer cells is regulated through non - canonical hh signaling . in other studies , rajurkar et al . showed that targeted ectopic expression of gli1 in the pancreatic cells accelerates pdac initiation by mutant kras . furthermore , inhibition of gli transcriptional activity by dominant negative gli3 reduced the incidence of kras - driven panins and pdac , indicating the importance of gli transcription factors in pancreatic tumorigenesis . recently , it has been found that gli1 promotes the growth and migration of pancreatic cancer cells via regulation of the transcription of eukaryotic translation initiation factor 5a2 ( eif5a2 ) . kras activating mutation is almost universal in sporadic pdac , and it is reported that the kras - mek - erk cascade increases gli transcriptional activity . ectopic expression of oncogenic kras in normal human pancreatic cell line hpde - c7 or bxpc3 , a pancreatic cancer cell line with wild type kras , increases transcription activity of gli molecules . depletion of oncogenic kras with specific mutant kras - targeted sirnas inhibits gli transcription activity , as indicated by expression of gli1 and ptch1 in pdac cell lines . it is not clear how the ras / raf / mek cascade affects gli1 transcriptional activity remains to be elucidated . it is known that gli transcriptional activity is regulated by the pattern of gli phosphorylation . ser130 of gli1 protein can be phosphorylated by erk2 , but it is not clear whether this mechanism is responsible for gli1 function in pancreatic cancer . in the mouse model of pancreatic cancer , after smo knockout , tgf treatment causes marked elevation of gli1 and gli3 . other ligand - independent hh signaling mechanisms in pancreatic cancer include altered expression of the co - receptor for hh ligands and epigenetic regulation of hh signaling molecules hip and ptch1 . taken together , increasing evidence indicates that the transcription activity of gli protein may be directly regulated by phosphorylation of the kras - mek - erk cascade in the tumor compartment . on the other hand , ligand - dependent hh signaling may be responsible for hh signaling activation in the tumor stroma . it becomes clear that hh signaling is activated in both the tumor stroma and in the tumor compartment in pancreatic cancer . however , the mechanisms underlying hh signaling activation in these two compartments are not the same . while ligand - dependent hh signaling is mainly responsible for stromal hh signaling , both canonical and non - canonical hh signaling occurs in the tumor compartment . in addition to different types of hh signaling activation , the roles of hh signaling for pancreatic cancer development , progression and metastasis are not well studied . the poorly understood biology of hh signaling in pancreatic cancer may account for the failed clinical trials using smo signaling inhibitors . with further efforts in deciphering the hh signaling mechanisms in pancreatic cancer , we predict a new wave of novel strategies to suppress hh signaling . to that end , we believe that efforts are needed to answer the following questions : 1 ) what is the role of stromal hh signaling for tumor development of pancreatic cancer ? 2 ) what is the best way to suppress hh signaling in the tumor compartment ? 3 ) in addition to tumor development , is hh signaling responsible for pancreatic cancer metastasis ? 4 ) does the current gem model ( kpc ) recapitulate all the features of pdac in the humans or only a subset of them ?

abstractpancreatic cancer , mostly pancreatic ductal adenocarcinoma ( pdac ) , is a leading cause of cancer - related death in the us , with a dismal median survival of 6 months . thus , there is an urgent unmet need to identify ways to diagnose and to treat this deadly cancer . although a number of genetic changes have been identified in pancreatic cancer , their mechanisms of action in tumor development , progression and metastasis are not completely understood . hedgehog signaling , which plays a major role in embryonic development and stem cell regulation , is known to be activated in pancreatic cancer ; however , specific inhibitors targeting the smoothened molecule failed to improve the condition of pancreatic cancer patients in clinical trials . furthermore , results regarding the role of hh signaling in pancreatic cancer are controversial with some reporting tumor promoting activities whereas others tumor suppressive actions . in this review , we will summarize what we know about hedgehog signaling in pancreatic cancer , and try to explain the contradicting roles of hedgehog signaling as well as the reason(s ) behind the failed clinical trials . in addition to the canonical hedgehog signaling , we will also discuss several non - canonical hedgehog signaling mechanisms .

The hedgehog signaling pathway Pancreatic cancer Shh signaling and pancreatic cancer Hh signaling in pancreas development Hh signaling in pancreatic cancer Paracrine hh signaling in tumor stroma Hh signaling in tumor cells Perspectives

in mammals , hedgehog ( hh ) signaling plays a crucial role in embryonic development , adult tissue homeostasis and pathogenesis of human diseases . in normal situation , hh signaling is regulated by one of the three ligands : sonic hedgehog ( shh ) , indian hedgehog ( ihh ) , and desert hedgehog ( dhh ) . hh ligands activate signaling in target cells by binding to the 12-pass transmembrane receptor patched ( ptc ) . in the absence of these ligands , ptc prevents the 7-transmembrane protein , smoothened ( smo ) , from transducing signal to downstream gli transcription factors , and the pathway is in the off state . primary cilium is a microtubule - based non - motile antenna - like structure that emanates from cell surface of virtually all mammalian cells . activation of gli proteins via the hh - ptc - smo route is regarded as the canonical hh signaling pathway . in addition to the canonical pathway , the molecules can bypass the ligand - receptor signaling axis to activate gli , and these types of regulation are regarded as non - canonical hh signaling . ras signaling , tgf , pi3k and pkc are reported to regulate hh signaling via non - canonical pathways . non - canonical hh signaling is often observed in malignant diseases and have been summarized in another recent published review . pancreatic cancer is a devastating malignant disease with a very high mortality . despite its low incidence ( 2% of all cancer cases ) , pancreatic cancer is the fourth leading cause of cancer - related deaths in the us and expected to become the second cause of cancer - related deaths in a few years . after several decades of efforts , the 5-year survival rate of pancreatic cancer remains around 5% , without dramatic improvement . by the time of diagnosis , more than 80% of cases are locally advanced or distally metastasized , and are not eligible for surgical resection , which is the most effective treatment option . even in patients with resected pancreatic cancer , the outcomes are not as good as other resected solid tumors . for pancreatic cancer patients , the most common histologic type of pancreatic cancer is pancreatic ductal adenocarcinoma ( pdac ) , accounting for > 90% of pancreatic cancer cases . the exact cellular origin of pdac is still not completely known . by histological studies and clinical observation , it is postulated that before the final formation of invasive cancer , there is a stepwise progression of precursor lesions , including pancreatic intraepithelial neoplasia ( panin ) , intraductal papillary mucinous neoplasms ( ipmn ) and mucinous cystic neoplasms ( mcn ) . panin , the most common precursor lesion of pdac , is a type of microscopic precursor lesion . based on the degree of cytonuclear and architectural atypia , panins are divided in three grades : panin-1 ( subdivided into panin-1a and panin-1b ) , panin-2 and panin-3/in situ carcinoma , reflecting a progressive increase in histologic grade . clinically , pancreatic cancer can be divided into four stages ( i , ii , iii , iv ) based on the tumor size , and appearance of lymph node or distal metastasis . the most common genetic event in pancreatic cancer is oncogenic kras mutation , which is almost universally present in pdac ( > 90% ) . since over 90% of low - grade panin ( panin-1 ) lesions also harbor oncogenic kras mutations , and mice conditionally expressing mutant kras develop panin , mutated kras is considered an early and initiating event in pdac development . molecular profiling studies revealed that during the panin - to - pdac progression , inactivating mutations of three tumor suppressor genes are commonly found : telomere shortening ( panin-1 ) p16/ cdkn2a ( some panin-1b and most panin-2 ) , tumor protein 53 ( tp53 , panin-3 ) , brca2 ( panin-3 ) and smad family member 4 ( smad4 , panin-3 ) ( fig . deficiency in the p16/cdkn2a axis is detectable in the early panin lesions ( 30% of panin-1b ) and in nearly all pdac , whereas inactivation of tp53 and smad4 is mainly found in panin-3 , and is associated with tumor progression ( fig . in addition to these four frequently mutated genes ( designated mountains in the genetic landscape of the pdac genomes ) , comprehensive genetic analysis has also uncovered alterations of numerous candidate cancer genes at low frequency ( designated hills ) , indicating the complexity and heterogeneity of pdac . development of pancreatic cancer is a multiple - step process , involving in formation of pancreatic intraepitheial lesions ( panin ) and carcinoma . accompanying the alterations in the tumor , accumulating changes in the stroma also occur , including expansion of fibroblasts , stellate cells , and an increase in tumor stromal fibers . another important feature of pancreatic cancer is the dense stroma , which is composed of fibroblasts , stellate cells , extracellular matrix and immune cells . the direct regulation of tgf signaling on pancreatic cancer desmoplasia has been reviewed elsewhere , and will not be repeated here . moreover , rooted from genetic alterations , many cytokines , growth factors , and their receptors as well as the associated signaling pathways are involved in the development and maintenance of pdac , reinforcing the heterogeneous features of this deadly disease . while genetic alterations during tumor development are well characterized , changes in pdac metastasis are not well studied . in the rest of the review , we will focus on the role of hh signaling in pancreatic cancer . ectopic expression of hh or aberrant activation of this pathway at the onset of pancreas organogenesis results in gain of tissues with duodenal properties and loss of pancreatic tissue , whereas the inactivation of hh pathway promotes the development of pancreatic tissue . overexpression of shh is observed in both pre - invasive and invasive epithelium of 70% of human pancreatic cancer samples , and detectable as early as panin1 and throughout all disease progression , but is absent in normal pancreas . conversely , aberrant hh ligand expression has been identified in the majority of pancreatic cancer cell lines . ectopic expression of oncogenic kras in normal human pancreatic ductal cells leads to increase of shh transcript , indicating that shh is a downstream effector of oncogenic kras in pancreatic cancer development . it was further shown that nf-b is constitutively active in pancreatic cancer , and shh is a target gene of nf-b . thus , it is possible that oncogenic kras promotes shh expression via nf-b signaling ( fig . as a result , shh can either activate hh signaling in the cancer cell or the stromal cell , through canonical hh signaling . in addition , other signaling pathways , such as kras , can also induce gli transcriptional activity ( non - canonical hh signaling ) in the tumor compartment . as a result of hh signaling activation , cancer cells will be more proliferative , more invasive and more resistant to apoptosis . conversely , hh signaling activation in the stromal cells can feedback to stimulate cancer cell proliferation . despite the above promising data , recent studies indicate that the roles of hh pathway for pancreatic cancer may not be that simple . initially , it was thought that overexpressed shh by cancer or pre - cancer cells promotes pdac by activation of hh signaling in the stroma or in the tumor proper , and application of hh inhibitors will bring hope for patients with pancreatic cancer . however , gene knockout of smo in the pancreas has no effects on kras - mediated pancreatic cancer development , and removal of stromal hh signaling actually accelerates kras - mediated tumor development , a result opposite to the prediction . below we will discuss activation and function of hh signaling in the stroma and cancer cells of pdac ( fig . ectopic expression of hh or aberrant activation of this pathway at the onset of pancreas organogenesis results in gain of tissues with duodenal properties and loss of pancreatic tissue , whereas the inactivation of hh pathway promotes the development of pancreatic tissue . overexpression of shh is observed in both pre - invasive and invasive epithelium of 70% of human pancreatic cancer samples , and detectable as early as panin1 and throughout all disease progression , but is absent in normal pancreas . conversely , aberrant hh ligand expression has been identified in the majority of pancreatic cancer cell lines . ectopic expression of oncogenic kras in normal human pancreatic ductal cells leads to increase of shh transcript , indicating that shh is a downstream effector of oncogenic kras in pancreatic cancer development . it was further shown that nf-b is constitutively active in pancreatic cancer , and shh is a target gene of nf-b . thus , it is possible that oncogenic kras promotes shh expression via nf-b signaling ( fig . as a result , shh can either activate hh signaling in the cancer cell or the stromal cell , through canonical hh signaling . in addition , other signaling pathways , such as kras , can also induce gli transcriptional activity ( non - canonical hh signaling ) in the tumor compartment . as a result of hh signaling activation , cancer cells will be more proliferative , more invasive and more resistant to apoptosis . conversely , hh signaling activation in the stromal cells can feedback to stimulate cancer cell proliferation . despite the above promising data , recent studies indicate that the roles of hh pathway for pancreatic cancer may not be that simple . initially , it was thought that overexpressed shh by cancer or pre - cancer cells promotes pdac by activation of hh signaling in the stroma or in the tumor proper , and application of hh inhibitors will bring hope for patients with pancreatic cancer . however , gene knockout of smo in the pancreas has no effects on kras - mediated pancreatic cancer development , and removal of stromal hh signaling actually accelerates kras - mediated tumor development , a result opposite to the prediction . below we will discuss activation and function of hh signaling in the stroma and cancer cells of pdac ( fig . shh ligand secreted from cancerous epithelial cells can activate smo - dependent signaling in adjacent stromal cells according to the canonical hh pathway , leading to desmoplasia . fibroblasts in tumor microenvironment , also named cancer - associated fibroblasts ( cafs ) , are widely considered to promote cancer development , and this theory is also evidenced in pdac . co - culture of fibroblasts , isolated from resected pancreatic adenocarcinoma samples , increased proliferation , migration , invasion , and colony formation of cancer cells . fibroblasts also increases gemcitabine resistance in vitro and promoted tumor growth and metastasis in vivo . furthermore , orthotopic xenograft of pancreatic tumor cell line ectopically expressing shh induces primary tumor size and promotes metastasis . taken together , all these data demonstrate that epithelium - derived shh is a major regulator of fibrosis in pdac , and the activated stroma promotes tumor in pdac progression . in another word , cancer or pre - cancer cells pharmacologic blockade of the canonical hh pathway with smo antagonist , such as cyclopamine , hhantag and shh ligand - blocking antibody 5e1 has been reported to reduce the growth and distal metastases of human pancreatic tumors in immunodeficient mice , and also in one genetically engineered mouse ( gem ) model . it is proposed that failure to treat this disease by chemotherapy is likely due to an inability of the drugs to penetrate the dense stroma to reach cancer cells . using a kpc mouse model ( pdx1-cre ; lsl - kras ; trp53 or pdx1-cre ; lsl - kras ; trp53 ) , olive and colleagues found that tumors contain an extensive stroma and poor vascular density and lead to the limitation of the chemotherapeutic agent delivery and reduction of the effectiveness of chemotherapy . these studies further support that elimination of desmoplasia by shh inhibitors in pdac will result in effective delivery of chemotherapeutic agents to the tumor , and thus better clinical outcomes . another phase ii clinical trial of smo inhibitor ipi-926 in com bination with gemcitabine on pdac was suspended because patients receiving the combination had a worse outcome when compared to the placebo group ( infinity corp reports , 2012 ) . furthermore , recent published studies from two groups revealed that either genetic ablation of shh in kpc mice or prolonged exposure to hh inhibitors led to more frequent adm and panin lesions , less well - differentiated , more proliferative and metastatic tumors compared with the control litter - mates . taken together , it is possible that the impact of stroma on pancreatic cancer is highly circumstantial , probably determined by temporal stage of cancer progression . further studies are definitely needed to delineate the biological function of stroma in pdac , and more importantly , smo inhibitor should be more carefully applied in cancer patients before better understanding of stromal functions for cancer development . the evidence for autocrine signaling is that in vivo hh signaling pathway components such as smo and ptc are also expressed in pdac and in pancreatic cancer cell lines . more recent studies revealed that hh signaling is restricted to the stromal compartment during pancreatic carcinogenesis and pdac cells do not respond to hh ligand . in the pdac gem mouse model based on oncogenic kras expression , conditional deletion of smo in the same cells has no effects on pancreas development or on the multistage development of pdac , indicating that the canonical hh signaling is indispensable for pdac progression . expression of smom2 , an oncogenic smoothened , using pdx1 promoter - driven cre recombinase does not result in hh signaling activation , and has no impact on krasg12v - induced tumor development . smom2 , however , is able to transduce hh signaling in several pancreatic cancer cell lines and orthotopic mouse models . additionally , in a subcutaneous xenograft model , yauch and colleagues showed that tumors from mouse xenografts displayed significant inhibition of tumor growth after treatment with a smo inhibitor , followed by decreased expression of mouse hh target genes without effects on human counterpart . taken all these data together , it seems that canonical ( ligand - dependent ) hh signaling is not activated in the tumor compartment of pdac . however , it is hard to rule out the possibility of non - canonical hh signaling in human pdac and a potential role for hh signaling in a minor subpopulation of epithelial tumor cells , such as cancer initiating cells . in our studies , we found that smo signaling inhibition in orthotopic xenografts of human pancreatic cancer almost completely suppresses hh signaling in the stromal cells but only reduces 50% of the hh signaling activity in cancer cells as indicated by hh target gene expression ( our unpublished data ) . these results indicate the coexistence of canonical and non - canonical hh signaling in pancreatic cancer cells . we also found that smo signaling inhibition in vitro reduces stem cell population , suggesting a role of ligand - dependent hh pathway in the maintenance of cancer stem cell population in pdac . recently , sharma and colleagues also demonstrated that nvp - bez-235 , another smoothened inhibitor , can also inhibit the self - renewal of pancreatic cancer stem cells ( cscs ) by suppressing the ligand dependent hh signaling pathway . although the involvement of the upstream part of the canonical hh signaling pathway in pancreatic cancer cells is controversial , gli proteins , the downstream transcription factors , do play a role in pancreatic cancer development . demonstrated that conditional deletion of smo does nt affect gli1 expression in cancer cells , indicating that gli transcription in cancer cells is regulated through non - canonical hh signaling . showed that targeted ectopic expression of gli1 in the pancreatic cells accelerates pdac initiation by mutant kras . furthermore , inhibition of gli transcriptional activity by dominant negative gli3 reduced the incidence of kras - driven panins and pdac , indicating the importance of gli transcription factors in pancreatic tumorigenesis . recently , it has been found that gli1 promotes the growth and migration of pancreatic cancer cells via regulation of the transcription of eukaryotic translation initiation factor 5a2 ( eif5a2 ) . kras activating mutation is almost universal in sporadic pdac , and it is reported that the kras - mek - erk cascade increases gli transcriptional activity . ectopic expression of oncogenic kras in normal human pancreatic cell line hpde - c7 or bxpc3 , a pancreatic cancer cell line with wild type kras , increases transcription activity of gli molecules . it is known that gli transcriptional activity is regulated by the pattern of gli phosphorylation . ser130 of gli1 protein can be phosphorylated by erk2 , but it is not clear whether this mechanism is responsible for gli1 function in pancreatic cancer . in the mouse model of pancreatic cancer , after smo knockout , tgf treatment causes marked elevation of gli1 and gli3 . other ligand - independent hh signaling mechanisms in pancreatic cancer include altered expression of the co - receptor for hh ligands and epigenetic regulation of hh signaling molecules hip and ptch1 . on the other hand , ligand - dependent hh signaling may be responsible for hh signaling activation in the tumor stroma . it becomes clear that hh signaling is activated in both the tumor stroma and in the tumor compartment in pancreatic cancer . however , the mechanisms underlying hh signaling activation in these two compartments are not the same . while ligand - dependent hh signaling is mainly responsible for stromal hh signaling , both canonical and non - canonical hh signaling occurs in the tumor compartment . in addition to different types of hh signaling activation , the roles of hh signaling for pancreatic cancer development , progression and metastasis are not well studied . the poorly understood biology of hh signaling in pancreatic cancer may account for the failed clinical trials using smo signaling inhibitors . with further efforts in deciphering the hh signaling mechanisms in pancreatic cancer , we predict a new wave of novel strategies to suppress hh signaling . to that end , we believe that efforts are needed to answer the following questions : 1 ) what is the role of stromal hh signaling for tumor development of pancreatic cancer ? 2 ) what is the best way to suppress hh signaling in the tumor compartment ? 3 ) in addition to tumor development , is hh signaling responsible for pancreatic cancer metastasis ?

as the size of the geriatric population increases , the number of elderly patients diagnosed with painful degenerative diseases of the lumbar spine requiring surgery is expected to rise concomitantly . many patients require lumbar spinal fusion surgery with instrumentation along with decompression to treat the degenerative lumbar disease . often , patient age is a major factor in deciding to which extent surgeries could or should be performed , and it is perceived that morbidity is increased with extensive surgeries in older patients32 ) . the geriatric population may be at increased risk for complications because of age and age - associated medical conditions . however , there is a lack of studies addressing the perioperative complications in elderly patients with decompression , arthrodesis of the lumbar spine , and the post operative outcomes of lumbar spinal fusion surgeries24 ) . a review of literature reveals controversy over the safety of lumbar spinal fusion surgery in the elderly due to varying findings on the risk . several researchers raised concerns over increased morbidity , cautioning against spinal surgery in the older population3,17 ) . the goal of this study was to evaluate ; 1 ) the complication rate in relation to perioperative complications , general factors such as age , sex , comorbidity , american society of anesthesiologists ( asa ) class , and fusion segments involved , and 2 ) examine the clinical outcomes of lumbar spinal fusion surgery for degenerative lumbar diseases for patients 70 years and older in contrast to patients younger than 65 year of age . in this study , the patient population of a specific age group was selected based on the type of surgeries the patients received in a specific time period . patient population was divided into two groups according to the definition of " geriatric " : 70 years and older ( group 1 ) , and younger than 65 years ( group 2 ) . lumbar spinal fusion surgeries were performed by 4 neurosurgeons at a single institute . we included patients with degenerative spine diseases , and excluded patients with age between 65 and 69 years , spine traumas , spinal revision surgeries , and spine tumors in order to avoid unexpected co - morbidities . data related to these patients were evaluated by a single observer using the standard hospital charts , outpatient notes , electronic medical records , operative reports , and preoperative and postoperative imaging studies . the following demographic variables were evaluated : age , sex , treatable or non - treatable medical co - morbidities , the preoperative asa classification of physical status , and segment of fusion . all patients were evaluated for co - morbidities such as cardiovascular , renal , pulmonary , hepatic problems and diabetes mellitus . co - morbidities were subdivided as follows : cardiovascular ( hypertension , arrhythmia , angina , coronary artery disease , and congestive heart failure ) , renal ( nephrotic cancer , ureter stone , renal cyst and acute or chronic renal failure ) , pulmonary ( lung abscess , tuberculosis , emphysema , chronic obstructive pulmonary disease , and asthma ) , hepatic ( common bile duct stones , hepatitis and liver cirrhosis ) and endocrine ( diabetes mellitus , hypothyroidism and hyperthyroidism ) . the standard surgical procedure was in steps sequenced as follows : partial hemilaminectomy , transpedicular screw fixation , and both posterior lumber interbody fusion with cage and posterolateral fusion . the mean operation time was 198.2 minutes , the mean estimated blood loss was 325.8 cc , and the mean transfusion was 185.7 cc . in general , surgeries were performed in degenerative cases when conservative treatment over three months failed to improve the patients ' symptoms . in this study , patients were ambulated from the second postoperative day with rigid molded plastic braces or lumbar corsets . initially , the patients were treated with analgesia for control of acute pain by intravenous injection , and then weaned to mild oral narcotic therapy and nsaids that were discontinued as quickly as possible . the term " complication " in this study a complication was any event requiring specific management during the perioperative period , including the intra - operative and post - operative periods . the patients enrolled in this study were followed - up for at least 12 months postoperatively . the frequency analysis , simple and multiple logistic regression analysis , and test were done using spss ver . logistic regression analyses were done to investigate whether complications were effected by age , sex , comorbidity , asa class , or fusion segments . fisher 's exact test was carried out to compare how the complications affected the macnab 's criteria results after final follow - up . in this study , the patient population of a specific age group was selected based on the type of surgeries the patients received in a specific time period . patient population was divided into two groups according to the definition of " geriatric " : 70 years and older ( group 1 ) , and younger than 65 years ( group 2 ) . lumbar spinal fusion surgeries were performed by 4 neurosurgeons at a single institute . we included patients with degenerative spine diseases , and excluded patients with age between 65 and 69 years , spine traumas , spinal revision surgeries , and spine tumors in order to avoid unexpected co - morbidities . data related to these patients were evaluated by a single observer using the standard hospital charts , outpatient notes , electronic medical records , operative reports , and preoperative and postoperative imaging studies . the following demographic variables were evaluated : age , sex , treatable or non - treatable medical co - morbidities , the preoperative asa classification of physical status , and segment of fusion . all patients were evaluated for co - morbidities such as cardiovascular , renal , pulmonary , hepatic problems and diabetes mellitus . co - morbidities were subdivided as follows : cardiovascular ( hypertension , arrhythmia , angina , coronary artery disease , and congestive heart failure ) , renal ( nephrotic cancer , ureter stone , renal cyst and acute or chronic renal failure ) , pulmonary ( lung abscess , tuberculosis , emphysema , chronic obstructive pulmonary disease , and asthma ) , hepatic ( common bile duct stones , hepatitis and liver cirrhosis ) and endocrine ( diabetes mellitus , hypothyroidism and hyperthyroidism ) . the standard surgical procedure was in steps sequenced as follows : partial hemilaminectomy , transpedicular screw fixation , and both posterior lumber interbody fusion with cage and posterolateral fusion . the mean operation time was 198.2 minutes , the mean estimated blood loss was 325.8 cc , and the mean transfusion was 185.7 cc . in general , surgeries were performed in degenerative cases when conservative treatment over three months failed to improve the patients ' symptoms . in this study , patients were ambulated from the second postoperative day with rigid molded plastic braces or lumbar corsets . initially , the patients were treated with analgesia for control of acute pain by intravenous injection , and then weaned to mild oral narcotic therapy and nsaids that were discontinued as quickly as possible . the term " complication " in this study is defined broadly and differently from published literature . in this study , a complication was any event requiring specific management during the perioperative period , including the intra - operative and post - operative periods . the patients enrolled in this study were followed - up for at least 12 months postoperatively . the frequency analysis , simple and multiple logistic regression analysis , and test were done using spss ver . logistic regression analyses were done to investigate whether complications were effected by age , sex , comorbidity , asa class , or fusion segments . fisher 's exact test was carried out to compare how the complications affected the macnab 's criteria results after final follow - up . during the five - year study period , the 485 patients met the inclusion criteria and underwent lumbar spinal fusion surgeries ( table 1 ) . of these , 156 patients ( 32.2% ) were male and 329 ( 67.8% ) were female , and there were 174 patients ( 35.9% ) in group 1 and 311 patients ( 64.1% ) in group 2 . the number of patients in the age group 70 - 75 , 75 - 79 , and 80 - 85 were 92 , 56 , and 26 , respectively . 204 ( 42.1% ) of the patients had comorbidities . the general condition of those patients varied by asa class : asa class i ( 303 patients , 62.5% ) , asa ii ( 149 patients , 30.7% ) and asa iii ( 33 patients , 6.8% ) . fusion segment levels were categorized as 1 segment ( 247 patients , 50.9% ) , 2 segments ( 179 patients , 36.9% ) , 3 segments ( 41 patients , 8.5% ) , 4 segments ( 18 patients , 3.7% ) . complications were encountered in 81 patients ( 16.7% ) . in group 1 , complications occurred in 35 patients ( 20.1% ) , and in group 2 , complications occurred in 51 patients ( 14.8% ) . among the observed comorbidities , the most common problem was hypertension , which was present preoperatively in 81 patients ( 39.7% ) . other comorbidities were cardiovascular diseases ( 32 patients , 15.7% ) , renal diseases ( 29 patients , 14.2% ) , pulmonary diseases ( 36 patients , 17.6% ) , endocrine disease ( 75 patients , 36.8% ) , and hepatic diseases ( 20 patients , 9.8% ) . in addition , 47 patients ( 23.0% ) had more than one comorbidity ( table 2 ) . underlying diseases were accompanied in 52.9% of the geriatric patients and there was a statistically significant association between age and underlying disease ( p=0.017 ) ( table 3 ) . the total peri - operative complication rate in all patients was 16.7% ; the peri - operative complication rate in age group 1 was 20.1% , while the complication rate in group 2 was 14.8% . the most common complication was wound dehiscence ( 25.9% ) , and the second most prevalent was ileus , found in 16.0% of cases . twenty - six of all complications required re - operation ( 3 cases of dura tearing , 10 cases of wound dehiscence , 2 cases of hematoma accumulation , 4 cases of superficial wound infection , 2 cases of deep wound infection , and 5 cases of screw malposition ) . all , except one patient , overcame the complications with proper treatment . the patient who presented with an acute myocardial infarction ( ami ) had a cardiac arrest on the third day after the surgery and died . the results of the logistic regression analysis are in table 5 , displaying results of univariate and multivariate analysis in order of age , sex , comorbidity , asa class , and fusion segment . age was the only statistically significantly factor in both univariate and multivariate analyses ( p=0.015 and 0.024 ) . no correlation was found to be statistically significant between complications and sex , comorbidity , asa class , fusion segment ( p>0.05 ) . based on the mean macnab 's criteria , post - operative outcome of 485 patients revealed as " excellent " in 280 patients ( 57.7% ) , " good " in 132 patients ( 27.2% ) , " fair " in 57 patients ( 11.8% ) , and " poor " in 16 patients ( 3.3% ) . for those patients who showed " excellent " or " good " results , patients had satisfactory improvements . on the other hand , " fair " or " poor " outcomes occurred in 73 patients ( 15.1% ) . patients in both age groups showed comparable post - operative outcomes ; 143 ( 82.2% ) patients in age group 1 , and 308 ( 86.5% ) patients in age group 2 showed " excellent " or " good " results . based on fisher 's exact test , which was done to find the correlation between occurrence of perioperative complications and post - operative outcome , there was no statistically significant relationship between perioperative complications and " excellent " or " good " group in macnab 's criteria ( table 6 ) . during the five - year study period , the 485 patients met the inclusion criteria and underwent lumbar spinal fusion surgeries ( table 1 ) . of these , 156 patients ( 32.2% ) were male and 329 ( 67.8% ) were female , and there were 174 patients ( 35.9% ) in group 1 and 311 patients ( 64.1% ) in group 2 . the number of patients in the age group 70 - 75 , 75 - 79 , and 80 - 85 were 92 , 56 , and 26 , respectively . 204 ( 42.1% ) of the patients had comorbidities . the general condition of those patients varied by asa class : asa class i ( 303 patients , 62.5% ) , asa ii ( 149 patients , 30.7% ) and asa iii ( 33 patients , 6.8% ) . fusion segment levels were categorized as 1 segment ( 247 patients , 50.9% ) , 2 segments ( 179 patients , 36.9% ) , 3 segments ( 41 patients , 8.5% ) , 4 segments ( 18 patients , 3.7% ) . complications were encountered in 81 patients ( 16.7% ) . in group 1 , complications occurred in 35 patients ( 20.1% ) , and in group 2 , complications occurred in 51 patients ( 14.8% ) . among the observed comorbidities , the most common problem was hypertension , which was present preoperatively in 81 patients ( 39.7% ) . other comorbidities were cardiovascular diseases ( 32 patients , 15.7% ) , renal diseases ( 29 patients , 14.2% ) , pulmonary diseases ( 36 patients , 17.6% ) , endocrine disease ( 75 patients , 36.8% ) , and hepatic diseases ( 20 patients , 9.8% ) . in addition , 47 patients ( 23.0% ) had more than one comorbidity ( table 2 ) . underlying diseases were accompanied in 52.9% of the geriatric patients and there was a statistically significant association between age and underlying disease ( p=0.017 ) ( table 3 ) . the total peri - operative complication rate in all patients was 16.7% ; the peri - operative complication rate in age group 1 was 20.1% , while the complication rate in group 2 was 14.8% . the most common complication was wound dehiscence ( 25.9% ) , and the second most prevalent was ileus , found in 16.0% of cases . twenty - six of all complications required re - operation ( 3 cases of dura tearing , 10 cases of wound dehiscence , 2 cases of hematoma accumulation , 4 cases of superficial wound infection , 2 cases of deep wound infection , and 5 cases of screw malposition ) . all , except one patient , overcame the complications with proper treatment . the patient who presented with an acute myocardial infarction ( ami ) had a cardiac arrest on the third day after the surgery and died . the results of the logistic regression analysis are in table 5 , displaying results of univariate and multivariate analysis in order of age , sex , comorbidity , asa class , and fusion segment . age was the only statistically significantly factor in both univariate and multivariate analyses ( p=0.015 and 0.024 ) . no correlation was found to be statistically significant between complications and sex , comorbidity , asa class , fusion segment ( p>0.05 ) . based on the mean macnab 's criteria , post - operative outcome of 485 patients revealed as " excellent " in 280 patients ( 57.7% ) , " good " in 132 patients ( 27.2% ) , " fair " in 57 patients ( 11.8% ) , and " poor " in 16 patients ( 3.3% ) . for those patients who showed " excellent " or " good " results , patients had satisfactory improvements . on the other hand , " fair " or " poor " outcomes occurred in 73 patients ( 15.1% ) . patients in both age groups showed comparable post - operative outcomes ; 143 ( 82.2% ) patients in age group 1 , and 308 ( 86.5% ) patients in age group 2 showed " excellent " or " good " results . based on fisher 's exact test , which was done to find the correlation between occurrence of perioperative complications and post - operative outcome , there was no statistically significant relationship between perioperative complications and " excellent " or " good " group in macnab 's criteria ( table 6 ) . the number of elderly persons in the republic of korea continues to grow ; by 2050 , 38.2% of the population is expected to be older than 65 years , greatly exceeding the worldwide mean of 16.2% . an aging population will consequently suffer from age - related degenerative diseases , such as disorders of the spine . advances in anesthesiology , spinal instrumentation , and postoperative care have made spinal procedures safer , with a decrease in morbidity and mortality , as well as continued improvements in patient outcomes6 ) . recognizing predictors of complications or poor outcome for the geriatric population is crucial for perioperative risk assessment and applying appropriate preventative procedures . if symptoms of degenerative spinal diseases persist after conservative treatments , surgical treatments may be considered5,9,15 ) . similar to several previous retrospective studies , the complication rate in this analysis was 16.7%2,8 ) . in a review of 105 spine surgery articles , nasser et al.22 ) reported an overall pooled complication incidence of 16.4% . at 2.6% incidence rate , re - operation after a spinal fusion surgery may be considered a failure of the initial surgery by the patient and the surgeon . it also represents an additional procedure in patients who are already at significant risk for complication6 ) . mok et al.21 ) recently evaluated the need for revision surgery in patients undergoing adult spine surgery , finding a cumulative rate of 25.8% , while cloyd et al.6 ) reported a re - operation rate of 27.4% in patients at least 65 years old who presented with a wide range of spine pathologies . recently , lee et al.20 ) reported that the rate of revision surgery due to complications was approximately 47% . in our study , the rate of revision surgery due to complications was approximately 40.6% , but age was not associated with the need for re - operation . okuda et al.23 ) found that patients older than 70 years who underwent posterior lumbar interbody fusions for spondylolisthesis had a complication rate of 16% , which was not significantly different from that in younger patients . kiliner et al.18 ) agreed that age did not affect the complication rates of posterior lumbar interbody fusion , but they did not report a complication rate separately for older patients . vitaz et al.33 ) suggested that elderly patients with lumbar spinal diseases can be surgically treated in the same manner as the younger patients . on the other hand , deyo et al.8 ) researched retrospective analysis of a statewide hospital discharge registry , in which more than 18000 hospitalization data were complied over a two - year period , excluding patients with nondegenerative pathologies , authors reported an overall complication rate of only 10.3% for the surgical treatment of degenerative lumbar spine disease , although the rate for patients older than 75 years was higher ( 18% ) . in a different study , cloyd et al.6 ) indicated an alarming 24.2% of elderly patients presented a major postoperative complications , and 53.2% showed at least one in - hospital complication ; the authors found that age was a risk factor for major postoperative complications . recently , lee et al.20 ) retrospectively investigated 489 patients with various lumbar spinal diseases who had lumbar spinal fusion surgeries and showed a difference between the older ( 70 years and older ) and younger patients ( under 65 years old ) ; the complication rate of the older group was 9% , while that of the younger group was 6% . our data further supports the statistically significant difference in peri - operative complication rate between the older and young groups ; the complication rate of the older group was 20.8% , while that of the younger group was 14.8% ( univariate analysis , p=0.015 ; multivariate analysis , p=0.024 ) . the mortality rate of 0.6% in the elderly group studied by deyo et al.8 ) was approximately three times greater than the rates commonly cited for lumbar diskectomy . silvers et al.31 ) reported a mortality rate of 0.8% for mortalities that occurred 1 to 2 months after the surgeries . in our study , one patient ( 0.2% ) died three days after the surgery due to an ami ( table 4 ) . the operation was performed on level 1 ( l4 - 5 ) , and duration was 196 minutes . the blood loss was 350 cc , comparatively less than the mean blood loss , and a transfusion was not necessary . early studies examining the effect of age on decompression with fusion for lumbar stenosis affirmed that the multiple comorbidities were associated with perioperative complications17,25,30 ) . katz et al.18 ) reported that cardiovascular , musculoskeletal , and other comorbidities led to poorer scores in most of the outcome measurements . however , for lumbar spinal fusion surgeries in the elderly , several recent studies have found the opposite to be true1 - 4,18 ) . in the study by lee et al.20 ) , perioperative complications were not significantly associated with comorbidities . the results of our study indicated that for lumbar spinal fusion surgeries , presence of comorbidities was not associated with perioperative complications ( univariate analysis , p=0.132 ; multivariate analysis , p=0.325 ) . also , our study revealed that asa classifications were not statistically significant ( univariate analysis , p=0.376 ; multivariate analysis , p=0.513 ) . elderly patients who are diagnosed with symptomatic spinal pathology may require single or expansive lumbar fusions to ensure adequate fusion and inhibit future instrumentation - related complications6 ) . several studies have proposed that longer fusion lengths may lead to an increased risk of complications in the elderly3,4,27 ) . carreon et al.3 ) found an or of 2.4 for postoperative complications in older patients undergoing lumbar arthrodesis with instrumentation . similarly , cassinelli et al.4 ) reported that fusion of four or more segments was a predictor of complications . the mean number of levels fused in these studies was 2.4 and 1.9 , respectively . on the other hand , daubs et al.7 ) studied patients 60 years and older who underwent spinal arthrodesis with a mean of 9.1 levels fused and found no association between the length of fusion and complication risk . moreover , glassman et al.10 ) advised that extending the fusion length may actually improve clinical outcomes in correcting poor sagittal balance or spinal cord compression . in our study , there was no significant association between the number of segments fused and the risk of perioperative complications in patients of all ages , including the elderly ( univariate analysis , p=0.297 ; multivariate analysis , p=0.446 ) . our last followed outcome based on macnab 's criteria concluded that 412 ( 85.0% ) out of 485 people had excellent or good results since they have resumed their daily activities with minimal , if any , pain . the results for macnab 's criteria in age group 1 ( 70 years and older ) and 2 ( younger than 65 years ) were 82.2% and 86.5% , showing only a small difference . these results are comparable with those in other reports of favorable outcomes , not only in the elderly , but in patients younger than 70 years as well . lee et al.19 ) , reported a study with 30 patients who underwent plif ( mean age 53.536.6 ) . the analysis of macnab 's criteria results showed 10% as " excellent " and 66.7% as " good " . in a similar study by sakeb and ahsan28 ) , a total of 52 cases ( 11 men and 41 women , mean age 46 years sd 05.88 , range 40 - 59 years ) were analyzed on the outcome one year after plif surgeries . the reported macnab 's criteria results were 55.77% " excellent " and 38.46% " good " . hur et al.14 ) examined 20 patients ( mean age 70 years ) who underwent laminectomy and fusion . the macnab 's criteria analysis at 6 months after operation had outcomes of 7% " excellent " and 86% " good " . sanderson and wood29 ) indicated excellent or good results in 81% of their patients 65 years of age or older during an average follow - up period of 42 months . silvers et al.31 ) reported that 93% of the patients in their study ( average age , 65 years ) had relief of pain , and that 5% returned to activity in the short - term outcome analysis . herkowitz and kurz12 ) also reported a 96% rate of excellent or good results during an average follow - up period of 3 years for patients with a mean age of 65 years ( range , 30 - 87 years ) who received decompression and arthrodesis for degenerative spondylolisthesis . johnsson et al.16 ) agreed with excellent or good results in approximately 60% of the patients in their study with a mean age 61.3 years ( range , 48 - 80 years ) . postacchini et al.26 ) reported excellent or good results for 67% of the patients during an average period of 8 years ( range , 4 - 21 years ) . lastly , hall et al.11 ) reported excellent or good results during a mean follow - up period of 4 years in 84% of patients , whose mean age was 63 years ( range , 32 - 83 years ) . first , as this is a retrospective analysis , for which the limitations are well known , results may underestimate the actual complication incidence . it is common in retrospective studies for investigator recall bias to be introduced , whereby the accuracy of medical records may lead to falsely low reported rates of complication13 ) . second , there are opportunities for several other biases . given the significant complexity of the patient population , the current study population may not represent the spine surgery population as a whole . therefore , these findings may not be applicable to the general spinal fusion surgery patients . increasing age was an important risk factor for perioperative complications in patients undergoing lumbar spinal fusion surgery , whereas other factors were not significant . however , patients ' satisfaction or return to daily activities when compared with younger patients does not show much difference . we recommend good clinical judgment as well as careful selection of geriatric patients for lumbar spinal fusion surgery .

objectivethe purpose of this study was to examine the efficacy and perioperative complications associated with lumbar spinal fusion surgery , focusing on geriatric patients in the republic of korea.methodswe retrospectively investigated 485 patients with degenerative spinal diseases who had lumbar spinal fusion surgeries between march 2006 and december 2010 at our institution . age , sex , comorbidity , american society of anesthesiologists ( asa ) class , fusion segments , perioperative complications , and outcomes were analyzed in this study . risk factors for complications and their association with age were analyzed.resultsin this study , 81 patients presented complications ( 16.7% ) . the rate of perioperative complications was significantly higher in patients 70 years or older than in other age groups ( univariate analysis , p=0.015 ; multivariate analysis , p=0.024 ) . the perioperative complications were not significantly associated with the other factors tested ( sex , comorbidity , asa class , and fusion segments ) . post - operative outcomes of lumbar spinal fusion surgeries for the patients were determined on the basis of macnab 's criteria ( average follow up period : 19.7 months ) , and 412 patients ( 85.0% ) were classified as having " excellent " or " good " results.conclusionincreasing age was an important risk factor for perioperative complications in patients undergoing lumbar spinal fusion surgery , whereas other factors were not significant . however , patients ' satisfaction or return to daily activities when compared with younger patients did not show much difference . we recommend good clinical judgment as well as careful selection of geriatric patients for lumbar spinal fusion surgery .

INTRODUCTION MATERIALS AND METHODS Patient population Co-morbidities Surgical treatment Postoperative care Complications Evaluation of outcome Statistical analysis RESULTS Patient population Comorbidities Complications Post-operative outcome DISCUSSION CONCLUSION

many patients require lumbar spinal fusion surgery with instrumentation along with decompression to treat the degenerative lumbar disease . however , there is a lack of studies addressing the perioperative complications in elderly patients with decompression , arthrodesis of the lumbar spine , and the post operative outcomes of lumbar spinal fusion surgeries24 ) . a review of literature reveals controversy over the safety of lumbar spinal fusion surgery in the elderly due to varying findings on the risk . the goal of this study was to evaluate ; 1 ) the complication rate in relation to perioperative complications , general factors such as age , sex , comorbidity , american society of anesthesiologists ( asa ) class , and fusion segments involved , and 2 ) examine the clinical outcomes of lumbar spinal fusion surgery for degenerative lumbar diseases for patients 70 years and older in contrast to patients younger than 65 year of age . in this study , the patient population of a specific age group was selected based on the type of surgeries the patients received in a specific time period . patient population was divided into two groups according to the definition of " geriatric " : 70 years and older ( group 1 ) , and younger than 65 years ( group 2 ) . lumbar spinal fusion surgeries were performed by 4 neurosurgeons at a single institute . we included patients with degenerative spine diseases , and excluded patients with age between 65 and 69 years , spine traumas , spinal revision surgeries , and spine tumors in order to avoid unexpected co - morbidities . data related to these patients were evaluated by a single observer using the standard hospital charts , outpatient notes , electronic medical records , operative reports , and preoperative and postoperative imaging studies . the following demographic variables were evaluated : age , sex , treatable or non - treatable medical co - morbidities , the preoperative asa classification of physical status , and segment of fusion . co - morbidities were subdivided as follows : cardiovascular ( hypertension , arrhythmia , angina , coronary artery disease , and congestive heart failure ) , renal ( nephrotic cancer , ureter stone , renal cyst and acute or chronic renal failure ) , pulmonary ( lung abscess , tuberculosis , emphysema , chronic obstructive pulmonary disease , and asthma ) , hepatic ( common bile duct stones , hepatitis and liver cirrhosis ) and endocrine ( diabetes mellitus , hypothyroidism and hyperthyroidism ) . in general , surgeries were performed in degenerative cases when conservative treatment over three months failed to improve the patients ' symptoms . in this study , patients were ambulated from the second postoperative day with rigid molded plastic braces or lumbar corsets . initially , the patients were treated with analgesia for control of acute pain by intravenous injection , and then weaned to mild oral narcotic therapy and nsaids that were discontinued as quickly as possible . the term " complication " in this study a complication was any event requiring specific management during the perioperative period , including the intra - operative and post - operative periods . the patients enrolled in this study were followed - up for at least 12 months postoperatively . logistic regression analyses were done to investigate whether complications were effected by age , sex , comorbidity , asa class , or fusion segments . fisher 's exact test was carried out to compare how the complications affected the macnab 's criteria results after final follow - up . in this study , the patient population of a specific age group was selected based on the type of surgeries the patients received in a specific time period . patient population was divided into two groups according to the definition of " geriatric " : 70 years and older ( group 1 ) , and younger than 65 years ( group 2 ) . lumbar spinal fusion surgeries were performed by 4 neurosurgeons at a single institute . we included patients with degenerative spine diseases , and excluded patients with age between 65 and 69 years , spine traumas , spinal revision surgeries , and spine tumors in order to avoid unexpected co - morbidities . the following demographic variables were evaluated : age , sex , treatable or non - treatable medical co - morbidities , the preoperative asa classification of physical status , and segment of fusion . co - morbidities were subdivided as follows : cardiovascular ( hypertension , arrhythmia , angina , coronary artery disease , and congestive heart failure ) , renal ( nephrotic cancer , ureter stone , renal cyst and acute or chronic renal failure ) , pulmonary ( lung abscess , tuberculosis , emphysema , chronic obstructive pulmonary disease , and asthma ) , hepatic ( common bile duct stones , hepatitis and liver cirrhosis ) and endocrine ( diabetes mellitus , hypothyroidism and hyperthyroidism ) . in general , surgeries were performed in degenerative cases when conservative treatment over three months failed to improve the patients ' symptoms . in this study , patients were ambulated from the second postoperative day with rigid molded plastic braces or lumbar corsets . initially , the patients were treated with analgesia for control of acute pain by intravenous injection , and then weaned to mild oral narcotic therapy and nsaids that were discontinued as quickly as possible . the term " complication " in this study is defined broadly and differently from published literature . in this study , a complication was any event requiring specific management during the perioperative period , including the intra - operative and post - operative periods . the patients enrolled in this study were followed - up for at least 12 months postoperatively . the frequency analysis , simple and multiple logistic regression analysis , and test were done using spss ver . logistic regression analyses were done to investigate whether complications were effected by age , sex , comorbidity , asa class , or fusion segments . fisher 's exact test was carried out to compare how the complications affected the macnab 's criteria results after final follow - up . during the five - year study period , the 485 patients met the inclusion criteria and underwent lumbar spinal fusion surgeries ( table 1 ) . of these , 156 patients ( 32.2% ) were male and 329 ( 67.8% ) were female , and there were 174 patients ( 35.9% ) in group 1 and 311 patients ( 64.1% ) in group 2 . the number of patients in the age group 70 - 75 , 75 - 79 , and 80 - 85 were 92 , 56 , and 26 , respectively . the general condition of those patients varied by asa class : asa class i ( 303 patients , 62.5% ) , asa ii ( 149 patients , 30.7% ) and asa iii ( 33 patients , 6.8% ) . fusion segment levels were categorized as 1 segment ( 247 patients , 50.9% ) , 2 segments ( 179 patients , 36.9% ) , 3 segments ( 41 patients , 8.5% ) , 4 segments ( 18 patients , 3.7% ) . complications were encountered in 81 patients ( 16.7% ) . in group 1 , complications occurred in 35 patients ( 20.1% ) , and in group 2 , complications occurred in 51 patients ( 14.8% ) . among the observed comorbidities , the most common problem was hypertension , which was present preoperatively in 81 patients ( 39.7% ) . other comorbidities were cardiovascular diseases ( 32 patients , 15.7% ) , renal diseases ( 29 patients , 14.2% ) , pulmonary diseases ( 36 patients , 17.6% ) , endocrine disease ( 75 patients , 36.8% ) , and hepatic diseases ( 20 patients , 9.8% ) . the most common complication was wound dehiscence ( 25.9% ) , and the second most prevalent was ileus , found in 16.0% of cases . the results of the logistic regression analysis are in table 5 , displaying results of univariate and multivariate analysis in order of age , sex , comorbidity , asa class , and fusion segment . age was the only statistically significantly factor in both univariate and multivariate analyses ( p=0.015 and 0.024 ) . no correlation was found to be statistically significant between complications and sex , comorbidity , asa class , fusion segment ( p>0.05 ) . based on the mean macnab 's criteria , post - operative outcome of 485 patients revealed as " excellent " in 280 patients ( 57.7% ) , " good " in 132 patients ( 27.2% ) , " fair " in 57 patients ( 11.8% ) , and " poor " in 16 patients ( 3.3% ) . for those patients who showed " excellent " or " good " results , patients had satisfactory improvements . on the other hand , " fair " or " poor " outcomes occurred in 73 patients ( 15.1% ) . patients in both age groups showed comparable post - operative outcomes ; 143 ( 82.2% ) patients in age group 1 , and 308 ( 86.5% ) patients in age group 2 showed " excellent " or " good " results . based on fisher 's exact test , which was done to find the correlation between occurrence of perioperative complications and post - operative outcome , there was no statistically significant relationship between perioperative complications and " excellent " or " good " group in macnab 's criteria ( table 6 ) . during the five - year study period , the 485 patients met the inclusion criteria and underwent lumbar spinal fusion surgeries ( table 1 ) . of these , 156 patients ( 32.2% ) were male and 329 ( 67.8% ) were female , and there were 174 patients ( 35.9% ) in group 1 and 311 patients ( 64.1% ) in group 2 . the number of patients in the age group 70 - 75 , 75 - 79 , and 80 - 85 were 92 , 56 , and 26 , respectively . the general condition of those patients varied by asa class : asa class i ( 303 patients , 62.5% ) , asa ii ( 149 patients , 30.7% ) and asa iii ( 33 patients , 6.8% ) . complications were encountered in 81 patients ( 16.7% ) . in group 1 , complications occurred in 35 patients ( 20.1% ) , and in group 2 , complications occurred in 51 patients ( 14.8% ) . among the observed comorbidities , the most common problem was hypertension , which was present preoperatively in 81 patients ( 39.7% ) . other comorbidities were cardiovascular diseases ( 32 patients , 15.7% ) , renal diseases ( 29 patients , 14.2% ) , pulmonary diseases ( 36 patients , 17.6% ) , endocrine disease ( 75 patients , 36.8% ) , and hepatic diseases ( 20 patients , 9.8% ) . in addition , 47 patients ( 23.0% ) had more than one comorbidity ( table 2 ) . the total peri - operative complication rate in all patients was 16.7% ; the peri - operative complication rate in age group 1 was 20.1% , while the complication rate in group 2 was 14.8% . the results of the logistic regression analysis are in table 5 , displaying results of univariate and multivariate analysis in order of age , sex , comorbidity , asa class , and fusion segment . age was the only statistically significantly factor in both univariate and multivariate analyses ( p=0.015 and 0.024 ) . no correlation was found to be statistically significant between complications and sex , comorbidity , asa class , fusion segment ( p>0.05 ) . based on the mean macnab 's criteria , post - operative outcome of 485 patients revealed as " excellent " in 280 patients ( 57.7% ) , " good " in 132 patients ( 27.2% ) , " fair " in 57 patients ( 11.8% ) , and " poor " in 16 patients ( 3.3% ) . for those patients who showed " excellent " or " good " results , patients had satisfactory improvements . on the other hand , " fair " or " poor " outcomes occurred in 73 patients ( 15.1% ) . patients in both age groups showed comparable post - operative outcomes ; 143 ( 82.2% ) patients in age group 1 , and 308 ( 86.5% ) patients in age group 2 showed " excellent " or " good " results . based on fisher 's exact test , which was done to find the correlation between occurrence of perioperative complications and post - operative outcome , there was no statistically significant relationship between perioperative complications and " excellent " or " good " group in macnab 's criteria ( table 6 ) . the number of elderly persons in the republic of korea continues to grow ; by 2050 , 38.2% of the population is expected to be older than 65 years , greatly exceeding the worldwide mean of 16.2% . advances in anesthesiology , spinal instrumentation , and postoperative care have made spinal procedures safer , with a decrease in morbidity and mortality , as well as continued improvements in patient outcomes6 ) . at 2.6% incidence rate , re - operation after a spinal fusion surgery may be considered a failure of the initial surgery by the patient and the surgeon . mok et al.21 ) recently evaluated the need for revision surgery in patients undergoing adult spine surgery , finding a cumulative rate of 25.8% , while cloyd et al.6 ) reported a re - operation rate of 27.4% in patients at least 65 years old who presented with a wide range of spine pathologies . recently , lee et al.20 ) reported that the rate of revision surgery due to complications was approximately 47% . in our study , the rate of revision surgery due to complications was approximately 40.6% , but age was not associated with the need for re - operation . okuda et al.23 ) found that patients older than 70 years who underwent posterior lumbar interbody fusions for spondylolisthesis had a complication rate of 16% , which was not significantly different from that in younger patients . vitaz et al.33 ) suggested that elderly patients with lumbar spinal diseases can be surgically treated in the same manner as the younger patients . on the other hand , deyo et al.8 ) researched retrospective analysis of a statewide hospital discharge registry , in which more than 18000 hospitalization data were complied over a two - year period , excluding patients with nondegenerative pathologies , authors reported an overall complication rate of only 10.3% for the surgical treatment of degenerative lumbar spine disease , although the rate for patients older than 75 years was higher ( 18% ) . in a different study , cloyd et al.6 ) indicated an alarming 24.2% of elderly patients presented a major postoperative complications , and 53.2% showed at least one in - hospital complication ; the authors found that age was a risk factor for major postoperative complications . recently , lee et al.20 ) retrospectively investigated 489 patients with various lumbar spinal diseases who had lumbar spinal fusion surgeries and showed a difference between the older ( 70 years and older ) and younger patients ( under 65 years old ) ; the complication rate of the older group was 9% , while that of the younger group was 6% . our data further supports the statistically significant difference in peri - operative complication rate between the older and young groups ; the complication rate of the older group was 20.8% , while that of the younger group was 14.8% ( univariate analysis , p=0.015 ; multivariate analysis , p=0.024 ) . the mortality rate of 0.6% in the elderly group studied by deyo et al.8 ) was approximately three times greater than the rates commonly cited for lumbar diskectomy . however , for lumbar spinal fusion surgeries in the elderly , several recent studies have found the opposite to be true1 - 4,18 ) . in the study by lee et al.20 ) , perioperative complications were not significantly associated with comorbidities . the results of our study indicated that for lumbar spinal fusion surgeries , presence of comorbidities was not associated with perioperative complications ( univariate analysis , p=0.132 ; multivariate analysis , p=0.325 ) . also , our study revealed that asa classifications were not statistically significant ( univariate analysis , p=0.376 ; multivariate analysis , p=0.513 ) . carreon et al.3 ) found an or of 2.4 for postoperative complications in older patients undergoing lumbar arthrodesis with instrumentation . in our study , there was no significant association between the number of segments fused and the risk of perioperative complications in patients of all ages , including the elderly ( univariate analysis , p=0.297 ; multivariate analysis , p=0.446 ) . our last followed outcome based on macnab 's criteria concluded that 412 ( 85.0% ) out of 485 people had excellent or good results since they have resumed their daily activities with minimal , if any , pain . the results for macnab 's criteria in age group 1 ( 70 years and older ) and 2 ( younger than 65 years ) were 82.2% and 86.5% , showing only a small difference . these results are comparable with those in other reports of favorable outcomes , not only in the elderly , but in patients younger than 70 years as well . the analysis of macnab 's criteria results showed 10% as " excellent " and 66.7% as " good " . in a similar study by sakeb and ahsan28 ) , a total of 52 cases ( 11 men and 41 women , mean age 46 years sd 05.88 , range 40 - 59 years ) were analyzed on the outcome one year after plif surgeries . the reported macnab 's criteria results were 55.77% " excellent " and 38.46% " good " . hur et al.14 ) examined 20 patients ( mean age 70 years ) who underwent laminectomy and fusion . the macnab 's criteria analysis at 6 months after operation had outcomes of 7% " excellent " and 86% " good " . sanderson and wood29 ) indicated excellent or good results in 81% of their patients 65 years of age or older during an average follow - up period of 42 months . silvers et al.31 ) reported that 93% of the patients in their study ( average age , 65 years ) had relief of pain , and that 5% returned to activity in the short - term outcome analysis . herkowitz and kurz12 ) also reported a 96% rate of excellent or good results during an average follow - up period of 3 years for patients with a mean age of 65 years ( range , 30 - 87 years ) who received decompression and arthrodesis for degenerative spondylolisthesis . johnsson et al.16 ) agreed with excellent or good results in approximately 60% of the patients in their study with a mean age 61.3 years ( range , 48 - 80 years ) . therefore , these findings may not be applicable to the general spinal fusion surgery patients . increasing age was an important risk factor for perioperative complications in patients undergoing lumbar spinal fusion surgery , whereas other factors were not significant . however , patients ' satisfaction or return to daily activities when compared with younger patients does not show much difference . we recommend good clinical judgment as well as careful selection of geriatric patients for lumbar spinal fusion surgery .

nursing science is engaged in practice - based roles and highlights the importance of professional values which have been founded on specific human values ( 1 ) . nursing is a combination of knowledge and skills for an effective professional care ( 2 ) . by regarding ethics as a form of knowledge these values are the standards for action and also provide a framework for evaluation ( 3 ) . nurses are expected to be aware of the ethical dimensions of nursing practice and apply them in their work ( 4 ) . ethical principles are among the fundamentals of nursing , so any form of care should meet patients rights based on these standards ( 4 ) . nurses are expected to provide physical , social , and psychological care ( 5 ) that should be moral and ethical as well . the new rapid changes caused by technology can influence these values ( 6 ) , and therefore professional and ethical standards must be set to fulfil professional obligations ( 3 ) new evidence has shown that these standards and values are not fully respected by most of the nurses ( 7 ) . on the other hand , it should be considered that nurses face complex professional and ethical issues in their workplace , such as different levels of personnel , developments in technology , and budget constraints ( 8 , 9 ) . they face many professional challenges while interacting with clients and organizations so they need to deal with them professionally to provide care with confidence and peace of mind ( 10 ) because of the moral and ethical sensitivity of their job ( 2 , 11 ) . if nurses do not engage actively with these daily ethical issues , they will fail to deliver the optimum standard of care ( 12 ) . to respond effectively to these dilemmas , nurses should have knowledge of their professional values ( 13 ) . they can be considered as resources to guide nurses when involved with ethical challenges ( 14 , 15 ) . among some studies on nurses professional values , mcneese - smith and crook stated that knowledge about these values can improve nurses practice , assist in team performance and cohesion , and aid in performance of counselling . chitty and black introduced some of the professional and ethical values from the nurses perspective such as : accountability and responsibility for own actions , balancing cure and care , benevolence , caring as a foundation for relationships , collaborative multidisciplinary practice , compassion , and competence ( 17 ) . in nursing education , studies have shown that professional values and moral ideals of nurses in the clinical environment are often considered as dilemmas ( 18 ) . assessed observation of professional ethics regarding medication and found out that most of the participants had a poor performance in medication process and respecting values ( 19 ) . arzani et al . aimed to evaluate nurses respect for professional and ethical values from their own perspective . they found that only 40% of nurses fully consider these values in their practice ( 20 ) . applying professional and ethical values in nursing nurses will face dilemmas , not only in nursing care , but also in relation to other health professions , their work place , and their own profession . among the barriers in applying professional ethics , however , most of the challenges are mainly about the nurse - patient interaction ( 21 ) . in addition to finding and implementing strategies to enhance nurses ability in ethical care , it is recommended to survey their perspective about applying ethical values in their profession . therefore , this study was designed with the aim of evaluating nurses perspective on applying professional values in isfahan hospitals in 2010 . this is a descriptive - analytical study that was conducted in selected hospitals in isfahan to survey application of professional values from nurses perspective . the hospitals in this study consisted of four educational hospitals , two hospitals affiliated to the social security organization , and two private hospitals . study samples were nurses in all roles who met the cited inclusion criteria of having a nursing bachelor s degree or higher , were listed in the category of formal nurse , contract nurse , corporate nurse , and planed nurses , were of iranian descent , and had graduated from official universities of iran . according to the number of nurses in each hospital that was assigned as a research area , and also based on the intended sample size of 250 nurses , a quota was determined for each study area in sampling method . all the questionnaires were given to the nurses in different shifts with prior coordination with the director of nursing . at first , permission from the research committee of the iranian nursing organization was obtained and offered to the hospital authorities . then , the aim of the study was explained to the subjects and their informed consent was obtained . since an interval was needed to take back the questionnaire , it was adjusted with the subjects at first . data were collected using a two - part questionnaire , including demographic characteristics and professional values data . the first part was a researcher made tool for gathering the demographic data of participants , and the second part was a standard tool titled nursing professional values scale ( npvs ) . weis and schank have designed and used this instrument in a study with the aim of describing nurses professional values ( 13 ) . this scale includes 26 items ; each item in this scale is a short descriptive phrase reflecting a specific code of ethics from the american nursing association in 1985 . this is a norm referenced with likert type format ranging from 1 ( not important ) to 5 ( most important ) , and ranges above 3 indicate strong professional orientation . the possible range of score is 26 to 150 , and therefore scores above 78 were considered as respecting professional and ethical values . the reliability and validity of this questionnaire was approved by weis and schank measuring the cronbach s alpha coefficient of 0.94 by using pre - test post - test method ( 22 ) . in another study by martin the content validity was established by experts in the field of ethics ( 23 ) . in a study by parvan et al . in iran , the reliability of this tool was approved with a cronbach s alpha that was 91% ( 24 ) . in the present study , due to cultural differences , the questionnaire was translated into persian and sent to six ethics experts for content validity . with a few changes , of the 250 distributed questionnaires , 162 were returned , whereas 12 were not fully completed . therefore , 150 questionnaires were analyzed by means of the statistical software spss version 16 . to determine the relationship between gender , level of education , work experience , different categories of employment , and place of employment and professional values , pearson correlation , independent t - test and anova test were applied . to show the relation between age and score of professional values , spearman correlation test was used . in this study , of the 150 participants , the mean age was 34.2 ( sd=7.3 ) , and the average years of employment as a nurse was 11 ( sd=7.9 ) years . no significant association was found between demographic characteristics such as nurses age ( p = 0.975 ) , gender ( p= 0.169 ) , education ( p= 0.150 ) , and work records ( p= 0.148 ) and their perspective on applying professional ethical values ( table 1 ) . the mean of professional values score among participants was 101.53 ( sd=18.12 ) , and considering the standard average of values score that is 78 for this questionnaire , it can be concluded that all the participants had a positive perspective toward applying professional values in nursing practice . no significant association was found between variables such as the type of hospital nurses position ( p= 0.258 ) and the type of their employment ( p= 0.196 ) and applying nursing professional values . the most prominent criteria of professional values that were noted by nurses were : protecting the health and safety of the public ( mean = 4.35 ) , protecting the moral and legal rights of patients ( mean = 4.21 ) , practice guided by principles of fidelity and respecting individuals ( mean = 4.13 ) , and respecting patients right to privacy ( mean = 4.33 ) ( table 2 ) . the overall results indicated that no significant association was present between demographic characteristics such as age , gender , education , and nurses work records and the nurses perspective on applying professional values . leduc and kotzer aimed to use npvs as an instrument to evaluate nurses perspective on applying professional ethical values in nursing students , newly graduated nurses , and seasoned professionals . they came to the conclusion that these three groups of nurses are the same in applying professional values . therefore , the claim that nursing experience can be effective in promoting professional values was not approved ( 25 ) . in their study , lynn et al . have found no significant difference between the professional and ethical values of senior baccalaureate and associate degree nursing students ( 26 ) . the similarity between the results of this study and other studies in this area shows that people who choose nursing as their job consider ethical values among the important and essential fundamentals of their profession . the results of this study revealed that the association between age and nurses perspective on applying professional values is reversed . it might mean that as the nurses get older , their respect for professional values will be decreased , although some studies have shown that no significant association is present between professional values scores and age of the samples ( 23 ) . in their study , parvan et.al have shown a significant negative correlation between age and the total score of values in nursing students . this means that as the nurses grow older , their respect for professional and ethical values decreases ( 24 ) . the most prominent criteria of professional ethics in this study were : protecting the health and safety of people , respecting the moral and legal rights of patients , practice guided by principles of fidelity and respecting individuals , and protecting patients right to privacy . shih et al . found out , however , that the most important criteria according to the perspective of nurses in taiwan are : caring for clients with a humanistic spirit , providing professionally competent and holistic care , fostering growth and discovering the meaning of life , experiencing the give - and - take of caring for others , receiving fair compensation , and raising the public s awareness of health promotion ( 27 ) . according to silen et al , nursing professional values as specified by thailand nurses association can be viewed as : sublime states of mind , loving kindness , compassion , and sympathetic joy and equanimity ( 28 ) . in the study conducted by weis and schank the most important professional values of nurses were related to the provision of nursing care and believing in the importance of the process of professionalization ( 22 ) . shahriari et al . have expressed that ethical values of nurses from the perspective of iranian nurses include maintaining patients dignity , commitment and work ethics , human communication , fairness , honesty and integrity , and striving to promote personal and professional competence ( 29 ) . parvan et al . showed the most important statements from the students perspective were maintaining confidentiality of patients and maintaining clinical competency . nursing faculty and students awareness of the need for strong professional values is important in preparation of nurses capable of managing patient care in an ethical and professional manner ( 24 ) . the difference between moral professional values in various studies reflects the differences in societies in terms of dominant values based on cultural , social , economic and religious situations . hence it is essential to identify nurses professional values as well as factors influencing disrespect of professional ethics and standards in patient care ( 7 ) . it is obvious that nurses can not avoid facing ethical dilemmas , but they should be capable enough to manage these challenges through receiving proper training . as a matter of fact , this is not exclusively related to nurses ; rather , all of the treatment team encounter such challenges and manage them based on their knowledge and training . in their study regarding the purpose of determining ethical conflicts in nurse practitioners and physician assistants in managed care , ulrich et al . some of the participants ( 44% ) believed that they had been well prepared for managing these challenges because they had been well trained ( 15 ) . thus , the role of education in preparing nurses and other health care providers for facing ethical dilemmas is further highlighted . the important point here is that despite the great importance of ethical issues , we still encounter many cases of non - compliance with the ethical values , and challenges in the field of patients rights . rezaei et al . have showed that in the emergency departments of a total of ten hospitals in lorestan ( province of western iran ) which were assessed for their compliance with professional ethics standards and patient s rights , these items have not been fully met ( 30 ) . the fact here is that it is not enough to train nurses and other health care providers to achieve desired outcomes ; following relevant feedbacks in the system is important as well . in this study , we addressed the most and the less important professional values specified by our nurses with different types of employment and different experiences . iranian nurses believe in respecting the legal and ethical rights of patients as the most prominent value . it is concluded that nurses professional values must be specified and assessed based on each country s circumstances to sensitize students and nurses about ethical issues and problems . in addition , identifying and removing barriers to professional ethics play an important role in applied professional values in nursing . this is a descriptive - analytical study that was conducted in selected hospitals in isfahan to survey application of professional values from nurses perspective . the hospitals in this study consisted of four educational hospitals , two hospitals affiliated to the social security organization , and two private hospitals . study samples were nurses in all roles who met the cited inclusion criteria of having a nursing bachelor s degree or higher , were listed in the category of formal nurse , contract nurse , corporate nurse , and planed nurses , were of iranian descent , and had graduated from official universities of iran . according to the number of nurses in each hospital that was assigned as a research area , and also based on the intended sample size of 250 nurses , a quota was determined for each study area in sampling method . all the questionnaires were given to the nurses in different shifts with prior coordination with the director of nursing . at first , permission from the research committee of the iranian nursing organization was obtained and offered to the hospital authorities . then , the aim of the study was explained to the subjects and their informed consent was obtained . since an interval was needed to take back the questionnaire , it was adjusted with the subjects at first . data were collected using a two - part questionnaire , including demographic characteristics and professional values data . the first part was a researcher made tool for gathering the demographic data of participants , and the second part was a standard tool titled nursing professional values scale ( npvs ) . weis and schank have designed and used this instrument in a study with the aim of describing nurses professional values ( 13 ) . this scale includes 26 items ; each item in this scale is a short descriptive phrase reflecting a specific code of ethics from the american nursing association in 1985 . this is a norm referenced with likert type format ranging from 1 ( not important ) to 5 ( most important ) , and ranges above 3 indicate strong professional orientation . the possible range of score is 26 to 150 , and therefore scores above 78 were considered as respecting professional and ethical values . the reliability and validity of this questionnaire was approved by weis and schank measuring the cronbach s alpha coefficient of 0.94 by using pre - test post - test method ( 22 ) . in another study by martin the content validity was established by experts in the field of ethics ( 23 ) . in a study by parvan et al . in iran , the reliability of this tool was approved with a cronbach s alpha that was 91% ( 24 ) . in the present study , due to cultural differences , the questionnaire was translated into persian and sent to six ethics experts for content validity . with a few changes , of the 250 distributed questionnaires , 162 were returned , whereas 12 were not fully completed . therefore , 150 questionnaires were analyzed by means of the statistical software spss version 16 . to determine the relationship between gender , level of education , work experience , different categories of employment , and place of employment and professional values , pearson correlation , independent t - test and anova test were applied . to show the relation between age and score of professional values , spearman correlation test was used . in this study , of the 150 participants , 84% were females and 88% had bachelor degree in nursing . the mean age was 34.2 ( sd=7.3 ) , and the average years of employment as a nurse was 11 ( sd=7.9 ) years . no significant association was found between demographic characteristics such as nurses age ( p = 0.975 ) , gender ( p= 0.169 ) , education ( p= 0.150 ) , and work records ( p= 0.148 ) and their perspective on applying professional ethical values ( table 1 ) . the mean of professional values score among participants was 101.53 ( sd=18.12 ) , and considering the standard average of values score that is 78 for this questionnaire , it can be concluded that all the participants had a positive perspective toward applying professional values in nursing practice . no significant association was found between variables such as the type of hospital nurses position ( p= 0.258 ) and the type of their employment ( p= 0.196 ) and applying nursing professional values . the most prominent criteria of professional values that were noted by nurses were : protecting the health and safety of the public ( mean = 4.35 ) , protecting the moral and legal rights of patients ( mean = 4.21 ) , practice guided by principles of fidelity and respecting individuals ( mean = 4.13 ) , and respecting patients right to privacy ( mean = 4.33 ) ( table 2 ) . the overall results indicated that no significant association was present between demographic characteristics such as age , gender , education , and nurses work records and the nurses perspective on applying professional values . leduc and kotzer aimed to use npvs as an instrument to evaluate nurses perspective on applying professional ethical values in nursing students , newly graduated nurses , and seasoned professionals . they came to the conclusion that these three groups of nurses are the same in applying professional values . therefore , the claim that nursing experience can be effective in promoting professional values was not approved ( 25 ) . in their study , lynn et al . have found no significant difference between the professional and ethical values of senior baccalaureate and associate degree nursing students ( 26 ) . the similarity between the results of this study and other studies in this area shows that people who choose nursing as their job consider ethical values among the important and essential fundamentals of their profession . the results of this study revealed that the association between age and nurses perspective on applying professional values is reversed . it might mean that as the nurses get older , their respect for professional values will be decreased , although some studies have shown that no significant association is present between professional values scores and age of the samples ( 23 ) . in their study , parvan et.al have shown a significant negative correlation between age and the total score of values in nursing students . this means that as the nurses grow older , their respect for professional and ethical values decreases ( 24 ) . the most prominent criteria of professional ethics in this study were : protecting the health and safety of people , respecting the moral and legal rights of patients , practice guided by principles of fidelity and respecting individuals , and protecting patients right to privacy . shih et al . found out , however , that the most important criteria according to the perspective of nurses in taiwan are : caring for clients with a humanistic spirit , providing professionally competent and holistic care , fostering growth and discovering the meaning of life , experiencing the give - and - take of caring for others , receiving fair compensation , and raising the public s awareness of health promotion ( 27 ) . according to silen et al , nursing professional values as specified by thailand nurses association can be viewed as : sublime states of mind , loving kindness , compassion , and sympathetic joy and equanimity ( 28 ) . in the study conducted by weis and schank the most important professional values of nurses were related to the provision of nursing care and believing in the importance of the process of professionalization ( 22 ) . shahriari et al . have expressed that ethical values of nurses from the perspective of iranian nurses include maintaining patients dignity , commitment and work ethics , human communication , fairness , honesty and integrity , and striving to promote personal and professional competence ( 29 ) . parvan et al . showed the most important statements from the students perspective were maintaining confidentiality of patients and maintaining clinical competency . nursing faculty and students awareness of the need for strong professional values is important in preparation of nurses capable of managing patient care in an ethical and professional manner ( 24 ) . the difference between moral professional values in various studies reflects the differences in societies in terms of dominant values based on cultural , social , economic and religious situations . hence it is essential to identify nurses professional values as well as factors influencing disrespect of professional ethics and standards in patient care ( 7 ) . it is obvious that nurses can not avoid facing ethical dilemmas , but they should be capable enough to manage these challenges through receiving proper training . as a matter of fact , this is not exclusively related to nurses ; rather , all of the treatment team encounter such challenges and manage them based on their knowledge and training . in their study regarding the purpose of determining ethical conflicts in nurse practitioners and physician assistants in managed care , ulrich et al . have found that nurses encounter ethical challenges more than physicians . some of the participants ( 44% ) believed that they had been well prepared for managing these challenges because they had been well trained ( 15 ) . thus , the role of education in preparing nurses and other health care providers for facing ethical dilemmas is further highlighted . the important point here is that despite the great importance of ethical issues , we still encounter many cases of non - compliance with the ethical values , and challenges in the field of patients rights . rezaei et al . have showed that in the emergency departments of a total of ten hospitals in lorestan ( province of western iran ) which were assessed for their compliance with professional ethics standards and patient s rights , these items have not been fully met ( 30 ) . the fact here is that it is not enough to train nurses and other health care providers to achieve desired outcomes ; following relevant feedbacks in the system is important as well . in this study , we addressed the most and the less important professional values specified by our nurses with different types of employment and different experiences . iranian nurses believe in respecting the legal and ethical rights of patients as the most prominent value . it is concluded that nurses professional values must be specified and assessed based on each country s circumstances to sensitize students and nurses about ethical issues and problems . in addition , identifying and removing barriers to professional ethics play an important role in applied professional values in nursing . more studies with greater sample size are needed for generalization of the outcomes .

applying professional values is an important issue in nursing practice . it is also crucial to find out nurses perspectives toward ethical and professional values in the clinical environment . for this purpose , we aimed to utilize a standard tool to survey perspectives on applying these values in nursing care in hospitals of isfahan , iran.this is a descriptive - analytical study in which 150 nurses working in hospitals of isfahan , iran in 2010 were recruited by quota convenience sampling , and their perspectives on professional values were assessed . data were collected by the nurses professional values scale ( npvs ) and analyzed using spss software version 16 applying descriptive and interpretive statistics.in the present study , 84% of the participants were female and 88% had bachelor s degree in nursing . the average age was 34.2 ( sd=7.3 ) , and the average years of working in a hospital were 11 ( sd=7.3 ) . no significant association was present between demographic characteristics such as age , sex , educational level or work records and applying professional and ethical values . results also demonstrated that respect for professional values is similar among nurses of different employment types.in this study , we addressed the most and the less important professional values specified by nurses with different types of employment and also different experiences . iranian nurses believe in respecting the legal and ethical rights of patients as the most prominent value in the nursing profession . we suggest that these professional values be specified and assessed based on iranian culture .

Introduction Method Results Discussion Conclusion

nursing science is engaged in practice - based roles and highlights the importance of professional values which have been founded on specific human values ( 1 ) . by regarding ethics as a form of knowledge these values are the standards for action and also provide a framework for evaluation ( 3 ) . nurses are expected to be aware of the ethical dimensions of nursing practice and apply them in their work ( 4 ) . nurses are expected to provide physical , social , and psychological care ( 5 ) that should be moral and ethical as well . the new rapid changes caused by technology can influence these values ( 6 ) , and therefore professional and ethical standards must be set to fulfil professional obligations ( 3 ) new evidence has shown that these standards and values are not fully respected by most of the nurses ( 7 ) . on the other hand , it should be considered that nurses face complex professional and ethical issues in their workplace , such as different levels of personnel , developments in technology , and budget constraints ( 8 , 9 ) . they face many professional challenges while interacting with clients and organizations so they need to deal with them professionally to provide care with confidence and peace of mind ( 10 ) because of the moral and ethical sensitivity of their job ( 2 , 11 ) . to respond effectively to these dilemmas , nurses should have knowledge of their professional values ( 13 ) . among some studies on nurses professional values , mcneese - smith and crook stated that knowledge about these values can improve nurses practice , assist in team performance and cohesion , and aid in performance of counselling . chitty and black introduced some of the professional and ethical values from the nurses perspective such as : accountability and responsibility for own actions , balancing cure and care , benevolence , caring as a foundation for relationships , collaborative multidisciplinary practice , compassion , and competence ( 17 ) . in nursing education , studies have shown that professional values and moral ideals of nurses in the clinical environment are often considered as dilemmas ( 18 ) . assessed observation of professional ethics regarding medication and found out that most of the participants had a poor performance in medication process and respecting values ( 19 ) . aimed to evaluate nurses respect for professional and ethical values from their own perspective . they found that only 40% of nurses fully consider these values in their practice ( 20 ) . applying professional and ethical values in nursing nurses will face dilemmas , not only in nursing care , but also in relation to other health professions , their work place , and their own profession . among the barriers in applying professional ethics , however , most of the challenges are mainly about the nurse - patient interaction ( 21 ) . in addition to finding and implementing strategies to enhance nurses ability in ethical care , it is recommended to survey their perspective about applying ethical values in their profession . therefore , this study was designed with the aim of evaluating nurses perspective on applying professional values in isfahan hospitals in 2010 . this is a descriptive - analytical study that was conducted in selected hospitals in isfahan to survey application of professional values from nurses perspective . the hospitals in this study consisted of four educational hospitals , two hospitals affiliated to the social security organization , and two private hospitals . study samples were nurses in all roles who met the cited inclusion criteria of having a nursing bachelor s degree or higher , were listed in the category of formal nurse , contract nurse , corporate nurse , and planed nurses , were of iranian descent , and had graduated from official universities of iran . according to the number of nurses in each hospital that was assigned as a research area , and also based on the intended sample size of 250 nurses , a quota was determined for each study area in sampling method . at first , permission from the research committee of the iranian nursing organization was obtained and offered to the hospital authorities . then , the aim of the study was explained to the subjects and their informed consent was obtained . data were collected using a two - part questionnaire , including demographic characteristics and professional values data . the first part was a researcher made tool for gathering the demographic data of participants , and the second part was a standard tool titled nursing professional values scale ( npvs ) . weis and schank have designed and used this instrument in a study with the aim of describing nurses professional values ( 13 ) . this scale includes 26 items ; each item in this scale is a short descriptive phrase reflecting a specific code of ethics from the american nursing association in 1985 . this is a norm referenced with likert type format ranging from 1 ( not important ) to 5 ( most important ) , and ranges above 3 indicate strong professional orientation . the possible range of score is 26 to 150 , and therefore scores above 78 were considered as respecting professional and ethical values . in another study by martin the content validity was established by experts in the field of ethics ( 23 ) . in a study by parvan et al . in the present study , due to cultural differences , the questionnaire was translated into persian and sent to six ethics experts for content validity . with a few changes , of the 250 distributed questionnaires , 162 were returned , whereas 12 were not fully completed . therefore , 150 questionnaires were analyzed by means of the statistical software spss version 16 . to determine the relationship between gender , level of education , work experience , different categories of employment , and place of employment and professional values , pearson correlation , independent t - test and anova test were applied . to show the relation between age and score of professional values , spearman correlation test was used . in this study , of the 150 participants , the mean age was 34.2 ( sd=7.3 ) , and the average years of employment as a nurse was 11 ( sd=7.9 ) years . no significant association was found between demographic characteristics such as nurses age ( p = 0.975 ) , gender ( p= 0.169 ) , education ( p= 0.150 ) , and work records ( p= 0.148 ) and their perspective on applying professional ethical values ( table 1 ) . the mean of professional values score among participants was 101.53 ( sd=18.12 ) , and considering the standard average of values score that is 78 for this questionnaire , it can be concluded that all the participants had a positive perspective toward applying professional values in nursing practice . no significant association was found between variables such as the type of hospital nurses position ( p= 0.258 ) and the type of their employment ( p= 0.196 ) and applying nursing professional values . the most prominent criteria of professional values that were noted by nurses were : protecting the health and safety of the public ( mean = 4.35 ) , protecting the moral and legal rights of patients ( mean = 4.21 ) , practice guided by principles of fidelity and respecting individuals ( mean = 4.13 ) , and respecting patients right to privacy ( mean = 4.33 ) ( table 2 ) . the overall results indicated that no significant association was present between demographic characteristics such as age , gender , education , and nurses work records and the nurses perspective on applying professional values . leduc and kotzer aimed to use npvs as an instrument to evaluate nurses perspective on applying professional ethical values in nursing students , newly graduated nurses , and seasoned professionals . they came to the conclusion that these three groups of nurses are the same in applying professional values . in their study , lynn et al . have found no significant difference between the professional and ethical values of senior baccalaureate and associate degree nursing students ( 26 ) . the similarity between the results of this study and other studies in this area shows that people who choose nursing as their job consider ethical values among the important and essential fundamentals of their profession . the results of this study revealed that the association between age and nurses perspective on applying professional values is reversed . it might mean that as the nurses get older , their respect for professional values will be decreased , although some studies have shown that no significant association is present between professional values scores and age of the samples ( 23 ) . in their study , parvan et.al have shown a significant negative correlation between age and the total score of values in nursing students . this means that as the nurses grow older , their respect for professional and ethical values decreases ( 24 ) . the most prominent criteria of professional ethics in this study were : protecting the health and safety of people , respecting the moral and legal rights of patients , practice guided by principles of fidelity and respecting individuals , and protecting patients right to privacy . found out , however , that the most important criteria according to the perspective of nurses in taiwan are : caring for clients with a humanistic spirit , providing professionally competent and holistic care , fostering growth and discovering the meaning of life , experiencing the give - and - take of caring for others , receiving fair compensation , and raising the public s awareness of health promotion ( 27 ) . according to silen et al , nursing professional values as specified by thailand nurses association can be viewed as : sublime states of mind , loving kindness , compassion , and sympathetic joy and equanimity ( 28 ) . in the study conducted by weis and schank the most important professional values of nurses were related to the provision of nursing care and believing in the importance of the process of professionalization ( 22 ) . have expressed that ethical values of nurses from the perspective of iranian nurses include maintaining patients dignity , commitment and work ethics , human communication , fairness , honesty and integrity , and striving to promote personal and professional competence ( 29 ) . showed the most important statements from the students perspective were maintaining confidentiality of patients and maintaining clinical competency . nursing faculty and students awareness of the need for strong professional values is important in preparation of nurses capable of managing patient care in an ethical and professional manner ( 24 ) . the difference between moral professional values in various studies reflects the differences in societies in terms of dominant values based on cultural , social , economic and religious situations . hence it is essential to identify nurses professional values as well as factors influencing disrespect of professional ethics and standards in patient care ( 7 ) . as a matter of fact , this is not exclusively related to nurses ; rather , all of the treatment team encounter such challenges and manage them based on their knowledge and training . some of the participants ( 44% ) believed that they had been well prepared for managing these challenges because they had been well trained ( 15 ) . the important point here is that despite the great importance of ethical issues , we still encounter many cases of non - compliance with the ethical values , and challenges in the field of patients rights . have showed that in the emergency departments of a total of ten hospitals in lorestan ( province of western iran ) which were assessed for their compliance with professional ethics standards and patient s rights , these items have not been fully met ( 30 ) . the fact here is that it is not enough to train nurses and other health care providers to achieve desired outcomes ; following relevant feedbacks in the system is important as well . in this study , we addressed the most and the less important professional values specified by our nurses with different types of employment and different experiences . iranian nurses believe in respecting the legal and ethical rights of patients as the most prominent value . it is concluded that nurses professional values must be specified and assessed based on each country s circumstances to sensitize students and nurses about ethical issues and problems . in addition , identifying and removing barriers to professional ethics play an important role in applied professional values in nursing . this is a descriptive - analytical study that was conducted in selected hospitals in isfahan to survey application of professional values from nurses perspective . the hospitals in this study consisted of four educational hospitals , two hospitals affiliated to the social security organization , and two private hospitals . study samples were nurses in all roles who met the cited inclusion criteria of having a nursing bachelor s degree or higher , were listed in the category of formal nurse , contract nurse , corporate nurse , and planed nurses , were of iranian descent , and had graduated from official universities of iran . according to the number of nurses in each hospital that was assigned as a research area , and also based on the intended sample size of 250 nurses , a quota was determined for each study area in sampling method . all the questionnaires were given to the nurses in different shifts with prior coordination with the director of nursing . at first , permission from the research committee of the iranian nursing organization was obtained and offered to the hospital authorities . then , the aim of the study was explained to the subjects and their informed consent was obtained . data were collected using a two - part questionnaire , including demographic characteristics and professional values data . the first part was a researcher made tool for gathering the demographic data of participants , and the second part was a standard tool titled nursing professional values scale ( npvs ) . weis and schank have designed and used this instrument in a study with the aim of describing nurses professional values ( 13 ) . this scale includes 26 items ; each item in this scale is a short descriptive phrase reflecting a specific code of ethics from the american nursing association in 1985 . this is a norm referenced with likert type format ranging from 1 ( not important ) to 5 ( most important ) , and ranges above 3 indicate strong professional orientation . the possible range of score is 26 to 150 , and therefore scores above 78 were considered as respecting professional and ethical values . in another study by martin the content validity was established by experts in the field of ethics ( 23 ) . in a study by parvan et al . in the present study , due to cultural differences , the questionnaire was translated into persian and sent to six ethics experts for content validity . with a few changes , of the 250 distributed questionnaires , 162 were returned , whereas 12 were not fully completed . therefore , 150 questionnaires were analyzed by means of the statistical software spss version 16 . to determine the relationship between gender , level of education , work experience , different categories of employment , and place of employment and professional values , pearson correlation , independent t - test and anova test were applied . to show the relation between age and score of professional values , spearman correlation test was used . in this study , of the 150 participants , 84% were females and 88% had bachelor degree in nursing . the mean age was 34.2 ( sd=7.3 ) , and the average years of employment as a nurse was 11 ( sd=7.9 ) years . no significant association was found between demographic characteristics such as nurses age ( p = 0.975 ) , gender ( p= 0.169 ) , education ( p= 0.150 ) , and work records ( p= 0.148 ) and their perspective on applying professional ethical values ( table 1 ) . the mean of professional values score among participants was 101.53 ( sd=18.12 ) , and considering the standard average of values score that is 78 for this questionnaire , it can be concluded that all the participants had a positive perspective toward applying professional values in nursing practice . no significant association was found between variables such as the type of hospital nurses position ( p= 0.258 ) and the type of their employment ( p= 0.196 ) and applying nursing professional values . the most prominent criteria of professional values that were noted by nurses were : protecting the health and safety of the public ( mean = 4.35 ) , protecting the moral and legal rights of patients ( mean = 4.21 ) , practice guided by principles of fidelity and respecting individuals ( mean = 4.13 ) , and respecting patients right to privacy ( mean = 4.33 ) ( table 2 ) . the overall results indicated that no significant association was present between demographic characteristics such as age , gender , education , and nurses work records and the nurses perspective on applying professional values . leduc and kotzer aimed to use npvs as an instrument to evaluate nurses perspective on applying professional ethical values in nursing students , newly graduated nurses , and seasoned professionals . they came to the conclusion that these three groups of nurses are the same in applying professional values . have found no significant difference between the professional and ethical values of senior baccalaureate and associate degree nursing students ( 26 ) . the similarity between the results of this study and other studies in this area shows that people who choose nursing as their job consider ethical values among the important and essential fundamentals of their profession . the results of this study revealed that the association between age and nurses perspective on applying professional values is reversed . it might mean that as the nurses get older , their respect for professional values will be decreased , although some studies have shown that no significant association is present between professional values scores and age of the samples ( 23 ) . in their study , parvan et.al have shown a significant negative correlation between age and the total score of values in nursing students . this means that as the nurses grow older , their respect for professional and ethical values decreases ( 24 ) . the most prominent criteria of professional ethics in this study were : protecting the health and safety of people , respecting the moral and legal rights of patients , practice guided by principles of fidelity and respecting individuals , and protecting patients right to privacy . found out , however , that the most important criteria according to the perspective of nurses in taiwan are : caring for clients with a humanistic spirit , providing professionally competent and holistic care , fostering growth and discovering the meaning of life , experiencing the give - and - take of caring for others , receiving fair compensation , and raising the public s awareness of health promotion ( 27 ) . according to silen et al , nursing professional values as specified by thailand nurses association can be viewed as : sublime states of mind , loving kindness , compassion , and sympathetic joy and equanimity ( 28 ) . in the study conducted by weis and schank the most important professional values of nurses were related to the provision of nursing care and believing in the importance of the process of professionalization ( 22 ) . have expressed that ethical values of nurses from the perspective of iranian nurses include maintaining patients dignity , commitment and work ethics , human communication , fairness , honesty and integrity , and striving to promote personal and professional competence ( 29 ) . showed the most important statements from the students perspective were maintaining confidentiality of patients and maintaining clinical competency . nursing faculty and students awareness of the need for strong professional values is important in preparation of nurses capable of managing patient care in an ethical and professional manner ( 24 ) . the difference between moral professional values in various studies reflects the differences in societies in terms of dominant values based on cultural , social , economic and religious situations . hence it is essential to identify nurses professional values as well as factors influencing disrespect of professional ethics and standards in patient care ( 7 ) . it is obvious that nurses can not avoid facing ethical dilemmas , but they should be capable enough to manage these challenges through receiving proper training . as a matter of fact , this is not exclusively related to nurses ; rather , all of the treatment team encounter such challenges and manage them based on their knowledge and training . some of the participants ( 44% ) believed that they had been well prepared for managing these challenges because they had been well trained ( 15 ) . the important point here is that despite the great importance of ethical issues , we still encounter many cases of non - compliance with the ethical values , and challenges in the field of patients rights . have showed that in the emergency departments of a total of ten hospitals in lorestan ( province of western iran ) which were assessed for their compliance with professional ethics standards and patient s rights , these items have not been fully met ( 30 ) . the fact here is that it is not enough to train nurses and other health care providers to achieve desired outcomes ; following relevant feedbacks in the system is important as well . in this study , we addressed the most and the less important professional values specified by our nurses with different types of employment and different experiences . iranian nurses believe in respecting the legal and ethical rights of patients as the most prominent value . it is concluded that nurses professional values must be specified and assessed based on each country s circumstances to sensitize students and nurses about ethical issues and problems . in addition , identifying and removing barriers to professional ethics play an important role in applied professional values in nursing .

osteocalcin ( oc ; also known as bone gla protein , bgp ) is a secreted 5-kda protein that is the ost prevalent noncollagenous protein of 49 amino acids in bone . it is synthesized exclusively in certain cells of the osteoblast lineage : mature osteoblasts and osteocytes . post - translational modification of oc ( gamma - carboxylation at 3 gla residues ) allows it to tightly bind to the calcium ions in hydroxyapatite ( ha ) . oc is largely found in the bone , but a small amount of oc circulates in the blood , and the serum oc level has been considered a marker of bone formation and remodeling . it has been postulated that oc normally functions in the bone to maintain the normal calcification and inhibit mineralization , perhaps in order to prevent osteocytes from becoming completely embedded in mineral . showed the esp , a gene expressed in osteoblasts , could encode a receptor - like protein tyrosine phosphatase termed ost - ptp . it was demonstrated that mice lacking esp ( esp/ ) displayed an increase in -cell proliferation , insulin secretion , and sensitivity , causing hyperinsulinemia and hypoglycemia . mice lacking oc ( ocn ) showed a decrease in -cell proliferation , insulin secretion and sensitivity , adiponectin expression and energy expenditure . ferron et al . also confirmed that oc had a role in promoting -cell proliferation , insulin secretion and sensitivity , adiponectin expression , and energy expenditure . they inferred that there was an interaction between bone and pancreas , especially between osteoblasts and adipocytes . recent studies have indicated that the skeleton as an endocrine organ plays important roles in the bone and energy metabolism depending on the insulin receptor on the osteoblasts . the insulin signaling in osteoblasts is necessary for whole - body glucose homeostasis because it can increase the oc activity . . however , differential patterns for regulating insulin secretion , insulin resistance , and lipid metabolism by osteocalcin in male and female t2 dm patients are still unknown . we compared the serum oc concentration between male and female dm patients , and examined the correlation of serum oc with glucose and lipid metabolism in males and females , respectively . we recruited 1200 patients from the shanghai tenth people s hospital during july 2011 to october 2012 . the inclusion criteria included : 1 ) patients diagnosed with type 2 diabetes mellitus ( t2 dm ) and 2 ) patients with an age range from 50 to 90 years ( all female patients were postmenopausal women ) . the exclusion criteria included : 1 ) patients with acute or severe chronic complications , 2 ) patients with past or current medications ( sex steroids , corticosteroids , bisphosphonates , calcitonin , serms ) known to affect bone metabolism , 3 ) patients with current cancer , laboratory evidence of kidney or liver diseases , hyperthyroid disease , parathyroid disease , or other diseases related to the endocrine system , 4 ) patients with acute or chronic infections , and 5 ) patients with current trauma or operations . a total of 739 patients who met these criteria were included in this study : 311 men with a mean age of 66.7610.24 years ( range : 50 to 90 years ) and 428 women with a mean age of 68.9310.46 years ( range : 50 to 90 years ) . the study protocol was approved by the local institutional review board , and all participants provided written informed consent . height ( accurate to 0.1 cm ) was measured at baseline using a wall - mounted stadiometer and body weight ( accurate to 100 g ) was measured using an electronic calibrated scale . body mass index ( bmi ) was calculated as weight / height ( kg / m ) . bone mineral density ( bmd ) was measured by using dexa with intra- and inter - assay coefficient of variation ( cv ) below 1% . the bmd of l1 - 4 , right hip , and femoral neck was measured . the average bmd was calculated as ( l1 - 4 bmd + right hip bmd + femoral neck bmd)/3 . blood samples were collected after 12-h overnight fasting for the detection of biochemical measurements , and the 75-g oral glucose tolerance test ( ogtt ) was performed according to the standardized clinical procedures . ( hba1c ) was determined by high - performance liquid chromatography ( hplc ) with the coefficient of variation ( cv ) of 1.11% . the plasma insulin ( biosource , nivelles , belgium ) and c - peptide ( immunotech , czech republic ) were determined by immunoradiometric assays with the intra- and inter - assay cvs of 1.62.2% and 6.16.5% and 2.33.0% and 3.55.1% , respectively . the total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) were all measured by using the enzymic method . the above parameters and the function of liver and kidney were tested with a routine laboratory biochemistry autoanalyzer . homeostasis model assessment of -cell function ( homa-%b ) , homeostasis model assessment of insulin sensitivity ( homa-%s ) , and homeostasis model assessment of insulin resistance ( homa - ir ) were calculated by fasting plasma glucose ( fpg ) and fasting c peptide ( fc - p ) . homa calculator software ( from the university of oxford ) was used to calculate the 3 parameters . all analyses were performed with statistical package for the social sciences version 13.0 ( spss , chicago , il , usa ) . spearman correlation coefficients were calculated to assess the correlations of oc with anthropometric variables , glucose , lipid profile , insulin secretion , and insulin sensitivity and resistance . multiple stepwise regression analysis was performed to determine the independent predictors of homa-%b , homa-%s , and homa - ir after adjustment for potential confounders . we recruited 1200 patients from the shanghai tenth people s hospital during july 2011 to october 2012 . the inclusion criteria included : 1 ) patients diagnosed with type 2 diabetes mellitus ( t2 dm ) and 2 ) patients with an age range from 50 to 90 years ( all female patients were postmenopausal women ) . the exclusion criteria included : 1 ) patients with acute or severe chronic complications , 2 ) patients with past or current medications ( sex steroids , corticosteroids , bisphosphonates , calcitonin , serms ) known to affect bone metabolism , 3 ) patients with current cancer , laboratory evidence of kidney or liver diseases , hyperthyroid disease , parathyroid disease , or other diseases related to the endocrine system , 4 ) patients with acute or chronic infections , and 5 ) patients with current trauma or operations . a total of 739 patients who met these criteria were included in this study : 311 men with a mean age of 66.7610.24 years ( range : 50 to 90 years ) and 428 women with a mean age of 68.9310.46 years ( range : 50 to 90 years ) . the study protocol was approved by the local institutional review board , and all participants provided written informed consent . height ( accurate to 0.1 cm ) was measured at baseline using a wall - mounted stadiometer and body weight ( accurate to 100 g ) was measured using an electronic calibrated scale . body mass index ( bmi ) was calculated as weight / height ( kg / m ) . bone mineral density ( bmd ) was measured by using dexa with intra- and inter - assay coefficient of variation ( cv ) below 1% . the bmd of l1 - 4 , right hip , and femoral neck was measured . the average bmd was calculated as ( l1 - 4 bmd + right hip bmd + femoral neck bmd)/3 . blood samples were collected after 12-h overnight fasting for the detection of biochemical measurements , and the 75-g oral glucose tolerance test ( ogtt ) was performed according to the standardized clinical procedures . ( hba1c ) was determined by high - performance liquid chromatography ( hplc ) with the coefficient of variation ( cv ) of 1.11% . the plasma insulin ( biosource , nivelles , belgium ) and c - peptide ( immunotech , czech republic ) were determined by immunoradiometric assays with the intra- and inter - assay cvs of 1.62.2% and 6.16.5% and 2.33.0% and 3.55.1% , respectively . the total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) were all measured by using the enzymic method . the above parameters and the function of liver and kidney were tested with a routine laboratory biochemistry autoanalyzer . homeostasis model assessment of -cell function ( homa-%b ) , homeostasis model assessment of insulin sensitivity ( homa-%s ) , and homeostasis model assessment of insulin resistance ( homa - ir ) were calculated by fasting plasma glucose ( fpg ) and fasting c peptide ( fc - p ) . homa calculator software ( from the university of oxford ) was used to calculate the 3 parameters . all analyses were performed with statistical package for the social sciences version 13.0 ( spss , chicago , il , usa ) . spearman correlation coefficients were calculated to assess the correlations of oc with anthropometric variables , glucose , lipid profile , insulin secretion , and insulin sensitivity and resistance . multiple stepwise regression analysis was performed to determine the independent predictors of homa-%b , homa-%s , and homa - ir after adjustment for potential confounders . a total of 739 t2 dm patients ( mean age 68.0110.41 years [ range : 50 to 90 years ] ; bmi : 24.603.51 patients were divided into males ( n=311 ) and females ( n=428 ) ( table 1 ) . the result showed a significant difference in the age , height , weight , course of disease , bmd , bgp , ca , p , fc - p , 2hc - p , fins , 2hins , tc , hdl , and ldl between males and females ( p<0.05 ) , but there were no significant difference in bmi , hba1c , fpg , 2-h pg , tg , homa-%b , homa-%s , and homa - ir between the 2 groups ( table 1 ) . the correlations of serum oc with glucose and lipid metabolic parameters are shown in table 2 . in males , the serum oc was negatively correlated with age ( r=0.187 , p<0.001 ) , hba1c ( r=0.277 , p<0.001 ) , fpg ( r=0.234 , p<0.001 ) , and 2-h pg ( r=0.255 , p<0.001 ) , but positively with 2-h c - p ( r=0.213 , p<0.001 ) , 2-h ins ( r=0.170 , p=0.002 ) , and homa-%b ( r=0.274 , p<0.001 ) . in females , serum oc was inversely correlated with bmi ( r=0.369 , p=0.029 ) , course of disease ( r=0.369 , p=0.029 ) , hba1c ( r=0.369 , p=0.029 ) , fpg ( r=0.369 , p=0.029 ) , tg ( r=0.369 , p=0.029 ) , and homa - ir ( r=0.354 , p=0.037 ) , but positively with 2-h c - p ( r=0.339 , p=0.047 ) , 2-h ins ( r=0.339 , p=0.047 ) , hdl ( r=0.339 , p=0.047 ) , homa-%b ( r=0.339 , p=0.047 ) , and homa-%s ( r=0.339 , p=0.047 ) . in all subjects , serum oc was negatively correlated with hba1c ( r=0.165 , p=0.001 ) , fpg ( r=0.151 , p=0.002 ) , and 2-h pg ( r=0.107 , p=0.028 ) , but positively with 2-h c - p ( r=0.115 , p=0.019 ) , 2-h ins ( r=0.106 , p=0.030 ) , hdl ( r=0.138 , p=0.005 ) , and homa-%b ( r=0.139 , p=0.004 ) ( figure 1 ) . to further determine which variables were independently associated with serum oc , multiple stepwise regression analysis was performed in men and women independently ( table 3 ) . results showed that course of disease , hba1c , and 2-h pg were independently associated with oc in males . bmi and fpg were independently correlated with osteocalcin in females . in all subjects , hba1c , fpg , and hdl were independently associated with oc . to further determine which variables were independently associated with homa-%b , results showed that in males hba1c ( =0.462 , p<0.001 ) was an independent predictor of homa-%b ; in females hba1c ( =0.554 , p<0.001 ) was an independent predictor of homa-%b ; in all subjects age ( =0.107 , p=0.002 ) , course of disease ( = 0.108 , p=0.002 ) , and hba1c ( = 0.520 , p<0.001 ) were independent predictors of homa-%b . to further determine which variables were independently associated with homa-%s , multivariate linear regression analysis was performed ( table 5 ) . results showed that in males bmi ( =0.224 , p<0.001 ) , hba1c ( =0.123 , p=0.031 ) , tc ( =0.135 , p=0.026 ) , and tg ( =0.271 , p<0.001 ) were independent predictors of homa-%s ; in females age ( =0.178 , p<0.001 ) , bmi ( =0.112 , p=0.019 ) , course of disease ( =0.138 , p=0.005 ) , tg ( =0.242 , p<0.001 ) , hdl ( =0.134 , p=0.007 ) , and ldl ( =0.147 , p=0.003 ) were independent predictors of homa-%s ; in all subjects age ( =0.142 , p<0.001 ) , bmi ( =0.172 , p<0.001 ) , course of disease ( =0.102 , p=0.007 ) , tc ( =0.128 , p=0.003 ) , tg ( =0.258 , p<0.001 ) , and hdl ( =0.096 , p=0.021 ) were independent predictors of homa-%s . to further determine which variables were independently associated with homa - ir , multivariate linear regression analysis was performed ( table 6 ) . results showed that in males age ( =0.144 , p=0.008 ) , bmi ( =0.114 , p=0.030 ) , course of disease ( =0.118 , p=0.030 ) , tc ( =0.187 , p=0.001 ) , and tg ( =0.491 , p<0.001 ) were independent predictors of homa - ir ; in females age ( =0.103 , p=0.033 ) , tg ( =0.221 , p<0.001 ) , hdl ( =0.174 , p=0.001 ) , and ldl ( =0.134 , p=0.008 ) were independent predictors of homa - ir ; in all subjects , age ( =0.122 , p=0.001 ) , bmi ( =0.100 , p=0.005 ) , tc ( =0.148 , p<0.001 ) , tg ( =0.360 , p<0.001 ) , and hdl ( =0.085 , p=0.039 ) were independent predictors of homa - ir . a total of 739 t2 dm patients ( mean age 68.0110.41 years [ range : 50 to 90 years ] ; bmi : 24.603.51 patients were divided into males ( n=311 ) and females ( n=428 ) ( table 1 ) . the result showed a significant difference in the age , height , weight , course of disease , bmd , bgp , ca , p , fc - p , 2hc - p , fins , 2hins , tc , hdl , and ldl between males and females ( p<0.05 ) , but there were no significant difference in bmi , hba1c , fpg , 2-h pg , tg , homa-%b , homa-%s , and homa - ir between the 2 groups ( table 1 ) . the correlations of serum oc with glucose and lipid metabolic parameters are shown in table 2 . in males , the serum oc was negatively correlated with age ( r=0.187 , p<0.001 ) , hba1c ( r=0.277 , p<0.001 ) , fpg ( r=0.234 , p<0.001 ) , and 2-h pg ( r=0.255 , p<0.001 ) , but positively with 2-h c - p ( r=0.213 , p<0.001 ) , 2-h ins ( r=0.170 , p=0.002 ) , and homa-%b ( r=0.274 , p<0.001 ) . in females , serum oc was inversely correlated with bmi ( r=0.369 , p=0.029 ) , course of disease ( r=0.369 , p=0.029 ) , hba1c ( r=0.369 , p=0.029 ) , fpg ( r=0.369 , p=0.029 ) , tg ( r=0.369 , p=0.029 ) , and homa - ir ( r=0.354 , p=0.037 ) , but positively with 2-h c - p ( r=0.339 , p=0.047 ) , 2-h ins ( r=0.339 , p=0.047 ) , hdl ( r=0.339 , p=0.047 ) , homa-%b ( r=0.339 , p=0.047 ) , and homa-%s ( r=0.339 , p=0.047 ) . in all subjects , serum oc was negatively correlated with hba1c ( r=0.165 , p=0.001 ) , fpg ( r=0.151 , p=0.002 ) , and 2-h pg ( r=0.107 , p=0.028 ) , but positively with 2-h c - p ( r=0.115 , p=0.019 ) , 2-h ins ( r=0.106 , p=0.030 ) , hdl ( r=0.138 , p=0.005 ) , and homa-%b ( r=0.139 , p=0.004 ) ( figure 1 ) . to further determine which variables were independently associated with serum oc , multiple stepwise regression analysis was performed in men and women independently ( table 3 ) . results showed that course of disease , hba1c , and 2-h pg were independently associated with oc in males . bmi and fpg were independently correlated with osteocalcin in females . in all subjects , hba1c , to further determine which variables were independently associated with homa-%b , multivariate linear regression analysis was performed ( table 4 ) . results showed that in males hba1c ( =0.462 , p<0.001 ) was an independent predictor of homa-%b ; in females hba1c ( =0.554 , p<0.001 ) was an independent predictor of homa-%b ; in all subjects age ( =0.107 , p=0.002 ) , course of disease ( = 0.108 , p=0.002 ) , and hba1c ( = 0.520 , p<0.001 ) were independent predictors of homa-%b . to further determine which variables were independently associated with homa-%s , multivariate linear regression analysis was performed ( table 5 ) . results showed that in males bmi ( =0.224 , p<0.001 ) , hba1c ( =0.123 , p=0.031 ) , tc ( =0.135 , p=0.026 ) , and tg ( =0.271 , p<0.001 ) were independent predictors of homa-%s ; in females age ( =0.178 , p<0.001 ) , bmi ( =0.112 , p=0.019 ) , course of disease ( =0.138 , p=0.005 ) , tg ( =0.242 , p<0.001 ) , hdl ( =0.134 , p=0.007 ) , and ldl ( =0.147 , p=0.003 ) were independent predictors of homa-%s ; in all subjects age ( =0.142 , p<0.001 ) , bmi ( =0.172 , p<0.001 ) , course of disease ( =0.102 , p=0.007 ) , tc ( =0.128 , p=0.003 ) , tg ( =0.258 , p<0.001 ) , and hdl ( =0.096 , p=0.021 ) were independent predictors of homa-%s . to further determine which variables were independently associated with homa - ir , multivariate linear regression analysis was performed ( table 6 ) . results showed that in males age ( =0.144 , p=0.008 ) , bmi ( =0.114 , p=0.030 ) , course of disease ( =0.118 , p=0.030 ) , tc ( =0.187 , p=0.001 ) , and tg ( =0.491 , p<0.001 ) were independent predictors of homa - ir ; in females age ( =0.103 , p=0.033 ) , tg ( =0.221 , p<0.001 ) , hdl ( =0.174 , p=0.001 ) , and ldl ( =0.134 , p=0.008 ) were independent predictors of homa - ir ; in all subjects , age ( =0.122 , p=0.001 ) , bmi ( =0.100 , p=0.005 ) , tc ( =0.148 , p<0.001 ) , tg ( =0.360 , p<0.001 ) , and hdl ( =0.085 , p=0.039 ) were independent predictors of homa - ir . this cross - sectional study showed that serum oc level in females was higher than that in males , which is consistent with results of a previous report . because oc is mainly secreted from osteoblasts during bone turnover , this may suggest that the bone turnover rate in females could be higher than that in middle - age and elderly male t2 dm patients . this is also supported by the lower bmd in the females than the males in the present study . several clinical trials have reported that serum oc level was significantly lower in t2 dm patients than in healthy controls . a recent study of aoki et al . showed the serum oc level was significantly higher in pre - diabetic patients than in healthy controls . these findings suggest that the oc level may change with the development of t2 dm . in the present study , after adjustment for confounding factors , serum oc was found to be negatively correlated with hba1c , fpg , and 2-h pg in males , and inversely with hba1c and fpg in females . many prospective studies and case - control studies have demonstrated that serum oc is inversely correlated with hba1c , fpg , and 2-h pg [ 20,21,28,3135 ] . the results of the above reports are consistent with our findings and support the view that oc may help to regulate glucose metabolism . the present study also showed that serum oc was positively correlated with 2-h c - p and 2-h ins in all individuals . in the study by akiko et al . , oc was found to stimulate glucagon - like peptide-1 ( glp-1 ) secretion , which could stimulate insulin secretion in response to food intake , suggesting that oc may stimulate postprandial insulin secretion . a recent study of bullo et al . revealed that serum oc was positively correlated with fins . these results suggest that oc may also stimulate fasting insulin secretion . in this paper , serum oc these results were in accordance with our outcomes manifesting that oc may induce insulin secretion both in men and women . this study shows that serum oc was positively correlated with homa-%s and negatively correlated with homa - ir in females , suggesting oc may help to improve insulin sensitivity and resistance in female patients . these findings indicated that the correlation between oc and insulin sensitivity and resistance was obvious in females but not so in males . a chinese study showed that serum oc was not associated with homa - ir in all participants . a korean study showed that serum oc was inversely correlated with homa - ir in postmenopausal women . however , some other studies in western countries , including the usa and sweden , showed that oc was negatively correlated with homa - ir in men and all subjects , and positively correlated with homa-%s in all subjects . it is speculated that differences in eating habits , physical features , and body composition ( percentage of total body fat ) may help to explain these diverse findings . our findings revealed that the serum oc was positively correlated with hdl in females and all participants , and negatively with tg in females . recent clinical studies also uncovered a relationship between oc and tg and hdl in all individuals . these recent results are consistent with our findings showing that oc may have relations with lipid metabolism . some studies in japan and sweden demonstrated that oc was related to tg and hdl in men ; however , in our study no relationship between oc and lipid metabolic indices was found in males . these results are inconsistent with our findings , showing that it is probably due to differences in region , dietary and living habit , some medication use , and some other factors in males . these results suggest that bone may regulate lipid metabolism via oc through mechanisms that need further study . recent evidence in animal models suggests that oc , a hormone secreted during bone turnover , exerts regulatory effects on glucose and lipid metabolism . consistent with this result , our results provide empirical evidence for the interactions of osteocalcin with markers of insulin secretion and sensitivity , and insulin resistance in humans . this evidence was confirmed by using 3 parallel statistical analyses based on multiple linear regression models . results showed that serum oc was not an independent factor influencing homa-%b , homa-%s , and homa - ir , and that hba1c was an independent related factor influencing homa-%b in all subjects . in addition , bmi and several parameters of lipid metabolism were the independent predictors influencing homa-%s and homa - ir in all subjects . these results indicate that the effect of oc on the pancreas may not be so strong . that is to say , in addition to oc , there seems to be other factors affecting insulin secretion , sensitivity , and resistance . first , this was a cross - sectional study showing only association but not causality . second , the influence of differences in gamma - carboxylation status of oc was not investigated . in summary , we found higher serum oc levels in female than male t2 dm patients . oc may have an important role in glucose metabolism , probably through enhancing insulin secretion in males and through both increasing insulin secretion and improving insulin resistance in females . the coupling mechanisms between bone metabolism and energy metabolism needs further study in the future .

backgroundosteocalcin has been reported to be relevant to glucose and lipid metabolism , indicating it may stimulate insulin secretion and improve insulin resistance . yet the difference between male and female patients is still not clear.we aimed to investigate the difference in serum osteocalcin , and its association with glucose , lipid metabolism , pancreatic function , insulin sensitivity , and resistance in male and female middle - aged and elderly type 2 diabetic ( t2 dm ) patients.material/methods739 t2 dm patients were included . after measurement of body mass index ( bmi ) , the levels of fasting plasma glucose ( fpg ) , insulin ( fins ) , c peptide ( fc - p ) , 2-h post - ogtt plasma glucose ( 2h - pg ) , hba1c , and osteocalcin were determined . homeostasis model assessment of -cell function ( homa-%b ) , homeostasis model assessment of insulin sensitivity ( homa-%s ) , and homeostasis model assessment of insulin resistance ( homa - ir ) were calculated.resultsfemales had higher osteocalcin concentration than males ( p<0.05 ) . in males , serum osteocalcin was negatively correlated with hba1c , fpg , and 2-h pg ( p<0.05 ) , but positively with 2-h post - ogtt c peptide ( 2hc - p ) , 2-h post - ogtt serum insulin ( 2h - ins ) , and homa-%b ( p<0.05 ) . in females , serum osteocalcin was negatively correlated with hba1c , fpg , triglyceride ( tg ) , and homa - ir ( p<0.05 ) , but positively with 2-h c - p , 2-h ins , homa-%b , homa-%s , and high - density lipoprotein ( hdl ) ( p<0.05 ) . in all subjects , serum osteocalcin was inversely correlated with hba1c , fpg , and 2-h pg ( p<0.05 ) , but positively with 2-h c - p , 2-h ins , hdl , and homa-%b ( p<0.05).conclusionsosteocalcin might improve glucose metabolism through enhancing insulin secretion in males , and through increasing insulin secretion and improving insulin resistance in females with t2 dm . osteocalcin probably also plays an important role in lipid metabolism .

Background Material and Methods Material Body size measurements BMD measurements Laboratory measurements Homeostasis model assessment Statistical analysis Results Baseline characteristics of patients and measurements between males and females Correlations of serum OC with anthropometric variables, glucose, lipid profile, insulin secretion, and insulin sensitivity and resistance Multivariate linear regression analysis: independent predictors of HOMA-%B Multivariate linear regression analysis: independent predictors of HOMA-%S Multivariate linear regression analysis: independent predictors of HOMA-IR Discussion Conclusions

it was demonstrated that mice lacking esp ( esp/ ) displayed an increase in -cell proliferation , insulin secretion , and sensitivity , causing hyperinsulinemia and hypoglycemia . mice lacking oc ( ocn ) showed a decrease in -cell proliferation , insulin secretion and sensitivity , adiponectin expression and energy expenditure . also confirmed that oc had a role in promoting -cell proliferation , insulin secretion and sensitivity , adiponectin expression , and energy expenditure . however , differential patterns for regulating insulin secretion , insulin resistance , and lipid metabolism by osteocalcin in male and female t2 dm patients are still unknown . we compared the serum oc concentration between male and female dm patients , and examined the correlation of serum oc with glucose and lipid metabolism in males and females , respectively . the inclusion criteria included : 1 ) patients diagnosed with type 2 diabetes mellitus ( t2 dm ) and 2 ) patients with an age range from 50 to 90 years ( all female patients were postmenopausal women ) . body mass index ( bmi ) was calculated as weight / height ( kg / m ) . the plasma insulin ( biosource , nivelles , belgium ) and c - peptide ( immunotech , czech republic ) were determined by immunoradiometric assays with the intra- and inter - assay cvs of 1.62.2% and 6.16.5% and 2.33.0% and 3.55.1% , respectively . the total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) were all measured by using the enzymic method . homeostasis model assessment of -cell function ( homa-%b ) , homeostasis model assessment of insulin sensitivity ( homa-%s ) , and homeostasis model assessment of insulin resistance ( homa - ir ) were calculated by fasting plasma glucose ( fpg ) and fasting c peptide ( fc - p ) . spearman correlation coefficients were calculated to assess the correlations of oc with anthropometric variables , glucose , lipid profile , insulin secretion , and insulin sensitivity and resistance . multiple stepwise regression analysis was performed to determine the independent predictors of homa-%b , homa-%s , and homa - ir after adjustment for potential confounders . the inclusion criteria included : 1 ) patients diagnosed with type 2 diabetes mellitus ( t2 dm ) and 2 ) patients with an age range from 50 to 90 years ( all female patients were postmenopausal women ) . body mass index ( bmi ) was calculated as weight / height ( kg / m ) . the plasma insulin ( biosource , nivelles , belgium ) and c - peptide ( immunotech , czech republic ) were determined by immunoradiometric assays with the intra- and inter - assay cvs of 1.62.2% and 6.16.5% and 2.33.0% and 3.55.1% , respectively . the total cholesterol ( tc ) , triglyceride ( tg ) , high - density lipoprotein ( hdl ) , and low - density lipoprotein ( ldl ) were all measured by using the enzymic method . homeostasis model assessment of -cell function ( homa-%b ) , homeostasis model assessment of insulin sensitivity ( homa-%s ) , and homeostasis model assessment of insulin resistance ( homa - ir ) were calculated by fasting plasma glucose ( fpg ) and fasting c peptide ( fc - p ) . spearman correlation coefficients were calculated to assess the correlations of oc with anthropometric variables , glucose , lipid profile , insulin secretion , and insulin sensitivity and resistance . multiple stepwise regression analysis was performed to determine the independent predictors of homa-%b , homa-%s , and homa - ir after adjustment for potential confounders . a total of 739 t2 dm patients ( mean age 68.0110.41 years [ range : 50 to 90 years ] ; bmi : 24.603.51 patients were divided into males ( n=311 ) and females ( n=428 ) ( table 1 ) . the result showed a significant difference in the age , height , weight , course of disease , bmd , bgp , ca , p , fc - p , 2hc - p , fins , 2hins , tc , hdl , and ldl between males and females ( p<0.05 ) , but there were no significant difference in bmi , hba1c , fpg , 2-h pg , tg , homa-%b , homa-%s , and homa - ir between the 2 groups ( table 1 ) . in males , the serum oc was negatively correlated with age ( r=0.187 , p<0.001 ) , hba1c ( r=0.277 , p<0.001 ) , fpg ( r=0.234 , p<0.001 ) , and 2-h pg ( r=0.255 , p<0.001 ) , but positively with 2-h c - p ( r=0.213 , p<0.001 ) , 2-h ins ( r=0.170 , p=0.002 ) , and homa-%b ( r=0.274 , p<0.001 ) . in females , serum oc was inversely correlated with bmi ( r=0.369 , p=0.029 ) , course of disease ( r=0.369 , p=0.029 ) , hba1c ( r=0.369 , p=0.029 ) , fpg ( r=0.369 , p=0.029 ) , tg ( r=0.369 , p=0.029 ) , and homa - ir ( r=0.354 , p=0.037 ) , but positively with 2-h c - p ( r=0.339 , p=0.047 ) , 2-h ins ( r=0.339 , p=0.047 ) , hdl ( r=0.339 , p=0.047 ) , homa-%b ( r=0.339 , p=0.047 ) , and homa-%s ( r=0.339 , p=0.047 ) . in all subjects , serum oc was negatively correlated with hba1c ( r=0.165 , p=0.001 ) , fpg ( r=0.151 , p=0.002 ) , and 2-h pg ( r=0.107 , p=0.028 ) , but positively with 2-h c - p ( r=0.115 , p=0.019 ) , 2-h ins ( r=0.106 , p=0.030 ) , hdl ( r=0.138 , p=0.005 ) , and homa-%b ( r=0.139 , p=0.004 ) ( figure 1 ) . results showed that course of disease , hba1c , and 2-h pg were independently associated with oc in males . in all subjects , hba1c , fpg , and hdl were independently associated with oc . to further determine which variables were independently associated with homa-%b , results showed that in males hba1c ( =0.462 , p<0.001 ) was an independent predictor of homa-%b ; in females hba1c ( =0.554 , p<0.001 ) was an independent predictor of homa-%b ; in all subjects age ( =0.107 , p=0.002 ) , course of disease ( = 0.108 , p=0.002 ) , and hba1c ( = 0.520 , p<0.001 ) were independent predictors of homa-%b . results showed that in males bmi ( =0.224 , p<0.001 ) , hba1c ( =0.123 , p=0.031 ) , tc ( =0.135 , p=0.026 ) , and tg ( =0.271 , p<0.001 ) were independent predictors of homa-%s ; in females age ( =0.178 , p<0.001 ) , bmi ( =0.112 , p=0.019 ) , course of disease ( =0.138 , p=0.005 ) , tg ( =0.242 , p<0.001 ) , hdl ( =0.134 , p=0.007 ) , and ldl ( =0.147 , p=0.003 ) were independent predictors of homa-%s ; in all subjects age ( =0.142 , p<0.001 ) , bmi ( =0.172 , p<0.001 ) , course of disease ( =0.102 , p=0.007 ) , tc ( =0.128 , p=0.003 ) , tg ( =0.258 , p<0.001 ) , and hdl ( =0.096 , p=0.021 ) were independent predictors of homa-%s . results showed that in males age ( =0.144 , p=0.008 ) , bmi ( =0.114 , p=0.030 ) , course of disease ( =0.118 , p=0.030 ) , tc ( =0.187 , p=0.001 ) , and tg ( =0.491 , p<0.001 ) were independent predictors of homa - ir ; in females age ( =0.103 , p=0.033 ) , tg ( =0.221 , p<0.001 ) , hdl ( =0.174 , p=0.001 ) , and ldl ( =0.134 , p=0.008 ) were independent predictors of homa - ir ; in all subjects , age ( =0.122 , p=0.001 ) , bmi ( =0.100 , p=0.005 ) , tc ( =0.148 , p<0.001 ) , tg ( =0.360 , p<0.001 ) , and hdl ( =0.085 , p=0.039 ) were independent predictors of homa - ir . a total of 739 t2 dm patients ( mean age 68.0110.41 years [ range : 50 to 90 years ] ; bmi : 24.603.51 patients were divided into males ( n=311 ) and females ( n=428 ) ( table 1 ) . the result showed a significant difference in the age , height , weight , course of disease , bmd , bgp , ca , p , fc - p , 2hc - p , fins , 2hins , tc , hdl , and ldl between males and females ( p<0.05 ) , but there were no significant difference in bmi , hba1c , fpg , 2-h pg , tg , homa-%b , homa-%s , and homa - ir between the 2 groups ( table 1 ) . in males , the serum oc was negatively correlated with age ( r=0.187 , p<0.001 ) , hba1c ( r=0.277 , p<0.001 ) , fpg ( r=0.234 , p<0.001 ) , and 2-h pg ( r=0.255 , p<0.001 ) , but positively with 2-h c - p ( r=0.213 , p<0.001 ) , 2-h ins ( r=0.170 , p=0.002 ) , and homa-%b ( r=0.274 , p<0.001 ) . in females , serum oc was inversely correlated with bmi ( r=0.369 , p=0.029 ) , course of disease ( r=0.369 , p=0.029 ) , hba1c ( r=0.369 , p=0.029 ) , fpg ( r=0.369 , p=0.029 ) , tg ( r=0.369 , p=0.029 ) , and homa - ir ( r=0.354 , p=0.037 ) , but positively with 2-h c - p ( r=0.339 , p=0.047 ) , 2-h ins ( r=0.339 , p=0.047 ) , hdl ( r=0.339 , p=0.047 ) , homa-%b ( r=0.339 , p=0.047 ) , and homa-%s ( r=0.339 , p=0.047 ) . in all subjects , serum oc was negatively correlated with hba1c ( r=0.165 , p=0.001 ) , fpg ( r=0.151 , p=0.002 ) , and 2-h pg ( r=0.107 , p=0.028 ) , but positively with 2-h c - p ( r=0.115 , p=0.019 ) , 2-h ins ( r=0.106 , p=0.030 ) , hdl ( r=0.138 , p=0.005 ) , and homa-%b ( r=0.139 , p=0.004 ) ( figure 1 ) . results showed that course of disease , hba1c , and 2-h pg were independently associated with oc in males . in all subjects , hba1c , to further determine which variables were independently associated with homa-%b , multivariate linear regression analysis was performed ( table 4 ) . results showed that in males hba1c ( =0.462 , p<0.001 ) was an independent predictor of homa-%b ; in females hba1c ( =0.554 , p<0.001 ) was an independent predictor of homa-%b ; in all subjects age ( =0.107 , p=0.002 ) , course of disease ( = 0.108 , p=0.002 ) , and hba1c ( = 0.520 , p<0.001 ) were independent predictors of homa-%b . results showed that in males bmi ( =0.224 , p<0.001 ) , hba1c ( =0.123 , p=0.031 ) , tc ( =0.135 , p=0.026 ) , and tg ( =0.271 , p<0.001 ) were independent predictors of homa-%s ; in females age ( =0.178 , p<0.001 ) , bmi ( =0.112 , p=0.019 ) , course of disease ( =0.138 , p=0.005 ) , tg ( =0.242 , p<0.001 ) , hdl ( =0.134 , p=0.007 ) , and ldl ( =0.147 , p=0.003 ) were independent predictors of homa-%s ; in all subjects age ( =0.142 , p<0.001 ) , bmi ( =0.172 , p<0.001 ) , course of disease ( =0.102 , p=0.007 ) , tc ( =0.128 , p=0.003 ) , tg ( =0.258 , p<0.001 ) , and hdl ( =0.096 , p=0.021 ) were independent predictors of homa-%s . results showed that in males age ( =0.144 , p=0.008 ) , bmi ( =0.114 , p=0.030 ) , course of disease ( =0.118 , p=0.030 ) , tc ( =0.187 , p=0.001 ) , and tg ( =0.491 , p<0.001 ) were independent predictors of homa - ir ; in females age ( =0.103 , p=0.033 ) , tg ( =0.221 , p<0.001 ) , hdl ( =0.174 , p=0.001 ) , and ldl ( =0.134 , p=0.008 ) were independent predictors of homa - ir ; in all subjects , age ( =0.122 , p=0.001 ) , bmi ( =0.100 , p=0.005 ) , tc ( =0.148 , p<0.001 ) , tg ( =0.360 , p<0.001 ) , and hdl ( =0.085 , p=0.039 ) were independent predictors of homa - ir . because oc is mainly secreted from osteoblasts during bone turnover , this may suggest that the bone turnover rate in females could be higher than that in middle - age and elderly male t2 dm patients . in the present study , after adjustment for confounding factors , serum oc was found to be negatively correlated with hba1c , fpg , and 2-h pg in males , and inversely with hba1c and fpg in females . many prospective studies and case - control studies have demonstrated that serum oc is inversely correlated with hba1c , fpg , and 2-h pg [ 20,21,28,3135 ] . the present study also showed that serum oc was positively correlated with 2-h c - p and 2-h ins in all individuals . this study shows that serum oc was positively correlated with homa-%s and negatively correlated with homa - ir in females , suggesting oc may help to improve insulin sensitivity and resistance in female patients . these findings indicated that the correlation between oc and insulin sensitivity and resistance was obvious in females but not so in males . a korean study showed that serum oc was inversely correlated with homa - ir in postmenopausal women . however , some other studies in western countries , including the usa and sweden , showed that oc was negatively correlated with homa - ir in men and all subjects , and positively correlated with homa-%s in all subjects . consistent with this result , our results provide empirical evidence for the interactions of osteocalcin with markers of insulin secretion and sensitivity , and insulin resistance in humans . results showed that serum oc was not an independent factor influencing homa-%b , homa-%s , and homa - ir , and that hba1c was an independent related factor influencing homa-%b in all subjects . in addition , bmi and several parameters of lipid metabolism were the independent predictors influencing homa-%s and homa - ir in all subjects . that is to say , in addition to oc , there seems to be other factors affecting insulin secretion , sensitivity , and resistance . oc may have an important role in glucose metabolism , probably through enhancing insulin secretion in males and through both increasing insulin secretion and improving insulin resistance in females .

jewett and williston ( 1971 ) recorded from the scalp of humans a series of seven quite consistent waves which are known as the auditory brainstem responses ( abr ) . as early response to acoustic clicks ( < 10 ms ) these waves , labeled i vii , are also found in animals , e.g. in cat ( jewett , 1970 ; melcher et al . , 1996 ) , rat ( hall , 1990 ) and guinea pig ( brown and patuzzi , 2010 ; malherbe et al . , the latencies of wave i vii are of physiological and clinical interest ( moller et al . , 1988 ; mason and herrmann , 1998 ) , especially the electrically evoked abr ( eabr ) in cochlear implantees ( abbas and brown , 1991 ; guiraud et al . , 2007 ; undurraga et al . , 2013 ) . each abr wave consists of a positive peak followed by a negative one called , e.g. p1 or peak i and n1 or in . ( 2009 ) reported an abr time difference between the peaks of wave i and wave iii of 2.13 0.14 ms ( n = 120 ears ) in normal hearing human subjects . it is generally accepted that waves i and ii are caused by the spiral ganglion cells ( sgc ) of the auditory nerve that connect the hair cells in the cochlea with the next neural processing unit , the cochlear nucleus , where wave iii is generated ( moller et al . , 1988 ) . i may correspond to the action potential ( ap ) conduction time from the hair cell to the cochlear nucleus . however , which functional segments of the sgcs produce peak i is still an open question . disagreements about peak i generators are consequences of different interpretations of the recorded potentials which result from transmembrane currents along the auditory nerve . from guinea pig experiments it was concluded that peak i originates from the sgc segment where aps are first produced ( matsushima , 1982 ) . other investigators found in humans a peak i preceding peak i. it is assumed that peak i is generated in the auditory nerve by the postsynaptic currents from hair cells ( hashimoto , 1982 ; hughes and fino , 1985 ; moore et al . according to another study on guinea pigs , peak i may be generated at a rather large distance along the sgc pathway ( brown and patuzzi , 2010 ) when the conducted aps are passing the dura mater at the proximal end of the internal auditory meatus ( iam , a canal in the temporal bone for the passage of the cranial nerves vii and viii ) . peak latencies can be similar or different for recording electrodes close to the current source ( near field ) and at greater distances ( far field ) , e.g. in the brain case or on the scalp . an electrode in the vicinity of a single axon records a passing ap as a positive / negative / positive sequence of potentials as the axonal transmembrane current influx is separated by outflow in the leading and trailing part of the conducted spike . the three related ( positive / negative / positive ) near field peaks broaden and decrease quickly in amplitude when the electrode is moved away from the axon . in the far field a stationary potential arises when many aps pass within a small time window a region where the conductance of the extracellular medium changes ( fig . 1 ; nakanishi , 1982 ; stegeman et al . , lead to contradictory hypotheses : ( i ) peak i may be caused by the distal iam ending ( tasaki et al . , 1954 ) , ( ii ) the low cerebrospinal fluid ( csf ) conductance between the proximal iam end and the higher conductance of the brainstem may cause two sequential peaks namely negative peak in ( this is the next abr minimum after peak i , which is also called n1 ) and peak ii when aps enter brainstem ( martin et al . , 1995 ) , ( iii ) brown and patuzzi ( 2010 ) suggest that a single electrical barrier , the dura mater , produces both peak i and peak in when the leading respectively the trailing dipole of the conducted transmembrane currents encounter this barrier . about 95% of sgcs are type i cells ( spoendlin , 1975 ) , which are innervated by inner hair cells ( ihc ) . in humans such a bipolar cell has a myelinated axon that is interrupted by a non - myelinated region around the soma . the non - myelinated somatic region is a human peculiarity , which is rarely found in cochleas of animals ( ota and kimura , 1980 ; nadol , 1988 ) . type ii sgcs , innervated by outer hair cells ( ohc ) , are non - myelinated , their axon and soma diameters are smaller and consequently signal conduction is slow ( about 1/9 ) in comparison to type i cells ( rattay et al . , 2013 ) . lack of insulation by internodes in axons of type ii cells demands essentially stronger transmembrane currents compared to axons of type i cells . concerning the origin of abr peak i , this fact needs an investigation as the higher ion currents could compensate for the small numbers of type ii cells . using a biophysical model ( fig . 2 , rattay et al . , 2001a ) we could show that spike conduction in mammalian sgcs is characterized by the following four phases : a postsynaptic delay , constant velocity in the peripheral axon , a presomatic delay and constant velocity in the central axon ( rattay et al . , 2013 ) . ap duration ( 330 s ) and axonal velocity of spike conduction were in good agreement with recorded mammalian peripheral nerve data . although this modeling study did not include signal delays for the synaptic transfer in the cochlear nucleus , the 2.24 ms spike conduction time along the average human type i cell is already longer than the time differences between abr peaks iii and i as found by esteves et al . ( 2009 ) , indicating that peak i appears later than the first elicited activities in the auditory nerve . on the other hand our computational evaluation of the membrane currents demonstrated that surprisingly high postsynaptic currents from ihc ribbon synapses with amplitudes of 400 pa ( grant et al . , 2010 ) outnumber threshold by a factor of at least 15 ( rattay et al . , 2013 ) . the goal of the following investigation is to analyze whether such strong postsynaptic currents in the peripheral terminals of sgcs could generate abr peak i or which other neural components could be the generators . alternatively , peak i could be a stationary potential , e.g. generated when spikes conducted along the auditory nerve arrive at the left dashed line in fig . 1 . a compartment model of a human type i cell is described in detail in rattay et al . ( 2001a ) . this model was used for intracellular and extracellular stimulation . according to fig . 2a , the cell consists of segments with active membranes with high ( marked red ) and low ion channel density ( pale pink ) . additionally , there are myelinated internodes ( gray ) where the membrane is assumed to be passive which means membrane currents , which depend on voltage sensitive ion channel gating mechanisms , are negligible . the high ion channel density , modeled as 10-fold hodgkin huxley membrane conductance , is needed for signal amplification along the neural pathway . this sodium channel density is comparable to nodes of ranvier in axons of the mammalian peripheral nerve system ( rattay and aberham , 1993 ; rattay et al . , 2003 ) . beside the peripheral terminal and the nodes of ranvier , the high sodium channel concentration is applied in the pre- and postsomatic compartment . according to the biophysical model approach , strong sodium current influx is especially needed in the presomatic region to load the large capacitance of the non - myelinated soma in order to conduct the spike across the soma into the central axon ( rattay et al . , 2001a ) . the high sodium channel density in the presomatic region is also confirmed experimentally in non - myelinated type i cells of the mouse ( hossain et al . , 2005 ) . applying kirchhoff 's law ( the sum of all currents is zero ) to the n - th compartment of the electric network of fig . 2b results in(1)(vi , nve , n)tcm , n+iion , n+vi , nvi , n1rn/2+rn1/2+vi , nvi , n+1rn/2+rn+1/2=0,where the first two terms are the transmembrane currents which are the postulated generator elements of the abr signal . they correspond to capacitance current and ion current across the membrane whereas the next two terms describe the intracellular current flow to the left and right compartment . introducing the reduced membrane voltage v = vi ve vrest ( vi , ve and vrest are the intracellular , extracellular and resting potential , respectively ) leads to the following system of differential equations ( rattay , 1999 ; rattay et al . ( 1 ) and ( 2 ) have a reduced form because of lack of neighbors . the membrane surface an of every compartment has to be calculated to find cm , n = ancm , n ( cm , n is the specific membrane capacitance ; note that the capacitance of n layers of membranes is proportional to 1/n ) . the ion membrane current is governed by the gating mechanisms of specific voltage sensitive ion channels . it consists of two components(3)iion = aniion , n+inoise , n , where iion is the ionic membrane current density and inoise , n represents ion channel current fluctuations in active compartments . the effective noise current is assumed to be proportional to the square root of the number of sodium channels within a compartment ( for details see rattay et al . , 2001a ) . in compartments with passive membranes ( internodes ) ( 1 ) and ( 2 ) is a current with constant conductance iion = gmanvn / n , where membrane conductance gm is 1 ms / cm and the number of insulating layers of cell membranes n is 40 and 80 for the peripheral and central axon , respectively ( rattay et al . , 2001a ) . using this method the temporal profiles of the transmembrane voltage and the transmembrane currents of each compartment were computed . the ion currents of active membranes were simulated with original hodgkin huxley kinetics ( hodgkin and huxley , 1952 ) at 27 c . at this temperature ap duration fits recorded data and , moreover , the model is appropriate for the non - myelinated regions of sgcs ( hartmann et al . , 1984 ; motz and rattay , 1986 ; rattay et al . , 2013 ) . further supports for this approach are found in the subsections proof of concept and in the discussion section . current injection into the first compartment p0 of the modeled neurons simulated the synaptic activation by hair cells . the shape and amplitude of the postsynaptic currents of type i cells were defined by an average amplitude i0 of 400 pa , 280 s for time to peak and a decay time constant of 370 s as recorded in adult p20 and p60 rats ( grant et al . , 2010 ) . the same shape but with smaller amplitude i0 of 26 pa was used for the stimulation of type ii cells corresponding to signal strengths recorded by weisz et al . when a cell is stimulated externally , the extracellular potentials cause virtual injected currents in every compartment ( rattay , 1999 , 2003 ) . in contrast to synaptic stimulation , the differing distances of the compartments to the stimulating electrode ( fig . 2c ) cause strong variations in the individual driving forces . an example of the resulting membrane voltage variations along the neural path is seen in the upstroke downstroke sequence during the application of the stimulus pulse in fig . 3a . in order to calculate the extracellular potentials ignoring capacitance effects of tissue , the extracellular potential was calculated as(4)ve=eielectrode/4r , where r is the center center distance between a compartment of interest and the spherical electrode . e = 300 ohm cm ( rattay et al . , 2001a ) was assumed to be the mean resistivity of the extracellular medium . within an infinite homogeneous extracellular medium eq . ( 4 ) describes the relationship between the current of a point source and the potential in its surrounding . ( 4 ) together with the superposition principle , a first approach for the vabr signal contribution of a single activated sgc was simulated as the sum of the contributions of both transmembrane current components from all compartments(5)vabr=e4n=1dim(iion , n+ic , n)/rn . as explained above , for every compartment the transmembrane current has a resistive component iion and a capacitance component ic = cmdv / dt , which is the first term in eq . the standard position of the recording electrode is shifted 2 mm from the stimulating electrode in orthogonal direction to the midmodiolar plane ( fig . standard compartment structure of the 32 mm long model neuron was the following : p0 , internode , p1 , internode , , p5 , internode , 3 presomatic compartments , soma ( compartment 16 ) , postsomatic compartment c0 , internode , central node c1 , 59 internode - node combinations , internode , resulting in dim = 135 compartments . as proof of the method we simulated an intracochlear recording of an electrically stimulated feline cochlea . 3c ) demonstrates the suitability of the simple membrane current model . in order to explain how the polarity of measured signals depends on the recording position and for the analysis of other far field effects , we used a simple elliptic closed head model where all current paths were closed within the head . the model consisted of the skull , the ear canal , the cochlea , the internal auditory meatus , the cerebrospinal fluid , the brain and the auditory nerve itself ( fig . 4 ) . using the finite element method ( fe ) , we calculated the electric field for selected recording positions when 1000 identical sgcs were synchronously activated . for this purpose part of the auditory nerve was modeled as an active cylinder with 0.5 mm diameter and 32 mm length ( denoted as active nerve in fig . the surface of the cylinder acted as a time dependent current source summarizing the transmembrane currents of 1000 active sgcs within the corresponding volume . in more detail , using comsol multiphysics 4.4 for mac os x 10.9.2 ( comsol inc . , burlington , ma , usa ) , the generated electrical potential in the volume conductor was calculated by a reduced form of maxwell 's equation(6)()=0,which depended on the conductivity of the medium . neumann type boundary conditions were applied,(7)()n = f(x , t),where n was the normal vector and f(x , t ) a predefined time and location depending function of current density . for all external surfaces the normal current density was restricted to zero ( i.e. , f(x , t ) = 0 for all x and t ) . along the surface of the active cylinder f(x , t ) represented the transmembrane currents of 1000 synchronously firing sgcs . the calculation was performed by an iterative time discrete solver for 2.5 ms in time steps of 0.025 ms . 4b ) were : bone 0.02 s / m , brain ( as gray matter ) 0.23 s / m , nerve fibers 0.6 s / m ( longitudinal ) and 0.083 s / m ( transversal ) , and cerebrospinal fluid 1.7 s / m ( rattay et al . the first part of the rising ap in the peripheral terminal p0 is closely related to the stimulus intensity . 280 s rise time and exponential decay with a time constant of 370 s ( fig . this strong postsynaptic current caused a short ap delay ( 110 s , fig . 5b ) whereas a drastically weaker synaptic stimulation ( 26 pa , fig . 5c ) had a delay of 670 s . in order to show the influence of stimulus strength , we applied this 26 pa stimulus intensity again to a type i cell , although it was the mean postsynaptic current recorded in rat type ii cells ( weisz et al . , 2012 ) . when ion current fluctuations are included , the model approach confirms the two important advantages of strong stimuli from inner hair cell ribbon synapses ( fig . 5d and e ) : a short ap delay and small jitter characterize quick and accurate signaling from ihcs to the cochlear nucleus . due to the strong stimulus , the ap peak at p0 of a type i cell was 21.7% larger than the conducted membrane voltage peak , e.g. at node p4 ( fig . the transmembrane current amplitudes at a node or internode in the peripheral axon were roughly comparable to that of the 400 pa stimulus ( fig . however , both transmembrane currents , iion and ic , at p0 exceeded that at p4 because of the bigger surface area of the active membrane ( fig . it is important to note that membrane currents are of both polarities and the effective difference between inward and outward currents of a compartment is often essentially smaller than the single components . the next investigation was a simplified approach to calculate the abr signal by assuming a recording electrode relatively close to the peripheral sgc section . 2c , that is 2 mm in orthogonal direction of the midmodiolar plane which contains the neural path of a target sgc and also a small spherical stimulating electrode . the stimulating electrode was not as close to the cochlear axis as usual with cochlear implants , where it should be as near as possible to the neural structures . the selected position of the stimulating electrode favors spike initiation of the peripheral ending quite similar to natural excitation . a 300 a square pulse of 100 s initiated a propagating spike at p0 a bit later than the 400 pa injected current ( fig . the conducted ap was delayed in the non - myelinated somatic region because of its high capacitance ( rattay et al . , 2001a ) before being conducted along the thicker central axon with the doubled velocity of the peripheral process ( rattay et al . , 2013 ) . the high capacitance of the soma demands enlarged sodium current influx in the presomatic region which consists of three compartments in order to achieve numerical accuracy . the high ion channel densities of these three presomatic compartments together with the low channel density within the large soma surface ( compare fig . 2a ) made this region the effective section for the ionic and the capacitance currents ( fig . the simulated membrane currents in the nodes and internodes of the central axon were exactly of doubled intensity compared to that of the peripheral axon when the number of insulating sheets , internodal length and fiber diameter is doubled . from morphometric analysis we did in fact find a constant ratio of 2 for human central / peripheral axon diameter whereas the feline ratio was 1.8 ( rattay et al . , 2013 ) . as expected , the model evaluations showed only small deviations from the theoretical prediction that membrane currents and spike velocity increase linear with the diameter of myelinated axons . the predicted velocity ratio of 2:1 was marginally distorted by irregularities at the endings of the rather short peripheral axon . although axonal membrane currents were of doubled amplitude in the central axon their impact was too small to cause abr peak i. evaluation of eq . ( 5 ) demonstrated that the largest abr wave i contribution of the simulated type i cell resulted from its soma region , whereas no part of the peripheral process had a substantial effect ( fig . enlargement of axon or soma diameters had minor influence on the shape of the recorded signal ( fig . the next computer experiment was used to analyze the contribution of unmyelinated type ii cells on abr wave i. for this purpose the peripheral process of the standard sgc was assumed to be unmyelinated and the region from p0 to the soma was modeled as homogeneous membrane with original hodgkin - huxley ion channel densities . the demyelination had three essential consequences : ( i ) ap conduction was slowed down to 23% ( compare figs . ( ii ) both types of transmembrane currents of an average compartment were more similar in shape and intensity than those of the node - internode combination where the ion current dominated in the node and the capacitance current in the internode ( fig . ( iii ) transmembrane current strengths were larger in a section , which was assumed earlier to be myelinated , as the total number of ion channels in this section increased by a large extent . nevertheless , the increased currents of the peripheral axon could not contribute as much as the soma region to the extracellularly recorded potential . the characteristic somatic contribution was even more obvious when the electrode distance was increased from 2 mm to 8 mm ( fig . 10a and b ) . using a passive membrane to simulate the soma , i.e. , excluding the nonlinear gating of ion channels , caused some reduction of the amplitude of the recorded signal in the near recording case ( fig . 10c ) but resulted in an almost identical voltage profile for active and passive soma membranes for distant recording ( fig . to emphasize the conclusions about the dominant role of sgc somas as generators of human abr peak i , the transmembrane currents from a bundle of 1000 synchronously firing type i cells were evaluated with a simplified head model ( fig . 4 ) . input data were the currents from 64 , 0.5 mm long segments that represented the total temporal current flux along the auditory nerve . each segment comprised the sum of all implicated ion and capacitance currents resulting from at least one node and one internode . 11a for t = 200 s , t = 750 s and t = 2200 s , representing times after stimulus onset when the spike is in the peripheral axon , in the soma region and in the central axon close to brainstem , respectively . about 300 s after the somatic spike passage , all somatic transmembrane currents vanished ( fig . 7b and c ) and the spatial shape of the central axon currents was the same as seen for t = 2200 s in fig . 11a . a simulated recording ( 2 mm from the surface of the active cylinder ) and the near field response ( 0.1 mm from the surface of the active cylinder ) transmembrane currents of the bypassing spikes dominated and caused the shape of the recorded signal ( as function of time ; fig . 11c ) to be similar to the mirrored picture of the transmembrane currents ( as functions of space ; fig . . moving the recording electrode in constant distance along the active cylinder caused the same three - phasic signal but with a delay corresponding to the traveling time of the spikes . for example , moving the electrode 3 mm to the right caused a delayed near field response ( dashed line in fig . the recorded signal at 2 mm distance showed a much weaker but broader version of the near field response , which is disturbed differently by far field components when the electrode was again moved by 3 mm ( fig . note , however , the constant time of peak i ( 750 s after stimulus onset ) for different electrode positions . 12a shows simulated intracochlear potentials under the following three conditions : ( i ) the quite realistic nerve geometry of fig . 2 but a homogeneous infinite medium , ( ii ) fe solution for rectified sgcs in an active cylinder and assuming the csf conductance value within the cochlea , ( iii ) same situation as in ( ii ) but the cochlea was simulated with the conductance of nerve tissue . in case ( i ) the peripheral axon was close to the recording electrode and consequently the first membrane currents caused stronger recorded amplitudes . in all three cases the strongest parts of the recorded signals appear nearly at the same time , i.e. , when the spike crosses the soma region as shown in the previous sections . this synchrony of the first peak demonstrates that it was not generated by a change in the conductance of the extracellular medium but it appeared just at the time when the transmembrane currents reach their maximum values . furthermore , in synchrony with the first negative intracochlear peak a positive peak could be recorded at the vertex and behind the ear ( fig . in contrast to the infinite model , the bony boundary of the skull hinders currents to leave the head surface . this was without impact on peak i time but caused about 5 times larger recorded potentials in the head model ( fig . . the almost synchronous initiation of a high number of spikes and their conduction in the auditory nerve cause a spatial - temporal pattern of currents within the head . at a fixed time after stimulus onset , e.g. at the time of peak i , the electrical situation in the head and on its surface is characterized by isopotentials . of specific interest is the isopotential equal zero because this time dependent surface is the border where the polarity changes . in our simple head model peak i has the same polarity behind the ear as within the cochlea ( magnified doted curve in fig . 12a ) , whereas at the time of peak i recording electrodes at vertex or ear are separated by the isopotential 0 from the intracochlear region . such isopotential lines ( on the surface of the head ) including isopotential zero are reported systematically for all human abr peaks ivii ( hughes and fino , 1985 ) . the phrase auditory brainstem response implies the assumption that all its constituent waves may be initiated in the brainstem . many studies in animals and clinical studies in humans were needed to improve , step by step , the knowledge of the individual abr peak generators . disagreements about the peak generators were caused by technical limitations such as lack of modern microelectrodes , ethical considerations for human experiments and confusing observations , e.g. along the pathway of the auditory nerve signal polarity changes at the proximal end of internal auditory meatus . according to this signal polarity effect , abr pioneer jewett using letters p and n for positive and negative peaks claimed in his 1970 paper : with the tongue as a reference point in the deeply anesthetized cat , recordings from the scalp or rostral brain locations obtained by repeated averaging , time - locked to an auditory click , showed four positive waves which were labeled p1p4 . p1 occurs simultaneously with n1 recorded at the round window and is probably generated by the viiith nerve . several investigators found convincing arguments that also p2 is generated by the auditory nerve when volleys of spikes in afferent fibers enter the brainstem ( moller , 1983 , 1988 ; martin et al . , 1995 ; brown and patuzzi , 2010 ) . although it is well accepted that stationary components in far field potentials arise when nerve signals cross the border between media with quite different electrical conductances ( stegeman et al . , 1987 ; martin et al . , 1995 ; brown and patuzzi , 2010 ) hypotheses about where abr peaks p1 and n1 are generated are controversial . moreover , it is not clear which of the abr peaks ivii are stationary potentials . using sophisticated magnetic resonance imaging techniques , it is possible to map electric conductance of all brain regions ( tuch et al . , 2001 ) . combined with finite element modeling such an approach could predict details of abr components , even in a patient specific manner . however , including this method is beyond the scope of the presented study . here , we used a simplified human head model in order to analyze which of the early abr peaks are stationary potentials . this analysis is based on our recent investigation on spike conduction in sgcs ( rattay et al . , 2013 ) , where we found nearly the same velocity diameter relationships for myelinated sgc axons as recorded in afferent peripheral mammalian axons ( boyd and kalu , 1979 ) . the 32.35 mm neural path length for the simulated spike conduction was an average value of medical imaging data measured from the peripheral terminals of sgcs to their entry points at the border to the brainstem . due to the similar conductances of the bone and dura mater the simulated conducted spikes generate abr n1 when crossing the ( bone + dura mater)/csf border and abr p2 when arriving at the csf / brainstem interface ( fig . according to our simulations peak i is generated in the soma region within the cochlea and not at the proximal end of the iam as supposed by brown and patuzzi ( 2010 ) . they substituted the propagating ap in the axon by two dipoles , which may generate p1 and n1 when each of the dipoles crosses the dura mater . according to this hypothesis latency difference n1p1 should be the same as the arrival time difference of the two dipoles at the conductance barrier . in our computational model this latency difference is calculated from the three gravity points of the half - waves of the three - phasic curve ( full line ) seen in fig . the first dipole is represented by gravity points 1 and 2 , the second by gravity points 2 and 3 . even a bit longer ap duration would cause a much shorter dipole latency difference as recorded in human . furthermore , the interpretation of brown and patuzzi ( 2010 ) is rooted in the assumption that each primary afferent neuron passes through an individual aperture in the highly electrically resistive dura mater sheath . yet , at least in human , the dura mater and the arachnoidal membrane invaginate into the iam from the porus to the fundus ( lescanne et al . , 2002 ) and at the transition zone between peripheral and central nervous system the nerve fibers cross the glia limitans not the dura mater ( fraher , 2002 ) , a glial barrier of much lower thickness than the dura . thus , the dura mater and the glia limitans are unlikely to be causative for stationary potentials . rather , the dura and the temporal bone , due to their comparable conductances , electrically form a single macroscopic compartment . furthermore , even though a tripole , generated by the ap , passes a conductance barrier an unipolar stationary potential is generated ( stegeman et al . , 1987 ) . thus , a single barrier alone can not be the cause of the successive peaks i and in as suggested by brown and patuzzi ( 2010 ) . reported latency differences n1p1 of 2.60 - 1.84 = 0.76 ms ( table 1 of martin et al . , 1995 ) and 2.32 1.67 = 0.65 ms ( table 3 of hughes and fino , 1985 ) are , however , rather close to our results of 0.80 ms ( fig . the same authors found latency differences of 1.32 ms and 1.30 ms for p2p1 , whereas our value was 1.42 ms ( fig . this discrepancy may mainly be caused by morphometric uncertainties about auditory nerve segment lengths in iam and csf . some authors could record the early abr peak i , which is assumed to result from postsynaptic currents of ihcs in type i sgcs ( hughes and fino , 1985 ; moore et al . , 1992 ; davis - gunter et al . , 2001 ; grant et al . our finite element approach was not detailed enough to see these signals in the simulated abr but we could clearly see it in the intra - cochlear model , e.g. marked by vertical arrows in fig . 7d , as a first negative peak in fig . 8b or in fig . 12 . our biophysical model approach underlines the assumption that the recorded peak i is caused by the surprisingly strong postsynaptic currents ( fig . i of 570 s ( left ears ) and 620 s ( right ears ; hughes and fino , 1985 ) are close to our intra - cochlear 580 s latency ( fig . 7 ) . these corresponding latencies i i and the observations that n1 recorded as a compound action potential ( cap ) of the auditory nerve at the round window appears simultaneously with abr p1 ( jewett , 1970 ; gersdorff , 1982 ; moller et al . , moreover , intra - cochlear recordings with cochlear implants showed slightly earlier n1 peaks than cap recordings at the round window ( miller et al . , 2004 ) . as non - human data are obtained from small animals , where commonly wave ii was not seen , it was hypothesized that they may have other peak generators as humans , eventually because the internal auditory meatus is essentially longer in man ( moller et al . , 1988 ) . regarding wave ii this hypothesis was disproved as the wave ii peak was recently shown to be generated as stationary potential in guinea pig ( brown and patuzzi , 2010 ) . concerning intracochlearly recorded n1 our single spiral ganglion cell simulation had quite similar temporal properties as found from cat experiments ( fig . 3 ) . according to this simulation , contrary to cat , in humans the strong transmembrane currents in the nonmyelinated soma region is seen to generate the first abr responses . the smaller number of type ii cells is compensated by large transmembrane currents typical for non - myelinated axons . however , for three reasons they can not rival type i cells concerning abr peak i generation . first , ap duration is assumed to be the same for both cell types and a dipole model is used where the total transmembrane currents are reduced to two spatially separated currents . in contrast to type i cells , the slow spike conduction velocity of non - myelinated axons causes a short dipole length . second , the postsynaptic currents from ohc ribbon synapses occur relatively rarely and at 26 pa they are strikingly smaller than the 400 pa amplitudes from ihcs ( grant et al . , 2010 ; weisz et al . , the simulated response to the weak stimulus created a postsynaptic delay in the order of 800 s for spike generation in type ii cells ( rattay et al . , 2013 ) , which together with the slow ap conduction , hinders an early contribution of the capacitance currents from somas of type ii cells . our analysis showed that the transmembrane currents of the peripheral axons are rather small ( fig . 6b and c ) and they can not be the generators of abr peak i. further evidence for this argument comes from eabr in cochlear implantees . as the electrical stimulating signals are several orders larger than the recorded ones , the first part of the neural response vanishes in the stimulus artifact . the value of 2.1 ms for the eabr peak iii latency ( starr and brackmann , 1979 ) correspond almost exactly with the 2.13 0.14 ms latency difference between peaks iii and i as recorded in normal hearing subjects ( esteves et al . , 2009 ) . similar short eabr peak iii delays are reported ( guiraud et al . , 2007 ) . in contrast to acoustically evoked auditory nerve excitations electrical stimulation causes no delay by mechanical components or by the synaptic processes at the hair cells as the electric field directly stimulates elements of the auditory nerve . in most cases spikes elicited by human cochlear implants are expected to be initiated in the central axons and not at the peripheral sgc ending as in fig . 6a where the electrode is quite far from the modiolus . moving the electrode closer to the modiolus favors more central sgc segments to be stimulated ( rattay et al . it is important to note that in people with hearing loss most of the peripheral axons degenerate whereas much more central axons seem to be intact and excitable ( felix et al . , 1997 ; nadol , 1997 ) . because of the rather long peripheral human axons , usually the stimulating electrode is distal to the soma and for such cases a theoretical analysis predicts that sgc without peripheral processes can not be excited with cathodic pulses ( rattay , 1999 ; rattay et al . , 2001a , b ) . this effect was confirmed by abr and cap analysis from cochlear implantees showing that the anodic part of biphasic pulses is more effective for excitation ( macherey et al . , 2008 ) . this result applies whether the cathodic or the anodic pulse is first and for various inter phase gaps ( undurraga et al . therefore , spikes are expected to be initiated in most cases in the distal segments of the central axon in cochlear implantees ( rattay et al . these elicited aps generate the large capacitance currents at the somas and the same electrical signaling as in natural hearing when the spikes are conducted along the central axons . thus the temporal coincidence of 2.1 ms for the eabr peak iii latency and the 2.1 ms for the difference between peaks iii and i in the healthy ear are strong indicators that the intense soma currents are the generator elements of peak i. in contrast to the human , nearly all studied mammalian type i cells are myelinated in the soma region in addition to their myelinated axons . thereby their somatic capacitance is significantly reduced ( rattay , 1990 ; rattay et al . , 2001a ) resulting in a weaker influence of the soma on the abr signal . however , the example of a feline type i cell ( fig . 2 ) demonstrates that the transmembrane currents in myelinated soma regions of type i cells may still be strong enough to be the generators of peak i in mammals too . the investigations were concentrated on the evaluation of the transmembrane currents of single representative type i and type ii cells and not on a model that simulates an abr signal as recorded at the scalp . a previously presented compartment model for sgcs was used to simulate the transmembrane currents ( rattay et al . , 2001a ) . important parameters for the choice of the membrane model were the ap duration and its rise time . different types of voltage - gated ion channels have been identified in sgc membranes based on rat and mouse data ( hossain et al . , 2005 ; yi et al . , 2010 ; negm and bruce , 2008 ) . however , most ion channel models for non - myelinated neural membrane segments in warm blooded animals , including the only available complete model for a sgc ( hossain et al . , 2005 ) , show ap durations longer than 1 ms ( hu et al . , 2009 ; rattay and wenger , 2010 ) . in contrast to such membrane models , the original hodgkin - huxley model with a temperature fit to 27 c resulted in aps with a duration of 330 s and a short rise time that was consistent with sgc spikes recorded within the feline cochlea ( fig . 3 ) . during the period of an ap the signal shape for the sum of ion currents is comparable with other models for myelinated peripheral axons but their ion current density amplitudes in the nodes of ranvier are a bit larger ( rattay and aberham , 1993 ; rattay et al . , resulting corrections from a more detailed modeling are not expected to contradict the dominant role of the somatic region in generating abr peak i.

early neural responses to acoustic signals can be electrically recorded as a series of waves , termed the auditory brainstem response ( abr ) . the latencies of the abr waves are important for clinical and neurophysiological evaluations . using a biophysical model of transmembrane currents along spiral ganglion cells , we show that in human ( i ) the non - myelinated somatic regions of type i cells , which innervate inner hair cells , predominantly contribute to peak i , ( ii ) the supra - strong postsynaptic stimulating current ( 400 pa ) and transmembrane currents of the myelinated peripheral axons of type i cells are an order smaller ; such postsynaptic currents correspond to the short latencies of a small recordable abr peak i , ( iii ) the abr signal involvement of the central axon of bipolar type i cells is more effective than their peripheral counterpart as the doubled diameter causes larger transmembrane currents and a larger spike dipole - length , ( iv ) non - myelinated fibers of type ii cells which innervate the outer hair cells generate essentially larger transmembrane currents but their abr contribution is small because of the small ratio type ii / type i cells , low firing rates and a short dipole length of spikes propagating slowly in non - myelinated fibers . using a finite element model of a simplified head , peaks in and ii ( where in is the negative peak after peak i ) are found to be stationary potentials when volleys of spikes cross the external electrical conductivity barrier at the bone&dura / csf and at the csf / brainstem interface whereas peaks i and i may be generated by strong local transmembrane currents as postsynaptic events at the distal ending and the soma region of type i cells , respectively . all simulated human inter - peak times ( i i , ii i , in i ) are close to published data .

Introduction Methods Results Discussion

jewett and williston ( 1971 ) recorded from the scalp of humans a series of seven quite consistent waves which are known as the auditory brainstem responses ( abr ) . it is generally accepted that waves i and ii are caused by the spiral ganglion cells ( sgc ) of the auditory nerve that connect the hair cells in the cochlea with the next neural processing unit , the cochlear nucleus , where wave iii is generated ( moller et al . i may correspond to the action potential ( ap ) conduction time from the hair cell to the cochlear nucleus . however , which functional segments of the sgcs produce peak i is still an open question . disagreements about peak i generators are consequences of different interpretations of the recorded potentials which result from transmembrane currents along the auditory nerve . other investigators found in humans a peak i preceding peak i. it is assumed that peak i is generated in the auditory nerve by the postsynaptic currents from hair cells ( hashimoto , 1982 ; hughes and fino , 1985 ; moore et al . according to another study on guinea pigs , peak i may be generated at a rather large distance along the sgc pathway ( brown and patuzzi , 2010 ) when the conducted aps are passing the dura mater at the proximal end of the internal auditory meatus ( iam , a canal in the temporal bone for the passage of the cranial nerves vii and viii ) . peak latencies can be similar or different for recording electrodes close to the current source ( near field ) and at greater distances ( far field ) , e.g. an electrode in the vicinity of a single axon records a passing ap as a positive / negative / positive sequence of potentials as the axonal transmembrane current influx is separated by outflow in the leading and trailing part of the conducted spike . , lead to contradictory hypotheses : ( i ) peak i may be caused by the distal iam ending ( tasaki et al . , 1954 ) , ( ii ) the low cerebrospinal fluid ( csf ) conductance between the proximal iam end and the higher conductance of the brainstem may cause two sequential peaks namely negative peak in ( this is the next abr minimum after peak i , which is also called n1 ) and peak ii when aps enter brainstem ( martin et al . , 1995 ) , ( iii ) brown and patuzzi ( 2010 ) suggest that a single electrical barrier , the dura mater , produces both peak i and peak in when the leading respectively the trailing dipole of the conducted transmembrane currents encounter this barrier . about 95% of sgcs are type i cells ( spoendlin , 1975 ) , which are innervated by inner hair cells ( ihc ) . in humans such a bipolar cell has a myelinated axon that is interrupted by a non - myelinated region around the soma . the non - myelinated somatic region is a human peculiarity , which is rarely found in cochleas of animals ( ota and kimura , 1980 ; nadol , 1988 ) . type ii sgcs , innervated by outer hair cells ( ohc ) , are non - myelinated , their axon and soma diameters are smaller and consequently signal conduction is slow ( about 1/9 ) in comparison to type i cells ( rattay et al . lack of insulation by internodes in axons of type ii cells demands essentially stronger transmembrane currents compared to axons of type i cells . concerning the origin of abr peak i , this fact needs an investigation as the higher ion currents could compensate for the small numbers of type ii cells . using a biophysical model ( fig . , 2001a ) we could show that spike conduction in mammalian sgcs is characterized by the following four phases : a postsynaptic delay , constant velocity in the peripheral axon , a presomatic delay and constant velocity in the central axon ( rattay et al . on the other hand our computational evaluation of the membrane currents demonstrated that surprisingly high postsynaptic currents from ihc ribbon synapses with amplitudes of 400 pa ( grant et al . the goal of the following investigation is to analyze whether such strong postsynaptic currents in the peripheral terminals of sgcs could generate abr peak i or which other neural components could be the generators . a compartment model of a human type i cell is described in detail in rattay et al . according to the biophysical model approach , strong sodium current influx is especially needed in the presomatic region to load the large capacitance of the non - myelinated soma in order to conduct the spike across the soma into the central axon ( rattay et al . the high sodium channel density in the presomatic region is also confirmed experimentally in non - myelinated type i cells of the mouse ( hossain et al . 2b results in(1)(vi , nve , n)tcm , n+iion , n+vi , nvi , n1rn/2+rn1/2+vi , nvi , n+1rn/2+rn+1/2=0,where the first two terms are the transmembrane currents which are the postulated generator elements of the abr signal . the effective noise current is assumed to be proportional to the square root of the number of sodium channels within a compartment ( for details see rattay et al . in compartments with passive membranes ( internodes ) ( 1 ) and ( 2 ) is a current with constant conductance iion = gmanvn / n , where membrane conductance gm is 1 ms / cm and the number of insulating layers of cell membranes n is 40 and 80 for the peripheral and central axon , respectively ( rattay et al . using this method the temporal profiles of the transmembrane voltage and the transmembrane currents of each compartment were computed . at this temperature ap duration fits recorded data and , moreover , the model is appropriate for the non - myelinated regions of sgcs ( hartmann et al . the shape and amplitude of the postsynaptic currents of type i cells were defined by an average amplitude i0 of 400 pa , 280 s for time to peak and a decay time constant of 370 s as recorded in adult p20 and p60 rats ( grant et al . the same shape but with smaller amplitude i0 of 26 pa was used for the stimulation of type ii cells corresponding to signal strengths recorded by weisz et al . ( 4 ) together with the superposition principle , a first approach for the vabr signal contribution of a single activated sgc was simulated as the sum of the contributions of both transmembrane current components from all compartments(5)vabr=e4n=1dim(iion , n+ic , n)/rn . as explained above , for every compartment the transmembrane current has a resistive component iion and a capacitance component ic = cmdv / dt , which is the first term in eq . the model consisted of the skull , the ear canal , the cochlea , the internal auditory meatus , the cerebrospinal fluid , the brain and the auditory nerve itself ( fig . the surface of the cylinder acted as a time dependent current source summarizing the transmembrane currents of 1000 active sgcs within the corresponding volume . along the surface of the active cylinder f(x , t ) represented the transmembrane currents of 1000 synchronously firing sgcs . in order to show the influence of stimulus strength , we applied this 26 pa stimulus intensity again to a type i cell , although it was the mean postsynaptic current recorded in rat type ii cells ( weisz et al . due to the strong stimulus , the ap peak at p0 of a type i cell was 21.7% larger than the conducted membrane voltage peak , e.g. however , both transmembrane currents , iion and ic , at p0 exceeded that at p4 because of the bigger surface area of the active membrane ( fig . it is important to note that membrane currents are of both polarities and the effective difference between inward and outward currents of a compartment is often essentially smaller than the single components . the next investigation was a simplified approach to calculate the abr signal by assuming a recording electrode relatively close to the peripheral sgc section . 2c , that is 2 mm in orthogonal direction of the midmodiolar plane which contains the neural path of a target sgc and also a small spherical stimulating electrode . the conducted ap was delayed in the non - myelinated somatic region because of its high capacitance ( rattay et al . , 2001a ) before being conducted along the thicker central axon with the doubled velocity of the peripheral process ( rattay et al . the simulated membrane currents in the nodes and internodes of the central axon were exactly of doubled intensity compared to that of the peripheral axon when the number of insulating sheets , internodal length and fiber diameter is doubled . although axonal membrane currents were of doubled amplitude in the central axon their impact was too small to cause abr peak i. evaluation of eq . ( 5 ) demonstrated that the largest abr wave i contribution of the simulated type i cell resulted from its soma region , whereas no part of the peripheral process had a substantial effect ( fig . the next computer experiment was used to analyze the contribution of unmyelinated type ii cells on abr wave i. for this purpose the peripheral process of the standard sgc was assumed to be unmyelinated and the region from p0 to the soma was modeled as homogeneous membrane with original hodgkin - huxley ion channel densities . ( ii ) both types of transmembrane currents of an average compartment were more similar in shape and intensity than those of the node - internode combination where the ion current dominated in the node and the capacitance current in the internode ( fig . ( iii ) transmembrane current strengths were larger in a section , which was assumed earlier to be myelinated , as the total number of ion channels in this section increased by a large extent . nevertheless , the increased currents of the peripheral axon could not contribute as much as the soma region to the extracellularly recorded potential . to emphasize the conclusions about the dominant role of sgc somas as generators of human abr peak i , the transmembrane currents from a bundle of 1000 synchronously firing type i cells were evaluated with a simplified head model ( fig . 11a for t = 200 s , t = 750 s and t = 2200 s , representing times after stimulus onset when the spike is in the peripheral axon , in the soma region and in the central axon close to brainstem , respectively . 7b and c ) and the spatial shape of the central axon currents was the same as seen for t = 2200 s in fig . a simulated recording ( 2 mm from the surface of the active cylinder ) and the near field response ( 0.1 mm from the surface of the active cylinder ) transmembrane currents of the bypassing spikes dominated and caused the shape of the recorded signal ( as function of time ; fig . 11c ) to be similar to the mirrored picture of the transmembrane currents ( as functions of space ; fig . 12a shows simulated intracochlear potentials under the following three conditions : ( i ) the quite realistic nerve geometry of fig . 2 but a homogeneous infinite medium , ( ii ) fe solution for rectified sgcs in an active cylinder and assuming the csf conductance value within the cochlea , ( iii ) same situation as in ( ii ) but the cochlea was simulated with the conductance of nerve tissue . in case ( i ) the peripheral axon was close to the recording electrode and consequently the first membrane currents caused stronger recorded amplitudes . this synchrony of the first peak demonstrates that it was not generated by a change in the conductance of the extracellular medium but it appeared just at the time when the transmembrane currents reach their maximum values . the almost synchronous initiation of a high number of spikes and their conduction in the auditory nerve cause a spatial - temporal pattern of currents within the head . at the time of peak i , the electrical situation in the head and on its surface is characterized by isopotentials . the phrase auditory brainstem response implies the assumption that all its constituent waves may be initiated in the brainstem . along the pathway of the auditory nerve signal polarity changes at the proximal end of internal auditory meatus . several investigators found convincing arguments that also p2 is generated by the auditory nerve when volleys of spikes in afferent fibers enter the brainstem ( moller , 1983 , 1988 ; martin et al . moreover , it is not clear which of the abr peaks ivii are stationary potentials . here , we used a simplified human head model in order to analyze which of the early abr peaks are stationary potentials . due to the similar conductances of the bone and dura mater the simulated conducted spikes generate abr n1 when crossing the ( bone + dura mater)/csf border and abr p2 when arriving at the csf / brainstem interface ( fig . according to our simulations peak i is generated in the soma region within the cochlea and not at the proximal end of the iam as supposed by brown and patuzzi ( 2010 ) . according to this hypothesis latency difference n1p1 should be the same as the arrival time difference of the two dipoles at the conductance barrier . yet , at least in human , the dura mater and the arachnoidal membrane invaginate into the iam from the porus to the fundus ( lescanne et al . , 2002 ) and at the transition zone between peripheral and central nervous system the nerve fibers cross the glia limitans not the dura mater ( fraher , 2002 ) , a glial barrier of much lower thickness than the dura . thus , the dura mater and the glia limitans are unlikely to be causative for stationary potentials . thus , a single barrier alone can not be the cause of the successive peaks i and in as suggested by brown and patuzzi ( 2010 ) . , 1995 ) and 2.32 1.67 = 0.65 ms ( table 3 of hughes and fino , 1985 ) are , however , rather close to our results of 0.80 ms ( fig . some authors could record the early abr peak i , which is assumed to result from postsynaptic currents of ihcs in type i sgcs ( hughes and fino , 1985 ; moore et al . our biophysical model approach underlines the assumption that the recorded peak i is caused by the surprisingly strong postsynaptic currents ( fig . i of 570 s ( left ears ) and 620 s ( right ears ; hughes and fino , 1985 ) are close to our intra - cochlear 580 s latency ( fig . these corresponding latencies i i and the observations that n1 recorded as a compound action potential ( cap ) of the auditory nerve at the round window appears simultaneously with abr p1 ( jewett , 1970 ; gersdorff , 1982 ; moller et al . regarding wave ii this hypothesis was disproved as the wave ii peak was recently shown to be generated as stationary potential in guinea pig ( brown and patuzzi , 2010 ) . according to this simulation , contrary to cat , in humans the strong transmembrane currents in the nonmyelinated soma region is seen to generate the first abr responses . the smaller number of type ii cells is compensated by large transmembrane currents typical for non - myelinated axons . however , for three reasons they can not rival type i cells concerning abr peak i generation . first , ap duration is assumed to be the same for both cell types and a dipole model is used where the total transmembrane currents are reduced to two spatially separated currents . in contrast to type i cells , the slow spike conduction velocity of non - myelinated axons causes a short dipole length . second , the postsynaptic currents from ohc ribbon synapses occur relatively rarely and at 26 pa they are strikingly smaller than the 400 pa amplitudes from ihcs ( grant et al . , the simulated response to the weak stimulus created a postsynaptic delay in the order of 800 s for spike generation in type ii cells ( rattay et al . , 2013 ) , which together with the slow ap conduction , hinders an early contribution of the capacitance currents from somas of type ii cells . our analysis showed that the transmembrane currents of the peripheral axons are rather small ( fig . 6b and c ) and they can not be the generators of abr peak i. further evidence for this argument comes from eabr in cochlear implantees . in contrast to acoustically evoked auditory nerve excitations electrical stimulation causes no delay by mechanical components or by the synaptic processes at the hair cells as the electric field directly stimulates elements of the auditory nerve . in most cases spikes elicited by human cochlear implants are expected to be initiated in the central axons and not at the peripheral sgc ending as in fig . it is important to note that in people with hearing loss most of the peripheral axons degenerate whereas much more central axons seem to be intact and excitable ( felix et al . because of the rather long peripheral human axons , usually the stimulating electrode is distal to the soma and for such cases a theoretical analysis predicts that sgc without peripheral processes can not be excited with cathodic pulses ( rattay , 1999 ; rattay et al . therefore , spikes are expected to be initiated in most cases in the distal segments of the central axon in cochlear implantees ( rattay et al . these elicited aps generate the large capacitance currents at the somas and the same electrical signaling as in natural hearing when the spikes are conducted along the central axons . thus the temporal coincidence of 2.1 ms for the eabr peak iii latency and the 2.1 ms for the difference between peaks iii and i in the healthy ear are strong indicators that the intense soma currents are the generator elements of peak i. in contrast to the human , nearly all studied mammalian type i cells are myelinated in the soma region in addition to their myelinated axons . , 2001a ) resulting in a weaker influence of the soma on the abr signal . 2 ) demonstrates that the transmembrane currents in myelinated soma regions of type i cells may still be strong enough to be the generators of peak i in mammals too . the investigations were concentrated on the evaluation of the transmembrane currents of single representative type i and type ii cells and not on a model that simulates an abr signal as recorded at the scalp . during the period of an ap the signal shape for the sum of ion currents is comparable with other models for myelinated peripheral axons but their ion current density amplitudes in the nodes of ranvier are a bit larger ( rattay and aberham , 1993 ; rattay et al . , resulting corrections from a more detailed modeling are not expected to contradict the dominant role of the somatic region in generating abr peak i.

administratively , the ladakh region in the state of jammu and kashmir ( india ) is divided into two districts : the western kargil district and the eastern leh district . this rain - shadow region is semiarid to arid , with winter temperatures dropping to below 25c . western and central leh are mostly rugged , relatively lower in elevation , and inhabited by agropastoralists . the eastern part of the district , called changthang , on the other hand , comprises high plateaus and rolling hills ( usually > 4000 m ) interspersed with lake and river basins that have moist patches of relatively dense graminoid vegetation . changthang covers approximately 20,000 km and is inhabited by the nomadic changpa community and tibetan refugees , both of whom primarily rear goat and sheep . according to the human census in 2001 conducted by the government , the population of the leh district was 117,637 , with the majority ( 85% ) being in the western region . the population is sparse in the changthang ( 13,444 ) with a density of approximately 0.7 km . ladakh is home to a diverse assemblage of wild carnivores , ungulates , and birds ( pfister 2004 ) . within ladakh , our survey covered most of this region , from the pangong tso in the north , along the eastern border with tibet , to hanle in the south ( figure 1 ) . we divided the entire region into five blocks based on their location and overall similarity in terms of topography and vegetation , and surveyed each block ( table 1 ) . n ; approximately 2800 km ) extends from the zanskar mountains in the south to the indus valley in the north into which the hanle river drains at loma . the higher reaches are primarily rolling mountains and plateaus , while the mid and lower portion is a wide valley fringed by a high range exceeding 5000 m on the east , and a lower , more gradual ridge on the west . the estimated viewscape is based on modeling for areas visible within 3 km on either side of the road ( see text for details ) . distribution of kiang in ladakh is also shown ( based on chundawat and qureshi 1999 ) . we also surveyed some additional adjacent areas with similar habitat.table 1density of kiang in the surveyed blocks of eastern ladakh , indiasurvey blocktransect length ( km)area ( km)no . kiangkiang density ( km)loma - demchok ( indus)822882490.86hanle valley66347780.23chushul - loma63299190.06parma valley7436090.03pongong tso80359100.03total36516533650.24 ( 95% cl 0.44 ) the five blocks surveyed for kiang in eastern ladakh during 2000 to 2003 . the estimated viewscape is based on modeling for areas visible within 3 km on either side of the road ( see text for details ) . distribution of kiang in ladakh is also shown ( based on chundawat and qureshi 1999 ) . density of kiang in the surveyed blocks of eastern ladakh , india through archival research and field observations , we documented the intensifying human we describe the perceptions of the ladakhi administration regarding this conflict , and the efforts they have undertaken to address it . through archival research , we also examine recent land - use changes in eastern ladakh , describe the growing pashmina or cashmere industry , and its fallouts for the kiang human conflict . literature was also surveyed for obtaining information on the past status and distribution of the kiang . we also obtained data on livestock populations from the sheep husbandry departments in nyoma and leh , to estimate the livestock density in eastern ladakh . we had informal conversations with over 50 herders to learn about their perceptions regarding conflicts . our data on the kiang population in ladakh come from rangewide surveys conducted in the year 2000 . the study area was surveyed from a slow - moving vehicle ( traveling at 1012 km / hour ) and areas on either side were periodically scanned using an 8 binocular and/or 20 spotting scope . on every sighting , animals were counted and classified , and habitat variables such as topographic feature , elevation , slope , aspect , dominant plant species , and approximate plant cover were recorded . classification of kiang by sex was not possible in most instances due to the similarity between the sexes , especially when seen from a distance . a total distance of 365 km was covered in the changthang , traveling on an average 6070 km per day . we also covered approximately 50 km on foot in the upper catchment of the hanle valley . subsequently , in the spring of 2001 , 2003 , and 2004 , we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the hanle valley , which had emerged as an area with moderate to high kiang density during the 2000 survey . density for kiang and livestock was calculated by estimating the area covered in each segment using geographical information system ( gis ) tools . the area surveyed along the roads viewscape in arcinfo , which effectively created polygons of all area visible from the road . the base map used for this estimation was the national imagery and mapping agency s vector map level 0 ( digital chart of the world ) edition : 5 . this was integrated with the digital elevation model ( resolution 90 m or 3 arc seconds ) shuttle radar topography mission ( srtm ) data from the space shuttle mission ( for the year 2000 ) obtained from ftp://e0mss21u.ecs.nasa.gov/srtm/ to generate maps of the study area with numerous layers of information that included elevation , aspect , and drainage . after creating the viewscape , we used a cutoff distance of 3 km on either side of the road , within which all visible areas were calculated . this was generally our maximum sighting distance for the kiang ( most rangefinders do not work at this distance ) . we estimated the extent of the surveyed areas ( through viewscapes ) for both the rangewide surveys as well as the region of our repeated kiang counts in hanle valley . as a preliminary exercise to understand the extent of forage consumption by kiang vis - - vis livestock , we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake ( as percent of body mass ) among herbivores ( foose 1982 ) . in the absence of population structure data for both kiang and livestock , we used adult body masses ( averaged between sexes ) from mishra and others ( 2002 ) . we used the established relationships for foregut fermenters ( goat , sheep , cattle , yak , cattle - yak hybrids ) and hindgut fermenters ( kiang , horses , donkeys ) , as appropriate . forage quality in the highly seasonal cold desert steppe of ladakh is expected to vary strongly between seasons , with high - protein , low - fiber forage being available in summer , and low - protein , high - fiber forage in winter . we therefore estimated forage consumption by each species separately for winter and summer , using the relationships for low - protein , high - fiber grass hay diet for winter ( 4.57.4% crude protein and 6570% cell wall ) and high - protein , low - fiber legume hay diet for summer ( 17.422.2% crude protein and 3156% cell wall ) ( foose 1982 ) . for hindgut fermenters , daily forage intake as a percent of body mass ( w ) in summer was estimated as 13.8w , and 6.95w in winter . similarly , for the ruminants ( goat , sheep , cow , dzo / dzomo , yak ) , it was estimated as 7.31w in summer and 4.04 w in winter ( foose 1982 ) . administratively , the ladakh region in the state of jammu and kashmir ( india ) is divided into two districts : the western kargil district and the eastern leh district . this rain - shadow region is semiarid to arid , with winter temperatures dropping to below 25c . western and central leh are mostly rugged , relatively lower in elevation , and inhabited by agropastoralists . the eastern part of the district , called changthang , on the other hand , comprises high plateaus and rolling hills ( usually > 4000 m ) interspersed with lake and river basins that have moist patches of relatively dense graminoid vegetation . changthang covers approximately 20,000 km and is inhabited by the nomadic changpa community and tibetan refugees , both of whom primarily rear goat and sheep . according to the human census in 2001 conducted by the government , the population of the leh district was 117,637 , with the majority ( 85% ) being in the western region . the population is sparse in the changthang ( 13,444 ) with a density of approximately 0.7 km . ladakh is home to a diverse assemblage of wild carnivores , ungulates , and birds ( pfister 2004 ) . within ladakh , our survey covered most of this region , from the pangong tso in the north , along the eastern border with tibet , to hanle in the south ( figure 1 ) . we divided the entire region into five blocks based on their location and overall similarity in terms of topography and vegetation , and surveyed each block ( table 1 ) . n ; approximately 2800 km ) extends from the zanskar mountains in the south to the indus valley in the north into which the hanle river drains at loma . the higher reaches are primarily rolling mountains and plateaus , while the mid and lower portion is a wide valley fringed by a high range exceeding 5000 m on the east , and a lower , more gradual ridge on the west . the estimated viewscape is based on modeling for areas visible within 3 km on either side of the road ( see text for details ) . distribution of kiang in ladakh is also shown ( based on chundawat and qureshi 1999 ) . we also surveyed some additional adjacent areas with similar habitat.table 1density of kiang in the surveyed blocks of eastern ladakh , indiasurvey blocktransect length ( km)area ( km)no . kiangkiang density ( km)loma - demchok ( indus)822882490.86hanle valley66347780.23chushul - loma63299190.06parma valley7436090.03pongong tso80359100.03total36516533650.24 ( 95% cl 0.44 ) the five blocks surveyed for kiang in eastern ladakh during 2000 to 2003 . the estimated viewscape is based on modeling for areas visible within 3 km on either side of the road ( see text for details ) . distribution of kiang in ladakh is also shown ( based on chundawat and qureshi 1999 ) . we describe the perceptions of the ladakhi administration regarding this conflict , and the efforts they have undertaken to address it . through archival research , we also examine recent land - use changes in eastern ladakh , describe the growing pashmina or cashmere industry , and its fallouts for the kiang human conflict . literature was also surveyed for obtaining information on the past status and distribution of the kiang . we also obtained data on livestock populations from the sheep husbandry departments in nyoma and leh , to estimate the livestock density in eastern ladakh . we had informal conversations with over 50 herders to learn about their perceptions regarding conflicts . our data on the kiang population in ladakh come from rangewide surveys conducted in the year 2000 . the study area was surveyed from a slow - moving vehicle ( traveling at 1012 km / hour ) and areas on either side were periodically scanned using an 8 binocular and/or 20 spotting scope . on every sighting , animals were counted and classified , and habitat variables such as topographic feature , elevation , slope , aspect , dominant plant species , and approximate plant cover were recorded . classification of kiang by sex was not possible in most instances due to the similarity between the sexes , especially when seen from a distance . a total distance of 365 km was covered in the changthang , traveling on an average 6070 km per day . we also covered approximately 50 km on foot in the upper catchment of the hanle valley . subsequently , in the spring of 2001 , 2003 , and 2004 , we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the hanle valley , which had emerged as an area with moderate to high kiang density during the 2000 survey . density for kiang and livestock was calculated by estimating the area covered in each segment using geographical information system ( gis ) tools . the area surveyed along the roads viewscape in arcinfo , which effectively created polygons of all area visible from the road . the base map used for this estimation was the national imagery and mapping agency s vector map level 0 ( digital chart of the world ) edition : 5 . this was integrated with the digital elevation model ( resolution 90 m or 3 arc seconds ) shuttle radar topography mission ( srtm ) data from the space shuttle mission ( for the year 2000 ) obtained from ftp://e0mss21u.ecs.nasa.gov/srtm/ to generate maps of the study area with numerous layers of information that included elevation , aspect , and drainage . after creating the viewscape , we used a cutoff distance of 3 km on either side of the road , within which all visible areas were calculated . this was generally our maximum sighting distance for the kiang ( most rangefinders do not work at this distance ) . we estimated the extent of the surveyed areas ( through viewscapes ) for both the rangewide surveys as well as the region of our repeated kiang counts in hanle valley . as a preliminary exercise to understand the extent of forage consumption by kiang vis - - vis livestock , we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake ( as percent of body mass ) among herbivores ( foose 1982 ) . in the absence of population structure data for both kiang and livestock , we used adult body masses ( averaged between sexes ) from mishra and others ( 2002 ) . we used the established relationships for foregut fermenters ( goat , sheep , cattle , yak , cattle - yak hybrids ) and hindgut fermenters ( kiang , horses , donkeys ) , as appropriate . forage quality in the highly seasonal cold desert steppe of ladakh is expected to vary strongly between seasons , with high - protein , low - fiber forage being available in summer , and low - protein , high - fiber forage in winter . we therefore estimated forage consumption by each species separately for winter and summer , using the relationships for low - protein , high - fiber grass hay diet for winter ( 4.57.4% crude protein and 6570% cell wall ) and high - protein , low - fiber legume hay diet for summer ( 17.422.2% crude protein and 3156% cell wall ) ( foose 1982 ) . for hindgut fermenters , daily forage intake as a percent of body mass ( w ) in summer was estimated as 13.8w , and 6.95w in winter . similarly , for the ruminants ( goat , sheep , cow , dzo / dzomo , yak ) , it was estimated as 7.31w in summer and 4.04 w in winter ( foose 1982 ) . kiang inhabits the open plains and rolling mountains of the changthang plateau in eastern ladakh at altitudes between 4000 m and 5500 m. it has a total estimated range of approximately 7400 km in ladakh ( chundawat and qureshi 1999 ; figure 1 ) . the species was common in ladakh during the earlier part of the 20th century ( stockley 1936 ) , but is believed to have declined substantially with the buildup of armed forces in ladakh during the war between india and china in 1962 ( fox and others 1991 ) . there are no population estimates available for the period before or immediately following this war , but it is believed that the population had recovered by the 1980s . the only published estimate of their population comes from the late 1980s when approximately 1500 animals were estimated to occur in ladakh ( fox and others 1991 ; shah 2002 ) . it thus appears that for at least a decade after the war , the kiang population remained relatively small in ladakh . the genesis of the present conflict can be traced back to the early 1980s , by which time their population had apparently recovered ( fox and others 1994 ) . the prevalent belief in ladakh during that time , and one that continues until today , is that the kiang population has drastically increased in eastern ladakh because many animals have moved from tibet to the indian side of the border to escape persecution ( fox and others 1991 ; anon . , there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock , and demands that the government should drive the kiang back into tibet the tibetan plateau was inhabited by nomadic hunting people 30,000 years ago , and nomadic pastoralists have been herding livestock on the plateau for at least two millennia ( schaller 1998 ) . the nomads , who became buddhists presumably in the last millennium , live in tents ( ribos ) in seasonal camps . in the past , the few nomadic groups inhabiting eastern ladakh belonging to the changpa community traveled distances exceeding 100 km during their seasonal migrations ( phuntsok 2000 ; hagalia 2004 ) . this has recently changed , and most seasonal movements are now restricted to within 4050 km ( hagalia 2004 ) . after the 1960s , there has been a steady increase in the population of the changpa and their livestock , presumably facilitated by access to better health care , provisions , and subsidies , both from the government as well as the army ( chaudhuri 2000 ; phuntsok 2001 ; bhatnagar and wangchuk 2001 ) . supplemental forage provided by the government in the event of severe winters now offsets most of the starvation - related livestock mortality , which in the past was perhaps an important factor limiting the livestock population . the 1962 war between india and china also led to the curtailment of movement of the changpa herders from eastern ladakh into parts of tibet , considerably reducing the availability of grazing land . many of their pastures such as skagzung , the primary winter pasture of the rupshu changpas ( figure 1 ) , became unavailable , effectively escalating the stocking density in eastern ladakh . furthermore , a large number of tibetan refugees have moved into eastern ladakh with their livestock herds since the late 1950s , thus contributing to a substantial increase in the number of herding families and the livestock population ( bhatnagar and wangchuk , 2001 ; hagalia 2004 ) . as per the government records , the livestock population in ladakh ( including changthang ) has doubled in about two decades , from approximately 212,500 in 1977 to 487,000 in 1999 ( richard 1999 ; bhatnagar and wangchuk 2001 ) . in 1999 , changthang constituted 44% of ladakh s livestock population , most of which ( 94% ) were sheep and goats . one of the most important livestock products from the region is the pashmina or cashmere , a fine luxury fiber derived from the underwool of the local changra goat . traditionally , more than 95% of cashmere from ladakh has been used for barter with or sale to traders from the neighboring kashmir region under a treaty with the erstwhile kingdom of kashmir dating from 1684 ( rizvi 1999 ) . over the last two decades , however , cashmere production in ladakh has been promoted by the government in a substantial way . this has included setting up of goat breeding farms , efforts at improving yield , and provisioning of veterinary services to nomads that has brought down the kid mortality rates to 23% from an estimated 3040% ( jina 1995 ) . however , the efforts at increasing the per animal cashmere yield have apparently had very limited success ( jina 1995 ) , suggesting that increasing cashmere production necessitates an increase in the goat population . more recently , with part support from the central government of india , the international fund for agricultural development and the undp , the ladakh administration has set up a cashmere de - hairing plant , expected to result in considerable added value to the cashmere from ladakh ( joshi and morup 2003 ; mital 2004 ) . presently , ladakh contributes merely 30,000 kg ( 0.37% ) to the global cashmere production and the administration is eager to increase ladakh s contribution in global trade . the recent intensification of human the human and livestock populations in kiang habitat have significantly increased in the last four decades due to factors ranging from better healthcare to the influx of refugees . furthermore , promotion of production and value of cashmere has meant that livestock rearing is fast becoming a cash - based enterprise that increasingly caters directly to the lucrative global cashmere market . herders are realizing the importance of every bit of pastureland in maximizing cashmere production and see kiang as a direct threat to pasture production . against this background , it is perhaps not surprising that people are fast losing their tolerance of the kiang . in our rangewide survey of the kiang , we surveyed an estimated area of 1653 km , which is 22% of the total range of the kiang in ladakh ( 7400 km ; chundawat and qureshi 1999 ) . the average density in the surveyed blocks was 0.24 ( 95% cl 0.44 ) kiang km , with the highest abundance along the upper indus from loma to demchok followed by the hanle valley ( table 1 , figure 1 ) . with an overall livestock population of 210,000 ( as per unpublished records from the nyoma office of the sheep husbandry department ( 2004 ) ) in the nyoma and durbuk blocks that comprise the changthang region of ladakh , the livestock density in the region was 11 km . thus , in terms of density , kiang represents only 2% of the total herbivore population ( kiang and livestock together ) in the changthang , with the bulk being contributed by goats ( 57% ) . the group sizes of kiang observed during the rangewide survey varied from 1 to 74 . the mean group size was 2.8 ( 95% cl 0.8 ) , with most of the sightings ( 55% ) being of solitary animals . most kiang were seen in open wide valleys ( 79% , n = 202 ) , followed by stony slopes ( 19% ) and very rarely in broken areas ( 2% ) . there were no sightings on slopes steeper than 30. we observed kiang between elevations of 4100 and 5500 m , with most sightings between 4200 and 4300 m. most kiang groups ( 73% ) were seen on sedge ( carex spp . and kobresia spp . ) and grass ( stipa spp . ) meadows in the valley bottom , which were occasionally interspersed with bushes of caragana sp . these meadows appear to be critical habitat for kiang during summer and winter as also reported by schaller ( 1998 ) , but are limited in extent to narrow discontinuous strips in moist areas along valleys . the remaining sightings were in areas that had sparse to dense artemisia steppe ( 25% ; with tanacitum sp . and sedum sp . being the other dominant plants ) and high altitude forb meadows ( 2% ; saussurea spp . , salsola sp . , and polygonum sp . as the dominant plants ) . at a landscape level covering entire eastern ladakh , of the total daily forage consumption by large herbivores , we estimated that kiang consume 1011% and livestock 8990% ( table 2 ) . among livestock , goats were estimated to consume the maximum forage ( 41% to 43% of the total forage consumed ) , followed by sheep ( 26% to 27% ) . table 2herbivore density and estimated forage consumption by kiang vis - - vis livestock in changthangspeciesbody weight(kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.241.6101.211livestock donkey900.040.110.11 horse2480.150.950.76 cattle1910.160.740.54 yak2980.261.591.110 yak cattle hybrids2270.010.03<10.02<1 sheep343.984.4272.926 goat336.446.9434.541livestock total112111.0314.5909.789total11.2716.0810.96body weights are taken from mishra ( 2001 ) and have been averaged between sexes . note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult . estimated forage consumption by kiang vis - - vis livestock in changthang body weights are taken from mishra ( 2001 ) and have been averaged between sexes . note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult . our subsequent estimate of the kiang population in the hanle valley ( the region between rongo and kalangtartar ) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang / km ( 95% cl 0.08 ) . we estimated that on an average , kiang remove only 3% to 4% of the total forage consumed in the hanle valley , with the greater part being consumed by goats ( 45% to 47% ) followed by yaks ( 20% to 21% ) and sheep ( 20% ) ( table 3 ) . table 3herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valleyspeciesbody weight ( kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.563.632.84livestock donkey9000000 horse2481.579.587.49 cattle1910.662.721.92 yak2983.9122.82016.521 yak cattle hybrids22700000 sheep3421.5223.72015.519 goat3350.6954.54735.545livestock total112178.34113.29776.796total78.9032.479.6 herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valley most wars in the last five decades have resulted in detrimental effects on wildlife ( blom 2000 ; dudley and others 2002 ; mishra and fitzherbert 2004 ) , and the war between india and china in 1962 was no exception , particularly from the viewpoint of kiang conservation . our review suggests that the kiang population in ladakh declined drastically in the years during and after the war between india and china in the 1960s , possibly due to hunting , land mines , and disturbances associated with the war . thus , for two decades during and after the war , a generation of herders presumably saw and became used to a very low density of kiang in ladakh s rangelands . not surprisingly , the genesis of kiang human conflict in ladakh can be traced back to the 1980s , the period by which the kiang population had recovered . it seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km has led to the perception to a generation of nomads that ladakh s rangelands are now overstocked with kiang . available estimates from three regions in tibet place the density of kiang between 0.15 and 0.39 kiang km ( schaller 1998 ) , suggesting that the kiang density in ladakh is comparable with those in tibet . furthermore , our results show that the share of forage consumed by the kiang vis - - vis livestock is relatively small ( 1011% in the whole of eastern ladakh , and 34% in hanle valley ) . thus , at a landscape level , the perception of the overabundance of kiang in ladakh , and of kiang compromising cashmere production , is clearly unfounded . nevertheless , there is variation in kiang density locally ( 0.03 to 0.86 kiang km recorded in our surveys ) . kiang occasionally congregate in large herds and can together remove considerable forage from the low - productivity trans - himalayan pastures . using the data in table 2 , a group of 70 kiang ( the largest group seen during the survey was 74 ) can be estimated to potentially consume 199254 kg dry forage in a single day . the peak standing biomass in sedge meadows , which seem to be important kiang habitats , has been estimated in the adjoining region of spiti at 857 kg ha ( mishra 2001 ) . these habitats seem critical for both kiang and livestock , but cover a small fraction of the landscape , with an estimated 150 km in the entire approximately 20,000 km of changthang . illustratively , this means that in a single day a large group of 70 kiang can potentially consume more than a quarter of the standing biomass from 1 ha of a key resource area . this consumption is also very similar to the estimated daily forage consumption by a herd of 200 goats ( approximately 215 kg ) . because of the long winters ( november to april ) and short summers ( july august ) , the potential for regrowth during summer is low , aggravating the impact . considering the potential of large kiang herds to remove significant amounts of forage , together with the fact that grazing areas have been reduced and the livestock population in ladakh more than doubled in the last 30 years , it is not difficult to see that at a localized scale of the valued sedge meadows , some herders must be facing serious forage competition from the kiang . given the intensifying commercialization of cashmere production in ladakh and its integration with global markets , this localized forage competition is , perhaps understandably , no longer tolerated , and the conflict needs to be addressed immediately . however , it must be borne in mind that the problem seems to be evident at local scales , and will need to be addressed at that scale . in the absence of any scientific understanding of the conflict so far , or efforts to document it , this local conflict has already begun to take on a serious political dimension . the politicians and the district administration in ladakh are blaming the kiang for compromising cashmere production , and the wildlife department is being looked upon to provide a solution . the only on - ground reaction of the district administration has been to fence large productive sedge and grass meadows from the kiang . in the hanle valley alone , an estimated 120 ha of meadows have been fenced already . three fourths of our total kiang sightings during the rangewide surveys were in such meadows , suggesting that this is an important habitat for the species , and continued fencing of this habitat may cause a decline in the kiang population of ladakh . although our review of the recent history of political and socioeconomic developments in kiang habitat and our estimates of density and forage consumption of kiang and livestock bring to light interesting dimensions of the kiang human conflict , the current level of understanding of the kiang s ecology and its relationships with livestock is still very preliminary . intensive research needs to be promoted to assist in better management of the species , and its conflicts with humans . monitoring programs need to assess kiang and livestock density , as well as the land use changes in kiang habitat . is locally concentrated in the key resource areas that contain the sedge meadows , and management strategies need to be devised at that local scale . surveys are needed to identify all high - conflict areas , and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang . it needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in ladakh . the kiang is a striking and conspicuous wild animal that may provide a potential for ecotourism . finally , after taking up the above interventions , it is important that fences on the sedge meadows to exclude kiang be removed and the practice of fencing discontinued because they lock out key resource areas from the kiangs range . without access to key resource areas , kiang and other wild herbivores , such as the tibetan gazelle , procapra picticaudata , kiang inhabits the open plains and rolling mountains of the changthang plateau in eastern ladakh at altitudes between 4000 m and 5500 m. it has a total estimated range of approximately 7400 km in ladakh ( chundawat and qureshi 1999 ; figure 1 ) . the species was common in ladakh during the earlier part of the 20th century ( stockley 1936 ) , but is believed to have declined substantially with the buildup of armed forces in ladakh during the war between india and china in 1962 ( fox and others 1991 ) . there are no population estimates available for the period before or immediately following this war , but it is believed that the population had recovered by the 1980s . the only published estimate of their population comes from the late 1980s when approximately 1500 animals were estimated to occur in ladakh ( fox and others 1991 ; shah 2002 ) . it thus appears that for at least a decade after the war , the kiang population remained relatively small in ladakh . the genesis of the present conflict can be traced back to the early 1980s , by which time their population had apparently recovered ( fox and others 1994 ) . the prevalent belief in ladakh during that time , and one that continues until today , is that the kiang population has drastically increased in eastern ladakh because many animals have moved from tibet to the indian side of the border to escape persecution ( fox and others 1991 ; anon . , there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock , and demands that the government should drive the kiang back into tibet the tibetan plateau was inhabited by nomadic hunting people 30,000 years ago , and nomadic pastoralists have been herding livestock on the plateau for at least two millennia ( schaller 1998 ) . the nomads , who became buddhists presumably in the last millennium , live in tents ( ribos ) in seasonal camps . in the past , the few nomadic groups inhabiting eastern ladakh belonging to the changpa community traveled distances exceeding 100 km during their seasonal migrations ( phuntsok 2000 ; hagalia 2004 ) . this has recently changed , and most seasonal movements are now restricted to within 4050 km ( hagalia 2004 ) . after the 1960s , there has been a steady increase in the population of the changpa and their livestock , presumably facilitated by access to better health care , provisions , and subsidies , both from the government as well as the army ( chaudhuri 2000 ; phuntsok 2001 ; bhatnagar and wangchuk 2001 ) . supplemental forage provided by the government in the event of severe winters now offsets most of the starvation - related livestock mortality , which in the past was perhaps an important factor limiting the livestock population . the 1962 war between india and china also led to the curtailment of movement of the changpa herders from eastern ladakh into parts of tibet , considerably reducing the availability of grazing land . many of their pastures such as skagzung , the primary winter pasture of the rupshu changpas ( figure 1 ) , became unavailable , effectively escalating the stocking density in eastern ladakh . furthermore , a large number of tibetan refugees have moved into eastern ladakh with their livestock herds since the late 1950s , thus contributing to a substantial increase in the number of herding families and the livestock population ( bhatnagar and wangchuk , 2001 ; hagalia 2004 ) . as per the government records , the livestock population in ladakh ( including changthang ) has doubled in about two decades , from approximately 212,500 in 1977 to 487,000 in 1999 ( richard 1999 ; bhatnagar and wangchuk 2001 ) . in 1999 , changthang constituted 44% of ladakh s livestock population , most of which ( 94% ) were sheep and goats . one of the most important livestock products from the region is the pashmina or cashmere , a fine luxury fiber derived from the underwool of the local changra goat . traditionally , more than 95% of cashmere from ladakh has been used for barter with or sale to traders from the neighboring kashmir region under a treaty with the erstwhile kingdom of kashmir dating from 1684 ( rizvi 1999 ) . over the last two decades , however , cashmere production in ladakh has been promoted by the government in a substantial way . this has included setting up of goat breeding farms , efforts at improving yield , and provisioning of veterinary services to nomads that has brought down the kid mortality rates to 23% from an estimated 3040% ( jina 1995 ) . however , the efforts at increasing the per animal cashmere yield have apparently had very limited success ( jina 1995 ) , suggesting that increasing cashmere production necessitates an increase in the goat population . more recently , with part support from the central government of india , the international fund for agricultural development and the undp , the ladakh administration has set up a cashmere de - hairing plant , expected to result in considerable added value to the cashmere from ladakh ( joshi and morup 2003 ; mital 2004 ) . presently , ladakh contributes merely 30,000 kg ( 0.37% ) to the global cashmere production and the administration is eager to increase ladakh s contribution in global trade . the recent intensification of human the human and livestock populations in kiang habitat have significantly increased in the last four decades due to factors ranging from better healthcare to the influx of refugees . furthermore , promotion of production and value of cashmere has meant that livestock rearing is fast becoming a cash - based enterprise that increasingly caters directly to the lucrative global cashmere market . herders are realizing the importance of every bit of pastureland in maximizing cashmere production and see kiang as a direct threat to pasture production . against this background , it is perhaps not surprising that people are fast losing their tolerance of the kiang . in our rangewide survey of the kiang , we surveyed an estimated area of 1653 km , which is 22% of the total range of the kiang in ladakh ( 7400 km ; chundawat and qureshi 1999 ) . the average density in the surveyed blocks was 0.24 ( 95% cl 0.44 ) kiang km , with the highest abundance along the upper indus from loma to demchok followed by the hanle valley ( table 1 , figure 1 ) . with an overall livestock population of 210,000 ( as per unpublished records from the nyoma office of the sheep husbandry department ( 2004 ) ) in the nyoma and durbuk blocks that comprise the changthang region of ladakh , the livestock density in the region was 11 km . thus , in terms of density , kiang represents only 2% of the total herbivore population ( kiang and livestock together ) in the changthang , with the bulk being contributed by goats ( 57% ) . the group sizes of kiang observed during the rangewide survey varied from 1 to 74 . the mean group size was 2.8 ( 95% cl 0.8 ) , with most of the sightings ( 55% ) being of solitary animals . most kiang were seen in open wide valleys ( 79% , n = 202 ) , followed by stony slopes ( 19% ) and very rarely in broken areas ( 2% ) . there were no sightings on slopes steeper than 30. we observed kiang between elevations of 4100 and 5500 m , with most sightings between 4200 and 4300 m. most kiang groups ( 73% ) were seen on sedge ( carex spp . and kobresia spp . ) and grass ( stipa spp . ) meadows in the valley bottom , which were occasionally interspersed with bushes of caragana sp . these meadows appear to be critical habitat for kiang during summer and winter as also reported by schaller ( 1998 ) , but are limited in extent to narrow discontinuous strips in moist areas along valleys . the remaining sightings were in areas that had sparse to dense artemisia steppe ( 25% ; with tanacitum sp . and sedum sp . being the other dominant plants ) and high altitude forb meadows ( 2% ; saussurea spp . , salsola sp . , and polygonum sp . as the dominant plants ) . at a landscape level covering entire eastern ladakh , of the total daily forage consumption by large herbivores , we estimated that kiang consume 1011% and livestock 8990% ( table 2 ) . among livestock , goats were estimated to consume the maximum forage ( 41% to 43% of the total forage consumed ) , followed by sheep ( 26% to 27% ) . table 2herbivore density and estimated forage consumption by kiang vis - - vis livestock in changthangspeciesbody weight(kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.241.6101.211livestock donkey900.040.110.11 horse2480.150.950.76 cattle1910.160.740.54 yak2980.261.591.110 yak cattle hybrids2270.010.03<10.02<1 sheep343.984.4272.926 goat336.446.9434.541livestock total112111.0314.5909.789total11.2716.0810.96body weights are taken from mishra ( 2001 ) and have been averaged between sexes . note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult . herbivore density and estimated forage consumption by kiang vis - - vis livestock in changthang body weights are taken from mishra ( 2001 ) and have been averaged between sexes . note that this will result in an overestimate of the offtake by kiang because we assumed that all kiang were adult . our subsequent estimate of the kiang population in the hanle valley ( the region between rongo and kalangtartar ) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang / km ( 95% cl 0.08 ) . we estimated that on an average , kiang remove only 3% to 4% of the total forage consumed in the hanle valley , with the greater part being consumed by goats ( 45% to 47% ) followed by yaks ( 20% to 21% ) and sheep ( 20% ) ( table 3 ) . table 3herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valleyspeciesbody weight ( kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.563.632.84livestock donkey9000000 horse2481.579.587.49 cattle1910.662.721.92 yak2983.9122.82016.521 yak cattle hybrids22700000 sheep3421.5223.72015.519 goat3350.6954.54735.545livestock total112178.34113.29776.796total78.9032.479.6 herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valley most wars in the last five decades have resulted in detrimental effects on wildlife ( blom 2000 ; dudley and others 2002 ; mishra and fitzherbert 2004 ) , and the war between india and china in 1962 was no exception , particularly from the viewpoint of kiang conservation . our review suggests that the kiang population in ladakh declined drastically in the years during and after the war between india and china in the 1960s , possibly due to hunting , land mines , and disturbances associated with the war . thus , for two decades during and after the war , a generation of herders presumably saw and became used to a very low density of kiang in ladakh s rangelands . not surprisingly , the genesis of kiang human conflict in ladakh can be traced back to the 1980s , the period by which the kiang population had recovered . it seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km has led to the perception to a generation of nomads that ladakh s rangelands are now overstocked with kiang . available estimates from three regions in tibet place the density of kiang between 0.15 and 0.39 kiang km ( schaller 1998 ) , suggesting that the kiang density in ladakh is comparable with those in tibet . furthermore , our results show that the share of forage consumed by the kiang vis - - vis livestock is relatively small ( 1011% in the whole of eastern ladakh , and 34% in hanle valley ) . thus , at a landscape level , the perception of the overabundance of kiang in ladakh , and of kiang compromising cashmere production , is clearly unfounded . nevertheless , there is variation in kiang density locally ( 0.03 to 0.86 kiang km recorded in our surveys ) . kiang occasionally congregate in large herds and can together remove considerable forage from the low - productivity trans - himalayan pastures . using the data in table 2 , a group of 70 kiang ( the largest group seen during the survey was 74 ) the peak standing biomass in sedge meadows , which seem to be important kiang habitats , has been estimated in the adjoining region of spiti at 857 kg ha ( mishra 2001 ) . these habitats seem critical for both kiang and livestock , but cover a small fraction of the landscape , with an estimated 150 km in the entire approximately 20,000 km of changthang . illustratively , this means that in a single day a large group of 70 kiang can potentially consume more than a quarter of the standing biomass from 1 ha of a key resource area . this consumption is also very similar to the estimated daily forage consumption by a herd of 200 goats ( approximately 215 kg ) . because of the long winters ( november to april ) and short summers ( july august ) , the potential for regrowth during summer is low , aggravating the impact . considering the potential of large kiang herds to remove significant amounts of forage , together with the fact that grazing areas have been reduced and the livestock population in ladakh more than doubled in the last 30 years , it is not difficult to see that at a localized scale of the valued sedge meadows , some herders must be facing serious forage competition from the kiang . given the intensifying commercialization of cashmere production in ladakh and its integration with global markets , this localized forage competition is , perhaps understandably , no longer tolerated , and the conflict needs to be addressed immediately . however , it must be borne in mind that the problem seems to be evident at local scales , and will need to be addressed at that scale . in the absence of any scientific understanding of the conflict so far , or efforts to document it , this local conflict has already begun to take on a serious political dimension . the politicians and the district administration in ladakh are blaming the kiang for compromising cashmere production , and the wildlife department is being looked upon to provide a solution . the only on - ground reaction of the district administration has been to fence large productive sedge and grass meadows from the kiang . in the hanle valley alone , an estimated 120 ha of meadows have been fenced already . three fourths of our total kiang sightings during the rangewide surveys were in such meadows , suggesting that this is an important habitat for the species , and continued fencing of this habitat may cause a decline in the kiang population of ladakh . although our review of the recent history of political and socioeconomic developments in kiang habitat and our estimates of density and forage consumption of kiang and livestock bring to light interesting dimensions of the kiang human conflict , the current level of understanding of the kiang s ecology and its relationships with livestock is still very preliminary . intensive research needs to be promoted to assist in better management of the species , and its conflicts with humans . monitoring programs need to assess kiang and livestock density , as well as the land use changes in kiang habitat . is locally concentrated in the key resource areas that contain the sedge meadows , and management strategies need to be devised at that local scale . surveys are needed to identify all high - conflict areas , and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang . it needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in ladakh . the kiang is a striking and conspicuous wild animal that may provide a potential for ecotourism . finally , after taking up the above interventions , it is important that fences on the sedge meadows to exclude kiang be removed and the practice of fencing discontinued because they lock out key resource areas from the kiangs range . without access to key resource areas , kiang and other wild herbivores , such as the tibetan gazelle , procapra picticaudata ,

an emerging conflict with trans - himalayan pastoral communities in ladakh s changthang plateau threatens the conservation prospects of the kiang ( equus kiang ) in india . it is locally believed that changthang s rangelands are overstocked with kiang , resulting in forage competition with livestock . here , we provide a review and preliminary data on the causes of this conflict . erosion of people s tolerance of the kiang can be attributed to factors such as the loss of traditional pastures during an indo - chinese war fought in 1962 , immigration of refugees from tibet , doubling of the livestock population in about 20 years , and increasing commercialization of cashmere ( pashmina ) production . the perception of kiang overstocking appears misplaced , because our range - wide density estimate of 0.24 kiang km2 ( 0.44 , 95% cl ) is comparable to kiang densities reported from tibet . a catastrophic decline during the war and subsequent recovery of the kiang population apparently led to the overstocking perception in ladakh . in the hanle valley , an important area for the kiang , its density was higher ( 0.56 km2 ) although even here , we estimated the total forage consumed by kiang to be only 34% compared to 9697% consumed by the large livestock population ( 78 km2 ) . our analysis nevertheless suggests that at a localized scale , some herders do face serious forage competition from kiang in key areas such as moist sedge meadows , and thus management strategies also need to be devised at this scale . in - depth socioeconomic surveys are needed to understand the full extent of the conflicts , and herder - centered participatory resolution needs to be facilitated to ensure that a sustainable solution for livelihoods and kiang conservation is achieved .

Methods Study Area Data Collection and Analysis Results and Discussion Genesis of the Conflict: Kiang Population Changes in Recent History Intensification of the Conflict: Changing Face of Pastoralism in Ladakh The Ecological Context: Current Kiang Population and Relationships with Livestock Synthesis of the Conflict: Is It Real or Perceived?

density of kiang in the surveyed blocks of eastern ladakh , india through archival research and field observations , we documented the intensifying human we describe the perceptions of the ladakhi administration regarding this conflict , and the efforts they have undertaken to address it . through archival research , we also examine recent land - use changes in eastern ladakh , describe the growing pashmina or cashmere industry , and its fallouts for the kiang human conflict . our data on the kiang population in ladakh come from rangewide surveys conducted in the year 2000 . we also covered approximately 50 km on foot in the upper catchment of the hanle valley . subsequently , in the spring of 2001 , 2003 , and 2004 , we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the hanle valley , which had emerged as an area with moderate to high kiang density during the 2000 survey . this was generally our maximum sighting distance for the kiang ( most rangefinders do not work at this distance ) . we estimated the extent of the surveyed areas ( through viewscapes ) for both the rangewide surveys as well as the region of our repeated kiang counts in hanle valley . as a preliminary exercise to understand the extent of forage consumption by kiang vis - - vis livestock , we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake ( as percent of body mass ) among herbivores ( foose 1982 ) . according to the human census in 2001 conducted by the government , the population of the leh district was 117,637 , with the majority ( 85% ) being in the western region . our data on the kiang population in ladakh come from rangewide surveys conducted in the year 2000 . we also covered approximately 50 km on foot in the upper catchment of the hanle valley . subsequently , in the spring of 2001 , 2003 , and 2004 , we conducted repeated vehicle counts of kiang along an approximately 100-km stretch in the hanle valley , which had emerged as an area with moderate to high kiang density during the 2000 survey . we estimated the extent of the surveyed areas ( through viewscapes ) for both the rangewide surveys as well as the region of our repeated kiang counts in hanle valley . as a preliminary exercise to understand the extent of forage consumption by kiang vis - - vis livestock , we estimated total daily forage consumption by kiang and livestock through established relationships between body mass and daily food intake ( as percent of body mass ) among herbivores ( foose 1982 ) . the species was common in ladakh during the earlier part of the 20th century ( stockley 1936 ) , but is believed to have declined substantially with the buildup of armed forces in ladakh during the war between india and china in 1962 ( fox and others 1991 ) . the prevalent belief in ladakh during that time , and one that continues until today , is that the kiang population has drastically increased in eastern ladakh because many animals have moved from tibet to the indian side of the border to escape persecution ( fox and others 1991 ; anon . , there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock , and demands that the government should drive the kiang back into tibet the tibetan plateau was inhabited by nomadic hunting people 30,000 years ago , and nomadic pastoralists have been herding livestock on the plateau for at least two millennia ( schaller 1998 ) . supplemental forage provided by the government in the event of severe winters now offsets most of the starvation - related livestock mortality , which in the past was perhaps an important factor limiting the livestock population . the 1962 war between india and china also led to the curtailment of movement of the changpa herders from eastern ladakh into parts of tibet , considerably reducing the availability of grazing land . as per the government records , the livestock population in ladakh ( including changthang ) has doubled in about two decades , from approximately 212,500 in 1977 to 487,000 in 1999 ( richard 1999 ; bhatnagar and wangchuk 2001 ) . against this background , it is perhaps not surprising that people are fast losing their tolerance of the kiang . in our rangewide survey of the kiang , we surveyed an estimated area of 1653 km , which is 22% of the total range of the kiang in ladakh ( 7400 km ; chundawat and qureshi 1999 ) . the average density in the surveyed blocks was 0.24 ( 95% cl 0.44 ) kiang km , with the highest abundance along the upper indus from loma to demchok followed by the hanle valley ( table 1 , figure 1 ) . with an overall livestock population of 210,000 ( as per unpublished records from the nyoma office of the sheep husbandry department ( 2004 ) ) in the nyoma and durbuk blocks that comprise the changthang region of ladakh , the livestock density in the region was 11 km . thus , in terms of density , kiang represents only 2% of the total herbivore population ( kiang and livestock together ) in the changthang , with the bulk being contributed by goats ( 57% ) . at a landscape level covering entire eastern ladakh , of the total daily forage consumption by large herbivores , we estimated that kiang consume 1011% and livestock 8990% ( table 2 ) . our subsequent estimate of the kiang population in the hanle valley ( the region between rongo and kalangtartar ) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang / km ( 95% cl 0.08 ) . we estimated that on an average , kiang remove only 3% to 4% of the total forage consumed in the hanle valley , with the greater part being consumed by goats ( 45% to 47% ) followed by yaks ( 20% to 21% ) and sheep ( 20% ) ( table 3 ) . table 3herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valleyspeciesbody weight ( kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.563.632.84livestock donkey9000000 horse2481.579.587.49 cattle1910.662.721.92 yak2983.9122.82016.521 yak cattle hybrids22700000 sheep3421.5223.72015.519 goat3350.6954.54735.545livestock total112178.34113.29776.796total78.9032.479.6 herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valley most wars in the last five decades have resulted in detrimental effects on wildlife ( blom 2000 ; dudley and others 2002 ; mishra and fitzherbert 2004 ) , and the war between india and china in 1962 was no exception , particularly from the viewpoint of kiang conservation . our review suggests that the kiang population in ladakh declined drastically in the years during and after the war between india and china in the 1960s , possibly due to hunting , land mines , and disturbances associated with the war . thus , for two decades during and after the war , a generation of herders presumably saw and became used to a very low density of kiang in ladakh s rangelands . not surprisingly , the genesis of kiang human conflict in ladakh can be traced back to the 1980s , the period by which the kiang population had recovered . it seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km has led to the perception to a generation of nomads that ladakh s rangelands are now overstocked with kiang . available estimates from three regions in tibet place the density of kiang between 0.15 and 0.39 kiang km ( schaller 1998 ) , suggesting that the kiang density in ladakh is comparable with those in tibet . furthermore , our results show that the share of forage consumed by the kiang vis - - vis livestock is relatively small ( 1011% in the whole of eastern ladakh , and 34% in hanle valley ) . thus , at a landscape level , the perception of the overabundance of kiang in ladakh , and of kiang compromising cashmere production , is clearly unfounded . considering the potential of large kiang herds to remove significant amounts of forage , together with the fact that grazing areas have been reduced and the livestock population in ladakh more than doubled in the last 30 years , it is not difficult to see that at a localized scale of the valued sedge meadows , some herders must be facing serious forage competition from the kiang . given the intensifying commercialization of cashmere production in ladakh and its integration with global markets , this localized forage competition is , perhaps understandably , no longer tolerated , and the conflict needs to be addressed immediately . three fourths of our total kiang sightings during the rangewide surveys were in such meadows , suggesting that this is an important habitat for the species , and continued fencing of this habitat may cause a decline in the kiang population of ladakh . is locally concentrated in the key resource areas that contain the sedge meadows , and management strategies need to be devised at that local scale . surveys are needed to identify all high - conflict areas , and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang . it needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in ladakh . without access to key resource areas , kiang and other wild herbivores , such as the tibetan gazelle , procapra picticaudata , kiang inhabits the open plains and rolling mountains of the changthang plateau in eastern ladakh at altitudes between 4000 m and 5500 m. it has a total estimated range of approximately 7400 km in ladakh ( chundawat and qureshi 1999 ; figure 1 ) . the species was common in ladakh during the earlier part of the 20th century ( stockley 1936 ) , but is believed to have declined substantially with the buildup of armed forces in ladakh during the war between india and china in 1962 ( fox and others 1991 ) . there are no population estimates available for the period before or immediately following this war , but it is believed that the population had recovered by the 1980s . the prevalent belief in ladakh during that time , and one that continues until today , is that the kiang population has drastically increased in eastern ladakh because many animals have moved from tibet to the indian side of the border to escape persecution ( fox and others 1991 ; anon . , there were complaints by herders regarding the extent of forage consumption by kiang and consequent competition with livestock , and demands that the government should drive the kiang back into tibet the tibetan plateau was inhabited by nomadic hunting people 30,000 years ago , and nomadic pastoralists have been herding livestock on the plateau for at least two millennia ( schaller 1998 ) . after the 1960s , there has been a steady increase in the population of the changpa and their livestock , presumably facilitated by access to better health care , provisions , and subsidies , both from the government as well as the army ( chaudhuri 2000 ; phuntsok 2001 ; bhatnagar and wangchuk 2001 ) . supplemental forage provided by the government in the event of severe winters now offsets most of the starvation - related livestock mortality , which in the past was perhaps an important factor limiting the livestock population . the 1962 war between india and china also led to the curtailment of movement of the changpa herders from eastern ladakh into parts of tibet , considerably reducing the availability of grazing land . furthermore , a large number of tibetan refugees have moved into eastern ladakh with their livestock herds since the late 1950s , thus contributing to a substantial increase in the number of herding families and the livestock population ( bhatnagar and wangchuk , 2001 ; hagalia 2004 ) . as per the government records , the livestock population in ladakh ( including changthang ) has doubled in about two decades , from approximately 212,500 in 1977 to 487,000 in 1999 ( richard 1999 ; bhatnagar and wangchuk 2001 ) . against this background , it is perhaps not surprising that people are fast losing their tolerance of the kiang . in our rangewide survey of the kiang , we surveyed an estimated area of 1653 km , which is 22% of the total range of the kiang in ladakh ( 7400 km ; chundawat and qureshi 1999 ) . the average density in the surveyed blocks was 0.24 ( 95% cl 0.44 ) kiang km , with the highest abundance along the upper indus from loma to demchok followed by the hanle valley ( table 1 , figure 1 ) . with an overall livestock population of 210,000 ( as per unpublished records from the nyoma office of the sheep husbandry department ( 2004 ) ) in the nyoma and durbuk blocks that comprise the changthang region of ladakh , the livestock density in the region was 11 km . thus , in terms of density , kiang represents only 2% of the total herbivore population ( kiang and livestock together ) in the changthang , with the bulk being contributed by goats ( 57% ) . at a landscape level covering entire eastern ladakh , of the total daily forage consumption by large herbivores , we estimated that kiang consume 1011% and livestock 8990% ( table 2 ) . among livestock , goats were estimated to consume the maximum forage ( 41% to 43% of the total forage consumed ) , followed by sheep ( 26% to 27% ) . our subsequent estimate of the kiang population in the hanle valley ( the region between rongo and kalangtartar ) based on repeated counts between 2001 and 2004 yielded a density of 0.56 kiang / km ( 95% cl 0.08 ) . we estimated that on an average , kiang remove only 3% to 4% of the total forage consumed in the hanle valley , with the greater part being consumed by goats ( 45% to 47% ) followed by yaks ( 20% to 21% ) and sheep ( 20% ) ( table 3 ) . table 3herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valleyspeciesbody weight ( kg)density(animals km)total forage consumption in summer ( kg km)percent forage consumption in summertotal forage consumption in winter ( kg km)percent forage consumption in winterkiang2750.563.632.84livestock donkey9000000 horse2481.579.587.49 cattle1910.662.721.92 yak2983.9122.82016.521 yak cattle hybrids22700000 sheep3421.5223.72015.519 goat3350.6954.54735.545livestock total112178.34113.29776.796total78.9032.479.6 herbivore density and estimated forage consumption by kiang vis - - vis livestock in the hanle valley most wars in the last five decades have resulted in detrimental effects on wildlife ( blom 2000 ; dudley and others 2002 ; mishra and fitzherbert 2004 ) , and the war between india and china in 1962 was no exception , particularly from the viewpoint of kiang conservation . our review suggests that the kiang population in ladakh declined drastically in the years during and after the war between india and china in the 1960s , possibly due to hunting , land mines , and disturbances associated with the war . thus , for two decades during and after the war , a generation of herders presumably saw and became used to a very low density of kiang in ladakh s rangelands . not surprisingly , the genesis of kiang human conflict in ladakh can be traced back to the 1980s , the period by which the kiang population had recovered . it seems that the recovery of the kiang from a very low density to the current estimated density of 0.24/km has led to the perception to a generation of nomads that ladakh s rangelands are now overstocked with kiang . available estimates from three regions in tibet place the density of kiang between 0.15 and 0.39 kiang km ( schaller 1998 ) , suggesting that the kiang density in ladakh is comparable with those in tibet . furthermore , our results show that the share of forage consumed by the kiang vis - - vis livestock is relatively small ( 1011% in the whole of eastern ladakh , and 34% in hanle valley ) . thus , at a landscape level , the perception of the overabundance of kiang in ladakh , and of kiang compromising cashmere production , is clearly unfounded . using the data in table 2 , a group of 70 kiang ( the largest group seen during the survey was 74 ) the peak standing biomass in sedge meadows , which seem to be important kiang habitats , has been estimated in the adjoining region of spiti at 857 kg ha ( mishra 2001 ) . considering the potential of large kiang herds to remove significant amounts of forage , together with the fact that grazing areas have been reduced and the livestock population in ladakh more than doubled in the last 30 years , it is not difficult to see that at a localized scale of the valued sedge meadows , some herders must be facing serious forage competition from the kiang . given the intensifying commercialization of cashmere production in ladakh and its integration with global markets , this localized forage competition is , perhaps understandably , no longer tolerated , and the conflict needs to be addressed immediately . three fourths of our total kiang sightings during the rangewide surveys were in such meadows , suggesting that this is an important habitat for the species , and continued fencing of this habitat may cause a decline in the kiang population of ladakh . intensive research needs to be promoted to assist in better management of the species , and its conflicts with humans . is locally concentrated in the key resource areas that contain the sedge meadows , and management strategies need to be devised at that local scale . surveys are needed to identify all high - conflict areas , and adequate compensatory mechanisms need to be worked out for herders who face genuine forage competition from the kiang . it needs to be examined whether the fact that herders share the forage with kiang could actually be used for adding value to the cashmere produced in ladakh .

hippocrates was quoted as saying all disease begins in the gut ' and over 2000 years later we are beginning to appreciate his sentiment . our body is colonised by a large number of microbes ( bacteria , fungi , archaea , viruses and protozoa ) ; which mostly reside within the gastrointestinal ( gi ) tract , are predominantly bacterial , and together these microbes collectively form the gut microbiota . often denoted previously as commensal organisms , we now know the gut microbiota acts in a symbiotic manner that is also beneficial for its host . interest in the gut microbiota , most notably the bacterial communities , has recently exploded , and we are beginning to uncover how crucial these microbes are to appropriate immune function and lifelong health , or conversely , susceptibility to inflammatory and infectious diseases . early in life the gi tract quickly becomes colonised by microbes , and the gut microbiota is purported to reach an adult state at around 3 years of age . bacteroidetes and firmicutes are the dominant phyla , making up more than 90% of the total microbial population in both mice and humans . other major phyla present in the gut include the proteobacteria , tenericutes , actinobacteria and verrucomicrobia . the gut microbiota has an important role in homoeostasis by controlling metabolic pathways , nutrient metabolism and the production of vitamins . furthermore , it has also been shown to be essential in the development and maturation of mucosal and systemic immune responses , and for the maintenance of intestinal epithelial barrier function . the importance of microbial signalling for immune development in the gi tract has also been demonstrated in germ - free ( gf ) mice , which have underdeveloped gut - associated lymphoid tissues ( galt ) including peyer 's patches , isolated lymphoid follicles and mesenteric lymph nodes . taken together , host microbiota interactions are critical for host immunity and health . here , we review the recent advances in gut microbiota analysis , and define the computational approaches that can be utilised to expand our ever - growing understanding of the role of the gut microbiota in health and disease . the immense interest and impact of the gut microbiota in current research can mainly be attributed to the recent advances in these applications to study microbial communities and their functions . we also detail key microbial cross talk with the immune system , resulting in critical instruction of appropriate immunity . finally , we have considered immune - mediated diseases where the relationship between the gut microbiota and disease susceptibility is currently most convincing , including inflammatory bowel disease ( ibd ) , obesity - related inflammatory disorders , allergic and infectious diseases . where applicable , the influence of key factors such as diet or antibiotic use on the composition of the gut microbiota is outlined . as well , we explore the use of gf mice , and gnotobiotic mouse models where gf mice are colonised with defined microbial communities , which provide a tool to elucidate the function of the gut microbiota in a disease setting . researchers now routinely use a wide - range of culture - dependent and high - throughput culture - independent methods to assess gut microbial communities . the recent advances of the culture - independent methods analysing the dna ( metagenomics ) , rna ( metatranscriptomics ) , proteins ( metaproteomics ) and metabolites ( metametabolomics ) present within the gut will be the focus of this section ( figure 1 ) . the development and reducing costs of next - generation sequencing , also known as high - throughput sequencing , combined with improved computational tools allowing for the analysis of large and complex data sets , have led to an enormous growth in the research field . each tool provides unique information to interpret the different species that live within the gut ; to provide understanding of how these species function ; and describe why certain species may contribute to disease , or respond differently to specific stimuli such as changes in diet . initial efforts to characterise the gut microbiome using next - generation sequencing of gut microbiota involved the use of a marker ' gene that serves as a unique fingerprint for different bacterial genera . amplicon sequencing is a pcr - based method that provides information about the different microorganisms present within a sample . as such , the bacterial communities within the gut can be assessed by sequencing the variable regions of the 16s ribosomal rna ( rrna ) encoding gene sequence found in bacteria and archaea , whereas eukaryotic organisms ( fungi , parasites and the host ) can be assessed by sequencing the 18s rrna encoding sequence . today , this method still provides an inexpensive way to compare bacterial communities between patients , and assess alterations over time within a patient . however , this 16s sequencing can not be routinely used to identify specific bacterial species as the 16s rrna gene typically lacks sufficient phylogenetic information to differentiate between taxa at the species level , and some bacterial genera can share greater than 99.5% of their 16s rrna sequences . metagenomic ( shotgun ) sequencing was later utilised to provide strain level resolution of all the species present within a microbial community . instead of employing an individual marker gene , a random subset of the dna present within the sample is sequenced , and the bacterial , viral and eukaryotic species associated with the sequences can then be reconstructed using high - powered bioinformatic tools . it has also been employed to provide information about the host gi tract through non - invasive sampling , as dna from the cells within the gi tract can be detected within faecal samples . despite being a very small proportion of the gut microbiome , even viruses present within the metagenome can be assessed using different sample preparation steps and metagenomic sequencing . using dna hybridisation prior to sequencing , further adaptations of metagenomic sequencing have included enrichment or capture of dna sequences specific to the species of interest . within a clinical setting , this approach could be adapted to identify specific pathogens or disease - associated species from within an individual 's gut microbiota without sequencing all of the dna within a sample , decreasing costs and the bioinformatic processing time for clinicians . in addition to identifying specific strains , metagenomic sequencing can also assess the functions that microorganisms can perform , as genes within the microbial genomes are linked to the ability of an organism to execute certain tasks . the functional capability of a bacterial community can also be predicted based on its 16s rrna amplicon profile by comparing against databases containing fully sequenced and annotated bacterial genomes . however , metagenomic dna sequencing remains the only way to identify functions from unknown species or unique microbial communities . metatranscriptomic approaches , or the sequencing of rna present within a sample , have also been applied to assess the current or ongoing functions of a metagenome within a specific period of time . although metagenomic sequencing will divulge the total functional capacity of a microbial community , metatranscriptomics reports genes that are being actively transcribed at a given time . further use of metatranscriptomic approaches linked with rna sequencing of the host will reveal more information about how distinctive microbial functions are linked to disease , and how they alter human health . metaproteomic studies examine the proteins that typically result from the microorganism , but can also result from inflammatory responses of the host . moreover , shotgun proteomic approaches have identified unique proteins present within the gut that were not predicted by metatranscriptomic approaches , highlighting the yet unknown nature of many interactions between the gut microbiota and the host . similarly , metametabolomic approaches also use a shotgun - like approach to examine a broad range of metabolic by - products and secretions of microorganisms that provide insight into active microbial functions and interplay between microbial species as well as the gut microbiota and the host . many gi diseases linked to the gut microbiota may be associated with altered proteins or metabolite production , rather than a depletion of certain microbes . once certain proteins and metabolites can be linked to microbiota - associated diseases , downstream applications will likely revolutionise our ability to quickly and efficiently detect alterations in the gut microbiota that are linked to disease phenotypes . this can provide previously unavailable insight into the metabolic pathways of the microbial community and how this intersects with the metabolism of the host . this knowledge could provide a basis for the crucial next step and the remaining challenge for this research field , to establish causal relationships between the gut microbiota and the host . colonisation of gf mice with mouse or human gut microbiota ( referred to as humanised mice ) has been used to demonstrate a causal role for the gut microbiota in immune development and disease susceptibility . it is likely that the emerging technological applications will move beyond the current focus of the bacterial communities present towards their function ; which holds the most promise for the identification of microbial or metabolite based therapeutic strategies for prevention and treatment of inflammatory and infectious diseases . the gi tract must remain non - responsive to food antigens and the gut microbiota , while being capable of responding quickly to invading pathogens . one of the first defence mechanisms afforded by the gut microbiota is limiting the access of pathogenic bacteria to the gut epithelium by competitive exclusion . the gut microbiota also promotes secretory immunoglobulin ( ig)a production that binds to microbes at mucosal surfaces , neutralises toxins and contributes to microbial tolerance . furthermore , bacterial molecular patterns are sensed by innate pattern recognition receptors ( prr ) such as toll - like receptors ( tlr ) or nod - like receptors , which have a role in the maintenance of intestinal epithelial cell ( iec ) homeostasis . the microbiota also contributes to the priming signal of the inflammasome pathway , leading to the transcription of cytokines such as tumour necrosis factor ( tnf)- and interleukin ( il)-6 , as well as pro - il-1 and pro - il-18 . the inflammasome , activated by a variety of pathogens including various viruses , can regulate the cleavage of these inactive precursors to active il-1 and il-18 . the gut microbiota also helps to maintain immune homeostasis by stimulating different arms of the t - cell response . segmented filamentous bacteria ( sfb ) are potent promoters of t helper ( th)17 cells in the intestine , which are important in protection against bacterial and fungal infections . sfb adhesion to iec results in the activation of the il-23 pathway , leading to il-22 production by group 3 innate lymphoid cells ( ilc3 ) and a subsequent th17 response . conversely , polysaccharide a from bacteroides fragilis was shown to stimulate the generation of foxp3 + regulatory t cells ( treg ) via tlr2 activation , and microbial - derived butyrate has also been shown to induce colonic treg . fermentation of dietary fibre in the colon by anaerobic bacteria generates short - chain fatty acids ( scfa ) including butyrate , acetate and propionate . as such , fermentable dietary fibre scfa signal via g - protein coupled receptors ( gpcr ) such as gpr41 , gpr43 and gpr109a on epithelial cells and immune cells . when scfa are taken up by host cells they serve as an energy source for metabolism . scfa were also shown to inhibit the transcription factor nuclear factor kappa b ( nf-b ) leading to reduced inflammatory cytokine production . furthermore , scfa have been demonstrated to promote mucous production by intestinal goblet cells , induce secretory iga and activate the inflammasome resulting in il-18 secretion . importantly , systemic scfa also reach other organs such as the bone marrow and lung via the circulation . when mac beneficially affect the host 's health by selectively changing the composition or activity of the gut microbiota , they can be referred to as prebiotics . probiotics are defined as live microorganisms , which when administered in adequate amounts confer a health benefit on the host . probiotics can interfere with the growth or survival of pathogens in the gut lumen , improve gut barrier function and immunity , or have an effect on the systemic immune system and organs . the molecular mechanisms by which probiotic strains act are still largely unknown and require a strain - specific approach as different probiotic strains can induce differential effects . both pre- and probiotics can be useful for the beneficial modulation of the gut microbiota , and their capability to enhance immune function has been studied for many years . no longer viewed as merely passengers , the gut microbiota has a critical role in the development and progression of ibd , and is the most extensively investigated with respect to the influence of the gut microbiota on disease susceptibility , relapse and remission . although the specific microbial changes appear to vary from study to study , some broad patterns are apparent . these include a loss of community diversity , increased representation of some gammaproteobacteria , and decreased relative abundance of several taxa within the firmicutes phylum . loss of faecal community diversity has been consistently shown in human ibd studies . the most prominent change in diversity associated with ibd is decreased abundance of the firmicutes phylum , which includes a loss of faecalibacterium prausnitzii , a bacterium with significant anti - inflammatory effects . other species with decreased representation in ibd include bacteroides fragilis , b. vulgatus , ruminococcus albus , r. callidus , and r. bromii , which were over 5-fold more abundant in healthy subjects compared with ibd patients . in addition to the loss of diversity and altered composition , recent metagenomic studies showed that the overall quantity of bacteria is also reduced in ibd . on average , 25% fewer microbial genes could be detected in the faecal community of ibd patients , pointing to lower functional as well as taxonomic diversity . indeed , some evidence suggests that the abundances of metabolic pathways are more consistently altered in ibd than the actual taxonomic abundances . conversely , specific taxa that are increased in ibd patients include enterobacteriaceae , such as escherichia / shigella . in particular , adherent - invasive e. coli strains have been identified within ileal biopsies from crohn 's disease patients , and are more prominent in this environment compared with faecal samples . the increase in enterobacteriaceae in the small intestine may reflect their capacity to use sialic acid and fucose liberated from mucous . under normal circumstances , these bacteria are likely to be outcompeted for this resource by the resident microbial community , but the alterations in the gut microbiota that occur with ibd may present the opportunity for these bacteria to flourish . other mucous associated bacteria suspected of having an important role in ibd are the sulphate - reducing bacteria such as desulfovibrio piger . in the gut , sulphate - reducing bacteria consume hydrogen to produce h2s by removing sulphate from sulfated mucous glycans . potential links between h2s and ibd include genotoxic properties and the chemical disruption of the mucous structure , as sulphides are potent reducers of disulphide bonds . specific groups of bacteria may also provide protection against ibd , by ameliorating the inflammatory response . further evidence for beneficial effects of gut microbiota in immune modulation are found in mice with impaired recognition of bacteria through deletion of tlr4 or its downstream signalling molecule myeloid differentiation primary response gene 88 ( myd88 ) , which experience worse symptoms of gi inflammation when exposed to dss compared with wild - type mice . in addition , activation of the inflammasome can occur via mac modulation of the gut microbiota as well as scfa administration , which promoted il-18-mediated epithelial repair following dss - induced gi inflammation . butyrate produced by the gut microbiota , most likely by members of the clostridia class , has also been shown to induce the expansion of treg in mice , ameliorating intestinal inflammation in an adoptive t - cell transfer model of colitis . other bacteria , such as f. prausnitzii , bifidobacterium and lactobacillus protect the host through a variety of mechanisms , including modulation of cytokine production and strengthening gut barrier function . the evidence for the efficacy of probiotic strains like bifidobacterium and lactobacillus in reducing the symptoms of crohn 's disease in humans remains unclear , although some beneficial effects have been shown in patients with ulcerative colitis . in addition , the gut microbiota may protect the host by outcompeting pathogenic bacteria that drive gi inflammation by preventing these pathogens from occupying niches . the incidence and prevalence of ibd is progressively rising , making the condition a global health problem with currently no permanent therapeutic treatment . one potential therapy of interest is faecal microbiota transplantation ( fmt ) , which has been particularly successful in treating c. difficile infections . however , so far , fmt for treating ibd has been shown to have variable efficacy , and long - term safety for fmt is largely unknown . risks include the possible transmission of infectious agents or antibiotic resistance genes within fmt , or the possible induction of other microbiota - associated diseases following fmt to a susceptible host . nonetheless , gut microbiota based treatments hold promise as a future therapy for ibd patients . the world is facing an epidemic of obesity - related metabolic abnormalities , collectively known as metabolic syndrome . the hallmarks of metabolic syndrome include hyperglycaemia , hyperlipidaemia , insulin resistance , obesity and hepatic steatosis . both lifestyle and genetic factors have a role in the development of obesity , and recently the gut microbiota has been suggested to be a key environmental factor that influences metabolic syndrome . the heritability of the gut microbiota has previously been examined linking the abundance of specific gut microbial taxa to host genetics . among the heritable taxa were members of the firmicutes phylum that are associated with leaness . thus , host genetics influence the composition of the human gut microbiota , and this can impact host metabolism . nonetheless , the diet has been suggested to be most influential in shaping the composition of the gut microbiota . an increase in the ratio of firmicutes to bacteroidetes , as well as a marked reduction in the bacterial diversity of the gut microbiota , have been associated with obesity in both genetic- and diet - induced mouse models and clinical obesity . moreover , diets that limit weight gain led to a reduction in the relative abundance of firmicutes and an increase in bacteroidetes in both mice and humans . however , other investigations have shown no association between obesity and the bacteroidetes to firmicutes ratio , or the diversity of the human gut microbiota , suggesting the relationship between obesity and the gut microbiota is highly complex and difficult to interpret . there is now a large body of evidence that supports the close link of obesity and insulin resistance with chronic , low - grade inflammation , and the gut microbiota has been implicated in the development of this inflammatory response . both diet and genetic factors can alter the gut microbiota and impair intestinal integrity , leading to metabolic endotoxemia , obesity , glucose intolerance and insulin resistance via activation of tlr4 and subsequent inflammatory cytokine production . moreover , restoration of the gut microbiota by the mac oligofructose resulted in amelioration of the inflammation as well as the associated metabolic abnormalities . other tlr responses have been linked to obesity - induced inflammation and insulin resistance as well . for example , tlr5 sensing of bacterial flagellin is required for gut microbial homeostasis . consequently , tlr5 deficient mice have altered gut microbiota and are prone to the development of metabolic syndrome . the gut microbiota provides enzymes that increase caloric harvest from mac and the resulting monosaccharides are absorbed or metabolised to scfa . these products get delivered to the liver , where lipogenesis results in hepatic triglyceride ( tg ) production . in addition , the gut microbiota acts to enhance lipoprotein lipase - directed deposition of these tg into adipocytes , promoting the storage of calories harvested from the diet into fat . fat storage is reduced in gf compared with conventional mice , and colonisation of these gf mice with gut microbiota results in a 60% increase in body fat within two weeks . gf mice also have increased host metabolism of fatty acids that protects against diet - induced obesity provoked by a prototypic high - fat , high - sugar western - style diet . furthermore , depletion of the gut microbiota using an antibiotic cocktail was sufficient to reduce adiposity and improve glucose tolerance and insulin sensitivity by promoting browning of adipose tissue in both diet and genetically induced obese mice . colonisation of gf mice with the gut microbiota from mice with either genetic or diet - induced obesity increased host fat deposition compared with colonisation with microbiota from lean mice . humanised mice from obese donor microbiota also showed more weight gain and fat deposition than humanised mice with lean donor microbiota . cohousing lean with obese humanised mice prevented the obese phenotype in these cage mates , and altered the microbiota to a more lean - like profile . the transfer of faeces from standard diet - fed compared with high - fat diet - fed donor mice was also sufficient to reduce the expression of inflammatory cytokines and lipogenic genes in adipose and hepatic tissue of mice with diet - induced obesity . these data suggest that there are available niches in the obese donor microbiota that can be filled by lean associated microbes . fmt from lean donors into humans with metabolic syndrome was also sufficient in the clinical setting to increase gut microbial diversity and improve insulin sensitivity . this opens the opportunity to determine specific bacterial species for transplantation and the treatment of metabolic abnormalities as a therapeutic approach in the future . some preclinical studies have reported beneficial effects of pre- and probiotics , yet the data from human randomised controlled trials are inconsistent , and more investigations are needed to inform clinical practice . epidemiologic studies imply that disruption of the gut microbiota early in life , via caesarean section as well as antibiotic therapy , increases the risk of being overweight later in childhood . indeed , antibiotic use during the first 6 months was associated with becoming overweight later in life among children of normal weight mothers . however , early administration of antibiotics reduced the risk of becoming overweight among children of overweight and obese mothers . taken together with the preclinical data , this demonstrates the complex relationship between the gut microbiota , antibiotic use and metabolic outcomes . in light of the high rates of childhood obesity , the use of antibiotics or pre- and probiotics to restore intestinal homeostasis and to potentially prevent obesity in children from overweight and obese mothers is an interesting concept . so far , there is no direct clinical evidence for a causal relationship between obesity and antibiotic use or delivery mode . yet , preclinical studies imply that even transient alterations in the infant gut microbiome could potentially affect metabolic homeostasis resulting in obesity . in mice , exposure to low dose antibiotics in early life resulted in an increased risk of obesity . low dose antibiotics do not deplete the microbiota but cause a shift in taxonomic composition towards an increased firmicutes to bacteroidetes ratio . there was a trend towards hyperglycaemia in antibiotic treated mice and genes related to hepatic lipogenesis were upregulated , but no phenotypic signs of hepatic steatosis were observed . in a more recent study , cox et al . showed that the period around birth was critical to alter host metabolism and adiposity . the metabolic phenotype persisted after cessation of the antibiotic treatment , even though the microbiota recovered . this study provides evidence for a critical window in early life when the gut microbiota can induce long - term alterations in host metabolism . taken together , the gut microbiota has a substantial effect on metabolic abnormalities by influencing the efficiency of energy harvest and storage , low - grade inflammation and browning of adipose tissue . notably , there are risk factors for these metabolic abnormalities beyond gut microbes , which include lifestyle factors such as energy intake and expenditure as well as genetic risk factors . this results in a complex interplay of these lifestyle , microbial and host factors that determine disease susceptibility . the integration of modern applications to assess microbiota in preclinical and clinical studies will help clarify this complex interplay and yield promise to identify novel treatments for beneficial modulation of the gut microbiota to improve metabolic health . allergic diseases are a global health concern , affecting over half a billion people worldwide . genetic and molecular risk factors are clearly associated with the development of allergy ; however , it is unlikely genetic factors can account for the rapid rise in allergic disease over the past 50 years . this indicates that environmental factors , such as the gut microbiota , may have a key role in the development of allergic disease . for example , reduced gut microbial diversity in infants was associated with increased allergic risk in school age children , and the development of food allergy was associated with altered gut microbiota in these infants . strikingly , a recent study has shown that lower abundance of bacteria such as bifidobacterium , akkermansia and faecalibacterium , along with higher abundance of particular fungi including candida and rhodotorula in neonates may predispose to allergy susceptibility by influencing t - cell differentiation . gf mice have been an effective way to address several questions regarding the relationship between the gut microbiota and allergic inflammation . colonisation of gf mice with gut microbiota in early life was sufficient to protect mice from allergic inflammation in the lung , whereas allergic airway inflammation ( aai ) persisted in mice colonised as adults . early life colonisation with gut microbiota also inhibited serum ige responses , and protected against sensitisation to food allergens . in addition , clostridia - induced il-22 has also been shown to reduce the uptake of dietary antigen into the systemic circulation . these investigations suggest that the gut microbiota is a key environmental factor in the regulation of allergic inflammation in the lung and gi tract . depletion of the gut microbiota using antibiotic administration in mice has also been employed to examine the impact on allergy susceptibility . vancomycin is not absorbed from the intestine , so when administered orally affects only the gut microbiota . oral administration of vancomycin in early life resulted in exacerbation of aai and serum ige , and exposure of neonatal mice to an oral antibiotic cocktail also supported food allergen sensitisation . these data show that antibiotic use increased the risk of allergy development later in life . mice lacking scfa receptors gpr41 or gpr43 developed more severe aai than wild - type littermates , and high mac diet - suppressed aai . dietary mac and scfa also promoted treg induction , oral tolerance and protected against experimental food allergy . further studies identify the lung microbiota as key player in suppressing aai . in mice , the neonatal airways contained predominantly firmicutes and gammaproteobacteria , and these neonatal mice were more susceptible to aai . an expansion of bacteroidetes in the lung and the development of treg cells with age were correlated with reduced aai . asthmatic patients have also been shown to have higher bacterial burden and diversity compared with controls . however , the function of the gut microbiota in atopic dermatitis ( ad ) is less clear . similarly , the impact of the gut microbiota in eosinophilic gi diseases , such as eosinophilic esophagitis , as well as other allergic disorders including allergic rhinitis and allergic conjunctivitis , are yet to be fully elucidated . importantly , these findings in mice are supported by epidemiological data that early life exposure to microbes can reduce the incidence of allergic disease . indeed , allergy is reduced in children exposed to a farm environment , and studies suggest microbial exposures in early life are a contributing factor . on the basis of these data , a large number of clinical studies have examined the efficacy of probiotics in the prevention and treatment of allergy . utilising probiotics for the prevention of ad has been extensively studied , yet there is still conflicting data . as well , more studies are needed to determine the potential for prebiotics in allergy prevention at present . two notable studies support beneficial modulation of the gut microbiota and associated metabolites in early life as an effective intervention strategy for allergic disease . the canadian healthy infant longitudinal development ( child ) study identified a transient alteration in the gut microbiota during the first 100 days of life in infants at risk of asthma , which was associated with reduced faecal acetate concentrations . this disturbance of the microbial composition in infants at risk was characterised by significantly lower abundance in the gut of lachnospira , veillonella , faecalibacterium and rothia bacterial genera . colonisation of gf mice with these specific gut microbiota ameliorated aai in the offspring , demonstrating a causal role for the bacterial communities in reducing allergy susceptibility . moreover , the gut microbiota of infants with cow 's milk allergy is altered in composition and diversity as compared with non - allergic infants . supplementation of extensively hydrolysed formula with a lactobacillus probiotic has been shown to accelerate tolerance to cow 's milk in infants with cow 's milk allergy , and was associated with increased abundance of faecal butyrate producing bacteria and butyrate levels following lactobacillus treatment . taken together , there is mounting evidence to support gut microbiota based treatments to reduce susceptibility to allergic disease . future research identifying the components of the gut microbiota that influence allergy avoidance or tolerance mechanisms is crucial to develop these strategies for widespread clinical use . nearly one third of deaths in low - income countries are attributable to infectious diseases . infection rates in the western world have reduced dramatically over the past century due to advances in hygiene , antibiotic use and vaccination . however , infectious disease still poses significant risk to vulnerable populations such as infants and the elderly . upper respiratory tract infections are the most common illness for which people seek medical care , and the burden on healthcare services is substantial . in addition , c. difficile is the leading healthcare - acquired infection , which has increased in severity and morbidity over the last decade . the gut microbiota provide critical signals for the development and function of the immune system that provides protection from invading intestinal pathogens . therefore , manipulation of the gut microbiota is an attractive therapeutic target to build immune capability to combat infectious diseases . interactions between epithelial cells and the gut microbiota are essential for intestinal homeostasis and barrier defences . defined gut microbial species have been shown to maintain tight junction integrity , which in turn limits salmonella typhimurium invasion . gut microbiota promote the secretory iga response that inactivates rotavirus and neutralises cholera toxin , and outcompete c. difficile colonisation thereby preventing infection . key gut microbial species also prime the development of cellular immune responses in the gi tract . sfb - induced th17 responses are sufficient to promote immunity against the intestinal pathogen citrobacter rodentium . moreover , damage induced by pathogens is limited by microbiota - induced treg . b. fragilis - induced il-10 producing treg limit helicobacter hepaticus infection , and bifidobacterium infantis - induced treg dampen excessive inflammation induced in s. typhimurium infection . the critical role for the gut microbiota in establishing immune defence against infection extends far beyond the local environment of the gi tract . in fact , recent evidence demonstrates that gut microbial signalling actively shapes the systemic immune response by controlling haematopoiesis in primary immune sites . microbiota derived signals maintain granulocyte / monocyte progenitor development in the bone marrow , which influenced multiple tissue - resident innate immune populations . absence of microbiota derived signals conferred susceptibility to systemic listeria monocytogenes and staphylococcus aureus infection , which was driven by defects in tissue - resident myeloid populations prior to infection . microbiota signalling can also calibrate the activation threshold of innate immune cells , that enhances the sensitivity to detect pathogen invasion . on infection with mouse cytomegalovirus and lymphocytic choriomeningitis virus , antiviral defences including type i interferon ( ifn ) are stimulated through prr ligation . in gf mice , type i ifn dependent priming of natural killer cells was reduced due to dampened prr signalling , and antiviral protection was lost . microbiota signalling has also been shown to enhance protection against influenza virus by facilitating inflammasome dependent il-1 and il-18 in the lung in a tlr dependent manner . reactive oxygen species - mediated defence of alveolar macrophages in respiratory klebsiella pneumonia infection was modulated by the gut microbiota as well . interestingly , only gut and not lung microbiota derived ligands activated this respiratory defence mechanism , demonstrating the importance of the gut microbiota in establishing immune defences at other microbiota colonised sites . the capacity to mount adaptive immune responses is influenced by gut microbial signals as well . this was clearly demonstrated in a mouse model of seasonal trivalent influenza vaccination ( tiv ) , where the absence of gut microbiota significantly attenuated the magnitude of vaccine - induced protective antibody response . a mechanistic role was elucidated , demonstrating tlr5 mediated sensing of the gut microbiota increased the magnitude of the antibody response . furthermore , these investigations found a correlation between tlr5 expression in peripheral blood mononuclear cells shortly after vaccination and the magnitude of tiv - induced antibody response in a clinical cohort . the authors suggest the observed variable efficacy of tiv between vaccines may in part be attributed to the host 's gut microbiota composition . this raises the possibility that beneficial modulation of the gut microbiota may be used as a therapeutic approach to improve the efficacy of vaccine - induced immunity . manipulating gut microbes to enhance immune function is not a new concept to be tested in the clinic . at present variability observed in the effectiveness of probiotic interventions in vaccination trials needs to be acknowledged . however , there is strong evidence across these clinical trials to demonstrate that probiotic supplementation in adults improves vaccine - induced immunity . for example , bifidobacterium lactis bl-04 and lactobacillus acidophilus la-14 significantly enhanced cholera toxin - specific igg induced by oral cholera vaccine , and lactobaccillus paracasei subsp . paracacei ( l. casei 431 ) strain significantly increased influenza - specific igg in response to parenteral tiv vaccination . moreover , l. casei 431 along with a number of other candidate strains , have also demonstrated a protective effect against acute upper respiratory tract infections . these studies provide clear evidence of a role for the gut microbiota in immunity to infection as well as vaccine - induced protective antibody responses . as most gut microbes ( especially anaerobes ) can not be cultured easily , the development of new culture - independent techniques have led to an increase in knowledge in the gut microbiota field . these modern technological applications are now used frequently in both pre- and clinical studies to address the role of the gut microbiota in health and disease ( figure 2 ) , and have substantially contributed to providing understanding of the complex interplay between gut microbial composition , host genetics and biological outcomes . studies in gf mice , which like newborns can be colonised by a diverse microbiota , demonstrate that a specific microbial composition can exert lasting effects on the immunity and disease outcomes of the host . it is only recently that investigators have begun to truly appreciate the importance of this , and translation of such findings into humans is challenging . so far there are only correlations between alterations in gut microbial composition and inflammatory and infectious diseases in humans . the humanised mouse models , when combined with current technologies , are a promising attempt to move beyond these correlations to provide causal roles for specific gut microbial functions and disease susceptibility . colonisation with the culture collections from a human donor can be used to determine which cultured members of the microbiota can transmit or prevent a disease phenotype , and these studies may identify viable microbial - based therapeutic targets . it also opens up possibilities to study the effects of dietary components on gut microbiota composition and disease outcome , or to evaluate the ability of bacterial strains with a therapeutic potential to invade established niches in gut communities . gf and gnotobiotic models provide a great tool to study the microbiota , but as with all animal models there are limitations around the translation to the clinical setting . these models might not completely recapitulate the human disease complexity or heterogeneity of the human gut microbiota . humanised mouse studies have the added complexity of the donor microbiota not having co - evolved with a mouse host , and studies have shown host - specific gut microbiota may be critical for an appropriately functioning immune system . nonetheless , these model systems are invaluable for the understanding of host microbiota interactions that contribute to disease . beneficial modulation of the gut microbiota has yielded encouraging results for the prevention and treatment of various inflammatory diseases in experimental models . further work is required to characterise strain - specific effects and efficacious dosing regimens for prebiotics and probiotics . alongside , fundamental research needs to be conducted to define the mechanisms by which the gut microbiota can enhance immune health . most likely non - bacterial microbes also have an important role in immune function and disease . their contribution remains an important area for future research , as this has been relatively unexplored to date . this is a fast - paced field of research and given the positive results so far , we are cautiously optimistic it will soon yield novel therapeutics that manipulate the microbiota to prevent or treat chronic inflammatory diseases and enhance protective immunity against pathogens .

the gut microbiota provides essential signals for the development and appropriate function of the immune system . through this critical contribution to immune fitness , the gut microbiota has a key role in health and disease . recent advances in the technological applications to study microbial communities and their functions have contributed to a rapid increase in host microbiota research . although it still remains difficult to define a so - called normal ' or healthy ' microbial composition , alterations in the gut microbiota have been shown to influence the susceptibility of the host to different diseases . current translational research combined with recent technological and computational advances have enabled in - depth study of the link between microbial composition and immune function , addressing the interplay between the gut microbiota and immune responses . as such , beneficial modulation of the gut microbiota is a promising clinical target for many prevalent diseases including inflammatory bowel disease , metabolic abnormalities such as obesity , reduced insulin sensitivity and low - grade inflammation , allergy and protective immunity against infections .

Introduction Technological Applications for Assessing Gut Microbiota Gut Microbiota and Local Immune Cell Cross-Talk Inflammatory Bowel Disease Obesity-Related Inflammatory Disorders Allergic Diseases Infectious Diseases Discussion and Future Directions

often denoted previously as commensal organisms , we now know the gut microbiota acts in a symbiotic manner that is also beneficial for its host . interest in the gut microbiota , most notably the bacterial communities , has recently exploded , and we are beginning to uncover how crucial these microbes are to appropriate immune function and lifelong health , or conversely , susceptibility to inflammatory and infectious diseases . early in life the gi tract quickly becomes colonised by microbes , and the gut microbiota is purported to reach an adult state at around 3 years of age . the gut microbiota has an important role in homoeostasis by controlling metabolic pathways , nutrient metabolism and the production of vitamins . furthermore , it has also been shown to be essential in the development and maturation of mucosal and systemic immune responses , and for the maintenance of intestinal epithelial barrier function . here , we review the recent advances in gut microbiota analysis , and define the computational approaches that can be utilised to expand our ever - growing understanding of the role of the gut microbiota in health and disease . the immense interest and impact of the gut microbiota in current research can mainly be attributed to the recent advances in these applications to study microbial communities and their functions . we also detail key microbial cross talk with the immune system , resulting in critical instruction of appropriate immunity . finally , we have considered immune - mediated diseases where the relationship between the gut microbiota and disease susceptibility is currently most convincing , including inflammatory bowel disease ( ibd ) , obesity - related inflammatory disorders , allergic and infectious diseases . where applicable , the influence of key factors such as diet or antibiotic use on the composition of the gut microbiota is outlined . as well , we explore the use of gf mice , and gnotobiotic mouse models where gf mice are colonised with defined microbial communities , which provide a tool to elucidate the function of the gut microbiota in a disease setting . the recent advances of the culture - independent methods analysing the dna ( metagenomics ) , rna ( metatranscriptomics ) , proteins ( metaproteomics ) and metabolites ( metametabolomics ) present within the gut will be the focus of this section ( figure 1 ) . the development and reducing costs of next - generation sequencing , also known as high - throughput sequencing , combined with improved computational tools allowing for the analysis of large and complex data sets , have led to an enormous growth in the research field . each tool provides unique information to interpret the different species that live within the gut ; to provide understanding of how these species function ; and describe why certain species may contribute to disease , or respond differently to specific stimuli such as changes in diet . initial efforts to characterise the gut microbiome using next - generation sequencing of gut microbiota involved the use of a marker ' gene that serves as a unique fingerprint for different bacterial genera . as such , the bacterial communities within the gut can be assessed by sequencing the variable regions of the 16s ribosomal rna ( rrna ) encoding gene sequence found in bacteria and archaea , whereas eukaryotic organisms ( fungi , parasites and the host ) can be assessed by sequencing the 18s rrna encoding sequence . within a clinical setting , this approach could be adapted to identify specific pathogens or disease - associated species from within an individual 's gut microbiota without sequencing all of the dna within a sample , decreasing costs and the bioinformatic processing time for clinicians . further use of metatranscriptomic approaches linked with rna sequencing of the host will reveal more information about how distinctive microbial functions are linked to disease , and how they alter human health . metaproteomic studies examine the proteins that typically result from the microorganism , but can also result from inflammatory responses of the host . moreover , shotgun proteomic approaches have identified unique proteins present within the gut that were not predicted by metatranscriptomic approaches , highlighting the yet unknown nature of many interactions between the gut microbiota and the host . similarly , metametabolomic approaches also use a shotgun - like approach to examine a broad range of metabolic by - products and secretions of microorganisms that provide insight into active microbial functions and interplay between microbial species as well as the gut microbiota and the host . many gi diseases linked to the gut microbiota may be associated with altered proteins or metabolite production , rather than a depletion of certain microbes . once certain proteins and metabolites can be linked to microbiota - associated diseases , downstream applications will likely revolutionise our ability to quickly and efficiently detect alterations in the gut microbiota that are linked to disease phenotypes . this knowledge could provide a basis for the crucial next step and the remaining challenge for this research field , to establish causal relationships between the gut microbiota and the host . colonisation of gf mice with mouse or human gut microbiota ( referred to as humanised mice ) has been used to demonstrate a causal role for the gut microbiota in immune development and disease susceptibility . it is likely that the emerging technological applications will move beyond the current focus of the bacterial communities present towards their function ; which holds the most promise for the identification of microbial or metabolite based therapeutic strategies for prevention and treatment of inflammatory and infectious diseases . the gi tract must remain non - responsive to food antigens and the gut microbiota , while being capable of responding quickly to invading pathogens . one of the first defence mechanisms afforded by the gut microbiota is limiting the access of pathogenic bacteria to the gut epithelium by competitive exclusion . the gut microbiota also promotes secretory immunoglobulin ( ig)a production that binds to microbes at mucosal surfaces , neutralises toxins and contributes to microbial tolerance . furthermore , bacterial molecular patterns are sensed by innate pattern recognition receptors ( prr ) such as toll - like receptors ( tlr ) or nod - like receptors , which have a role in the maintenance of intestinal epithelial cell ( iec ) homeostasis . the microbiota also contributes to the priming signal of the inflammasome pathway , leading to the transcription of cytokines such as tumour necrosis factor ( tnf)- and interleukin ( il)-6 , as well as pro - il-1 and pro - il-18 . the gut microbiota also helps to maintain immune homeostasis by stimulating different arms of the t - cell response . sfb adhesion to iec results in the activation of the il-23 pathway , leading to il-22 production by group 3 innate lymphoid cells ( ilc3 ) and a subsequent th17 response . conversely , polysaccharide a from bacteroides fragilis was shown to stimulate the generation of foxp3 + regulatory t cells ( treg ) via tlr2 activation , and microbial - derived butyrate has also been shown to induce colonic treg . as such , fermentable dietary fibre scfa signal via g - protein coupled receptors ( gpcr ) such as gpr41 , gpr43 and gpr109a on epithelial cells and immune cells . when mac beneficially affect the host 's health by selectively changing the composition or activity of the gut microbiota , they can be referred to as prebiotics . probiotics can interfere with the growth or survival of pathogens in the gut lumen , improve gut barrier function and immunity , or have an effect on the systemic immune system and organs . both pre- and probiotics can be useful for the beneficial modulation of the gut microbiota , and their capability to enhance immune function has been studied for many years . no longer viewed as merely passengers , the gut microbiota has a critical role in the development and progression of ibd , and is the most extensively investigated with respect to the influence of the gut microbiota on disease susceptibility , relapse and remission . under normal circumstances , these bacteria are likely to be outcompeted for this resource by the resident microbial community , but the alterations in the gut microbiota that occur with ibd may present the opportunity for these bacteria to flourish . other mucous associated bacteria suspected of having an important role in ibd are the sulphate - reducing bacteria such as desulfovibrio piger . in the gut , sulphate - reducing bacteria consume hydrogen to produce h2s by removing sulphate from sulfated mucous glycans . in addition , activation of the inflammasome can occur via mac modulation of the gut microbiota as well as scfa administration , which promoted il-18-mediated epithelial repair following dss - induced gi inflammation . butyrate produced by the gut microbiota , most likely by members of the clostridia class , has also been shown to induce the expansion of treg in mice , ameliorating intestinal inflammation in an adoptive t - cell transfer model of colitis . other bacteria , such as f. prausnitzii , bifidobacterium and lactobacillus protect the host through a variety of mechanisms , including modulation of cytokine production and strengthening gut barrier function . the evidence for the efficacy of probiotic strains like bifidobacterium and lactobacillus in reducing the symptoms of crohn 's disease in humans remains unclear , although some beneficial effects have been shown in patients with ulcerative colitis . in addition , the gut microbiota may protect the host by outcompeting pathogenic bacteria that drive gi inflammation by preventing these pathogens from occupying niches . however , so far , fmt for treating ibd has been shown to have variable efficacy , and long - term safety for fmt is largely unknown . both lifestyle and genetic factors have a role in the development of obesity , and recently the gut microbiota has been suggested to be a key environmental factor that influences metabolic syndrome . the heritability of the gut microbiota has previously been examined linking the abundance of specific gut microbial taxa to host genetics . thus , host genetics influence the composition of the human gut microbiota , and this can impact host metabolism . nonetheless , the diet has been suggested to be most influential in shaping the composition of the gut microbiota . an increase in the ratio of firmicutes to bacteroidetes , as well as a marked reduction in the bacterial diversity of the gut microbiota , have been associated with obesity in both genetic- and diet - induced mouse models and clinical obesity . moreover , diets that limit weight gain led to a reduction in the relative abundance of firmicutes and an increase in bacteroidetes in both mice and humans . however , other investigations have shown no association between obesity and the bacteroidetes to firmicutes ratio , or the diversity of the human gut microbiota , suggesting the relationship between obesity and the gut microbiota is highly complex and difficult to interpret . there is now a large body of evidence that supports the close link of obesity and insulin resistance with chronic , low - grade inflammation , and the gut microbiota has been implicated in the development of this inflammatory response . both diet and genetic factors can alter the gut microbiota and impair intestinal integrity , leading to metabolic endotoxemia , obesity , glucose intolerance and insulin resistance via activation of tlr4 and subsequent inflammatory cytokine production . moreover , restoration of the gut microbiota by the mac oligofructose resulted in amelioration of the inflammation as well as the associated metabolic abnormalities . consequently , tlr5 deficient mice have altered gut microbiota and are prone to the development of metabolic syndrome . the gut microbiota provides enzymes that increase caloric harvest from mac and the resulting monosaccharides are absorbed or metabolised to scfa . in addition , the gut microbiota acts to enhance lipoprotein lipase - directed deposition of these tg into adipocytes , promoting the storage of calories harvested from the diet into fat . fat storage is reduced in gf compared with conventional mice , and colonisation of these gf mice with gut microbiota results in a 60% increase in body fat within two weeks . furthermore , depletion of the gut microbiota using an antibiotic cocktail was sufficient to reduce adiposity and improve glucose tolerance and insulin sensitivity by promoting browning of adipose tissue in both diet and genetically induced obese mice . fmt from lean donors into humans with metabolic syndrome was also sufficient in the clinical setting to increase gut microbial diversity and improve insulin sensitivity . epidemiologic studies imply that disruption of the gut microbiota early in life , via caesarean section as well as antibiotic therapy , increases the risk of being overweight later in childhood . taken together with the preclinical data , this demonstrates the complex relationship between the gut microbiota , antibiotic use and metabolic outcomes . in light of the high rates of childhood obesity , the use of antibiotics or pre- and probiotics to restore intestinal homeostasis and to potentially prevent obesity in children from overweight and obese mothers is an interesting concept . yet , preclinical studies imply that even transient alterations in the infant gut microbiome could potentially affect metabolic homeostasis resulting in obesity . this study provides evidence for a critical window in early life when the gut microbiota can induce long - term alterations in host metabolism . taken together , the gut microbiota has a substantial effect on metabolic abnormalities by influencing the efficiency of energy harvest and storage , low - grade inflammation and browning of adipose tissue . notably , there are risk factors for these metabolic abnormalities beyond gut microbes , which include lifestyle factors such as energy intake and expenditure as well as genetic risk factors . the integration of modern applications to assess microbiota in preclinical and clinical studies will help clarify this complex interplay and yield promise to identify novel treatments for beneficial modulation of the gut microbiota to improve metabolic health . genetic and molecular risk factors are clearly associated with the development of allergy ; however , it is unlikely genetic factors can account for the rapid rise in allergic disease over the past 50 years . this indicates that environmental factors , such as the gut microbiota , may have a key role in the development of allergic disease . for example , reduced gut microbial diversity in infants was associated with increased allergic risk in school age children , and the development of food allergy was associated with altered gut microbiota in these infants . gf mice have been an effective way to address several questions regarding the relationship between the gut microbiota and allergic inflammation . colonisation of gf mice with gut microbiota in early life was sufficient to protect mice from allergic inflammation in the lung , whereas allergic airway inflammation ( aai ) persisted in mice colonised as adults . in addition , clostridia - induced il-22 has also been shown to reduce the uptake of dietary antigen into the systemic circulation . these investigations suggest that the gut microbiota is a key environmental factor in the regulation of allergic inflammation in the lung and gi tract . depletion of the gut microbiota using antibiotic administration in mice has also been employed to examine the impact on allergy susceptibility . vancomycin is not absorbed from the intestine , so when administered orally affects only the gut microbiota . an expansion of bacteroidetes in the lung and the development of treg cells with age were correlated with reduced aai . asthmatic patients have also been shown to have higher bacterial burden and diversity compared with controls . however , the function of the gut microbiota in atopic dermatitis ( ad ) is less clear . similarly , the impact of the gut microbiota in eosinophilic gi diseases , such as eosinophilic esophagitis , as well as other allergic disorders including allergic rhinitis and allergic conjunctivitis , are yet to be fully elucidated . indeed , allergy is reduced in children exposed to a farm environment , and studies suggest microbial exposures in early life are a contributing factor . two notable studies support beneficial modulation of the gut microbiota and associated metabolites in early life as an effective intervention strategy for allergic disease . the canadian healthy infant longitudinal development ( child ) study identified a transient alteration in the gut microbiota during the first 100 days of life in infants at risk of asthma , which was associated with reduced faecal acetate concentrations . this disturbance of the microbial composition in infants at risk was characterised by significantly lower abundance in the gut of lachnospira , veillonella , faecalibacterium and rothia bacterial genera . colonisation of gf mice with these specific gut microbiota ameliorated aai in the offspring , demonstrating a causal role for the bacterial communities in reducing allergy susceptibility . moreover , the gut microbiota of infants with cow 's milk allergy is altered in composition and diversity as compared with non - allergic infants . future research identifying the components of the gut microbiota that influence allergy avoidance or tolerance mechanisms is crucial to develop these strategies for widespread clinical use . infection rates in the western world have reduced dramatically over the past century due to advances in hygiene , antibiotic use and vaccination . the gut microbiota provide critical signals for the development and function of the immune system that provides protection from invading intestinal pathogens . therefore , manipulation of the gut microbiota is an attractive therapeutic target to build immune capability to combat infectious diseases . interactions between epithelial cells and the gut microbiota are essential for intestinal homeostasis and barrier defences . defined gut microbial species have been shown to maintain tight junction integrity , which in turn limits salmonella typhimurium invasion . key gut microbial species also prime the development of cellular immune responses in the gi tract . the critical role for the gut microbiota in establishing immune defence against infection extends far beyond the local environment of the gi tract . microbiota signalling has also been shown to enhance protection against influenza virus by facilitating inflammasome dependent il-1 and il-18 in the lung in a tlr dependent manner . interestingly , only gut and not lung microbiota derived ligands activated this respiratory defence mechanism , demonstrating the importance of the gut microbiota in establishing immune defences at other microbiota colonised sites . a mechanistic role was elucidated , demonstrating tlr5 mediated sensing of the gut microbiota increased the magnitude of the antibody response . the authors suggest the observed variable efficacy of tiv between vaccines may in part be attributed to the host 's gut microbiota composition . this raises the possibility that beneficial modulation of the gut microbiota may be used as a therapeutic approach to improve the efficacy of vaccine - induced immunity . manipulating gut microbes to enhance immune function is not a new concept to be tested in the clinic . these studies provide clear evidence of a role for the gut microbiota in immunity to infection as well as vaccine - induced protective antibody responses . as most gut microbes ( especially anaerobes ) can not be cultured easily , the development of new culture - independent techniques have led to an increase in knowledge in the gut microbiota field . these modern technological applications are now used frequently in both pre- and clinical studies to address the role of the gut microbiota in health and disease ( figure 2 ) , and have substantially contributed to providing understanding of the complex interplay between gut microbial composition , host genetics and biological outcomes . studies in gf mice , which like newborns can be colonised by a diverse microbiota , demonstrate that a specific microbial composition can exert lasting effects on the immunity and disease outcomes of the host . so far there are only correlations between alterations in gut microbial composition and inflammatory and infectious diseases in humans . the humanised mouse models , when combined with current technologies , are a promising attempt to move beyond these correlations to provide causal roles for specific gut microbial functions and disease susceptibility . it also opens up possibilities to study the effects of dietary components on gut microbiota composition and disease outcome , or to evaluate the ability of bacterial strains with a therapeutic potential to invade established niches in gut communities . these models might not completely recapitulate the human disease complexity or heterogeneity of the human gut microbiota . humanised mouse studies have the added complexity of the donor microbiota not having co - evolved with a mouse host , and studies have shown host - specific gut microbiota may be critical for an appropriately functioning immune system . beneficial modulation of the gut microbiota has yielded encouraging results for the prevention and treatment of various inflammatory diseases in experimental models . alongside , fundamental research needs to be conducted to define the mechanisms by which the gut microbiota can enhance immune health . most likely non - bacterial microbes also have an important role in immune function and disease . this is a fast - paced field of research and given the positive results so far , we are cautiously optimistic it will soon yield novel therapeutics that manipulate the microbiota to prevent or treat chronic inflammatory diseases and enhance protective immunity against pathogens .

in this article , we explore the potential of the right to enjoy the benefits of scientific progress and its applications ( rebsp ) , a right included in article 27 of the universal declaration of human rights ( udhr ) and article 15 of the international covenant on economic , social and cultural rights ( icescr ) , as an important lever for a rights - based approach to improving treatment for neglected diseases . we use the example of multidrug - resistant tuberculosis ( mdr - tb ) , a disease with disproportionate impacts on the poor and marginalized , to highlight relevant questions for further discussion . better understanding of the rebsp may well facilitate greater likelihood that patients and communities will realize their rights to health , and greater clarity on obligations of state and non - state actors in responding to the crisis that is mdr - tb in the world today . while the rebsp is slowly receiving increased attention from un human rights bodies and academics , it remains largely overlooked , so greater attention to this right , in relation to the right to health , is timely . today s world is increasingly turning to science and technology for solutions to persistent socioeconomic and development problems , and the human dimension of science therefore demands increased attention . mdr - tb is both a major global health challenge and a clear illustration of how the rebsp might interface with the right to health . about half a million new cases are estimated to occur each year , with the majority the result of direct transmission . yet only one patient in five has access to currently recommended treatment , and only half of these patients are cured effectively with that treatment . the reasons for lack of effective treatment are multifactorial and include poor access to mdr - tb diagnosis , lengthy and complicated treatment regimens , and the high cost of drugs and poor effectiveness of the regimen itself . currently recommended regimens for mdr - tb consist of older drugs previously discarded for first - line tb treatment , and repurposed drugs , that is , drugs developed for indications other than tb . these drugs are combined in multi - drug regimens based primarily on expert advice and limited observational data , and result in cure for 60% of patients even under ideal conditions . clearly a more effective and more accessible treatment regimen for mdr - tb is required , both to cure individuals and to prevent ongoing community transmission of this airborne infectious disease . human rights barriers to effective tb treatment include discrimination and exclusion of sick people , often in combination with poverty . could an approach based on the right to enjoy the benefits of scientific progress change that situation ? we explore in this article how the rebsp might address a number of relevant questions . for example , could it be leveraged to facilitate access to existing or repurposed medicines , that is , the existing products of scientific progress ? could it help to spur the development of novel medicines not yet existent in the drug pipeline ? given that the development of a new medicine is based on scientific research , who has obligations to engage in scientific research ? following development of a new medicine , who has the responsibility to ensure that the new medicine is trialled appropriately and then made accessible to those who need it most ? below we map the origins and contours of the rebsp and its relationship to the right to health ; then we review the current burden of mdr - tb and status of mdr - tb drug treatment ; we describe actions needed to improve access to cure for mdr - tb ; we explore responsibilities for implementation of the rebsp and the role of legal / political action in ensuring human rights ; and then we close by proposing future steps to test whether the rebsp can be drawn upon to improve availability of and access to new medicines and thereby improved treatment for mdr - tb patients . while the focus of this paper is on treatment for mdr - tb , we recognize that similar arguments may be made with respect to other aspects of mdr - tb management , including diagnostics , and , indeed , to many other conditions . the right to enjoy the benefits of scientific progress as a human right has remained rather obscure and unexplored in human rights discourse . an early commentator argued in favor of establishing a duty for the community to make funding available for scientific research , as a means for alleviating wants of mankind , for the development of mankind , and for the pursuit of truth . besides inclusion in article 27.1 of the udhr and article 15.1b of the icescr , it is also part of article 15 of unesco s universal declaration on bioethics and human rights . in the period of the drafting of the udhr and the icescr between 1946 and 1966 , there was much optimism about the potential contribution of science to improving human life and living conditions of people . at the same time , there was awareness and concern about the collusion of science in the nazi genocide during world war ii . as a result , these human rights instruments explicitly stated that people should benefit from scientific progress . an important issue in this respect is whether the rebsp only refers to sharing the concrete benefits of scientific progress among people or whether it also includes the development of science as such . this is crucial from the perspective of investing in scientific research for the benefit of the health situation of poor people , which is less profitable for the corporate sector . the present authors agree with the latter view as advocated by chapman , because it does justice to one of the key features of human rights : non - discriminatory and equal access to benefits . science as a concept relates to obtaining and expanding knowledge and understanding processes and phenomena which occur in nature and society . the link with the realization of human rights can be found in the terms progress and benefits in articles 27 and 15 . progress recognizes that science develops in certain ways , sometimes linear and sequential , and sometimes through qualitative shifts , towards creating positive effects for human beings , society at large , and life on earth in general . what was the extent of scientific knowledge decades ago is recognized as being superseded by newer advances in knowledge generated from ongoing scientific research hence the idea of progress. the term benefits has a qualitative connotation and includes effects that positively impact on humans and society . both terms therefore link to the notion of progressive realization of economic , social , and cultural rights as laid out in article 2.1 of the icescr in the sense that progress of science that generates benefits allows for the achievement of higher and more advanced levels of welfare and wellbeing aimed at the full realization of rights over time . the rebsp , especially the notions of science and progress , should therefore be seen as a vehicle for achieving other rights , such as the right to health , rather than an end in itself . although the rebsp has been phrased as an individual right , we submit that , similar to the right to health , it has a collective dimension . indeed , it is almost irrevocably social in its expression , rather than individualized . for example , in discussing how remedies and accountability operate under the right to health , the general comment notes that regardless of whether groups as such can seek remedies as distinct holders of rights , state parties are bound by both the collective and individual dimensions of article 12 . collective rights are critical in the field of health ; modern public health policy relies heavily on prevention and promotion which are approaches directed primarily to groups . regardless of whether groups as such can seek remedies as distinct holders of rights , state parties are bound by both the collective and individual dimensions of article 12 . collective rights are critical in the field of health ; modern public health policy relies heavily on prevention and promotion which are approaches directed primarily to groups . this means that , in the same way , enjoying scientific progress will not benefit one person only , or a limited group of people . . this will particularly apply when progress in pharmaceutical research leads to greater availability and accessibility of drugs for the treatment of mdr - tb . a human rights approach which focuses on strengthening access for the most vulnerable and marginalized groups may contribute to achieving equity and thus give clear expression to the collective dimension of the right . there are clear links with the right to the highest attainable standard of health ( icescr , article 12 ) which includes the right to have access to essential drugs for the treatment of life - threatening diseases . this means that existing drugs need to be made accessible without prohibitive cost barriers to those in need . the rebsp can potentially take this further and suggest that essential drugs need to be given this , there is likely to be an underlying tension between , on the one hand , the rebsp and , on the other hand , the protection of intellectual property rights ( patents ) of inventors and producers ( companies and private research institutes ) . for example , exclusivity of pharmaceutical test data , as provided for under the trips agreement , may act as a barrier for making the results of scientific progress generally available . a uk government report suggested that the current system of intellectual property protections and patents has failed to stimulate research and development for neglected diseases , primarily as there is no profitable market for such drugs . there is consequently a greater need to invest public monies in the development of such drugs on public health , human rights , and moral grounds , or remove the need to recoup the costs of drug development through direct sales of that drug by providing other mechanisms for cost recovery . one such mechanism is the priority review voucher program which enables any pharmaceutical company that develops a new treatment for a neglected disease ( including tb ) to receive priority review from the us food and drug administration for another product . a priority review voucher commits the fda to reviewing a drug within six months , instead of the usual aim of ten months . while the authors of the original priority - review proposal suggest that a voucher could be worth more than us$ 100 million , a recent sale of a voucher ( which is transferrable ) was recorded at 350 million usd . a priority review voucher was awarded to janssen in 2012 for registering bedaquiline , potentially allowing the company to offset the costs of drug development for bedaquiline . this , and similar strategies have the potential to achieve delinkage and might be useful to explore in the future . of course , states can also leverage enhanced access to medicines through public - private partnerships , as has been effected by the stop tb partnership s global drug facility . however , these are initiatives limited to specific drugs on a case - by - case basis outside of any framework that provides strong obligations on any partners . to date , there have been no cases before courts where the rebsp has been directly invoked aimed at forcing a government to make an available medicine accessible for the treatment of a particular disease . similarly , there have been no cases where the rebsp has been used to leverage the development of new drugs for neglected diseases . this is due to its unknown nature , but also to the fact that it has not been recognized as a justiciable right that individuals can invoke directly . one rare example is a case from argentina in which the right was not explicitly stated , but was implicitly at stake . a group of argentinian ngos brought the case , asking the state to manufacture a vaccine with known efficacy for the prevention of argentine hemorrhagic fever , which affects people living in the high - risk pampas and who did not have easy access to preventive treatment . the private sector had failed to produce a drug , because it was not profitable to do so . the judgement was based , inter alia , on the right to health as included in the icescr . the number of international cases may increase in the future when the optional protocol to the icescr becomes more well - known as an international remedy for domestic victims of violations of the rights in the covenant . from a more general perspective , therefore , it is apparent that the rebsp does not entitle individuals to direct enforceable benefits of scientific progress , but rather entails a right for people to have a legislative and policy framework adopted and implemented which aims at making the benefits of scientific progress available and accessible both through encouraging new scientific discoveries , and through removing barriers for existing scientific knowledge to be used for public benefit . steps to be taken in this regard must be deliberate , concrete , and as targeted as possible . this leads to the question of who has obligations or responsibilities to realize this right and what the nature of these obligations is , which we explore below using dr - tb to illustrate the general case . the majority of people in the world suffering from tb are poor and/or marginalized , and it disproportionately affects low - middle income countries with limited health care resources . despite some progress in tb control , close to 10 million people are estimated to develop tb each year , of whom more than a third are not diagnosed and therefore not treated . the figures for mdr - tb are even more stark : only a quarter of estimated cases are detected , and fewer have access to treatment . while eastern europe has the world s highest proportion of mdr - tb among all tb cases up to 40% in some settings the largest burden in terms of numbers lies in countries such as china , india , and south africa , due to large populations and high tb incidence . while lack of effective tools for diagnosis of tb drug resistance remains a major barrier to effective mdr - tb treatment globally , most tb drugs in current use were developed between 1943 and 1963 , and the current regimens involving combinations of first - line tb drugs have been in place for about 40 years . the reliance on the dots ( directly observed treatment , short - course ) strategy , with extremely limited drug susceptibility testing and a limited armamentarium of available tb drugs , has left global tb programs exceptionally vulnerable to the rise of mdr - tb . even with two new drugs recently approved for tb treatment ( principally for mdr - tb treatment ) , the pipeline for new tb drugs remains poor . prior to the approval of bedaquiline and delamanid , the most recent new tb drug to reach the market was rifampicin in the 1960s . despite recent improvements , investment in tb drug research falls far short of what is estimated to be required , with the us$255 million in 2013 amounting to just a third of the annual target set in the global plan to stop tb . similarly , investment in global tb control falls short by approximately us$1.4 billion of that estimated to be required for effective implementation , including that required for mdr - tb treatment . given the disproportionate burden of tb , and mdr - tb among the poor , the lack of investment in global strategies to combat tb and the consequent lack of a profitable market , there is a distinct lack of interest in tb drug development in the private sector . this results in two kinds of access problems : the lack of development of new drugs , on the one hand , and the inability to access new drugs ( or newly repurposed drugs ) for treating dr - tb . both represent challenges for thinking through any entitlement to benefit from scientific progress . mdr - tb treatment currently requires at least four or five drugs , often with considerable side effects , for at least 18 months . treatment includes an injectable agent , given daily , for between four and six months . even under ideal conditions , treatment is successful for approximately 60% of patients ; high mortality , treatment failure , and failure to complete the treatment regimen are common . it is therefore not surprising that the development of bedaquiline and delamanid , both new classes of drugs with tb activity , has raised hopes for a new , more efficacious , and more mdr - tb - tolerable treatment regimen . despite regulatory approvals in a range of countries for both new drugs and available guidance on use from who , access to these drugs for mdr - tb patients has remained extremely limited . while bedaquiline received conditional approval from the us food and drug administration in 2012 , and additional approvals in the european union , south africa , india , and other countries , less than 1,000 patients had received the drug by 2015 . the situation for delamanid is even more dramatic ; by february 2016 , only 180 patients have been able to access the drug , primarily under compassionate use criteria . delamanid was registered in europe , korea , and japan in 2014 , but no further registrations are pending , including in the countries contributing the majority of patients to the clinical trials on which drug registration was based . the reasons behind poor accessibility of these new drugs are multiple : high cost , potential side - effects leading to excess mortality , lack of systems for pharmacovigilance , resistance development , and , finally , lack of guidance on effective use of these new drugs for mdr - tb treatment in national tb programs . with bedaquiline priced at us$900 for a six - month treatment course , cost would appear to be a clear barrier , but even under a usaid - funded program to support bedaquiline access where the drug was donated by the pharmaceutical company and is provided free to national tb programs uptake has remained slow . current guidance from who suggests that both bedaquiline and delamanid should be used as additional drugs in mdr - tb treatment , in cases where a treatment regimen can not be constructed with existing drugs . in reality , this means that these new drugs are added to the existing , poorly tolerated , and lengthy treatment regimen , resulting in limited improvements to overall treatment success . encouragingly , there are a small number of clinical trials underway and in planning that aim to incorporate bedaquiline , delamanid , or both into shorter , more efficacious mdr - tb treatment regimens . these regimens will require a combination of new , repurposed , and existing drugs in order to maximize efficacy . however , funding and capacity to conduct trials that are not directly linked to drug registration , that is , not funded by pharmaceutical companies , is limited . if the few trials currently underway or in planning produce promising results , these regimens then need to be made accessible in routine tb programs . while treatment regimens may be efficacious under clinical trial conditions , this may not translate into appropriate patient outcomes under programmatic conditions . development and testing of new regimens for example , of different duration and combinations in relation to likely adherence would need to be implemented to ensure the medication is suitable for programmatic use , particularly in high - burden settings . as a result , ideally a continuous pipeline of new drug development should feed a constantly evolving clinical trial environment , the results of which are then operationalized in order to provide treatment for those in need . thus , both access to existing drugs and the development of new drugs for mdr - tb are integrally related to each other , as well as representing key challenges for the rebsp . all along this pathway , important obstacles may be encountered , which , if translated into rights language , can generate particular obligations and responsibilities for different parties ( table 1 ) . the right to enjoy the benefits of scientific progress : actors , duties and responsibilities related to multidrug - resistant tb ( mdr - tb ) . establish an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases desist from using ip provisions when domestic state has thoughtfully and appropriately sought alternative routes . engage actively in translating csr commitment into rights responsibilities for ip issues lobby for an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases actively promote an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases an overarching science policy reflecting the rebsp strengthen research capacity in lmics through technology and knowledge transfer without ip hindrances . respond to responsibilities outlined in guidelines for transnational corporations by pursuing relevant research conducting research relevant to mdr - tb nicrease public accountability for decision making as to what is funded and how direct state funding for research on mdr - tb subsidies or tax advantages to encourage private sector desist from ever - greening practices that remove potentially effective drugs from a development pipeline university to develop and implement systems to support responsive research contribute to funding for r&d on new medicines state adherence to commitments for funding r&d investment in human resource capacity to do research translate responsibilities through technology transfer to lmics capacity development of researchers to deliver research outputs needed . engage with ( donor ) states , ngos , and international organizations to promote and organize r&d on new medicines subsidies or tax advantages to encourage private sector allocate university resources to promote relevant research soliciting partnership agreements to support research regulatory action to ring - fence public sources ( such as tax ) investment in human resource capacity to do research expedited regulatory approval for trials expedited regulatory approval for appropriate indication ensuring sufficient pharma capacity for drug manufacture ensuring sufficient pharma capacity for drug manufacture exploratory research to identify candidate drugs for repurposing mobilizing resources globally for development and operational research negotiations on cost with pharma ( tender agreements , with forecasting ) ensure rights to a drug are available ( give away rights ) if pharma has no interest in manufacture setting up global consortia for purchasing fast - track or expedited processes for neglected diseases pursue registration of drugs vigorously following successful demonstration of effectiveness providing expertise for advice in regulatory decision - making permitting operational studies ; flexibility in registering drugs in post - rct phase engage with government regulatory authorities desist from imposing forms of patent protection on lmics that inhibit access to essential medicines desist from lobbying home country governments for excessive patent protection active research dissemination ; university commitment of resources for dissemination development of treatment protocols for up - scaled implementation contribute to funding through donor and multi - stakeholder structures contribute to funding through donor and multi - stakeholder structures for accelerated drug - development for dr - tb training of staff in research dissemination support operational research contribute to mobilizing multi - stakeholder partnerships to support implementation public education on tb science to strengthen civil society voice public education on tb science abstain from activities that delay , postpone , or block availability of new drugs prioritize concrete and targeted measures aimed at reaching vulnerable members of society additional actors may include the world health organization , whose stewardship role obliges it to facilitate and coordinate cooperation for dr - tb drug development , and civil society , whose roles include holding state and other actors accountable , including bringing cases to court where appropriate , promoting public information and awareness , and mobilizing community voice . the obligations and responsibilities with regard to developing regimens for existing repurposed drugs largely include those that pertain to different actors with regard to basic science identification of new drugs and establishing their efficacy and effectiveness , and are not repeated in this row only matters that are specific for access to existing drugs . when identifying the types of obligations that give substance to the rebsp , it makes sense to distinguish between different duty - holders and actors ( table 1 ) . states who have voluntarily accepted to become legally bound by the icescr through ratification are the main duty - bearers . but is the state also the most appropriate actor to implement obligations resulting from article 15.1b ? in the 1960s , when the icescr was adopted , governments were centrally involved in funding research and the direct development of science , which was seen as a public good . this has changed drastically in present times , where the role and influence of the private sector ( corporations and private research institutes ) and non - state consortia has increased enormously and market forces often determine for which purposes both private and public resources will be spent . at the same time , the availability of public resources for research into new medicines has diminished . the typology of human rights obligations ( respect , protect , and fulfill ) provides an appropriate framework for explanation . obligations to respect the rebsp entail that both the domestic state and the home state of a pharmaceutical company must not introduce obstacles in its intellectual property laws and policy that would prevent institutes for research and development ( r&d ) from focusing on neglected diseases . the obligation to protect means that a state must regulate the conduct of corporations and prevent these from using their corporate power to frustrate the availability and accessibility of medicines that are less profitable . the human rights obligation to fulfill can be divided into obligations to facilitate and to promote . a state has an obligation to create an enabling environment for the development and implementation of a science policy which lays out principles that should underlie scientific research and establish an approach for research prioritization and diffusion of research findings . this is an example of an obligation to facilitate which is aimed at creating institutional arrangements to support the implementation of a right . with a view to making medicines for neglected disease available and accessible , states have an obligation to facilitate in the sense of stimulating research for new medicines by companies , research institutes , and universities , whether in the form of joint ventures or directly in the public sphere . this can be done , for example , by providing subsidies or tax advantages to incentivize research into neglected diseases , investing in the human resources base needed for research , and routing research grant funding into prioritized focus areas , whether through direct state funding of research , through partnership agreements with earmarked research funders , or through regulatory interventions to mobilize research funding . direct funding for research is typically expressed in the form of a state commitment to allocate a minimum percentage of gross domestic product ( gdp ) to r&d . for example , both the european union and the obama administration have set spending targets for research and innovation at 3% of gdp . in 2006 , the african union adopted the khartoum declaration , which committed states to allocate a minimum of 1% of gdp to r&d . this was subsequently reinforced in the african union s science , technology and innovation strategy for africa 2024 . in practice , though , such commitments may be overly optimistic for low- and middle - income countries ( lmic ) . for example , of the 30 african countries that adopted the abuja declaration in 2001 to allocate 15% of total government expenditure to health , only one had reached this target 10 years later . the obligation to promote has a long - term nature : it aims at providing information and convincing key players of the need to make essential medicines available . it may be combined with public awareness - raising campaigns aimed at empowering the general public to make informed choices about their health , or at providing civil society and the public with information to pressure key stakeholders to act , as has been illustrated amply in the hiv treatment access arena . additionally , once evidence of effectiveness is obtained , states should ensure that the regulatory approvals are expedited and systems for distribution put in place to put drugs into circulation as quickly as possible . for example , while south africa s medicine control council approved a clinical access program for the new tb drug bedaquiline in january 2013 , which removed delays in registering the drug , the cumbersome approval process for patients to access the drug still caused considerable delay in treatment initiation for extensively drug - resistant tb ( xdr - tb ) . even when regulatory approvals put drugs rapidly into the public domain , obstacles posed by patents that create unaffordable prices need addressing . msf has shown how the patent held in south africa for linezolid has prevented generic competition , kept the price high , and resulted in a failure to access linezolid for most patients in need , denying them the opportunity for possible cure . this challenges governments to pursue active purchasing programs able to negotiate prices down or , if needed , resort to the provisions of trips that allow for parallel importation , generic substitution , or other exceptions to the exclusive rights of patent holders . for example , india s patent act provides for the possibility to declare inventions not patentable with a view to prevent evergreening to ensure that patent protection is not granted for minor inventions . moreover , a complex set of operational questions requires guidelines for how best to implement new regimens for mdr - tb that maximize access to new drugs of better efficacy , lower toxicity , and shorter duration , while avoiding development of further resistance through indiscriminate and poorly coordinated use of these medications . guidelines to manage this competing pressure to open up access to new drugs while avoiding use patterns that increase the risk of new drugs developing resistance may require careful balancing of individual rights of access to care with the broader social good of protecting public health consistent with the siracusa principles . in south africa , for example , state flexibility in allowing for off - schedule use of delamanid for compassionate use , and for operational research , has opened the possibility for salvage regimens to be operationally tested and to provide preliminary evidence for future programmatic guidelines , thus shortening the time to wider upscaling of treatment options in future . thereafter , training of staff in new protocols should enjoy similar prioritization to ensure front - line staff is empowered to implement the new protocols carefully and avoid programmatic obstacles to implementation . thus , states , as a part of their human rights obligations , should accelerate not only the research process but all the subsequent steps needed to see a new drug included in standard treatment protocols . it is noted that patents regimes may frustrate or delay this endeavor in many countries . from a normative perspective , therefore , the rebsp entails a right for individuals to have a legislative and policy framework adopted that enables the development and manufacturing of a new medicine . this means both that the policy should not impede or interfere with such drug development and/or repurposing for mdr - tb treatment ( respect ) , but also that it should ensure corporations do not delay new knowledge for medicines for neglected diseases from becoming available ( protect ) and put in place positive measures to encourage drug development and/or repurposing ( fulfill ) . such a policy framework must be reasonable in conception and implementation , which means that it must be coordinated and coherent with a view to focusing on the need to make new and repurposed medicines available for diseases that disproportionately affect the poor , who would otherwise be denied access . for patients with mdr - tb , therefore , such a framework is informed by a human rights approach by which the state would be expected to prioritize measures in its science policy that give preference to drug development and/or repurposing for neglected diseases that disproportionately affect poor and vulnerable members of society . in an era characterized by economic globalization , however , it is self - evident that making essential medicines available is a process involving multiple actors : not only pharmaceutical companies , but also their home states , international organizations , and private funders ( table 1 ) . lack of availability of essential medicines for the treatment of neglected diseases is a global problem that demands a global response . it has been argued that especially governments in the global north who have the capacity and resources have an obligation to contribute to the fulfillment of human rights outside of their national boundaries . through processes of economic globalization , actions and omissions by actors in one state may affect the human rights of people in other countries . however , traditionally , human rights law only applied on the territory of states that have ratified human rights treaties . with a view to filling this normative gap , there is debate over the establishment of a human rights framework that applies beyond national borders . this is the idea of extraterritorial human rights obligations aimed at realizing the universal scope of human rights , a concept elaborated in the maastricht principles on the extraterritorial obligations of states in the area of economic , social and cultural rights . drawn from international law , these principles also aim to clarify the content of extraterritorial state obligations for contributing to the fulfillment of the right to health in countries with serious public health problems . an extraterritorial human rights obligation for states in the global north resulting from the rebsp would logically take two forms . first , states should desist from policies that undermine the rights of lmic populations to enjoy the benefits of scientific progress ( respect ) . one example is overzealous insistence on patent protection and pursuance of pharmaceutical company pricing protections in recipient countries at the expense of local access to medicines ; this was demonstrated in developed countries behavior in the wto in the lead - up to the doha declaration . more recently , it is also evident in the conclusion of numerous bi- and multilateral ( trips - plus ) trade agreements subsequent to doha which continue to undermine doha trips flexibilities and the ability of poorer countries to develop their own ip regimes . patent protection has been shown to be associated with inappropriately high costs of medications which then inhibit governments from adopting efficacious but costly medicines in their regimens . notably , paragraph 3.b of the sustainable development goals recognizes the need to support r&d for vaccines and medicines that primarily affect developing countries and to provide access to such medicines under the doha declaration . second , developed countries might well be expected to realize this obligation by strengthening the research infrastructure and capacity in the global south through promoting the transfer of scientific knowledge free from intellectual property rights . a division and coordination of domestic and extraterritorial human rights obligations between states is required , depending on the level of development , the domestic health situation and tb prevalence , and the capacity and resources of more wealthy states to assist . third , another potential avenue is that represented by funds set up for drug and vaccine development as global collaborations with contributions from rich countries , the corporate sector , and donor organizations . in this regard , there is a need to mobilize and share common but differentiated responsibilities of all relevant stakeholders , both governmental and non - governmental , for international cooperation aimed at giving effect to the right to essential medicines . however , thus far , states from the north appear unwilling to accept that they also have human rights obligations beyond their national borders on the basis that such a commitment would be contrary to their interests and would limit their policy freedom abroad . ironically , these same states emphasize the importance of lmics complying with intellectual property standards so as to protect the interests of their pharmaceutical industries abroad . the bilateral and multilateral trips - plus agreements are a good example of this trend . although corporations do not have human rights obligations , they do have special functions and responsibilities to engage in activities for the promotion of public health . in this regard , corporate social responsibility can be made concrete by giving substance to the implementation of the rebsp . as lee and hunt have argued , pharmaceutical companies have a shared right to health responsibility to engage in r&d for neglected diseases that affect poor people in poor countries . it is what society may expect from having a social license to operate for the general welfare , not just for profit . people who suffer from mdr - tb have only one option for accessing drugs , so their treatment success is partly reliant on the actions of these companies . these companies may therefore be said to perform a public function that the state is unable to execute . there is also a body of work developing that outlines the responsibilities of corporations for meeting human rights standards . initially framed as norms for transnational corporations equivalent to rights obligations of governments , but subsequently reframed as guiding principles after considerable resistance to the accountability associated with the norms approach , these principles recognize the role of business enterprises as specialized organs of society performing specialized functions , required to comply with all applicable laws and to respect human rights . both arguments point to the need for pharmaceutical companies to operate within a human rights framework , albeit with lesser levels of accountability than governments . while efforts have been directed to identifying the role of corporations in a rights - based approach to health , there are many other non - state actors involved . universities and research institutions , typically funded with public monies , but with relative autonomy from the state , are instrumental in conducting both the basic and the applied research needed to bring new drugs into the development pipeline , and to identify drugs with repurpose uses . in the same way that for - profit corporations may have legitimate expectations placed upon them as a social license to operate , universities are increasingly being called on to demonstrate their social responsiveness . here one might find a trade - off in balancing academic freedom for the researcher to pursue whatever line of inquiry they find academically compelling ( academic freedom itself being a right protected in article 15.3 of the icescr ) against the expectation that universities focus their research in ways that prioritize the most urgent societal challenges . clearly , researchers can not be compelled to engage in research to develop new tb drugs , but , in practice , generous funding streams and academic reward systems responding to priority areas such as mdr - tb could and do influence researcher behavior and direct researcher focus . for example , the influence of donor funding in prioritizing hiv and tb - related research has been shown in the pattern of research conducted in many settings across the world . however , it is typically in the translation of scientific research where universities lag , and where the rebsp highlights important gaps . ensuring that scientific findings reach audiences who could act to ensure the implementation of the findings is often a responsibility omitted from university reward systems , and often unfunded by donors . such audiences are typically policy - making structures both in the executive and legislature , but should also include relevant civil society actors , whose agency may be key to realizing rights and to ensuring the policies and programs for implementation of new scientific knowledge . the experience in the hiv field is instructive , where very strong civil society action spurred research and development of new drugs , raised awareness of entitlements to treatment among those most affected by the hiv pandemic , and directly impacted on drug pricing for arvs to treat hiv . the close alliance of scientists with the treatment access movement in south africa played a key role in a number of important public health advances and in advancing access to the benefits of scientific research related to hiv . thus , the obligation on researchers to engage actively in the dissemination of their findings and in the conveying of scientific information to non - scientific audiences represents not only good scientific practice , but a strengthening of the roles of universities and research institutions to help realise the rebsp . if taken seriously , universities would then need to invest in training researchers in better public engagement in science , rewarding scientific activities that enhance popular understanding of science , and dissemination activities aimed at ensuring research findings are translated into practical change . the who was previously seen as the undisputed steward of matters affecting health on a global scale ; this has changed radically in the past 20 years , with the growth of large private and public - private consortia and agencies whose funding mandates are so large that they dwarf that of who . questions have been raised about the accountability of these foundations and to what extent they have turned global health governance into a terrain dominated by rich and powerful interests . in the same way private corporations are being asked hard questions regarding accountability and standards , we believe the same principles should apply to these newer agencies of global health governance . they have a key role in realizing the right to health and in ensuring people s rebsp , which , rather than being framed as philanthropy or largesse , should be seen within the unfolding context of the extended purview of rights frameworks beyond national boundaries and to non - state actors . civil society organizations ( csos ) and patient groups should persuade corporations to take up their social responsibility in this area . to some extent , we have seen that collaboration between the corporate sector and university research institutes may help to bridge the gap between basic science and clinical research to advance innovative medicines for poor patients , for which we already have some multi - stakeholder models such as the drugs for neglected diseases initiative and critical path to tb drug regimens . financing mechanisms , such as illustrated in the tb alliance , are also putting teeth to multi - stakeholder initiatives to accelerate drug development , which will enhance availability and accessibility consistent with the rebsp . nonetheless , questions about the implications of locating such hugely important policy initiatives outside of a framework of state sovereignty ( and the associated system of human rights - based accountability ) remain potential threats to a full realization of the rebsp . it is clear that the rebsp does not provide an individual entitlement to claim a right to have a new drug developed for a neglected disease such as mdr - tb . however , it does provide a number of important levers complementary to right to health claims . it sets up entitlements for a system of science development and dissemination that could and should benefit the most marginalized in society , and represents a collective claim for the use of science for social benefits . inasmuch as the market fails to address neglected diseases such as mdr - tb , the rebsp provides a potential counterbalance to frame a positive obligation on states to both marshal their own resources and to coordinate the actions of multiple other actors towards this goal . spanning the full gamut , these are obligations of respect ( to refrain from policies that violate rights ) , protect ( regulate the behavior of private actors ) and fulfill ( generate an enabling policy environment for priority research ) . at the same time , non - state actors can be brought within the ambit of contributing to addressing one of the major public heath crises of the current period without holding the same level of accountability as states but still recognizing obligations at a level of soft law responsibilities . we believe that there are enough grounds to warrant testing the rebsp framework further in different case settings both in relation to neglected diseases and other access - to - health contexts . for example , new technologies carry the promise of screening for a range of non - communicable diseases or for new forms of safer production that reduce risks for workers and consumers from hazardous chemicals . developing the conceptualization of the rebsp and advancing the evidence base for its role may provide powerful practical tools for human rights advocates , civil society activists , and health and human rights researchers to tackle neglected diseases and address wider health challenges across the globe .

abstractthe right to enjoy the benefits of scientific progress ( rebsp ) is a little - known but potentially valuable right that can contribute to rights - based approaches to addressing multidrug - resistant tb ( mdr - tb ) . we argue that better understanding of the rebsp may help to advance legal and civil society action for health rights . while the rebsp does not provide an individual entitlement to have a new drug developed for mdr - tb , it sets up entitlements to expect a state to establish a legislative and policy framework aimed at developing scientific capacity to address the most important health issues and at disseminating the outcomes of scientific research . by making scientific findings available and accessible , people can be enabled to claim the use of science for social benefits . inasmuch as the market fails to address neglected diseases such as mdr - tb , the rebsp provides a potential counterbalance to frame a positive obligation on states to both marshal their own resources and to coordinate the actions of multiple other actors towards this goal , including non - state actors . while the latter do not hold the same level of accountability as states , the rebsp can still enable the recognition of obligations at a level of soft law responsibilities .

Introduction Situating the right to enjoy the benefits of scientific progress as a human right MDR-TB burden and the status of drug treatment What is needed to improve MDR-TBtreatment? The REBSP: Obligations and responsibilities in relation to MDR-TB Rights claims in a globalizing society Conclusion

in this article , we explore the potential of the right to enjoy the benefits of scientific progress and its applications ( rebsp ) , a right included in article 27 of the universal declaration of human rights ( udhr ) and article 15 of the international covenant on economic , social and cultural rights ( icescr ) , as an important lever for a rights - based approach to improving treatment for neglected diseases . we use the example of multidrug - resistant tuberculosis ( mdr - tb ) , a disease with disproportionate impacts on the poor and marginalized , to highlight relevant questions for further discussion . better understanding of the rebsp may well facilitate greater likelihood that patients and communities will realize their rights to health , and greater clarity on obligations of state and non - state actors in responding to the crisis that is mdr - tb in the world today . while the rebsp is slowly receiving increased attention from un human rights bodies and academics , it remains largely overlooked , so greater attention to this right , in relation to the right to health , is timely . mdr - tb is both a major global health challenge and a clear illustration of how the rebsp might interface with the right to health . the reasons for lack of effective treatment are multifactorial and include poor access to mdr - tb diagnosis , lengthy and complicated treatment regimens , and the high cost of drugs and poor effectiveness of the regimen itself . currently recommended regimens for mdr - tb consist of older drugs previously discarded for first - line tb treatment , and repurposed drugs , that is , drugs developed for indications other than tb . clearly a more effective and more accessible treatment regimen for mdr - tb is required , both to cure individuals and to prevent ongoing community transmission of this airborne infectious disease . could an approach based on the right to enjoy the benefits of scientific progress change that situation ? for example , could it be leveraged to facilitate access to existing or repurposed medicines , that is , the existing products of scientific progress ? given that the development of a new medicine is based on scientific research , who has obligations to engage in scientific research ? below we map the origins and contours of the rebsp and its relationship to the right to health ; then we review the current burden of mdr - tb and status of mdr - tb drug treatment ; we describe actions needed to improve access to cure for mdr - tb ; we explore responsibilities for implementation of the rebsp and the role of legal / political action in ensuring human rights ; and then we close by proposing future steps to test whether the rebsp can be drawn upon to improve availability of and access to new medicines and thereby improved treatment for mdr - tb patients . while the focus of this paper is on treatment for mdr - tb , we recognize that similar arguments may be made with respect to other aspects of mdr - tb management , including diagnostics , and , indeed , to many other conditions . the right to enjoy the benefits of scientific progress as a human right has remained rather obscure and unexplored in human rights discourse . in the period of the drafting of the udhr and the icescr between 1946 and 1966 , there was much optimism about the potential contribution of science to improving human life and living conditions of people . an important issue in this respect is whether the rebsp only refers to sharing the concrete benefits of scientific progress among people or whether it also includes the development of science as such . this is crucial from the perspective of investing in scientific research for the benefit of the health situation of poor people , which is less profitable for the corporate sector . the present authors agree with the latter view as advocated by chapman , because it does justice to one of the key features of human rights : non - discriminatory and equal access to benefits . both terms therefore link to the notion of progressive realization of economic , social , and cultural rights as laid out in article 2.1 of the icescr in the sense that progress of science that generates benefits allows for the achievement of higher and more advanced levels of welfare and wellbeing aimed at the full realization of rights over time . the rebsp , especially the notions of science and progress , should therefore be seen as a vehicle for achieving other rights , such as the right to health , rather than an end in itself . although the rebsp has been phrased as an individual right , we submit that , similar to the right to health , it has a collective dimension . a human rights approach which focuses on strengthening access for the most vulnerable and marginalized groups may contribute to achieving equity and thus give clear expression to the collective dimension of the right . there are clear links with the right to the highest attainable standard of health ( icescr , article 12 ) which includes the right to have access to essential drugs for the treatment of life - threatening diseases . the rebsp can potentially take this further and suggest that essential drugs need to be given this , there is likely to be an underlying tension between , on the one hand , the rebsp and , on the other hand , the protection of intellectual property rights ( patents ) of inventors and producers ( companies and private research institutes ) . similarly , there have been no cases where the rebsp has been used to leverage the development of new drugs for neglected diseases . from a more general perspective , therefore , it is apparent that the rebsp does not entitle individuals to direct enforceable benefits of scientific progress , but rather entails a right for people to have a legislative and policy framework adopted and implemented which aims at making the benefits of scientific progress available and accessible both through encouraging new scientific discoveries , and through removing barriers for existing scientific knowledge to be used for public benefit . the figures for mdr - tb are even more stark : only a quarter of estimated cases are detected , and fewer have access to treatment . while eastern europe has the world s highest proportion of mdr - tb among all tb cases up to 40% in some settings the largest burden in terms of numbers lies in countries such as china , india , and south africa , due to large populations and high tb incidence . while lack of effective tools for diagnosis of tb drug resistance remains a major barrier to effective mdr - tb treatment globally , most tb drugs in current use were developed between 1943 and 1963 , and the current regimens involving combinations of first - line tb drugs have been in place for about 40 years . the reliance on the dots ( directly observed treatment , short - course ) strategy , with extremely limited drug susceptibility testing and a limited armamentarium of available tb drugs , has left global tb programs exceptionally vulnerable to the rise of mdr - tb . even with two new drugs recently approved for tb treatment ( principally for mdr - tb treatment ) , the pipeline for new tb drugs remains poor . prior to the approval of bedaquiline and delamanid , the most recent new tb drug to reach the market was rifampicin in the 1960s . similarly , investment in global tb control falls short by approximately us$1.4 billion of that estimated to be required for effective implementation , including that required for mdr - tb treatment . given the disproportionate burden of tb , and mdr - tb among the poor , the lack of investment in global strategies to combat tb and the consequent lack of a profitable market , there is a distinct lack of interest in tb drug development in the private sector . it is therefore not surprising that the development of bedaquiline and delamanid , both new classes of drugs with tb activity , has raised hopes for a new , more efficacious , and more mdr - tb - tolerable treatment regimen . despite regulatory approvals in a range of countries for both new drugs and available guidance on use from who , access to these drugs for mdr - tb patients has remained extremely limited . the reasons behind poor accessibility of these new drugs are multiple : high cost , potential side - effects leading to excess mortality , lack of systems for pharmacovigilance , resistance development , and , finally , lack of guidance on effective use of these new drugs for mdr - tb treatment in national tb programs . current guidance from who suggests that both bedaquiline and delamanid should be used as additional drugs in mdr - tb treatment , in cases where a treatment regimen can not be constructed with existing drugs . thus , both access to existing drugs and the development of new drugs for mdr - tb are integrally related to each other , as well as representing key challenges for the rebsp . the right to enjoy the benefits of scientific progress : actors , duties and responsibilities related to multidrug - resistant tb ( mdr - tb ) . establish an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases desist from using ip provisions when domestic state has thoughtfully and appropriately sought alternative routes . engage actively in translating csr commitment into rights responsibilities for ip issues lobby for an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases actively promote an ip system that rewards r&d but does not place obstacles to the rebsp for neglected diseases an overarching science policy reflecting the rebsp strengthen research capacity in lmics through technology and knowledge transfer without ip hindrances . respond to responsibilities outlined in guidelines for transnational corporations by pursuing relevant research conducting research relevant to mdr - tb nicrease public accountability for decision making as to what is funded and how direct state funding for research on mdr - tb subsidies or tax advantages to encourage private sector desist from ever - greening practices that remove potentially effective drugs from a development pipeline university to develop and implement systems to support responsive research contribute to funding for r&d on new medicines state adherence to commitments for funding r&d investment in human resource capacity to do research translate responsibilities through technology transfer to lmics capacity development of researchers to deliver research outputs needed . engage with ( donor ) states , ngos , and international organizations to promote and organize r&d on new medicines subsidies or tax advantages to encourage private sector allocate university resources to promote relevant research soliciting partnership agreements to support research regulatory action to ring - fence public sources ( such as tax ) investment in human resource capacity to do research expedited regulatory approval for trials expedited regulatory approval for appropriate indication ensuring sufficient pharma capacity for drug manufacture ensuring sufficient pharma capacity for drug manufacture exploratory research to identify candidate drugs for repurposing mobilizing resources globally for development and operational research negotiations on cost with pharma ( tender agreements , with forecasting ) ensure rights to a drug are available ( give away rights ) if pharma has no interest in manufacture setting up global consortia for purchasing fast - track or expedited processes for neglected diseases pursue registration of drugs vigorously following successful demonstration of effectiveness providing expertise for advice in regulatory decision - making permitting operational studies ; flexibility in registering drugs in post - rct phase engage with government regulatory authorities desist from imposing forms of patent protection on lmics that inhibit access to essential medicines desist from lobbying home country governments for excessive patent protection active research dissemination ; university commitment of resources for dissemination development of treatment protocols for up - scaled implementation contribute to funding through donor and multi - stakeholder structures contribute to funding through donor and multi - stakeholder structures for accelerated drug - development for dr - tb training of staff in research dissemination support operational research contribute to mobilizing multi - stakeholder partnerships to support implementation public education on tb science to strengthen civil society voice public education on tb science abstain from activities that delay , postpone , or block availability of new drugs prioritize concrete and targeted measures aimed at reaching vulnerable members of society additional actors may include the world health organization , whose stewardship role obliges it to facilitate and coordinate cooperation for dr - tb drug development , and civil society , whose roles include holding state and other actors accountable , including bringing cases to court where appropriate , promoting public information and awareness , and mobilizing community voice . when identifying the types of obligations that give substance to the rebsp , it makes sense to distinguish between different duty - holders and actors ( table 1 ) . this has changed drastically in present times , where the role and influence of the private sector ( corporations and private research institutes ) and non - state consortia has increased enormously and market forces often determine for which purposes both private and public resources will be spent . obligations to respect the rebsp entail that both the domestic state and the home state of a pharmaceutical company must not introduce obstacles in its intellectual property laws and policy that would prevent institutes for research and development ( r&d ) from focusing on neglected diseases . with a view to making medicines for neglected disease available and accessible , states have an obligation to facilitate in the sense of stimulating research for new medicines by companies , research institutes , and universities , whether in the form of joint ventures or directly in the public sphere . this can be done , for example , by providing subsidies or tax advantages to incentivize research into neglected diseases , investing in the human resources base needed for research , and routing research grant funding into prioritized focus areas , whether through direct state funding of research , through partnership agreements with earmarked research funders , or through regulatory interventions to mobilize research funding . in 2006 , the african union adopted the khartoum declaration , which committed states to allocate a minimum of 1% of gdp to r&d . for example , while south africa s medicine control council approved a clinical access program for the new tb drug bedaquiline in january 2013 , which removed delays in registering the drug , the cumbersome approval process for patients to access the drug still caused considerable delay in treatment initiation for extensively drug - resistant tb ( xdr - tb ) . moreover , a complex set of operational questions requires guidelines for how best to implement new regimens for mdr - tb that maximize access to new drugs of better efficacy , lower toxicity , and shorter duration , while avoiding development of further resistance through indiscriminate and poorly coordinated use of these medications . thus , states , as a part of their human rights obligations , should accelerate not only the research process but all the subsequent steps needed to see a new drug included in standard treatment protocols . from a normative perspective , therefore , the rebsp entails a right for individuals to have a legislative and policy framework adopted that enables the development and manufacturing of a new medicine . this means both that the policy should not impede or interfere with such drug development and/or repurposing for mdr - tb treatment ( respect ) , but also that it should ensure corporations do not delay new knowledge for medicines for neglected diseases from becoming available ( protect ) and put in place positive measures to encourage drug development and/or repurposing ( fulfill ) . for patients with mdr - tb , therefore , such a framework is informed by a human rights approach by which the state would be expected to prioritize measures in its science policy that give preference to drug development and/or repurposing for neglected diseases that disproportionately affect poor and vulnerable members of society . in an era characterized by economic globalization , however , it is self - evident that making essential medicines available is a process involving multiple actors : not only pharmaceutical companies , but also their home states , international organizations , and private funders ( table 1 ) . first , states should desist from policies that undermine the rights of lmic populations to enjoy the benefits of scientific progress ( respect ) . more recently , it is also evident in the conclusion of numerous bi- and multilateral ( trips - plus ) trade agreements subsequent to doha which continue to undermine doha trips flexibilities and the ability of poorer countries to develop their own ip regimes . a division and coordination of domestic and extraterritorial human rights obligations between states is required , depending on the level of development , the domestic health situation and tb prevalence , and the capacity and resources of more wealthy states to assist . in this regard , there is a need to mobilize and share common but differentiated responsibilities of all relevant stakeholders , both governmental and non - governmental , for international cooperation aimed at giving effect to the right to essential medicines . in this regard , corporate social responsibility can be made concrete by giving substance to the implementation of the rebsp . as lee and hunt have argued , pharmaceutical companies have a shared right to health responsibility to engage in r&d for neglected diseases that affect poor people in poor countries . people who suffer from mdr - tb have only one option for accessing drugs , so their treatment success is partly reliant on the actions of these companies . while efforts have been directed to identifying the role of corporations in a rights - based approach to health , there are many other non - state actors involved . clearly , researchers can not be compelled to engage in research to develop new tb drugs , but , in practice , generous funding streams and academic reward systems responding to priority areas such as mdr - tb could and do influence researcher behavior and direct researcher focus . however , it is typically in the translation of scientific research where universities lag , and where the rebsp highlights important gaps . the experience in the hiv field is instructive , where very strong civil society action spurred research and development of new drugs , raised awareness of entitlements to treatment among those most affected by the hiv pandemic , and directly impacted on drug pricing for arvs to treat hiv . the close alliance of scientists with the treatment access movement in south africa played a key role in a number of important public health advances and in advancing access to the benefits of scientific research related to hiv . thus , the obligation on researchers to engage actively in the dissemination of their findings and in the conveying of scientific information to non - scientific audiences represents not only good scientific practice , but a strengthening of the roles of universities and research institutions to help realise the rebsp . if taken seriously , universities would then need to invest in training researchers in better public engagement in science , rewarding scientific activities that enhance popular understanding of science , and dissemination activities aimed at ensuring research findings are translated into practical change . they have a key role in realizing the right to health and in ensuring people s rebsp , which , rather than being framed as philanthropy or largesse , should be seen within the unfolding context of the extended purview of rights frameworks beyond national boundaries and to non - state actors . to some extent , we have seen that collaboration between the corporate sector and university research institutes may help to bridge the gap between basic science and clinical research to advance innovative medicines for poor patients , for which we already have some multi - stakeholder models such as the drugs for neglected diseases initiative and critical path to tb drug regimens . financing mechanisms , such as illustrated in the tb alliance , are also putting teeth to multi - stakeholder initiatives to accelerate drug development , which will enhance availability and accessibility consistent with the rebsp . nonetheless , questions about the implications of locating such hugely important policy initiatives outside of a framework of state sovereignty ( and the associated system of human rights - based accountability ) remain potential threats to a full realization of the rebsp . it is clear that the rebsp does not provide an individual entitlement to claim a right to have a new drug developed for a neglected disease such as mdr - tb . it sets up entitlements for a system of science development and dissemination that could and should benefit the most marginalized in society , and represents a collective claim for the use of science for social benefits . inasmuch as the market fails to address neglected diseases such as mdr - tb , the rebsp provides a potential counterbalance to frame a positive obligation on states to both marshal their own resources and to coordinate the actions of multiple other actors towards this goal . at the same time , non - state actors can be brought within the ambit of contributing to addressing one of the major public heath crises of the current period without holding the same level of accountability as states but still recognizing obligations at a level of soft law responsibilities . developing the conceptualization of the rebsp and advancing the evidence base for its role may provide powerful practical tools for human rights advocates , civil society activists , and health and human rights researchers to tackle neglected diseases and address wider health challenges across the globe .

the existence of coupling between neuronal activity , metabolic and hemodynamic responses is a prerequisite for brain function research employing non - invasive neuroimaging techniques such as functional magnetic resonance imaging ( fmri ) , positron emission tomography ( pet ) and near - infrared optical imaging ( nioi ) , which can visualize stimulus - induced activation areas in the human brain ( see rev . ) . although the mechanism of the coupling between these physiological parameters remains to be elucidated despite numerous investigations conducted over past decades ( see rev . ) , a tight coupling has been assumed to elucidate brain function based on the data obtained using these techniques ( see rev . ) . within the past some dozen years , it has been reported that changes in rcbf in response to visual stimuli are accompanied by smaller changes in the regional metabolic rate of o2 ( rcmro2 ) in the human visual cortex ( e.g. , rcbf : rcmro2 = 10:1 or 2:1 ) . this implies that the oxygen supply is not precisely matched with the demand ( referred to as " overcompensation " or " decoupling between rcbf and rcmro2 " ) . more recently , the use of optical techniques to monitor the visual cortex of animals has shown that ( 1 ) after onset of a stimulus , the concentration of deoxy - hb increases first at a focal region in the cortex co - localized with neuronal activation and increased o2 consumption , and ( 2 ) this is followed by a decrease in deoxy - hb and a large increase in oxy - hb , which is caused by a delayed but large and less localized increase in rcbf [ 6 - 8 ] . in the course of our studies , we found a decoupling between rcbf and rcbv during visual stimulation in the human occipital cortex , although it has been empirically appreciated that an increase in rcbf accompanies an increase in rcbv . the relationship between the two was determined using whole - head measurement , which is often used for the analysis of stimulus - induced changes in rcbf and rcbv . on the basis of our results , we propose that some mechanism regulates regional blood flow ( rcbf ) and blood volume ( rcbv ) above a certain level of neuronal activity . if the mechanism works as a general rule during regional brain activation , it should occur regardless of type of stimuli , area of cortex , or animal species . in the present study , we performed two different experiments to test the above hypothesis . first , the relationship between neuronal activity and hemodynamic responses was examined in the human occipital cortex using two types of visual stimuli ( a black and white annular checkerboard and a flash - photo stimulus ) . secondly , we investigated the relationship in the rat somatosensory cortex when the peripheral nerve was stimulated electrically . here all subjects had normal or corrected - to - normal vision and provided written informed consent . four of the six subjects participated in nioi measurements only . a flash - photo stimulator ( sls-2141 , nihon kohden kogyo co. ltd , japan ) was used to provide visual stimuli ( temporal frequency at 0.5 , 1 , 8 and 21 hz ) . the time sequence of the experiments consisted of [ control ( 30 sec ) + stimulation ( 30 sec ) ] . each of four different frequencies was shown four times in pseudo - random order . during the experiments , subjects sat still on a chair and were required to keep their eyes closed lightly . a black and white annular checkerboard , with a central fixation point and gray background , was used as the visual stimulus ( perimacular annulus , 1.2 to 5.8 degrees ; angle of each wedge , 10 degrees ; number of layers , 5 ; temporal frequency , 0.5 , 1.4 , 4.7 , and 14 hz ) . the time sequence of the experiments consisted of [ control ( 28 sec ) + stimulation ( 28 sec ) ] . each of four different reversal frequencies was shown four times in pseudo - random order . during the control and stimulation periods , subjects were required to fixate on a fixation cross in the middle of the checkerboard , and to lie still on his back on a patient table of mri . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . the system consisted of six optical source units , each having three laser diodes ( 780 , 805 , and 830 nm ) and six photomultiplier tubes . the source units and detector tubes were connected to glass - fiber bundles for the illumination of incident light and for the collection of reflected light from the head . combinations of 16 nearest - neighbor pairs of input and output fibers were used to obtain a topographical image covering a 76 76 mm area in the occipital region of the head . the pixel size of the nioi was estimated to be 20 20 mm at a source - detector distance of 27 mm . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . the system consisted of one optical source unit , with three laser diodes ( 780 , 805 , and 830 nm ) , and one photomultiplier tube . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . as follows . = 1.855abs780 - 0.239abs805 - 1.095abs830 a 1.5 t mri scanner ( magnetom vision ; siemens , germany ) was used to obtain blood oxygen level - dependent contrast functional images . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . areas of significant activation were determined using spm99 . motion correction and spatial smoothing ( three - dimensional gaussian kernel , 11 mm full width at half maximum ) areas of activation were determined by a statistical threshold of p < 0.0001 ( voxel level ) corrected for multiple comparison for the entire search volume ) . to enable the fmri and optical signals to be compared , the time series of the fmri signals were processed as follows . after removing motion artifacts from all the t2 * -weighted images using automated image registration ( air , version 3.0 , the time courses of the fmri signals from the region of interest were obtained using avs / express version 3.2 ( advanced visual systems inc . all animal experiments were conducted in accordance with our institutional guidelines for the care and use of laboratory animals . male wistar rats weighing 190220 g were purchased from slc ( shizuoka , japan ) and allowed free access to food and water . they were then tracheotomized , immobilized with pancuronium bromide ( 2 mg / kg / h ) , and artificially ventilated with room air . a craniotomy ( 4 5 mm ) was performed on the left hemisphere and the dura mater was removed to expose the somatosensory cortex . a pair of needle electrodes was inserted underneath the skin of the plantar and ankle region in the contralateral hindlimb . except as otherwise noted , the posterior tibial nerve ( in part , the peroneal nerve ) was electrically stimulated with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at 5 hz . no major changes were detected in the mean arterial blood pressure ( 110 mmhg 5 s.d . ) during and after posterior tibial ( pt ) nerve stimulation . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . signal changes from the surface of the cortex ( semiglobular , 500 m in diameter ) were collected at a time constant of 0.5 sec . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . the blood - flow rate in individual microvessels was calculated as v * (d/2 ) . another five rats were used to acquire digitized images through transmission filters at 577.3 ( 1.2 ) nm with a charge - coupled device ( ccd ) camera . the magnitude of absorption change at 577 nm ( a577 ) was calculated in each pixel and color - coded to produce a false - color map . the exposed cortex was stained with a voltage - sensitive dye jpw-1114 ( molecular probes , usa ) ( 0.5 mg / ml for 90 min ) . the hindlimb was electrically stimulated 21 times with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at a frequency of 0.33 hz . the fluorescence associated with membrane potential changes was measured with a high - speed ccd imaging system , micam01 ( brain vision inc . 84 images were acquired at 2 msec intervals , and 21 series of time - course images were averaged . all subjects had normal or corrected - to - normal vision and provided written informed consent . four of the six subjects participated in nioi measurements only . a flash - photo stimulator ( sls-2141 , nihon kohden kogyo co. ltd , japan ) was used to provide visual stimuli ( temporal frequency at 0.5 , 1 , 8 and 21 hz ) . the time sequence of the experiments consisted of [ control ( 30 sec ) + stimulation ( 30 sec ) ] . each of four different frequencies was shown four times in pseudo - random order . during the experiments , subjects sat still on a chair and were required to keep their eyes closed lightly . a black and white annular checkerboard , with a central fixation point and gray background , was used as the visual stimulus ( perimacular annulus , 1.2 to 5.8 degrees ; angle of each wedge , 10 degrees ; number of layers , 5 ; temporal frequency , 0.5 , 1.4 , 4.7 , and 14 hz ) . the time sequence of the experiments consisted of [ control ( 28 sec ) + stimulation ( 28 sec ) ] . each of four different reversal frequencies was shown four times in pseudo - random order . during the control and stimulation periods , subjects were required to fixate on a fixation cross in the middle of the checkerboard , and to lie still on his back on a patient table of mri . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . the system consisted of six optical source units , each having three laser diodes ( 780 , 805 , and 830 nm ) and six photomultiplier tubes . the source units and detector tubes were connected to glass - fiber bundles for the illumination of incident light and for the collection of reflected light from the head . combinations of 16 nearest - neighbor pairs of input and output fibers were used to obtain a topographical image covering a 76 76 mm area in the occipital region of the head . the pixel size of the nioi was estimated to be 20 20 mm at a source - detector distance of 27 mm . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . the system consisted of one optical source unit , with three laser diodes ( 780 , 805 , and 830 nm ) , and one photomultiplier tube . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . as follows . = 1.855abs780 - 0.239abs805 - 1.095abs830 a 1.5 t mri scanner ( magnetom vision ; siemens , germany ) was used to obtain blood oxygen level - dependent contrast functional images . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . areas of significant activation were determined using spm99 . motion correction and spatial smoothing ( three - dimensional gaussian kernel , 11 mm full width at half maximum ) areas of activation were determined by a statistical threshold of p < 0.0001 ( voxel level ) corrected for multiple comparison for the entire search volume ) . to enable the fmri and optical signals to be compared , the time series of the fmri signals were processed as follows . after removing motion artifacts from all the t2 * -weighted images using automated image registration ( air , version 3.0 , the time courses of the fmri signals from the region of interest were obtained using avs / express version 3.2 ( advanced visual systems inc . all subjects had normal or corrected - to - normal vision and provided written informed consent . four of the six subjects participated in nioi measurements only . a flash - photo stimulator ( sls-2141 , nihon kohden kogyo co. ltd , japan ) was used to provide visual stimuli ( temporal frequency at 0.5 , 1 , 8 and 21 hz ) . the time sequence of the experiments consisted of [ control ( 30 sec ) + stimulation ( 30 sec ) ] . each of four different frequencies was shown four times in pseudo - random order . during the experiments , subjects sat still on a chair and were required to keep their eyes closed lightly . a black and white annular checkerboard , with a central fixation point and gray background , was used as the visual stimulus ( perimacular annulus , 1.2 to 5.8 degrees ; angle of each wedge , 10 degrees ; number of layers , 5 ; temporal frequency , 0.5 , 1.4 , 4.7 , and 14 hz ) . the time sequence of the experiments consisted of [ control ( 28 sec ) + stimulation ( 28 sec ) ] . each of four different reversal frequencies was shown four times in pseudo - random order . during the control and stimulation periods , subjects were required to fixate on a fixation cross in the middle of the checkerboard , and to lie still on his back on a patient table of mri . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . the system consisted of six optical source units , each having three laser diodes ( 780 , 805 , and 830 nm ) and six photomultiplier tubes . the source units and detector tubes were connected to glass - fiber bundles for the illumination of incident light and for the collection of reflected light from the head . combinations of 16 nearest - neighbor pairs of input and output fibers were used to obtain a topographical image covering a 76 76 mm area in the occipital region of the head . the pixel size of the nioi was estimated to be 20 20 mm at a source - detector distance of 27 mm . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . the system consisted of one optical source unit , with three laser diodes ( 780 , 805 , and 830 nm ) , and one photomultiplier tube . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . as follows . a 1.5 t mri scanner ( magnetom vision ; siemens , germany ) was used to obtain blood oxygen level - dependent contrast functional images . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . areas of significant activation were determined using spm99 . motion correction and spatial smoothing ( three - dimensional gaussian kernel , 11 mm full width at half maximum ) areas of activation were determined by a statistical threshold of p < 0.0001 ( voxel level ) corrected for multiple comparison for the entire search volume ) . to enable the fmri and optical signals to be compared , the time series of the fmri signals were processed as follows . after removing motion artifacts from all the t2 * -weighted images using automated image registration ( air , version 3.0 , the time courses of the fmri signals from the region of interest were obtained using avs / express version 3.2 ( advanced visual systems inc . all animal experiments were conducted in accordance with our institutional guidelines for the care and use of laboratory animals . male wistar rats weighing 190220 g were purchased from slc ( shizuoka , japan ) and allowed free access to food and water . they were then tracheotomized , immobilized with pancuronium bromide ( 2 mg / kg / h ) , and artificially ventilated with room air . a craniotomy ( 4 5 mm ) was performed on the left hemisphere and the dura mater was removed to expose the somatosensory cortex . a pair of needle electrodes was inserted underneath the skin of the plantar and ankle region in the contralateral hindlimb . except as otherwise noted , the posterior tibial nerve ( in part , the peroneal nerve ) was electrically stimulated with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at 5 hz . no major changes were detected in the mean arterial blood pressure ( 110 mmhg 5 s.d . ) during and after posterior tibial ( pt ) nerve stimulation . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . , japan ) combined with a microscopy system . signal changes from the surface of the cortex ( semiglobular , 500 m in diameter ) were collected at a time constant of 0.5 sec . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . the blood - flow rate in individual microvessels was calculated as v * (d/2 ) . another five rats were used to acquire digitized images through transmission filters at 577.3 ( 1.2 ) nm with a charge - coupled device ( ccd ) camera . the magnitude of absorption change at 577 nm ( a577 ) was calculated in each pixel and color - coded to produce a false - color map . the exposed cortex was stained with a voltage - sensitive dye jpw-1114 ( molecular probes , usa ) ( 0.5 mg / ml for 90 min ) . the hindlimb was electrically stimulated 21 times with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at a frequency of 0.33 hz . the fluorescence associated with membrane potential changes was measured with a high - speed ccd imaging system , micam01 ( brain vision inc . , 84 images were acquired at 2 msec intervals , and 21 series of time - course images were averaged . all animal experiments were conducted in accordance with our institutional guidelines for the care and use of laboratory animals . male wistar rats weighing 190220 g were purchased from slc ( shizuoka , japan ) and allowed free access to food and water . they were then tracheotomized , immobilized with pancuronium bromide ( 2 mg / kg / h ) , and artificially ventilated with room air . a craniotomy ( 4 5 mm ) was performed on the left hemisphere and the dura mater was removed to expose the somatosensory cortex . a pair of needle electrodes was inserted underneath the skin of the plantar and ankle region in the contralateral hindlimb . except as otherwise noted , the posterior tibial nerve ( in part , the peroneal nerve ) was electrically stimulated with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at 5 hz . no major changes were detected in the mean arterial blood pressure ( 110 mmhg 5 s.d . ) during and after posterior tibial ( pt ) nerve stimulation . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . , signal changes from the surface of the cortex ( semiglobular , 500 m in diameter ) were collected at a time constant of 0.5 sec . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . the blood - flow rate in individual microvessels was calculated as v * (d/2 ) . another five rats were used to acquire digitized images through transmission filters at 577.3 ( 1.2 ) nm with a charge - coupled device ( ccd ) camera . the magnitude of absorption change at 577 nm ( a577 ) was calculated in each pixel and color - coded to produce a false - color map . the exposed cortex was stained with a voltage - sensitive dye jpw-1114 ( molecular probes , usa ) ( 0.5 mg / ml for 90 min ) . the hindlimb was electrically stimulated 21 times with a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at a frequency of 0.33 hz . the fluorescence associated with membrane potential changes was measured with a high - speed ccd imaging system , micam01 ( brain vision inc . , 84 images were acquired at 2 msec intervals , and 21 series of time - course images were averaged . figure 1 shows the hemodynamic responses measured using nioi and/or fmri in human occipital cortex during visual stimulation . using flash - photo stimulation , the hb species concentrations measured with nioi increased from their basal levels , but the increases were minimum at a temporal frequency of 8 hz ( fig . this result was the opposite to results obtained using pet or fmri , which showed the maximum increase in rcbf in the visual cortex occurred at around 8 hz . to investigate this discrepancy between changes in hb species concentrations and rcbf , we performed simultaneous nioi and fmri measurements on two subjects using a black / white annular checkerboard ( because we were unable to use our photo stimulator in the mri system ) ( fig . changes in the blood oxygenation level - dependent ( bold ) fmri signals of individual subjects showed a maximum at stimulus frequencies around 1.44.7 hz , while [oxy - hb ] , [deoxy - hb ] , and [total - hb ] measured with nioi showed a minimum around these frequencies . it should be noted that the frequency of stimulation using flickering checkerboards is considered to be twice the white - to - white or black - to - black frequency of the checkerboard , if the visual stimulus basically comes from pattern reversal . therefore , the present results obtained using a checkerboard stimulus are consistent with our nioi results and the results of other studies obtained using flash - photo stimulation [ 16 - 18 ] . moreover , the use of this stimulus highlighted the following two findings : ( 1 ) changes in [ deoxy - hb ] for the checkerboard stimulation decreased from the basal level ( [deoxy ] < 0 ) , whereas those for the flash - photo stimulation increased from the basal level ( [deoxy ] > 0 ) , and ( 2 ) the responses of the hb parameters and bold signals dissociated at around 8 hz . one possible explanation for the former finding is that the difference in the [ deoxy - hb ] reflects differences in the metabolic and circulatory conditions in the visual cortex during the resting state . the subjects were asked to close their eyes during the flash - photo stimulation , whereas during the checkerboard stimulation they were asked to keep them open . it has been reported that the metabolic rate of glucose in the visual cortex ( cmrglc ) decreased from the basal level when subjects closed their eyes , whereas during the checkerboard stimulation ( with the eyes open ) it increased above the basal level . as for the second finding , the result indicates that the dissociation between rcbf and rcbv occurs at a temporal frequency around 8 hz , since the temporal frequency dependence of the bold signal responses corresponded well with the rcbf responses [ 16 - 18 ] , whereas the responses of the hb parameters , especially [ total - hb ] , reflected changes in the rcbv . moreover , it has been reported that electrical and cmro2 responses showed a maximum at a frequency around 4 hz ( i.e. , 8 pattern reversals / sec ) when a checkerboard was used for visual stimulation . these results suggest that there are physiological requirements for the dissociation of stimulus - induced responses of rcbf and rcbv above a certain level of neuronal activity . , [oxy - hb ] ( arbitrary unit ) ; , [total - hb ] ; x , [deoxy - hb ] ( b ) simultaneous measurement with fmri and nioi . the data show subject a ( left ) , b ( right ) , respectively . a black and white annular , bold signal ( % ) ; , [oxy - hb ] ( arbitrary unit ) ; , [total - hb ] ; x , [deoxy - hb ] if the above dissociation phenomenon applies generally to any type of regional brain activation , it should occur regardless of stimulus type , cortical area , or animal species . to test the above hypothesis , we examined stimulus - induced hemodynamic responses in the somatosensory cortex of rats during electrical stimulation of the peripheral nerve ( see materials and methods ) . figure 2 shows the spatiotemporal profile of stimulus - induced activation in the somatosensory cortex of the rat . figure 2a shows a ccd image over the left parietal cortex viewed through the cranial window . the vessel labeled a is a second - order ( parietal ) branch of the middle cerebral artery ( mca ) , and vessels b and c are its tributaries ( third - order branches ) . vessel b predominantly supplies the hindlimb somatosensory area and vessel c predominantly supplies the trunk area ( left branch ) . one of the tributaries of vessel c formed an interarterial anastomosis with the anterior cerebral artery ( aca ) , denoted by a broken line . the stimulus - induced maximal neuronal activation obtained with changes in the membrane potential was localized in the hindpaw area at 28 msec after stimulus onset ( fig . in contrast , figure 2c shows an absorbance change at a wavelength of 577 nm ( a577 ) , which mainly reflects [ oxy - hb ] , indicating that the change in rcbf spread beyond the hindlimb area at 6 sec after stimulus onset . this wide distribution of cbf correlated well with the widespread increase in intravascular po2 measured using albumin - bound oxygen - sensitive phosphorescence dye , although the maximal increases in rcbf and po2 were observed over the hindlimb area ( fig . the bottom right corner of the figure shows 0.5 mm lateral and 0.5 mm caudal from the bregma . vessels traced with red solid line denote parietal branches of the mca , and broken lines denote parietal branches of the anterior cerebral artery ( aca ) . the map was obtained above 50% of threshold ( 0.15 f / f ( % ) 0.3 ) at 28 msec after stimulus onset . time courses of changes in membrane potential at the numbered pixels are superimposed ( 1 and 2 , hindpaw area ; 3 , hindlimb area ) . ( c ) spatial distribution of stimulus - induced absorption change ( a577 ) at 6 sec after stimulus onset . ( d ) maximal spatial distribution of intravascular po2 at 7 sec after stimulus onset . the somatosensory area could be divided into the following four regions : upper right , predominantly hindlimb area ; lower right , predominantly motor area ; upper and lower left , predominantly trunk area . figure 3a shows representative temporal profiles of changes in the hemodynamic parameters measured using ldf in the maximally activated hindpaw area ( marked with a white circle in figs . 2c and 2d ) , in which maximal changes in rbc flow ( rrbcflow ) , rbc velocity ( rrbcveloc ) , and rbc number ( rrbcmass ) were observed 5~7 sec after stimulus onset . to investigate the relationship between stimulus intensity and degree of change in these hemodynamic parameters , the ldf measurements were performed in the activated hindpaw area at various current intensities and stimulus periods ( fig . since it has been reported that electrical stimulation of the peripheral nerve of rats showed a maximal response ( without tetanus ) of the hemodynamic parameters at around 5 hz , the stimulus frequency was kept at 5 hz during this experiment . the index of stimulus intensity ( horizontal axis in fig . 3b ) was assumed as a function of a product of the current intensity and stimulus period . at a lower stimulus intensity ( si 2 ) , rrbcflow , rrbcveloc , and rrbcmass all increased ( p < 0.001 ) due to functional hyperemia , while at a higher stimulus intensity ( si > 2 ) , rrbcflow and rrbcveloc increased , while rrbcmass decreased ( p < 0.001 ) . these results demonstrated that the dissociative response between rcbf and rcbv in the human visual cortex ( fig . 1 ) also occurred in the somatosensory cortex of rats in response to different stimuli . this finding strongly suggests that grubb 's relationship between cbf and cbv ( cbv = 0.8*cbf ) does not always apply , especially to the relationship between regional changes in cbf and cbv ( probably above a certain level of neuronal activation ) . in addition , the saturation of the increase in rrbcflow indicates that the maximal level of increase in o2 inflow into the activated area remains at a certain level ( about 30% , see fig . ( a ) typical example of temporal profiles of stimulus - induced changes in hemodynamic parameters measured using ldf . measurements focused on the hindpaw area shown with a white circle ( 500 m in diameter ) in figs 2(c ) and 2(d ) . vertical axis denotes percent increases in tissue blood flow ( rrbcflow ) , red blood cell velocity ( rrbcveloc ) , and number of red blood cells ( rrbcmass ) in the hindpaw area measured with ldf . horizontal axis denotes index of stimulus intensity as a function of stimulus duration ( 2 , 4 or 8 sec ) and current intensity ( 2.5 , 5 , 7.5 or 10 ma ) . other stimulus conditions were pulse duration of 0.5 msec and stimulus frequency of 5 hz . statistical analysis was done using a bonferroni multiple comparison test , and p < 0.05 was regarded as statistically significant . figure 4a shows changes in the blood flow rate and diameter ( inset ) of the pial arterioles supplying the blood to the hindpaw area ( see fig . the blood flow and diameter of the afferent vessel a and its tributaries ( b , which supplies the hindlimb area , and c , which supplies the trunk area ) increased just after the onset of stimulation ( fig . the tissue blood flow in the hindpaw area measured using the laser - doppler flowmeter increased in accordance with the increase in blood flow in vessel b measured using the fldam . the blood flow and diameter of vessel b showed long - lasting increases , probably due to the metabolic effect of the neuronal activation in the hindpaw area . it should be noted that vessel c does not supply blood to the activated area , but the change in blood flow in vessel c was almost the same as that in vessel b. these results suggest that the increases in the diameter and blood flow in vessel c are regulated so that vessel c plays an active role as an " escape route " to prevent excess inflow of o2 into vessel b. the increase in po2 more than 10 mmhg may be undesirable in the capillary bed and venules in the activated area ( cf . , fig . these results may account for the finding that the maximal level of increase in o2 inflow into the activated area remained at a certain level ( i.e. , the asymptotic increase in rrbcflow in fig . 3 ) . the stimulus conditions used in this study ( a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at 5 hz ) considerably exceeded motor and sensory thresholds , which may have been sufficient to excite a , a , and a axons and c - fibers . in turn , this may have been enough to elicit the maximal increase in rrbcflow and a maximal level of o2 inflow in the hindpaw area . the diameter and blood flow of the afferent vessel a and its tributaries , b and c , increased just after onset of the stimulation due to a fast and transient neurogenic regulation . the blood supply to the activated area was maintained by a delayed and lasting metabolic factor . vessel c does not supply blood to the activated area , but the change in blood flow in vessel c ( 27% increase ) was almost the same as that in vessel b ( 30% increase ) , suggesting that vessel c plays an active role as an " escape route " to prevent excess o2inflow into vessel b and the activated area . this regulation could be achieved by a dissociation between rcbf and rcbv responses , called a flow - mass regulatory mechanism . a third mechanism , possibly a high - o2 sensing mechanism and/or fluid shear stress , may act as a regulator for the mechanism in addition to the neurogenic and metabolic regulation . stimulus - induced changes in pial arteriolar blood flow and diameter in the somatosensory cortex . ( a ) comparison of the time course of rcbf measured using ldf and blood - flow rate in individual pial arterioles measured using fldam . vessels labeled a are second - order branches of mca ( 32.7 m 14.3 sd in diameter ) , those labeled b are third - order branches ( 25.0 m 11.3 sd ) supplying the hindlimb ( hindpaw ) somatosensory area , and those labeled c are third - order branches ( 24.3 m 11.1 sd ) predominantly supplying the trunk area ( see fig . averaged values from 15 trials of 5 rats are shown ; the errors for each data point are less than 10% . ( b ) illustration of blood - flow regulation in the somatosensory cortex during pt nerve stimulation . f.i . our results indicate the existence of a flow(rcbf)-mass(rcbv ) regulatory mechanism , and that changes in blood flow during brain activation are not tightly regulated to supply only to an the activated area . this loose regulation serves to prevent intense functional hyperemia and excess o2 inflow into the focally activated area . a high - o2 sensing mechanism and/or fluid shear stress are proposed as a regulator of the flow - mass regulatory mechanism , which may elicit a neurogenic modulation of vascular tonus in the activated cortex . collapse of the flow - mass regulatory mechanism ( e.g. , arteriosclerosis ) would result in excess inflow of oxygen into the focally activated area , triggering the production of harmful reactive oxygen species and leading to the accumulation of irreversible neuronal damage ( fig . relationship between o2 inflow and rcbf in the activated area is shown by red line . during moderate neuronal activation , rcbf , rcbv and o2 inflow increase in the activated area ( functional hyperemia ) . above a certain level of neuronal activation , dissociation of increase in rcbf and rcbv takes place to prevent excess o2 inflow into the focally activated area ( flow - mass regulation ) . normal regulation of rcbf and rcbv is probably achieved by the action of both regulations ( i.e. , functional hyperemia and flow - mass regulation ) . further , we hypothesize that when flow - mass regulation is disrupted ( e.g. , arteriosclerosis ) , rcbf , rcbv , and o2 inflow may increase to undesirable levels in the activated area , leading to the production of harmful reactive oxygen species resulting in accumulation of neuronal damage . as conceived , designed and coordinated the study , participated in all the human and animal experiments , and drafted the manuscript . js participated in the design and coordination of the study , and carried out the fldam measurements . this research was supported by the breakthrough 21 project of the ministry of post and telecommunications of japan , and in part by grants - in - aid from the nissan science foundation of japan .

backgroundthe correlation between regional changes in neuronal activity and changes in hemodynamics is a major issue for noninvasive neuroimaging techniques such as functional magnetic resonance imaging ( fmri ) and near - infrared optical imaging ( nioi ) . a tight coupling of these changes has been assumed to elucidate brain function from data obtained with those techniques . in the present study , we investigated the relationship between neuronal activity and hemodynamic responses in the occipital cortex of humans during visual stimulation and in the somatosensory cortex of rats during peripheral nerve stimulation.methodsthe temporal frequency dependence of macroscopic hemodynamic responses on visual stimuli was investigated in the occipital cortex of humans by simultaneous measurements made using fmri and nioi . the stimulus - intensity dependence of both microscopic hemodynamic changes and changes in neuronal activity in response to peripheral nerve stimulation was investigated in animal models by analyzing membrane potential ( fluorescence ) , hemodynamic parameters ( visible spectra and laser - doppler flowmetry ) , and vessel diameter ( image analyzer).resultsabove a certain level of stimulus - intensity , increases in regional cerebral blood flow ( rcbf ) were accompanied by a decrease in regional cerebral blood volume ( rcbv ) , i.e. , dissociation of rcbf and rcbv responses occurred in both the human and animal experiments . furthermore , the animal experiments revealed that the distribution of increased rcbf and o2 spread well beyond the area of neuronal activation , and that the increases showed saturation in the activated area.conclusionsthese results suggest that above a certain level of neuronal activity , a regulatory mechanism between regional cerebral blood flow ( rcbf ) and rcbv acts to prevent excess o2 inflow into the focally activated area .

Background Methods Human Experiments Subjects Optical Measurements and Analysis fMRI Measurements and Analysis Animal Experiments Animal Preparation Measurement of Hemodynamic Parameters Measurements of Activation Area and Blood-Flow Distribution Results and Discussion Authors' contributions Acknowledgements

the existence of coupling between neuronal activity , metabolic and hemodynamic responses is a prerequisite for brain function research employing non - invasive neuroimaging techniques such as functional magnetic resonance imaging ( fmri ) , positron emission tomography ( pet ) and near - infrared optical imaging ( nioi ) , which can visualize stimulus - induced activation areas in the human brain ( see rev . ) , a tight coupling has been assumed to elucidate brain function based on the data obtained using these techniques ( see rev . ) within the past some dozen years , it has been reported that changes in rcbf in response to visual stimuli are accompanied by smaller changes in the regional metabolic rate of o2 ( rcmro2 ) in the human visual cortex ( e.g. more recently , the use of optical techniques to monitor the visual cortex of animals has shown that ( 1 ) after onset of a stimulus , the concentration of deoxy - hb increases first at a focal region in the cortex co - localized with neuronal activation and increased o2 consumption , and ( 2 ) this is followed by a decrease in deoxy - hb and a large increase in oxy - hb , which is caused by a delayed but large and less localized increase in rcbf [ 6 - 8 ] . in the course of our studies , we found a decoupling between rcbf and rcbv during visual stimulation in the human occipital cortex , although it has been empirically appreciated that an increase in rcbf accompanies an increase in rcbv . the relationship between the two was determined using whole - head measurement , which is often used for the analysis of stimulus - induced changes in rcbf and rcbv . on the basis of our results , we propose that some mechanism regulates regional blood flow ( rcbf ) and blood volume ( rcbv ) above a certain level of neuronal activity . in the present study , we performed two different experiments to test the above hypothesis . first , the relationship between neuronal activity and hemodynamic responses was examined in the human occipital cortex using two types of visual stimuli ( a black and white annular checkerboard and a flash - photo stimulus ) . secondly , we investigated the relationship in the rat somatosensory cortex when the peripheral nerve was stimulated electrically . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . the system consisted of one optical source unit , with three laser diodes ( 780 , 805 , and 830 nm ) , and one photomultiplier tube . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . to enable the fmri and optical signals to be compared , the time series of the fmri signals were processed as follows . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . a flash - photo stimulator ( sls-2141 , nihon kohden kogyo co. ltd , japan ) was used to provide visual stimuli ( temporal frequency at 0.5 , 1 , 8 and 21 hz ) . during the control and stimulation periods , subjects were required to fixate on a fixation cross in the middle of the checkerboard , and to lie still on his back on a patient table of mri . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . during the control and stimulation periods , subjects were required to fixate on a fixation cross in the middle of the checkerboard , and to lie still on his back on a patient table of mri . a 16-channel near - infrared optical imaging system , optim_a , was used to obtain optical images of changes in concentration of hemoglobin ( hb ) species in the occipital cortex for simultaneous measurement with fmri . a single - channel near - infrared spectroscopy ( om-100a , shimadzu co. , japan ) was used to monitor changes in concentrations of hb species during the flash - photo stimulation . the system consisted of one optical source unit , with three laser diodes ( 780 , 805 , and 830 nm ) , and one photomultiplier tube . changes in the hb species concentration , expressed in an arbitrary unit , ( [oxy - hb ] , [deoxy - hb ] , and [total - hb ] + [deoxy - hb ] ) ) from the control conditions were calculated based on a modified lambert - beer law , using the extinction coefficient of chromophores reported by matcher et al . functional images weighted with the apparent transverse relaxation time ( t2 * ) were obtained with an echo planar imaging ( epi ) sequence ( repetition time ( tr ) , 4000 msec ; echo time ( te ) , 55.24 msec ; flip angle ( fa ) , 90 ; field of view ( fov ) , 256 256 mm ; matrix size , 64 64 ; slice thickness , 4 mm ) . they were then tracheotomized , immobilized with pancuronium bromide ( 2 mg / kg / h ) , and artificially ventilated with room air . a craniotomy ( 4 5 mm ) was performed on the left hemisphere and the dura mater was removed to expose the somatosensory cortex . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . regional changes in red - blood - cell flow ( rrbcflow ) , velocity ( rrbcveloc ) , and content ( rrbcmass ) were measured in 12 rats using a laser - doppler tissue flowmeter ( ldf ) ( flo - ce1 , omega flow inc . in separate experiments ( 5 rats ) , changes in the diameters ( d ) of second- and third- branches of the middle cerebral artery ( mca ) and rbc velocity ( v ) in these single pial arterioles were measured using a fiber - optic laser - doppler anemometer microscope ( fldam ) . figure 1 shows the hemodynamic responses measured using nioi and/or fmri in human occipital cortex during visual stimulation . using flash - photo stimulation , the hb species concentrations measured with nioi increased from their basal levels , but the increases were minimum at a temporal frequency of 8 hz ( fig . to investigate this discrepancy between changes in hb species concentrations and rcbf , we performed simultaneous nioi and fmri measurements on two subjects using a black / white annular checkerboard ( because we were unable to use our photo stimulator in the mri system ) ( fig . changes in the blood oxygenation level - dependent ( bold ) fmri signals of individual subjects showed a maximum at stimulus frequencies around 1.44.7 hz , while [oxy - hb ] , [deoxy - hb ] , and [total - hb ] measured with nioi showed a minimum around these frequencies . moreover , the use of this stimulus highlighted the following two findings : ( 1 ) changes in [ deoxy - hb ] for the checkerboard stimulation decreased from the basal level ( [deoxy ] < 0 ) , whereas those for the flash - photo stimulation increased from the basal level ( [deoxy ] > 0 ) , and ( 2 ) the responses of the hb parameters and bold signals dissociated at around 8 hz . one possible explanation for the former finding is that the difference in the [ deoxy - hb ] reflects differences in the metabolic and circulatory conditions in the visual cortex during the resting state . it has been reported that the metabolic rate of glucose in the visual cortex ( cmrglc ) decreased from the basal level when subjects closed their eyes , whereas during the checkerboard stimulation ( with the eyes open ) it increased above the basal level . as for the second finding , the result indicates that the dissociation between rcbf and rcbv occurs at a temporal frequency around 8 hz , since the temporal frequency dependence of the bold signal responses corresponded well with the rcbf responses [ 16 - 18 ] , whereas the responses of the hb parameters , especially [ total - hb ] , reflected changes in the rcbv . moreover , it has been reported that electrical and cmro2 responses showed a maximum at a frequency around 4 hz ( i.e. these results suggest that there are physiological requirements for the dissociation of stimulus - induced responses of rcbf and rcbv above a certain level of neuronal activity . , [oxy - hb ] ( arbitrary unit ) ; , [total - hb ] ; x , [deoxy - hb ] ( b ) simultaneous measurement with fmri and nioi . to test the above hypothesis , we examined stimulus - induced hemodynamic responses in the somatosensory cortex of rats during electrical stimulation of the peripheral nerve ( see materials and methods ) . figure 2 shows the spatiotemporal profile of stimulus - induced activation in the somatosensory cortex of the rat . the vessel labeled a is a second - order ( parietal ) branch of the middle cerebral artery ( mca ) , and vessels b and c are its tributaries ( third - order branches ) . the stimulus - induced maximal neuronal activation obtained with changes in the membrane potential was localized in the hindpaw area at 28 msec after stimulus onset ( fig . in contrast , figure 2c shows an absorbance change at a wavelength of 577 nm ( a577 ) , which mainly reflects [ oxy - hb ] , indicating that the change in rcbf spread beyond the hindlimb area at 6 sec after stimulus onset . this wide distribution of cbf correlated well with the widespread increase in intravascular po2 measured using albumin - bound oxygen - sensitive phosphorescence dye , although the maximal increases in rcbf and po2 were observed over the hindlimb area ( fig . time courses of changes in membrane potential at the numbered pixels are superimposed ( 1 and 2 , hindpaw area ; 3 , hindlimb area ) . ( c ) spatial distribution of stimulus - induced absorption change ( a577 ) at 6 sec after stimulus onset . figure 3a shows representative temporal profiles of changes in the hemodynamic parameters measured using ldf in the maximally activated hindpaw area ( marked with a white circle in figs . 2c and 2d ) , in which maximal changes in rbc flow ( rrbcflow ) , rbc velocity ( rrbcveloc ) , and rbc number ( rrbcmass ) were observed 5~7 sec after stimulus onset . to investigate the relationship between stimulus intensity and degree of change in these hemodynamic parameters , the ldf measurements were performed in the activated hindpaw area at various current intensities and stimulus periods ( fig . since it has been reported that electrical stimulation of the peripheral nerve of rats showed a maximal response ( without tetanus ) of the hemodynamic parameters at around 5 hz , the stimulus frequency was kept at 5 hz during this experiment . these results demonstrated that the dissociative response between rcbf and rcbv in the human visual cortex ( fig . 1 ) also occurred in the somatosensory cortex of rats in response to different stimuli . this finding strongly suggests that grubb 's relationship between cbf and cbv ( cbv = 0.8*cbf ) does not always apply , especially to the relationship between regional changes in cbf and cbv ( probably above a certain level of neuronal activation ) . in addition , the saturation of the increase in rrbcflow indicates that the maximal level of increase in o2 inflow into the activated area remains at a certain level ( about 30% , see fig . ( a ) typical example of temporal profiles of stimulus - induced changes in hemodynamic parameters measured using ldf . vertical axis denotes percent increases in tissue blood flow ( rrbcflow ) , red blood cell velocity ( rrbcveloc ) , and number of red blood cells ( rrbcmass ) in the hindpaw area measured with ldf . figure 4a shows changes in the blood flow rate and diameter ( inset ) of the pial arterioles supplying the blood to the hindpaw area ( see fig . the tissue blood flow in the hindpaw area measured using the laser - doppler flowmeter increased in accordance with the increase in blood flow in vessel b measured using the fldam . the blood flow and diameter of vessel b showed long - lasting increases , probably due to the metabolic effect of the neuronal activation in the hindpaw area . it should be noted that vessel c does not supply blood to the activated area , but the change in blood flow in vessel c was almost the same as that in vessel b. these results suggest that the increases in the diameter and blood flow in vessel c are regulated so that vessel c plays an active role as an " escape route " to prevent excess inflow of o2 into vessel b. the increase in po2 more than 10 mmhg may be undesirable in the capillary bed and venules in the activated area ( cf . these results may account for the finding that the maximal level of increase in o2 inflow into the activated area remained at a certain level ( i.e. the stimulus conditions used in this study ( a rectangular pulse of 3.8 ma intensity and 0.5 msec duration at 5 hz ) considerably exceeded motor and sensory thresholds , which may have been sufficient to excite a , a , and a axons and c - fibers . in turn , this may have been enough to elicit the maximal increase in rrbcflow and a maximal level of o2 inflow in the hindpaw area . the blood supply to the activated area was maintained by a delayed and lasting metabolic factor . vessel c does not supply blood to the activated area , but the change in blood flow in vessel c ( 27% increase ) was almost the same as that in vessel b ( 30% increase ) , suggesting that vessel c plays an active role as an " escape route " to prevent excess o2inflow into vessel b and the activated area . this regulation could be achieved by a dissociation between rcbf and rcbv responses , called a flow - mass regulatory mechanism . stimulus - induced changes in pial arteriolar blood flow and diameter in the somatosensory cortex . vessels labeled a are second - order branches of mca ( 32.7 m 14.3 sd in diameter ) , those labeled b are third - order branches ( 25.0 m 11.3 sd ) supplying the hindlimb ( hindpaw ) somatosensory area , and those labeled c are third - order branches ( 24.3 m 11.1 sd ) predominantly supplying the trunk area ( see fig . ( b ) illustration of blood - flow regulation in the somatosensory cortex during pt nerve stimulation . our results indicate the existence of a flow(rcbf)-mass(rcbv ) regulatory mechanism , and that changes in blood flow during brain activation are not tightly regulated to supply only to an the activated area . this loose regulation serves to prevent intense functional hyperemia and excess o2 inflow into the focally activated area . a high - o2 sensing mechanism and/or fluid shear stress are proposed as a regulator of the flow - mass regulatory mechanism , which may elicit a neurogenic modulation of vascular tonus in the activated cortex . , arteriosclerosis ) would result in excess inflow of oxygen into the focally activated area , triggering the production of harmful reactive oxygen species and leading to the accumulation of irreversible neuronal damage ( fig . relationship between o2 inflow and rcbf in the activated area is shown by red line . during moderate neuronal activation , rcbf , rcbv and o2 inflow increase in the activated area ( functional hyperemia ) . above a certain level of neuronal activation , dissociation of increase in rcbf and rcbv takes place to prevent excess o2 inflow into the focally activated area ( flow - mass regulation ) . normal regulation of rcbf and rcbv is probably achieved by the action of both regulations ( i.e. , arteriosclerosis ) , rcbf , rcbv , and o2 inflow may increase to undesirable levels in the activated area , leading to the production of harmful reactive oxygen species resulting in accumulation of neuronal damage . as conceived , designed and coordinated the study , participated in all the human and animal experiments , and drafted the manuscript . js participated in the design and coordination of the study , and carried out the fldam measurements .

. the fumes produced in most metallurgical processes may be harmful if inhaled . characteristic properties of the particles , especially particle size and chemical composition , influence their impact on human health . the ultrafine and nano - sized particles may , due to their very large surface area be more reactive than the more widely studied , larger particulate matters . they may also behave differently in the respiratory system . as the chemical composition of particles originating from different industries and processes may vary greatly , it is important to characterize the fumes and link them to their origin to enable enhanced risk assessments and enable capture . moreover , the fumes from industrial plants are a major contributor to the so - called diffuse emissions which are receiving increased attention from authorities as they may influence the air quality of the local , urban communities as well as the environment and climate at large . a great deal of efforts and investments are currently directed towards improvements in ventilation and filter systems , designed to collect and control the off - gases and fumes from the various metallurgical and mechanical process operations . from the corporate point of view , a process optimized to minimize fume formation would not only improve the indoor air quality and decrease the environmental impact of the plant ; it would also minimize material and energy losses associated with the production . to achieve this , more knowledge about the origin and characteristics of the fumes are necessary . there are several measurement techniques which can be used to characterize and quantify the airborne particulate matter in an industrial plant . the measurements reported on in this article were performed concurrently with gravimetric filters and laser measurements ( laserdust by norsk elektro optikk as , oslo , norway ) . the major advantage of the electric low pressure impactor ( elpi , dekati ltd . , tampere , finland ) is the detection and quantification of particles as small as 7 nm . the elpi also enables real - time assessment of the particle size distribution ( psd ) of aerosols as well as subsequent analysis of the collected aerosol particles by electron microscopy . the elpi has previously been used to assess particulate emissions from traffic , combustion , welding fume , and aluminum electrolysis . this article aims to describe how the novel technology of the electric low pressure impactor can be used to assess indoor air quality surrounding ferroalloy production processes and to establish real - time particle size distributions of aerosols at a ferrosilicon and a silicomanganese production site . the term fume , as used in this article , designates airborne particulate matter ( pm ) formed by the evaporation of liquid metal . fume from different locations in silicomanganese ( simn ) and ferrosilicon ( fesi ) production plants was collected and analyzed using an elpi ( dekati ltd . , tampere , finland ) which is described below . for proper function of the elpi a particle density should be assumed for the mathematical conversion of the as - obtained electrical current values to a number concentration ( number of particles per volume of air ) . in these studies , the assumed density values were that of amorphous silica ( sio2 , 2.2 g / cm ) ; this assumption is motivated and discussed in the following sections of this article . the density value has a relatively weak influence on the number concentration as it merely affects the size range intervals for each impactor stage ( the cut - off values between the different size fractions ) . the same assumed density was also used in a second step when the number concentrations were algebraically converted to mass concentrations . the number - to - mass conversion was performed using the filter functions in the elpivi software ( dekati ltd . , tampere , finland ) and the density has a large influence on this conversion , which in turn introduces significant error sources as discussed in further sections . the particles are collected in the elpi on aluminium foil substrates , one for each size fraction as detailed in table i. these particle samples were subsequently analyzed by scanning electron microscopy ( sem ) , transmission electron microscopy ( tem ) , and energy dispersive spectroscopy ( eds ) . the amount of fume particles collected by the elpi was not , however , sufficient to quantitatively analyze the chemical and/or phase compositions by other methods . ( m ) 10.0070.0280.02 20.0280.0540.04 30.0540.0910.07 40.0910.1530.12 50.1530.2590.20 60.2590.3790.31 70.3790.6090.48 80.6090.9420.76 90.9421.591.22101.592.381.95112.383.973.07123.979.856.25 in the ferrosilicon plant , a number of dust samples were collected from the off - gas channel above the furnace ( where the gas and dust from the furnace top ventilation system are collected and removed ) simultaneously to the elpi measurements . these samples were more substantial and could be analyzed by both eds and inductively coupled plasma optical emission spectroscopy ( icp - oes ) . while the samples from the off - gas channel are not directly comparable to the tapping area fume , they may still serve as a reference for the furnace fume of that specific plant . dust samples were also collected by gravimetric filters in the roof exhaust of the silicomanganese plant . these samples were used as a reference for diffuse , fugitive , particulate emissions from this specific plant . they were not substantial enough for wet chemical analysis but were analyzed by sem and eds in the same way as the elpi substrates . the elpi classifies aerosols according to their aerodynamic diameter , dp and collects real - time particle size measurements in the size range of 7 nm 10 m ; a particle size distribution is established based on the size fractions of the impactor stages . these stages and their aerodynamic diameter intervals with corresponding geometric mean diameter values are detailed in table i. the elpi was constructed at the tampere university of technology ( tampere , finland ) for the purpose of monitoring particle size distributions of airborne particulate matter . detailed descriptions of the elpi function and its principles of operation are given in the literature . the aerosols analyzed in this study were collected on greased aluminium foil substrates , as shown in figure 1 . this enables subsequent analysis by electron microscopy and energy dispersive spectroscopy but the amount of material collected is too small to allow more quantitative and/or phase compositional analysis . figure 1 . substrate with fume sample collected by the elpi the operating principle of the elpi is illustrated in figure 2a ; it includes particle charging in a corona charger ; inertial classification in a cascade impactor ; and electrical detection of the collected particles by a multichannel electrometer . a vacuum pump is used to control the airflow ( 10 l / min ) through the instrument . ( a ) sketch of the main components of the elpi and ( b ) illustration of the particle flow in the impactor the particles enter the elpi by use of the vacuum pump . the particles then enter the impactor unit which consists of 12 stages and a filter stage . the particles are then separated into different stages depending on their aerodynamic behavior . as a charged particle is collected in an impactor stage , it produces an electrical current recorded by the electrometer . the air stream passes through a nozzle in a jet plate upon entering each impactor stage . after the nozzle , the stream makes a sharp turn which allows the air to pass between the jet plate and the collection plate . the large particles , which can not turn so abruptly , will be deposited on top of the collection plate . smaller particles are more agile and will therefore follow the air stream to the next stage . the radius of the streamlines decreases for each impactor stage . each stage has a certain probability for errors , such as losses of particles due to diffusion and bounce - off effects . the instrument signal is mathematically treated to correct for these errors . in an impactor , the size fractionation of aerosols is based on the aerodynamic diameter ( dp ) . it is defined as the diameter of a unit density spherical particle having the same settling velocity as the actual particle . another widely used definition is the stokes diameter ( ds ) , which is the diameter of a spherical particle having the same bulk density and settling velocity as the actual particle . the aerodynamic diameter is the output parameter of the elpi measurements , and the stokes diameter is used to calculate the cut - off values between the different size fractions . naturally , most aerosols are not perfectly spherical and the morphology ( particle shape ) of the particles and/or particulate agglomerates may vary greatly , which in turn will influence their aerodynamic properties through their inherent inertia . figure 3 . the positions of the elpi at the ( a ) fesi furnace tapping area and ( b ) simn furnace top figure 4 . sem micrographs of ( a ) fesi furnace fumes with a geometric mean aerodynamic diameter of 0.31 m and ( b ) simn furnace fumes with a geometric mean aerodynamic diameter of 0.31 m the alloys ferrosilicon and silicomanganese are both produced in electric arc furnaces . raw materials include crushed or pre - sintered minerals and ores , and sometimes scrap iron or slag from ferromanganese production , in addition to carbonaceous reductants ( coke and coal ) . the raw materials are fed at the top of the furnace . inside the furnace , the raw materials react and form a raw metal which is tapped from the furnace into a transportable ladle . the tapped metal is generally refined in the ladle by a slag process a thorough description of the production processes for fesi and simn can be found in the textbooks by schei and olsen , respectively . the major source of fume is the furnace itself , yet modern furnace design in combination with powerful ventilation and filter systems are efficiently removing most of the furnace top gases and fumes . typically , the tapping , refining , and casting operations are the main sources of fume inside the plant and of the so - called diffuse emissions from the plant . in the fesi plant , the elpi was set up in the furnace tapping area ( see figure 3a ) and the duration of the measurements was approximately 3 hr . the fesi furnace was tapped continuously and substantial gassing and fuming from the tapping hole was observed during the measurement . tem micrographs of ( a ) fesi furnace fumes with a geometric mean aerodynamic diameter of 0.07 m and ( b ) simn furnace fumes with a geometric mean aerodynamic diameter of 0.04 m figure 6 . number concentration particle size distributions from ( a ) the first and ( b ) the second measurement at the tapping area of a ferrosilicon furnace in the simn plant , the elpi was set up on the top furnace floor ( see figure 3b ) . the elpi measurements were performed inside and outside a fume collecting curtain to assess the effect of the curtain on the indoor air quality as experienced by the workers . number concentration particle size distributions from ( a ) outside and ( b ) inside a fume collecting curtain at the top of a silicomanganese furnace the elpi classifies aerosols according to their aerodynamic diameter , dp and collects real - time particle size measurements in the size range of 7 nm 10 m ; a particle size distribution is established based on the size fractions of the impactor stages . these stages and their aerodynamic diameter intervals with corresponding geometric mean diameter values are detailed in table i. the elpi was constructed at the tampere university of technology ( tampere , finland ) for the purpose of monitoring particle size distributions of airborne particulate matter . detailed descriptions of the elpi function and its principles of operation are given in the literature . the aerosols analyzed in this study were collected on greased aluminium foil substrates , as shown in figure 1 . this enables subsequent analysis by electron microscopy and energy dispersive spectroscopy but the amount of material collected is too small to allow more quantitative and/or phase compositional analysis . figure 1 . substrate with fume sample collected by the elpi the operating principle of the elpi is illustrated in figure 2a ; it includes particle charging in a corona charger ; inertial classification in a cascade impactor ; and electrical detection of the collected particles by a multichannel electrometer . a vacuum pump is used to control the airflow ( 10 l / min ) through the instrument . ( a ) sketch of the main components of the elpi and ( b ) illustration of the particle flow in the impactor the particles enter the elpi by use of the vacuum pump . the particles then enter the impactor unit which consists of 12 stages and a filter stage . the particles are then separated into different stages depending on their aerodynamic behavior . as a charged particle is collected in an impactor stage , it produces an electrical current recorded by the electrometer . the air stream passes through a nozzle in a jet plate upon entering each impactor stage . after the nozzle , the stream makes a sharp turn which allows the air to pass between the jet plate and the collection plate . the large particles , which can not turn so abruptly , will be deposited on top of the collection plate . smaller particles are more agile and will therefore follow the air stream to the next stage . each stage has a certain probability for errors , such as losses of particles due to diffusion and bounce - off effects . the instrument signal is mathematically treated to correct for these errors . in an impactor , the size fractionation of aerosols is based on the aerodynamic diameter ( dp ) . it is defined as the diameter of a unit density spherical particle having the same settling velocity as the actual particle . another widely used definition is the stokes diameter ( ds ) , which is the diameter of a spherical particle having the same bulk density and settling velocity as the actual particle . the aerodynamic diameter is the output parameter of the elpi measurements , and the stokes diameter is used to calculate the cut - off values between the different size fractions . naturally , most aerosols are not perfectly spherical and the morphology ( particle shape ) of the particles and/or particulate agglomerates may vary greatly , which in turn will influence their aerodynamic properties through their inherent inertia . figure 3 . the positions of the elpi at the ( a ) fesi furnace tapping area and ( b ) simn furnace top figure 4 . sem micrographs of ( a ) fesi furnace fumes with a geometric mean aerodynamic diameter of 0.31 m and ( b ) simn furnace fumes with a geometric mean aerodynamic diameter of 0.31 m raw materials include crushed or pre - sintered minerals and ores , and sometimes scrap iron or slag from ferromanganese production , in addition to carbonaceous reductants ( coke and coal ) . the raw materials are fed at the top of the furnace . inside the furnace , the raw materials react and form a raw metal which is tapped from the furnace into a transportable ladle . the tapped metal is generally refined in the ladle by a slag process a thorough description of the production processes for fesi and simn can be found in the textbooks by schei and olsen , respectively . the major source of fume is the furnace itself , yet modern furnace design in combination with powerful ventilation and filter systems are efficiently removing most of the furnace top gases and fumes . typically , the tapping , refining , and casting operations are the main sources of fume inside the plant and of the so - called diffuse emissions from the plant . in the fesi plant , the elpi was set up in the furnace tapping area ( see figure 3a ) and the duration of the measurements was approximately 3 hr . the fesi furnace was tapped continuously and substantial gassing and fuming from the tapping hole was observed during the measurement . tem micrographs of ( a ) fesi furnace fumes with a geometric mean aerodynamic diameter of 0.07 m and ( b ) simn furnace fumes with a geometric mean aerodynamic diameter of 0.04 m figure 6 . number concentration particle size distributions from ( a ) the first and ( b ) the second measurement at the tapping area of a ferrosilicon furnace in the simn plant , the elpi was set up on the top furnace floor ( see figure 3b ) . the elpi measurements were performed inside and outside a fume collecting curtain to assess the effect of the curtain on the indoor air quality as experienced by the workers . figure 7 . number concentration particle size distributions from ( a ) outside and ( b ) inside a fume collecting curtain at the top of a silicomanganese furnace scanning electron micrographs of the fesi and simn fumes are shown in figure 4 . the particles are from the elpi stages which have geometric mean aerodynamic diameters of 0.31 m ( 0.2590.379 m ) . the morphologies of the particles in both plants were corroborated by sem analysis of off - gas fume and roof exhaust fume , respectively . the particles consist of agglomerated spheres and this morphology is typical for particles in the size range from 0.09 to 0.94 m . the presence of agglomerates in fesi fume has previously been reported by dingsyr et al . they applied the notions of primary and secondary agglomerates to describe the fume particles ; the former being particles where the protoparticles ( in our case , the spheres ) are held together by material bridges and the latter being primary agglomerates attached to each other by weak van der waals forces . secondary agglomerates are not likely to be recorded by the elpi as the may disengage in the charger unit . primary agglomerates on the other hand will remain agglomerated and it is therefore the primary agglomerate sizes , and not the spherical protoparticles sizes , which are recorded by the elpi . hence , the average particle diameter recorded by the elpi differs from particle size measurements by sem and brunauer - emmett - teller ( bet ) surface adsorption , whereas it corresponds well to the values obtained by accelerated sedimentation . mass concentration particle size distribution for the : ( a ) fesi fume in figure 6a and ( b ) simn fume in figure 7a transmission electron micrographs are show in figure 5 . the micrographs show particles from the elpi stages with geometric mean aerodynamic diameters of 0.07 and 0.04 m , for the fesi and simn fumes , respectively . these results are representative for the particles collected in the stages with size ranging from 0.03 to 0.09 m . no tem results were obtained for the particles in the smallest fraction due to the difficulties associated with tem sample preparation . the eds elemental analysis , summarized in table ii , specifies that the fume from the fesi furnace is dominated by si and o which indicates that the spheres likely consist primarily of sio2 . the fume from the fesi furnace also includes significant levels of fe , k , al , mg , na , and ca , which is in good agreement with the chemical composition of the off - gas fume collected simultaneously at the plant and also with fesi fumes observed by others . the smallest fractions , analyzed by tem , had lower levels of trace elements than the larger fractions analyzed in sem . for fesi , fe was the only contaminant detected in the fume and fe may be included in the cu - grid used as sample holder ( c and cu are the main constituents of the sample holder ) . elemental analysis of the fumes as obtained by eds fesi fumesimn fumemajor elementssi , osi , mn , ominor elementsfe , k , al , mg , na , camg , ca , al , ktrace elements na , fe , zn , cu , cl the chemical composition of the simn fume particles is more complex and varying than the fesi fume , which was also corroborated by the gravimetric filter samples collected simultaneously at the plant . it consists primarily of si , mn , and o. certain size fractions , most notably those with aerodynamic diameters larger than 1 m , contain significant levels of mg , ca , al , and k and also traces of other contaminants such as na , fe , zn , cu , and cl . the relatively high levels of alkali ( na and k ) are also found in laboratory scale experiments which will be published elsewhere . the morphology and chemical composition of the fume seem to be in fair accordance with previous reports . for simn fume , the smallest fractions display greater dominance of si than the larger fractions and the ultrafine particles contain significantly lesser amounts of mn and trace elements than the larger - size fractions . it is also possible that the ultrafine particles originate from sources outside the fume curtain ; as this particular simn plant is hosting other ferroalloy processes , which are only partially separated from the simn furnace hall , it is possible that some intermixing of particles through air exchange has occurred . figures 6 and 7 show the particle size distributions ( psd ) , in terms of number concentration , as a function of the aerodynamic particle diameter , dp , as obtained by the elpi . the average aerodynamic diameters of the fume particles , as recorded by the elpi , are 0.17 and 0.10 m for the fesi and the simn fume particles , respectively . using the terminology applied by preining , the particle diameter can be used to classify aerosols as fine ( dp < 1 m ) , ultrafine ( dp < 100 nm ) , and nano - sized ( dp < 30 nm ) . for the fesi particles 99% ( percent by numbers ) are so - called fine aerosols ; 58% are ultrafine aerosols and 25% are nano - size aerosols . for simn fume , almost 100% of the particles collected are fine aerosols ; 78% are ultrafine and 40% are nano - sized . the graphs in figures 6 and 7 show that the two psds taken at each plant are relatively similar in shape . still , two measurements at each site are not statistically sufficient to assess the characteristics of the airborne particulate matter in the plant . to assess the temporal variation of the indoor air quality and potential diffuse emissions of a plant multiple and frequent measurements would have to be undertaken . this work shows that such a project is feasible , by use of an elpi , although it is beyond the scope of this particular study . in figure 8 , the same particle size distributions as above are recalculated as mass concentrations . from the eds results , the fume from both furnaces was found to be mainly sio2 and therefore a density of 2.2 g / cm was assumed in the mass concentration calculations . for the simn , however , this is not an ideal assumption as the chemical composition of the particles from the simn production is quite diverse and the phase composition remains largely unknown . it is , however , a useful approximation to compare the number concentration distributions ( figures 6a and 7a ) with the mass concentration distributions ( figures 8a and 8b ) , since most administrative standards for airborne particulate matter are given in mass concentration units . for the fesi furnace , the percentages by mass are as follows : 88 wt% are fine , 5 wt% are ultrafine , and merely 1 wt% are nano - sized . for the simn furnace these numbers are 72 wt% , 66 wt% , and 11 wt% , respectively . the electric low pressure impactor has been successfully applied to assess the particle size distribution of fume particles in the indoor air surrounding two ferroalloy production processes . the aerosol sizes recorded by the elpi correspond to the aerodynamic diameters of the so - called primary agglomerates . particle size distributions in the range of 7 nm to 10 m have been established for both sites . the majority of the particles are smaller than 1 m and approximately 5 wt% are so called ultrafine aerosols . the samples collected by elpi were not substantial enough for in - depth chemical analysis . the fume consisted primarily of spherical , amorphous silica protoparticles with trace levels of alkali and other contaminants . this work shows that the elpi has great potential use for the assessment of airborne particulate matter from ferroalloy production . for a thorough pm assessment , number concentration particle size distributions were measured in - situ by the elpi and mass concentration particle size distributions were calculated from these results . the differences between the particle size distributions based on number and mass concentrations are significant and may be attributed to the very small masses of the ultrafine and nano - sized particles . the funding for these studies was provided by the norwegian ferroalloy producers research association ( fff ) and the norwegian research council ( nfr ) through the project fugitive emissions of materials and energy

the present article presents a comprehensive evaluation of the potential use of an electrical low pressure impactor ( elpi ) in the ferroalloy industry with respect to indoor air quality and fugitive emission control . the elpi was used to assess particulate emission properties , particularly of the fine particles ( dp 1 m ) , which in turn may enable more satisfactory risk assessments for the indoor working conditions in the ferroalloy industry . an elpi has been applied to characterize the fume in two different ferroalloy plants , one producing silicomanganese ( simn ) alloys and one producing ferrosilicon ( fesi ) alloys . the impactor classifies the particles according to their aerodynamic diameter and gives real - time particle size distributions ( psd ) . the psd based on both number and mass concentrations are shown and compared . collected particles have also been analyzed by transmission and scanning electron microscopy with energy dispersive spectroscopy . from the elpi classification , particle size distributions in the range 7 nm 10 m have been established for industrial simn and fesi fumes . due to the extremely low masses of the ultrafine particles , the number and mass concentration psd are significantly different . the average aerodynamic diameters for the fesi and the simn fume particles were 0.17 and 0.10 m , respectively . based on this work , the elpi is identified as a valuable tool for the evaluation of airborne particulate matter in the indoor air of metallurgical production sites . the method is well suited for real - time assessment of morphology ( particle shape ) , particle size , and particle size distribution of aerosols .

INTRODUCTION EXPERIMENTAL METHOD The Electrical Low Pressure Impactor (ELPI) The Ferroalloy Plants RESULTS AND DISCUSSION CONCLUSION SUPPLEMENTAL MATERIAL FUNDING

characteristic properties of the particles , especially particle size and chemical composition , influence their impact on human health . the ultrafine and nano - sized particles may , due to their very large surface area be more reactive than the more widely studied , larger particulate matters . as the chemical composition of particles originating from different industries and processes may vary greatly , it is important to characterize the fumes and link them to their origin to enable enhanced risk assessments and enable capture . moreover , the fumes from industrial plants are a major contributor to the so - called diffuse emissions which are receiving increased attention from authorities as they may influence the air quality of the local , urban communities as well as the environment and climate at large . a great deal of efforts and investments are currently directed towards improvements in ventilation and filter systems , designed to collect and control the off - gases and fumes from the various metallurgical and mechanical process operations . from the corporate point of view , a process optimized to minimize fume formation would not only improve the indoor air quality and decrease the environmental impact of the plant ; it would also minimize material and energy losses associated with the production . there are several measurement techniques which can be used to characterize and quantify the airborne particulate matter in an industrial plant . the major advantage of the electric low pressure impactor ( elpi , dekati ltd . the elpi also enables real - time assessment of the particle size distribution ( psd ) of aerosols as well as subsequent analysis of the collected aerosol particles by electron microscopy . the elpi has previously been used to assess particulate emissions from traffic , combustion , welding fume , and aluminum electrolysis . this article aims to describe how the novel technology of the electric low pressure impactor can be used to assess indoor air quality surrounding ferroalloy production processes and to establish real - time particle size distributions of aerosols at a ferrosilicon and a silicomanganese production site . fume from different locations in silicomanganese ( simn ) and ferrosilicon ( fesi ) production plants was collected and analyzed using an elpi ( dekati ltd . for proper function of the elpi a particle density should be assumed for the mathematical conversion of the as - obtained electrical current values to a number concentration ( number of particles per volume of air ) . in these studies , the assumed density values were that of amorphous silica ( sio2 , 2.2 g / cm ) ; this assumption is motivated and discussed in the following sections of this article . the same assumed density was also used in a second step when the number concentrations were algebraically converted to mass concentrations . the number - to - mass conversion was performed using the filter functions in the elpivi software ( dekati ltd . , tampere , finland ) and the density has a large influence on this conversion , which in turn introduces significant error sources as discussed in further sections . the particles are collected in the elpi on aluminium foil substrates , one for each size fraction as detailed in table i. these particle samples were subsequently analyzed by scanning electron microscopy ( sem ) , transmission electron microscopy ( tem ) , and energy dispersive spectroscopy ( eds ) . the amount of fume particles collected by the elpi was not , however , sufficient to quantitatively analyze the chemical and/or phase compositions by other methods . ( m ) 10.0070.0280.02 20.0280.0540.04 30.0540.0910.07 40.0910.1530.12 50.1530.2590.20 60.2590.3790.31 70.3790.6090.48 80.6090.9420.76 90.9421.591.22101.592.381.95112.383.973.07123.979.856.25 in the ferrosilicon plant , a number of dust samples were collected from the off - gas channel above the furnace ( where the gas and dust from the furnace top ventilation system are collected and removed ) simultaneously to the elpi measurements . while the samples from the off - gas channel are not directly comparable to the tapping area fume , they may still serve as a reference for the furnace fume of that specific plant . they were not substantial enough for wet chemical analysis but were analyzed by sem and eds in the same way as the elpi substrates . the elpi classifies aerosols according to their aerodynamic diameter , dp and collects real - time particle size measurements in the size range of 7 nm 10 m ; a particle size distribution is established based on the size fractions of the impactor stages . these stages and their aerodynamic diameter intervals with corresponding geometric mean diameter values are detailed in table i. the elpi was constructed at the tampere university of technology ( tampere , finland ) for the purpose of monitoring particle size distributions of airborne particulate matter . detailed descriptions of the elpi function and its principles of operation are given in the literature . this enables subsequent analysis by electron microscopy and energy dispersive spectroscopy but the amount of material collected is too small to allow more quantitative and/or phase compositional analysis . substrate with fume sample collected by the elpi the operating principle of the elpi is illustrated in figure 2a ; it includes particle charging in a corona charger ; inertial classification in a cascade impactor ; and electrical detection of the collected particles by a multichannel electrometer . ( a ) sketch of the main components of the elpi and ( b ) illustration of the particle flow in the impactor the particles enter the elpi by use of the vacuum pump . the particles then enter the impactor unit which consists of 12 stages and a filter stage . the particles are then separated into different stages depending on their aerodynamic behavior . as a charged particle is collected in an impactor stage , it produces an electrical current recorded by the electrometer . after the nozzle , the stream makes a sharp turn which allows the air to pass between the jet plate and the collection plate . the large particles , which can not turn so abruptly , will be deposited on top of the collection plate . in an impactor , the size fractionation of aerosols is based on the aerodynamic diameter ( dp ) . another widely used definition is the stokes diameter ( ds ) , which is the diameter of a spherical particle having the same bulk density and settling velocity as the actual particle . the aerodynamic diameter is the output parameter of the elpi measurements , and the stokes diameter is used to calculate the cut - off values between the different size fractions . naturally , most aerosols are not perfectly spherical and the morphology ( particle shape ) of the particles and/or particulate agglomerates may vary greatly , which in turn will influence their aerodynamic properties through their inherent inertia . the positions of the elpi at the ( a ) fesi furnace tapping area and ( b ) simn furnace top figure 4 . inside the furnace , the raw materials react and form a raw metal which is tapped from the furnace into a transportable ladle . the tapped metal is generally refined in the ladle by a slag process a thorough description of the production processes for fesi and simn can be found in the textbooks by schei and olsen , respectively . typically , the tapping , refining , and casting operations are the main sources of fume inside the plant and of the so - called diffuse emissions from the plant . in the fesi plant , the elpi was set up in the furnace tapping area ( see figure 3a ) and the duration of the measurements was approximately 3 hr . number concentration particle size distributions from ( a ) the first and ( b ) the second measurement at the tapping area of a ferrosilicon furnace in the simn plant , the elpi was set up on the top furnace floor ( see figure 3b ) . the elpi measurements were performed inside and outside a fume collecting curtain to assess the effect of the curtain on the indoor air quality as experienced by the workers . number concentration particle size distributions from ( a ) outside and ( b ) inside a fume collecting curtain at the top of a silicomanganese furnace the elpi classifies aerosols according to their aerodynamic diameter , dp and collects real - time particle size measurements in the size range of 7 nm 10 m ; a particle size distribution is established based on the size fractions of the impactor stages . these stages and their aerodynamic diameter intervals with corresponding geometric mean diameter values are detailed in table i. the elpi was constructed at the tampere university of technology ( tampere , finland ) for the purpose of monitoring particle size distributions of airborne particulate matter . detailed descriptions of the elpi function and its principles of operation are given in the literature . this enables subsequent analysis by electron microscopy and energy dispersive spectroscopy but the amount of material collected is too small to allow more quantitative and/or phase compositional analysis . substrate with fume sample collected by the elpi the operating principle of the elpi is illustrated in figure 2a ; it includes particle charging in a corona charger ; inertial classification in a cascade impactor ; and electrical detection of the collected particles by a multichannel electrometer . ( a ) sketch of the main components of the elpi and ( b ) illustration of the particle flow in the impactor the particles enter the elpi by use of the vacuum pump . the particles then enter the impactor unit which consists of 12 stages and a filter stage . the particles are then separated into different stages depending on their aerodynamic behavior . as a charged particle is collected in an impactor stage , it produces an electrical current recorded by the electrometer . after the nozzle , the stream makes a sharp turn which allows the air to pass between the jet plate and the collection plate . the large particles , which can not turn so abruptly , will be deposited on top of the collection plate . in an impactor , the size fractionation of aerosols is based on the aerodynamic diameter ( dp ) . the aerodynamic diameter is the output parameter of the elpi measurements , and the stokes diameter is used to calculate the cut - off values between the different size fractions . naturally , most aerosols are not perfectly spherical and the morphology ( particle shape ) of the particles and/or particulate agglomerates may vary greatly , which in turn will influence their aerodynamic properties through their inherent inertia . sem micrographs of ( a ) fesi furnace fumes with a geometric mean aerodynamic diameter of 0.31 m and ( b ) simn furnace fumes with a geometric mean aerodynamic diameter of 0.31 m raw materials include crushed or pre - sintered minerals and ores , and sometimes scrap iron or slag from ferromanganese production , in addition to carbonaceous reductants ( coke and coal ) . inside the furnace , the raw materials react and form a raw metal which is tapped from the furnace into a transportable ladle . the tapped metal is generally refined in the ladle by a slag process a thorough description of the production processes for fesi and simn can be found in the textbooks by schei and olsen , respectively . typically , the tapping , refining , and casting operations are the main sources of fume inside the plant and of the so - called diffuse emissions from the plant . in the fesi plant , the elpi was set up in the furnace tapping area ( see figure 3a ) and the duration of the measurements was approximately 3 hr . number concentration particle size distributions from ( a ) the first and ( b ) the second measurement at the tapping area of a ferrosilicon furnace in the simn plant , the elpi was set up on the top furnace floor ( see figure 3b ) . the elpi measurements were performed inside and outside a fume collecting curtain to assess the effect of the curtain on the indoor air quality as experienced by the workers . number concentration particle size distributions from ( a ) outside and ( b ) inside a fume collecting curtain at the top of a silicomanganese furnace scanning electron micrographs of the fesi and simn fumes are shown in figure 4 . the particles are from the elpi stages which have geometric mean aerodynamic diameters of 0.31 m ( 0.2590.379 m ) . the morphologies of the particles in both plants were corroborated by sem analysis of off - gas fume and roof exhaust fume , respectively . the particles consist of agglomerated spheres and this morphology is typical for particles in the size range from 0.09 to 0.94 m . they applied the notions of primary and secondary agglomerates to describe the fume particles ; the former being particles where the protoparticles ( in our case , the spheres ) are held together by material bridges and the latter being primary agglomerates attached to each other by weak van der waals forces . primary agglomerates on the other hand will remain agglomerated and it is therefore the primary agglomerate sizes , and not the spherical protoparticles sizes , which are recorded by the elpi . hence , the average particle diameter recorded by the elpi differs from particle size measurements by sem and brunauer - emmett - teller ( bet ) surface adsorption , whereas it corresponds well to the values obtained by accelerated sedimentation . mass concentration particle size distribution for the : ( a ) fesi fume in figure 6a and ( b ) simn fume in figure 7a transmission electron micrographs are show in figure 5 . the micrographs show particles from the elpi stages with geometric mean aerodynamic diameters of 0.07 and 0.04 m , for the fesi and simn fumes , respectively . these results are representative for the particles collected in the stages with size ranging from 0.03 to 0.09 m . no tem results were obtained for the particles in the smallest fraction due to the difficulties associated with tem sample preparation . the eds elemental analysis , summarized in table ii , specifies that the fume from the fesi furnace is dominated by si and o which indicates that the spheres likely consist primarily of sio2 . the fume from the fesi furnace also includes significant levels of fe , k , al , mg , na , and ca , which is in good agreement with the chemical composition of the off - gas fume collected simultaneously at the plant and also with fesi fumes observed by others . for fesi , fe was the only contaminant detected in the fume and fe may be included in the cu - grid used as sample holder ( c and cu are the main constituents of the sample holder ) . elemental analysis of the fumes as obtained by eds fesi fumesimn fumemajor elementssi , osi , mn , ominor elementsfe , k , al , mg , na , camg , ca , al , ktrace elements na , fe , zn , cu , cl the chemical composition of the simn fume particles is more complex and varying than the fesi fume , which was also corroborated by the gravimetric filter samples collected simultaneously at the plant . it consists primarily of si , mn , and o. certain size fractions , most notably those with aerodynamic diameters larger than 1 m , contain significant levels of mg , ca , al , and k and also traces of other contaminants such as na , fe , zn , cu , and cl . the morphology and chemical composition of the fume seem to be in fair accordance with previous reports . for simn fume , the smallest fractions display greater dominance of si than the larger fractions and the ultrafine particles contain significantly lesser amounts of mn and trace elements than the larger - size fractions . it is also possible that the ultrafine particles originate from sources outside the fume curtain ; as this particular simn plant is hosting other ferroalloy processes , which are only partially separated from the simn furnace hall , it is possible that some intermixing of particles through air exchange has occurred . figures 6 and 7 show the particle size distributions ( psd ) , in terms of number concentration , as a function of the aerodynamic particle diameter , dp , as obtained by the elpi . the average aerodynamic diameters of the fume particles , as recorded by the elpi , are 0.17 and 0.10 m for the fesi and the simn fume particles , respectively . using the terminology applied by preining , the particle diameter can be used to classify aerosols as fine ( dp < 1 m ) , ultrafine ( dp < 100 nm ) , and nano - sized ( dp < 30 nm ) . for simn fume , almost 100% of the particles collected are fine aerosols ; 78% are ultrafine and 40% are nano - sized . still , two measurements at each site are not statistically sufficient to assess the characteristics of the airborne particulate matter in the plant . to assess the temporal variation of the indoor air quality and potential diffuse emissions of a plant multiple and frequent measurements would have to be undertaken . this work shows that such a project is feasible , by use of an elpi , although it is beyond the scope of this particular study . in figure 8 , the same particle size distributions as above are recalculated as mass concentrations . from the eds results , the fume from both furnaces was found to be mainly sio2 and therefore a density of 2.2 g / cm was assumed in the mass concentration calculations . for the simn , however , this is not an ideal assumption as the chemical composition of the particles from the simn production is quite diverse and the phase composition remains largely unknown . it is , however , a useful approximation to compare the number concentration distributions ( figures 6a and 7a ) with the mass concentration distributions ( figures 8a and 8b ) , since most administrative standards for airborne particulate matter are given in mass concentration units . for the fesi furnace , the percentages by mass are as follows : 88 wt% are fine , 5 wt% are ultrafine , and merely 1 wt% are nano - sized . for the simn furnace these numbers are 72 wt% , 66 wt% , and 11 wt% , respectively . the electric low pressure impactor has been successfully applied to assess the particle size distribution of fume particles in the indoor air surrounding two ferroalloy production processes . the aerosol sizes recorded by the elpi correspond to the aerodynamic diameters of the so - called primary agglomerates . particle size distributions in the range of 7 nm to 10 m have been established for both sites . the majority of the particles are smaller than 1 m and approximately 5 wt% are so called ultrafine aerosols . this work shows that the elpi has great potential use for the assessment of airborne particulate matter from ferroalloy production . for a thorough pm assessment , number concentration particle size distributions were measured in - situ by the elpi and mass concentration particle size distributions were calculated from these results . the differences between the particle size distributions based on number and mass concentrations are significant and may be attributed to the very small masses of the ultrafine and nano - sized particles .

fungi and oomycetes are responsible for many of the most devastating plant diseases that lead to very significant losses in the agricultural sector worldwide . approximately 100,000 species of fungi and oomycetes have been described , but only a very small proportion of these are pathogenic . however , phylogenetic studies have shown that disease - causing pathogens are not necessarily closely related to each other . in fact , they are spread throughout all taxonomic groups of fungi , often showing a close evolutionary relationship to nonpathogenic species . it therefore seems likely that phytopathogenicity has evolved as a trait many times during fungal and oomycete evolution . a significant effort has been made into the identification of pathogenicity determinants such as individual genes that are essential for a pathogen to invade a host plant successfully but that are dispensable for saprophytic growth . despite the different origin and different site on the phylogenetic tree of the true fungi and oomycetes , it has been shown that a range of secreted proteins known as effectors are important for establishing infection of the host plant . these secreted proteins can suppress plant defenses and subvert cellular processes to suit the needs of invading pathogens . they include a number of secreted proteinases , transcriptional factors , and components of signal transduction pathways . in fungi , the proteinases can play either a general nutritive role or specific roles in cell metabolism or as pathogenicity or virulence factors . in fungi aspartate , cysteine , metallo- , serine , and threonine proteinases , as well as uncharacterized classes of proteinases , have been identified . a total of 282,061 predicted proteins were grouped into 23,724 clusters , and only 16 clusters contained proteins that were found in all 34 species of fungi but which were absent from some species of oomycetes . this number of fungal - specific clusters is surprisingly low considering the phylogenetic distance between the oomycetes and fungi . pathogenicity factors have been defined as genes that are essential for successful completion of the pathogenic life cycle but dispensable for saprophytic growth . proteinases that are secreted by fungi ( especially pathogens ) allow them to adapt to different living conditions . fungi have expanded their repertoire of peptidases throughout evolution to take advantages of different protein sources . proteinases are divided into six main classes serine , cysteine , threonine , aspartic , glutamic , and metallopeptidases , according to the nature of functional groups in the active center of the enzyme molecule . extracellular proteolytic enzymes of the fungi are represented to a large degree by serine proteinases . there are two major families of serine proteinases that are present in fungi : subtilisin ( s8 ) and chymotrypsin ( s1 ) families . the chymotrypsin family includes trypsins and chymotrypsins and most of these enzymes in fungi are trypsins . according to preliminary observations , the production of trypsin - like enzymes is characteristic of plant pathogens whereas extracellular endoproteolytic activity of saprotrophs is provided mainly with subtilisin - like enzymes [ 79 ] . the most essential enzymes for pathogenesis can well be those that allow the fungus to penetrate the protective cutin layer of the plant epidermis and disrupt the pectin matrix of the plant cell wall in which cellulose fibrils are embedded . there seems to be a number of proteinase and peptidase domains that are more common in the genomes of phytopathogens [ 6 , 10 ] . proteinases are also implicated in many pathological conditions in animals and plants and are considered as important virulence factors of many pathogens , including viruses , bacteria , fungi , and parasites . proteinases from microorganisms display many unique characteristics in terms of catalytic mechanism , substrate specificity , activation mechanism , thermostability , and ph optimum . fungi could represent another source of proteinases with novel characteristics , as proteinases play a major role in the physiology , morphogenesis , and metabolism of fungi . in view of these functions , proteinases of several catalytic classes the purpose of this study was a comparative analysis of proteinases secreted by phytophthora infestans ( mont . ) de bary , rhizoctonia solani , and fusarium culmorum that are causal agents of several potato diseases . we thought that the data obtained from this study may help clarify the question whether their composition depends on the phylogenetic position of the pathogen . the isolates of phytophthora infestans ( mont . ) de bary , rhizoctonia solani kuhn ( ag-3 ) 153 , and fusarium culmorum ( w. g. sm . ) sacc . were supplied by potato , vegetable , and fruit scientific and practical center of the national academy of sciences of belorussia . the cultures were maintained on oatmeal agar and stored at room temperature ( 21c ) . culture media were tested for suitability to give good growth as well as for adequate enzyme production . the following media were tested : ( i ) per 100 ml : kh2po4 ( 0.15 g ) ; mgso47h2o2 ( 0.025 g ) ; feso47h2o2 ( 1 mg ) ; thiamine ( 1 mg ) and riboflavin ( 1 mg ) ; ( ii ) the medium i plus yeast extract ( 1 g ) . 41 filter paper , washed with a small quantity of warm distilled water , heated overnight in an oven at about 90 2c , cooled in a desiccator , and weighed . culture medium was inoculated in erlenmeyer flasks ( 500 ml ) by introducing 15 ml of spore suspension into 150 ml of the culture medium . exoproteinases were isolated from the culture medium after 12 days of growth of the microorganism . proteins were precipitated with ( nh4)2so4 at 80% ( w / v ) of saturation . the precipitate was separated by centrifugation at 10000 g for 30 min at 4c . the precipitate was dissolved in water , desalted by gel chromatography on sephadex g-25 , and used for the enzyme assays . proteolytic enzyme activity was determined by the kunitz method using 1% casein , 0.5% azocasein , and 0.5% hemoglobin as substrates . time of azocasein hydrolysis was 30 min , and for casein and hemoglobin it was 1 h. the activity of cysteine proteinases was evaluated in the presence of 25 mm l - cysteine and 1 mm edta according to the modified kunitz method . one unit of proteolytic activity ( u ) is the amount of enzyme that leads to an increase in optical density in 0.1 at 366 nm ( with azocasein ) and at 280 nm ( with casein and hemoglobin ) within 1 min . amidase enzyme activity was determined by the method of erlanger et al . using synthetic and p - nitroanilide substrates : n-benzoyl - l - arginine p - nitroanilide ( bapna ) , n - carbobenzyloxy - l - alanyl - l - alanyl - l - leucine p - nitroanilide ( z - aalpna , bachem , switzerland ) , and n - succinyl - l - phenylalanine p - nitroanilide ( suc - fpna ) , n - succinyl - glycyl - glycyl - l - phenylalanine p - nitroanilide ( suc - ggfpna ) , l - leucine p - nitroanilide ( lpna ) , n - acetyl - l - alanyl - l - alanyl - l - alanyl p - nitroanilide ( ac - aaapna , bachem , switzerland ) . one unit of amidase activity ( au ) was the amount of enzyme that hydrolyzed 1 nmol of the substrate in 1 min . in the inhibitor analysis the following inhibitors were used : iodoacetamide ( iaa , 1 mm ) , chloromethylketone tosyl - l - lysine ( cmktl , 1 mm ) , chloromethylketone tosyl - l - phenylalanine ( cmktp , 1 mm ) , ethylenediaminetetraacetic acid disodium salt ( edta , 4.0 mm ) , dl - dithiothreitol ( dtt , 1 mm ) , phenylmethanesulfonyl fluoride ( pmsf , 1 mm ) , p - chloro - mercurybenzoate ( pcmb , 1 mm ) , and diisopropyl fluorophosphate ( difp , 0.2 mm ) . electrophoresis in 20% polyacrylamide gel in the presence of sodium dodecyl sulfate ( sds - page ) and - mercaptoethanol was performed by the method of laemmli . gels were stained with 0.1% solution of coomassie r-250 in 20% ethanol with 5% formaldehyde . to obtain the zymograms sds - page electrophoresis was carried out in the presence of copolymerized substrate ( gelatin 0.1% ) by the method of heussen and dowdle . protein samples ( not more 50 mcg ) were applied without prior heating . upon electrophoresis ending gels were washed with triton x-100 ( 2.5% ) under vigorous stirring , rinsed with 0.1 m glycine - naoh buffer ph 7.8 , and incubated overnight in the same buffer at room temperature . gels were then stained with 0.1% amidoschwarz in ethanol : acetic acid : water ( 3 : 1 : 6 ) for 1 h and washed with the same solution without the dye . proteins with proteolytic activity were detected as colorless bands against a deep blue colored background of the stained gelatin . protein content was determined by with bsa as a standard using bradford 's modified method . all experiments and assays were carried out at least in triplicate and the results are presented as mean values obtained with an indication of the standard deviation . the chemicals were used of the following companies : azocasein and hemoglobin ( sigma chemicals co. , usa ) , casein ( biolar , latvia ) , the synthetic substrates mentioned ( sigma , if other not indicated ) , the synthetic inhibitors mentioned ( sigma ) , and lmw calibration kit ( sigma ) . de bary , r. solani , and f. culmorum were tested for the activities of the exoproteinases . the influence of several environmental factors on the production of extracellular proteinases of these microorganisms was studied systematically in controlled batch cultures . not all of the defined media tested in the study gave production of the examined enzymes , although they all supported fairly good growth ( see figure 1 ) . so we did not observe some changes in yield of proteinases secreted into the culture medium when it was inoculated with these isolates into the semisynthetic culture medium containing kh2po4 , mgso4 , feso4 , thiamin , and riboflavin . as the studied pathogen isolates caused the most devastating diseases of potato , we added to the culture media the heat - stable potato tuber proteins . this initiated the secretion of proteinases by fungi r. solani and f. culmorum ( figures 1(b ) and 1(c ) ) . in the case of p. infestans the exoproteinase activity remained low and practically unchanged during the growth of the culture , although we observed the biomass increasing ( figure 1(d ) ) . it was shown that the addition of kno3 into the medium lead to a significant decrease in the exoproteolytic activity , indicating the suppression of secretion and possibly synthesis of the exoenzymes . as exoproteinase secretion was inhibited in the presence of nitrate , there was reason to believe that mineral nitrogen regulates adaptation of the pathogens to the environment by a mechanism that , according to the authors of , can be attributed to catabolic repression . to study the effect of organic nitrogen on the exoproteinase secretion of the pathogens , yeast extract was extra added into the culture medium . when the yeast extract was added to the culture medium a noticeable increase in the exoproteinase secretion was observed , which was accompanied by accelerated growth of mycelium ( figures 1(b ) , 1(c ) , and 1(d ) ) . the yeast extract as an additional source of nutritional substrates for the microorganism apparently acted as an inducer . it is important to point out that the oomycete was able to secret exoproteinases only in the presence of yeast extract . therefore , we conclude p. infestans is more exacting in its nutrition for the enzyme production than for growth . it is interesting that there was an observed interdependence for several factors studied . amongst others , based on the observed interactions , the selection of environmental factors to increase protease activity is not straightforward , as unexpected antagonistic or synergistic effects can occur . there were some differences in the effect of the environmental parameters on the various proteinase - related phenotypes . not all of the defined media tested in the present study gave production of the examined enzymes , although they all supported fairly good growth . total proteinase activity increased with the degree of evolutionary development of the isolates , which originated from phylogenetically distant fungi belonging to the kingdom true fungi ascomycetes ( f. culmorum ) and basidiomycetes ( r. solani ) , while p. infestans belong to the phylum oomycota ( figure 1(a ) ) . traditionally , due to their filamentous growth habit , oomycetes have been classified in the kingdom fungi . however , modern molecular and biochemical analyses suggest that oomycetes have little taxonomic affinity with filamentous fungi but are more closely related to brown algae ( heterokonts ) in the stramenopiles , one of several major eukaryotic kingdoms [ 2022 ] . in a series of initial screening experiments of the factors investigated , only medium ph and nitrogen concentration particular strongly affected the extracellular proteinase activities . it was found that the medium ph ranged from slightly acidic to neutral and reached a constant value of 7.27.4 after 12 days ( figure 1 ) during the growth of the isolates . the capacity of pathogenic fungi to support the medium ph did not depend on the composition of the culture media . the dependence of synthesis and secretion of exoproteinases on the medium ph has also been found in some microorganisms . consequently , the ph can be attributed as one of the factors controlling these processes . however , fungi normally avoid natural habitats with unsuitable ph , possibly because of the metabolic costs of this type of adjustments in competition with more specifically ph - adapted microorganisms . finally , the proteolytic enzyme activity is known to be strongly ph dependent , so in order to have effective protein degradation the ph optimum of the proteolytic enzymes should ideally match the ph of their habitat . we observed that the ph values of the medium affected the growth of the isolates ( figure 1 , curves 3 ) . it was shown that the secretion of proteinases depended on the cultivation temperature . when the culture was grown at 28c a decrease in the proteolytic amidase activity the temperature of 21c was optimal for the production of exoproteinases by the isolates , and apparently corresponded to the temperature regime of its habitat in the natural environment . this temperature optimum for the studied pathogen and its inability to grow at higher temperatures may reflect its distribution in natural habitats that are buffered against higher temperatures . reported temperature optimum of 2025c for several strains of pythium attrantheridium isolated from cavity spot lesions on carrots and on apple and cherry seedlings . similarly , in vitro growth of pythium splendens , a species that causes a root disease of carambola in southern florida , decreases at temperatures above 30c . the exoenzymes secreted by fungi were the most effective at neutral and slightly alkaline ph values . so , exoproteinases of f. culmorum were characterized by the maximum of proteolytic activity at ph 8.0 , and of r. solani at ph 8.5 . the highest level of exoproteinase activity of p. infestans was observed at neutral ph values and was characterized by a maximum at ph 7.0 . the second some increase of the enzyme activity of p. infestans was exhibited at slightly alkaline ph indicating the presence of proteinases with ph optimum of action in the region from 8 to 9 . it was indicated by sds - page that all studied isolates secreted three or more proteins with proteolytic activity ( figure 2 ) . fusarium culmorum and p. infestans predominantly produced 29- and 49-kda proteinases , and r. solani secreted a 67- and 22-kda proteinases ( figure 2 ) . exoproteinases of all three pathogens showed low activity toward casein and hemoglobin , while activity in azocasein assay was much higher ( table 1 ) . there are clear catalytic differences between subtilisins and trypsins , in their substrate specificities can allow their distinction . most effectively hydrolyzed bapna ( a substrate for trypsin - like proteinases ) and to a lesser extent z - aalpna ( a substrate for subtilisin - like proteinases ) . at the same time , the exoproteinases did not act on the substrates for chymotrypsin- and elastase - like proteinases ( suc - ggfpna and ac - aaapna , resp . ) , as well as for aminopeptidases ( lpna ) . the enzymes secreted by f. culmorum hydrolyzed z - aalpna very efficiently and to a lesser extent bapna . they showed low activity toward substrates for chymotrypsin- and elastase - like proteinases , and for aminopeptidases as well . for the exoenzymes secreted by r. solani the profile depended on the culture medium composition ( table 3 ) . bapna was hydrolyzed most efficiently if the yeast extract was absent , but z - aalpna was significantly less efficiently hydrolyzed . specific substrates for chymotrypsin- and elastase - like proteinases and for aminopeptidases were hydrolyzed poorly if at all . the addition of yeast extract led to a change in the proteinase spectrum : z - aalpna was hydrolyzed most effectively , but bapna was much more poorly hydrolyzed ( more than five times , see table 3 ) . analysis of the data on the effect of synthetic substrates specific to certain groups of proteinases to the exoenzyme activity of the oomycete and the fungi indicated that p. infestans secreted predominantly serine and metalloproteinases , and the enzymes of serine type are trypsin- and subtilisin - like proteinases . in the case of f. culmorum the exoproteinase profile of p. infestans and f. culmorum were not dependent on medium composition . when r. solani was grown without yeast extract , trypsin - like serine proteinases were secreted mainly , including sh - dependent serine enzymes . the subtilisin - like proteinase activity was significantly lower ( table 3 ) . in the presence of yeast extract the composition of r. solani exoenzymes was enriched in subtilisin - like proteinases whereas the content of trypsin - like enzymes was decreased significantly ( table 3 ) . the presence of a reducing agent ( l - cysteine with edta ) did not affect the proteolytic activity of the fungal exoproteinases in the azocasein assay , which indicated the absence of cysteine exoproteinases in the growth medium . the interaction of different synthetic inhibitors with exoproteinases secreted by the three microorganisms was studied ( table 4 ) . edta , which has often been used as an indicator of metalloproteinases , had some effect only upon the total proteolytic activity of the exoenzymes of p. infestans . the proteolytic activity of p. infestans exoproteinases was reduced by almost twofold in the presence of l - cysteine with edta . this indicated the presence of exometalloproteases whose activity was inhibited by edta ( table 4 ) . this was confirmed by increase in the p. infestans exoproteinase activity by 50% in the presence of 1 mm cacl2 at ph 7.0 . it is well known that metalloproteinases of microorganisms are activated in the presence of calcium ions . analysis of chloromethylketone treatment showed that the serine proteinases were trypsin - like ( table 4 ) . so the data obtained in the experiments with synthetic inhibitors confirmed that the oomycete p. infestans secreted serine- and metalloproteinases predominantly and that serine - type enzymes were trypsin- and subtilisin - like proteinases . treatment with mercuric chloride significantly reduced the amidase proteolytic activities of the exoenzyme ( table 4 ) . that serves as once more confirmation of the presence of serine proteinases in the culture medium of f. culmorum . the results of the azocasein assays ( table 4 ) show that inhibitions of aspartate and cysteine proteinases were small or absent . the interaction of exoproteinases secreted by r. solani and f. culmorum with the natural protein serine proteinase inhibitors isolated from potato tubers and legume seeds was also studied ( figure 3 ) . the activity of r. solani exoproteinases was inhibited most effectively by specific trypsin inhibitors from potato tubers and from honey locust seeds as well as soybean kunitz trypsin inhibitor ( skti ) ( figure 3(a ) , curve 1 , 3 , and 4 ) . the interaction of potato chymotrypsin inhibitor i with exoproteinases was much weaker ( figure 3(a ) , curve 2 ) . the mentioned assumption about the trypsin - like activity of r. solani exoproteinases grown without yeast extract was confirmed . skti and soybean bowman - birk inhibitor ( sbbi ) acted on exoproteinases of f. culmorum much more weakly ( figure 3(b ) , curve 3 and 6 ) . however , the specific subtilisin inhibitor from potato tubers inhibited effectively their activity , reducing it by more than 60% ( figure 3(b ) , curve 5 ) . this indicates that the subtilisin - like enzymes constitute a significant part of the f. culmorum exoproteinases . similar results were obtained in the study of protein inhibitor action on the enzymes secreted by p. infestans ( data not presented ) . the data obtained in the experiments confirmed belonging of exoproteinases of the fungi and oomycete to the chymotrypsin clan of proteolytic enzymes . it should be noted that potato subtilisin inhibitor suppressed in vitro growth and development of f. culmorum macroconidia and p. infestans zoospores . we can assume that the secreted exoproteinases of the phytopathogenic microorganisms are a factor of their pathogenicity . thus , the inhibitor analysis of the major extracellular proteinases of pathogenic fungi r. solani and f. culmorum and the oomycete p. infestans showed that they belong to the group of serine proteinases mainly . the substrate specificity of the proteinases and their sensitivity to synthetic and natural inhibitors suggested that the enzymes of f. culmorum and p. infestans are trypsin - like and subtilisin - like proteinases . the exoenzymes of r. solani depended on the culture medium composition , especially on the form of nitrogen nutrition . when r. solani grows as a saprobe the exoenzyme was represented by subtilisin - like proteinases . although r. solani has been isolated only from potato tissues , it can be thought to persist as a saprobe that lives on plant debris , as evidenced by the increase in subtilisin - like activity . the secretion of the trypsin - like proteinases in culture can be due to their participation in tissue degradation or aid to infection by destroying pathogenesis - related proteins or other nonstructural molecules . it is intriguing to speculate that exoproteolytic competence of the ascomycete r. solani allowed growth on a greater variety of living and nonliving proteinaceous substrates . in reviewing the protease data it is important to recognize that only those enzymes that retain activity after sample electrophoresis are displayed by sds - substrate - page , whereas the azocasein assays report the sum of the activities of all of the proteases present in the sample . this explains the differences under the condition of the electrophoretic separation ( figure 2 ) and the effects of inhibitors on total protease activity shown in table 4 . this may have been a component allowing niche differentiation between the ascomycetes and the basidiomycetes , which will have adapted the former to pathogenicity to animals or may have derived from adaptation to pathogenicity . in any event , the fact that two families of subtilisins radiated in the early ascomycetes suggest that these fungi had a lifestyle that selected for multiple proteinase activities . the range of nutritional sources utilized by a certain fungus is regarded as a consequence of diverse molecular , cellular , and ecological factors . many of the enzymes secreted by pathogenic fungi can affect their relationships with plant and animal hosts . this suggests that differences in the properties of the enzymes provide selective advantages in different habitats . the serine proteinases are very widespread in nature and are involved in a wide variety of biological processes . enzymes belonging to this class vary significantly in substrate specificity , which can correspond to the requirements of fungal ecological niches . according to the opinion of hu and st . leger r. j. , the fragmentary distribution of trypsins among fungi indicates that their phylogenetic distribution may be greater in the early fungi than in modern ones . our data suggest that different nutritional sources can be important for the differential production of serine proteinases . the multiple subtilisins in pathogenic fungi could play different roles in pathogenesis , increase adaptability and host range , or have different functions in survival in various ecological habitats outside the host . like subtilisins , thus , there are several mechanisms available for different strains to adapt enzyme activities to their specific needs on their particular hosts . the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that tradeoffs may prevent the simultaneous upregulation of both classes of enzymes . it appears total proteinase activity increases with the degree of evolutionary advancement of the fungus . these exceptional phylogenies could reflect convergent evolution through which phylogenetically distinct enzymes evolved to share significant similarity , perhaps by targeting similar substrates . despite few morphological similarities , phylogenetic analysis have shown that there are more similarities in the exoproteinase composition between the oomycete p. infestans and the ascomycete f. culmorum although they are more distant relatively than the distance between the ascomycete and basidiomycete r. solani . our study also suggests that the in vitro behavior of these species can not be directly related to the ecological niche from which they have been isolated . this difference between the proteinases can reflect the physiological difference between their nutritional environments ( saprotroph and phytopathogen ) . however , differences in the properties of the proteinases found in the studied organisms were unlikely to be caused by variations in food substrate composition only as all our experiments involved the same culture media . it seems likely , therefore , that the proteinase compositions that we observed have a significant genetic component .

proteinases secreted by the oomycete phytophthora infestans ( mont . ) de bary , rhizoctonia solani , and fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen . the substrate specificity of the extracellular proteinases of f. culmorum , r. solani , and p. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin - like and subtilisin - like enzymes regardless of culture medium composition . the relation of trypsin - like and subtilisin - like enzymes is dependent on the culture medium composition , especially on the form of nitrogen nutrition , particularly in the case of the exoenzymes secreted by r. solani . phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives . our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis , increasing the adaptability and host range , or could have different functions in survival in various ecological habitats outside the host .

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

fungi and oomycetes are responsible for many of the most devastating plant diseases that lead to very significant losses in the agricultural sector worldwide . approximately 100,000 species of fungi and oomycetes have been described , but only a very small proportion of these are pathogenic . despite the different origin and different site on the phylogenetic tree of the true fungi and oomycetes , it has been shown that a range of secreted proteins known as effectors are important for establishing infection of the host plant . in fungi aspartate , cysteine , metallo- , serine , and threonine proteinases , as well as uncharacterized classes of proteinases , have been identified . a total of 282,061 predicted proteins were grouped into 23,724 clusters , and only 16 clusters contained proteins that were found in all 34 species of fungi but which were absent from some species of oomycetes . pathogenicity factors have been defined as genes that are essential for successful completion of the pathogenic life cycle but dispensable for saprophytic growth . proteinases that are secreted by fungi ( especially pathogens ) allow them to adapt to different living conditions . proteinases are divided into six main classes serine , cysteine , threonine , aspartic , glutamic , and metallopeptidases , according to the nature of functional groups in the active center of the enzyme molecule . according to preliminary observations , the production of trypsin - like enzymes is characteristic of plant pathogens whereas extracellular endoproteolytic activity of saprotrophs is provided mainly with subtilisin - like enzymes [ 79 ] . there seems to be a number of proteinase and peptidase domains that are more common in the genomes of phytopathogens [ 6 , 10 ] . proteinases from microorganisms display many unique characteristics in terms of catalytic mechanism , substrate specificity , activation mechanism , thermostability , and ph optimum . fungi could represent another source of proteinases with novel characteristics , as proteinases play a major role in the physiology , morphogenesis , and metabolism of fungi . in view of these functions , proteinases of several catalytic classes the purpose of this study was a comparative analysis of proteinases secreted by phytophthora infestans ( mont . ) de bary , rhizoctonia solani , and fusarium culmorum that are causal agents of several potato diseases . we thought that the data obtained from this study may help clarify the question whether their composition depends on the phylogenetic position of the pathogen . the isolates of phytophthora infestans ( mont . ) de bary , rhizoctonia solani kuhn ( ag-3 ) 153 , and fusarium culmorum ( w. g. sm . ) were supplied by potato , vegetable , and fruit scientific and practical center of the national academy of sciences of belorussia . culture medium was inoculated in erlenmeyer flasks ( 500 ml ) by introducing 15 ml of spore suspension into 150 ml of the culture medium . exoproteinases were isolated from the culture medium after 12 days of growth of the microorganism . proteolytic enzyme activity was determined by the kunitz method using 1% casein , 0.5% azocasein , and 0.5% hemoglobin as substrates . time of azocasein hydrolysis was 30 min , and for casein and hemoglobin it was 1 h. the activity of cysteine proteinases was evaluated in the presence of 25 mm l - cysteine and 1 mm edta according to the modified kunitz method . using synthetic and p - nitroanilide substrates : n-benzoyl - l - arginine p - nitroanilide ( bapna ) , n - carbobenzyloxy - l - alanyl - l - alanyl - l - leucine p - nitroanilide ( z - aalpna , bachem , switzerland ) , and n - succinyl - l - phenylalanine p - nitroanilide ( suc - fpna ) , n - succinyl - glycyl - glycyl - l - phenylalanine p - nitroanilide ( suc - ggfpna ) , l - leucine p - nitroanilide ( lpna ) , n - acetyl - l - alanyl - l - alanyl - l - alanyl p - nitroanilide ( ac - aaapna , bachem , switzerland ) . in the inhibitor analysis the following inhibitors were used : iodoacetamide ( iaa , 1 mm ) , chloromethylketone tosyl - l - lysine ( cmktl , 1 mm ) , chloromethylketone tosyl - l - phenylalanine ( cmktp , 1 mm ) , ethylenediaminetetraacetic acid disodium salt ( edta , 4.0 mm ) , dl - dithiothreitol ( dtt , 1 mm ) , phenylmethanesulfonyl fluoride ( pmsf , 1 mm ) , p - chloro - mercurybenzoate ( pcmb , 1 mm ) , and diisopropyl fluorophosphate ( difp , 0.2 mm ) . electrophoresis in 20% polyacrylamide gel in the presence of sodium dodecyl sulfate ( sds - page ) and - mercaptoethanol was performed by the method of laemmli . to obtain the zymograms sds - page electrophoresis was carried out in the presence of copolymerized substrate ( gelatin 0.1% ) by the method of heussen and dowdle . upon electrophoresis ending gels were washed with triton x-100 ( 2.5% ) under vigorous stirring , rinsed with 0.1 m glycine - naoh buffer ph 7.8 , and incubated overnight in the same buffer at room temperature . all experiments and assays were carried out at least in triplicate and the results are presented as mean values obtained with an indication of the standard deviation . the chemicals were used of the following companies : azocasein and hemoglobin ( sigma chemicals co. , usa ) , casein ( biolar , latvia ) , the synthetic substrates mentioned ( sigma , if other not indicated ) , the synthetic inhibitors mentioned ( sigma ) , and lmw calibration kit ( sigma ) . de bary , r. solani , and f. culmorum were tested for the activities of the exoproteinases . the influence of several environmental factors on the production of extracellular proteinases of these microorganisms was studied systematically in controlled batch cultures . not all of the defined media tested in the study gave production of the examined enzymes , although they all supported fairly good growth ( see figure 1 ) . so we did not observe some changes in yield of proteinases secreted into the culture medium when it was inoculated with these isolates into the semisynthetic culture medium containing kh2po4 , mgso4 , feso4 , thiamin , and riboflavin . as the studied pathogen isolates caused the most devastating diseases of potato , we added to the culture media the heat - stable potato tuber proteins . this initiated the secretion of proteinases by fungi r. solani and f. culmorum ( figures 1(b ) and 1(c ) ) . in the case of p. infestans the exoproteinase activity remained low and practically unchanged during the growth of the culture , although we observed the biomass increasing ( figure 1(d ) ) . it was shown that the addition of kno3 into the medium lead to a significant decrease in the exoproteolytic activity , indicating the suppression of secretion and possibly synthesis of the exoenzymes . as exoproteinase secretion was inhibited in the presence of nitrate , there was reason to believe that mineral nitrogen regulates adaptation of the pathogens to the environment by a mechanism that , according to the authors of , can be attributed to catabolic repression . to study the effect of organic nitrogen on the exoproteinase secretion of the pathogens , yeast extract was extra added into the culture medium . when the yeast extract was added to the culture medium a noticeable increase in the exoproteinase secretion was observed , which was accompanied by accelerated growth of mycelium ( figures 1(b ) , 1(c ) , and 1(d ) ) . it is important to point out that the oomycete was able to secret exoproteinases only in the presence of yeast extract . there were some differences in the effect of the environmental parameters on the various proteinase - related phenotypes . not all of the defined media tested in the present study gave production of the examined enzymes , although they all supported fairly good growth . total proteinase activity increased with the degree of evolutionary development of the isolates , which originated from phylogenetically distant fungi belonging to the kingdom true fungi ascomycetes ( f. culmorum ) and basidiomycetes ( r. solani ) , while p. infestans belong to the phylum oomycota ( figure 1(a ) ) . traditionally , due to their filamentous growth habit , oomycetes have been classified in the kingdom fungi . however , modern molecular and biochemical analyses suggest that oomycetes have little taxonomic affinity with filamentous fungi but are more closely related to brown algae ( heterokonts ) in the stramenopiles , one of several major eukaryotic kingdoms [ 2022 ] . in a series of initial screening experiments of the factors investigated , only medium ph and nitrogen concentration particular strongly affected the extracellular proteinase activities . it was found that the medium ph ranged from slightly acidic to neutral and reached a constant value of 7.27.4 after 12 days ( figure 1 ) during the growth of the isolates . the capacity of pathogenic fungi to support the medium ph did not depend on the composition of the culture media . we observed that the ph values of the medium affected the growth of the isolates ( figure 1 , curves 3 ) . it was shown that the secretion of proteinases depended on the cultivation temperature . when the culture was grown at 28c a decrease in the proteolytic amidase activity the temperature of 21c was optimal for the production of exoproteinases by the isolates , and apparently corresponded to the temperature regime of its habitat in the natural environment . the exoenzymes secreted by fungi were the most effective at neutral and slightly alkaline ph values . so , exoproteinases of f. culmorum were characterized by the maximum of proteolytic activity at ph 8.0 , and of r. solani at ph 8.5 . the second some increase of the enzyme activity of p. infestans was exhibited at slightly alkaline ph indicating the presence of proteinases with ph optimum of action in the region from 8 to 9 . fusarium culmorum and p. infestans predominantly produced 29- and 49-kda proteinases , and r. solani secreted a 67- and 22-kda proteinases ( figure 2 ) . most effectively hydrolyzed bapna ( a substrate for trypsin - like proteinases ) and to a lesser extent z - aalpna ( a substrate for subtilisin - like proteinases ) . at the same time , the exoproteinases did not act on the substrates for chymotrypsin- and elastase - like proteinases ( suc - ggfpna and ac - aaapna , resp . ) the enzymes secreted by f. culmorum hydrolyzed z - aalpna very efficiently and to a lesser extent bapna . they showed low activity toward substrates for chymotrypsin- and elastase - like proteinases , and for aminopeptidases as well . for the exoenzymes secreted by r. solani the profile depended on the culture medium composition ( table 3 ) . analysis of the data on the effect of synthetic substrates specific to certain groups of proteinases to the exoenzyme activity of the oomycete and the fungi indicated that p. infestans secreted predominantly serine and metalloproteinases , and the enzymes of serine type are trypsin- and subtilisin - like proteinases . in the case of f. culmorum the exoproteinase profile of p. infestans and f. culmorum were not dependent on medium composition . when r. solani was grown without yeast extract , trypsin - like serine proteinases were secreted mainly , including sh - dependent serine enzymes . the subtilisin - like proteinase activity was significantly lower ( table 3 ) . in the presence of yeast extract the composition of r. solani exoenzymes was enriched in subtilisin - like proteinases whereas the content of trypsin - like enzymes was decreased significantly ( table 3 ) . the presence of a reducing agent ( l - cysteine with edta ) did not affect the proteolytic activity of the fungal exoproteinases in the azocasein assay , which indicated the absence of cysteine exoproteinases in the growth medium . the interaction of different synthetic inhibitors with exoproteinases secreted by the three microorganisms was studied ( table 4 ) . edta , which has often been used as an indicator of metalloproteinases , had some effect only upon the total proteolytic activity of the exoenzymes of p. infestans . the proteolytic activity of p. infestans exoproteinases was reduced by almost twofold in the presence of l - cysteine with edta . this was confirmed by increase in the p. infestans exoproteinase activity by 50% in the presence of 1 mm cacl2 at ph 7.0 . analysis of chloromethylketone treatment showed that the serine proteinases were trypsin - like ( table 4 ) . so the data obtained in the experiments with synthetic inhibitors confirmed that the oomycete p. infestans secreted serine- and metalloproteinases predominantly and that serine - type enzymes were trypsin- and subtilisin - like proteinases . treatment with mercuric chloride significantly reduced the amidase proteolytic activities of the exoenzyme ( table 4 ) . that serves as once more confirmation of the presence of serine proteinases in the culture medium of f. culmorum . the interaction of exoproteinases secreted by r. solani and f. culmorum with the natural protein serine proteinase inhibitors isolated from potato tubers and legume seeds was also studied ( figure 3 ) . the activity of r. solani exoproteinases was inhibited most effectively by specific trypsin inhibitors from potato tubers and from honey locust seeds as well as soybean kunitz trypsin inhibitor ( skti ) ( figure 3(a ) , curve 1 , 3 , and 4 ) . the mentioned assumption about the trypsin - like activity of r. solani exoproteinases grown without yeast extract was confirmed . skti and soybean bowman - birk inhibitor ( sbbi ) acted on exoproteinases of f. culmorum much more weakly ( figure 3(b ) , curve 3 and 6 ) . this indicates that the subtilisin - like enzymes constitute a significant part of the f. culmorum exoproteinases . similar results were obtained in the study of protein inhibitor action on the enzymes secreted by p. infestans ( data not presented ) . the data obtained in the experiments confirmed belonging of exoproteinases of the fungi and oomycete to the chymotrypsin clan of proteolytic enzymes . it should be noted that potato subtilisin inhibitor suppressed in vitro growth and development of f. culmorum macroconidia and p. infestans zoospores . we can assume that the secreted exoproteinases of the phytopathogenic microorganisms are a factor of their pathogenicity . thus , the inhibitor analysis of the major extracellular proteinases of pathogenic fungi r. solani and f. culmorum and the oomycete p. infestans showed that they belong to the group of serine proteinases mainly . the substrate specificity of the proteinases and their sensitivity to synthetic and natural inhibitors suggested that the enzymes of f. culmorum and p. infestans are trypsin - like and subtilisin - like proteinases . the exoenzymes of r. solani depended on the culture medium composition , especially on the form of nitrogen nutrition . when r. solani grows as a saprobe the exoenzyme was represented by subtilisin - like proteinases . although r. solani has been isolated only from potato tissues , it can be thought to persist as a saprobe that lives on plant debris , as evidenced by the increase in subtilisin - like activity . the secretion of the trypsin - like proteinases in culture can be due to their participation in tissue degradation or aid to infection by destroying pathogenesis - related proteins or other nonstructural molecules . it is intriguing to speculate that exoproteolytic competence of the ascomycete r. solani allowed growth on a greater variety of living and nonliving proteinaceous substrates . this may have been a component allowing niche differentiation between the ascomycetes and the basidiomycetes , which will have adapted the former to pathogenicity to animals or may have derived from adaptation to pathogenicity . in any event , the fact that two families of subtilisins radiated in the early ascomycetes suggest that these fungi had a lifestyle that selected for multiple proteinase activities . many of the enzymes secreted by pathogenic fungi can affect their relationships with plant and animal hosts . this suggests that differences in the properties of the enzymes provide selective advantages in different habitats . enzymes belonging to this class vary significantly in substrate specificity , which can correspond to the requirements of fungal ecological niches . our data suggest that different nutritional sources can be important for the differential production of serine proteinases . the multiple subtilisins in pathogenic fungi could play different roles in pathogenesis , increase adaptability and host range , or have different functions in survival in various ecological habitats outside the host . the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that tradeoffs may prevent the simultaneous upregulation of both classes of enzymes . despite few morphological similarities , phylogenetic analysis have shown that there are more similarities in the exoproteinase composition between the oomycete p. infestans and the ascomycete f. culmorum although they are more distant relatively than the distance between the ascomycete and basidiomycete r. solani . our study also suggests that the in vitro behavior of these species can not be directly related to the ecological niche from which they have been isolated . however , differences in the properties of the proteinases found in the studied organisms were unlikely to be caused by variations in food substrate composition only as all our experiments involved the same culture media .

the online version of this article ( doi:10.1007/s10393 - 010 - 0324-z ) contains supplementary material , which is available to authorized users . each new introduction of the highly pathogenic avian influenza h5n1 virus ( hpai h5n1 hereafter ) is a novel natural experiment providing clues about conditions that promote the virus s establishment and persistence . despite the large literature on the hpai h5n1 panzootic , surprisingly few studies have so far investigated the spatial factors associated with disease presence . much of this work has focused on a limited number of countries : thailand , vietnam , china , romania , and nigeria ( gilbert et al . , 2006 , 2007 ; , 2006 ; pfeiffer et al . , 2007 ; cecchi et al . , 2008 ; ward et al . , free - grazing domestic ducks ( gilbert et al . , 2006 , 2007 ; pfeiffer et al . , 2007 ) and floodplain agriculture or wetland - related risk factors ( oyana et al . , 2006 ; gilbert et al . , 2007 ; cecchi et al . 2008 ) appear to be fundamental drivers for h5n1 persistence , and have been used to produce regional risk maps ( gilbert et al . , regional ecological niche modeling using parameters of surface reflectance and landform has been done for west africa ( williams et al . , 2008 ) , the middle east and northeastern africa ( williams and peterson , 2009 ) , and the indian subcontinent ( adhikari et al . , 2009 ) . these three papers all found that hpai h5n1 cases were occurring under predictable ecological conditions , and found that , in most cases , predicted disease presence coincided with areas that had marked seasonal variation . however , to date , there have been few attempts at analyzing global patterns of hpai h5n1 distribution , with the exception of work by kilpatrick et al . ( 2006 ) , which characterized the global risk of virus introduction based on large - distance poultry trade and migratory birds movement patterns . our aim here was to identify where the virus would stand the best chance of becoming established and promoting secondary spread , should it be introduced . the approach requires quantitatively testing for matches between hpai h5n1 persistence and the agro - ecological conditions that the virus experienced during each new invasion . these conditions may be defined in terms of poultry population composition , production structure , veterinary resources , or other factors that influence persistence and viral evolution ( peng , 1994 ; abo et al . , 1998 ) . areas with large populations of free - grazing ducks , high densities of backyard poultry , and large numbers of poultry farms ( e.g. , vietnam ) are likely to present selection regimes that differ from those in countries with heavily industrialized poultry production ( e.g. , japan ) , or from countries with negligible poultry densities ( e.g. , sudan ) . not only would such analysis test the importance of risk factors previously identified , but it may also reveal the importance of factors that are broadly homogeneous within countries but may vary across countries . for example , an indicator such as minimum temperature may not so much contrast different regions at risk within laos , but it would do so at the global scale . in addition , identifying and characterizing conditions of persistence opens a new line of study in evolutionary epidemiology , namely tracking viral evolutionary responses under different agro - ecological conditions . first , quantifying hpai h5n1 persistence across countries is difficult given major discrepancies in surveillance and reporting . the absence of hpai in countries that fail to report h5n1 outbreaks can not be reasonably inferred for those countries lacking veterinary resources and surveillance capacity . second , a global data set by definition compiles data from countries characterized by very different epidemiological conditions , because disease persistence entails different processes under different sets of circumstances . for example , china has for several years adopted mass vaccination in poultry countrywide , and it appears difficult to differentiate if reported hpai h5n1 cases reflect incomplete vaccination coverage or a field virus evading the vaccines applied . 2008 ) predict a high hpai risk for the tibetan highlands and the half - desert ecology of xinjiang province and only a moderate high - risk level for the guangdong and fujian provinces where hpai genetic diversity appears quite large ( wallace et al . , 2007 ) . this article attempts to overcome these obstacles to analyze the agro - ecological conditions associated with hpai h5n1 globally . first , the indicators that presumably best relate to different measures of hpai h5n1 persistence are inventoried and validated . second , the countries with similar agro - ecological conditions are statistically clustered into niches . finally , the agro - ecological distances between countries worldwide and the niche with the greatest human h5n1 risk are mapped . the analyses included all countries and chinese , indonesian , and indian provinces where hpai h5n1 has been registered between january 2004 and december 2008 ( n = 61 for the all - countries set , n = 124 for the all - countries and chinese , indonesian , and indian provinces set ; full list in supporting information ) . china , indonesia , and india were included at the province - level because of the countries sizes and importance to hpai h5n1 epidemiology ( vijaykrishna et al . , 2008 ; we therefore had two sets of spatial units for our analyses : one with only national level data ( n = 61 ) , and another one with 58 countries and 66 provinces from china , india , and indonesia . in order to account for heterogeneities in reporting and epidemiological situation , first , the number of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season number since the recorded introduction of hpai h5n1 ( prs , numeric , from 0 to 1 ) . second , the presence of human cases for more than 1 year over the study period ( hcp , binary ) . third , although it may represent a transient epidemic , i.e. , not true persistence , the presence of human cases ( hc , binary ) was also considered as a complementary index . this was based on the assumption that , in most instances , human cases were indicative of a relatively extensive virus circulation , i.e. , agro - ecological conditions permitting an easy transmission.table 1list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variablescodedefinitionunithcpresence of human casesbinary ( 0 or 1)hcppresence of human cases for more than 1 year over the study periodbinary ( 0 or 1)prsno . of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season no . since the recorded introduction of hpai h5n1proportion ( between 0 and 1)agpopdnagricultural population densitypeople / kmagpopdnoiagricultural population density by output / input chickensqrt(agpopdn oich)chdnchicken densityheads / kmchmtdnchicken meat densitykg / kmduchdnduck density by chicken densitysqrt(dudn chdn)dudnduck densityheads / kmoichchicken production output / inputmeat kg / headpopdntotal human population densitypeople / kmpppcpurchasing power per capitausd / persontmaxmaximum temperaturectminminimum temperaturectrngrange of temperaturecurpopdnurban population densitypeople / kmurrtproportion of urban populationproportion list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variables data on human cases of hpai h5n1 were compiled from who ( 2009 ) . the presence of hpai h5n1 in poultry and wild birds between autumn 2003 and autumn 2008 was established by season from the oie wahid ( world organization for animal health , 2009 ) and fao empres - i databases ( fao , 2009a ) . the first series of indicators described the poultry setting : chicken density ( chdn , heads / km ) : although this variable was not found significantly related to hpai h5n1 persistence in previous work ( gilbert et al . , 2008 ; ward et al . , 2008 ) , chicken production and trade appear to drive the expansion phase of h5n1 invasion ( slingenbergh and gilbert , 2008);chicken production output / input ( oich , meat kg / head ) : chicken density alone is a necessary but insufficient marker of the environment in which poultry - associated hpai may spread . the more industrialized the production , the more kg of meat is produced per poultry head in stock on an annual basis;duck density ( dudn , heads / km ) : duck density was previously shown to be a significant risk factor for hpai h5n1 ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) and is a marker of potential disease reservoirs;duck density by chicken density ( duchdn ; estimated as sqrt[dudn chdn ] ) : the product identifies areas where synergistic interactions between chicken and duck populations may take place;chicken meat density ( chmtdn , kg / km ) : the variable is an indicator of the density of broiler industry plants . chicken density ( chdn , heads / km ) : although this variable was not found significantly related to hpai h5n1 persistence in previous work ( gilbert et al . , 2008 ; ward et al . , 2008 ) , chicken production and trade appear to drive the expansion phase of h5n1 invasion ( slingenbergh and gilbert , 2008 ) ; chicken production output / input ( oich , meat kg / head ) : chicken density alone is a necessary but insufficient marker of the environment in which poultry - associated hpai may spread . the more industrialized the production , the more kg of meat is produced per poultry head in stock on an annual basis ; duck density ( dudn , heads / km ) : duck density was previously shown to be a significant risk factor for hpai h5n1 ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) and is a marker of potential disease reservoirs ; duck density by chicken density ( duchdn ; estimated as sqrt[dudn chdn ] ) : the product identifies areas where synergistic interactions between chicken and duck populations may take place ; chicken meat density ( chmtdn , kg / km ) : the variable is an indicator of the density of broiler industry plants . in addition to the above , a number of socio - demographic indicators were included : agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) , with agricultural population density indicative of the density of smallholders;urban population density ( urpopdn , people / km ) and proportion of urban population ( urrt ) : expansions in poultry production in periurban areas worldwide have been reported to play a role in hpai epidemiology ( kapan et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation;total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface;purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard;agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . coexistence of old and new forms of poultry production may enhance viral circulation by increasing the mix of host types and farming sectors across local landscapes ( wallace et al . an increase in such diversity increases the kinds of ecological refugia in which livestock influenza can avoid extirpation . agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . 2008 ) , with agricultural population density indicative of the density of smallholders ; urban population density ( urpopdn , people / km ) and proportion of urban population ( urrt ) : expansions in poultry production in periurban areas worldwide have been reported to play a role in hpai epidemiology ( kapan et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation ; total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface ; purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard ; agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . coexistence of old and new forms of poultry production may enhance viral circulation by increasing the mix of host types and farming sectors across local landscapes ( wallace et al . an increase in such diversity increases the kinds of ecological refugia in which livestock influenza can avoid extirpation . finally , we also included indicators of climate ( table 1 ) : i ) minimum , maximum , and range of temperature ( tmin , tmax , trng , c ) : temperature may be indicative of virus persistence in the environment ( brown et al . national level agricultural and human demography indicators were collected from faostat data from 2007 ( fao , 2009b ) , purchasing power per capita was collected from the international monetary fund ( 2009 ) , and temperature data were collected from the tyndall center ( mitchell et al . , 2004 ) . subnational data for china were collected from animal husbandry yearbook data for chinese provinces ( editorial department of china animal husbandry yearbook , 2005 ) , and subnational data for india and indonesia were obtained from glipha ( fao , 2009c ) , statistics indonesia of the republic of indonesia ( badan pusat statistik , 2009 ) , and office of the registrar general and census commissioner , india ( 2009 ) . countries worldwide were included in the database so that the persistence models developed could be extrapolated to areas where hpai h5n1 has not yet spread . duck data were not available for many countries . because of the importance of ducks in hpai h5n1 epidemiology ( gilbert et al . , 2006 ) , we developed a model to predict national level duck numbers for countries for which this variable was missing ( see supplementary information ) . for mapping purposes , we masked areas where poultry production is considered to be impossible , i.e. , desert regions , using a suitability mask derived from the gridded livestock of the world database ( wint and robinson , 2007 ) . exploratory data analysis consisted of testing all explanatory indicators for normality , log - transforming those variables requiring so ( chdn , dudn , duchdn , chmtdn , agpopdn , urpopdn , popdn , and agpopdnoi ) . we aimed to quantify the statistical associations between the 14 predictors and the three different measures of hpai h5n1 persistence ( hc , hcp , and prs ) . linear models ( standard multiple linear regression for prs , and multiple logistic regressions for hc and hcp ) were chosen because : i ) they allow testing the statistical relationship between a set of independent variables and a quantitative ( prs ) , or binary independent variable ( hc and hcp ) using the same modeling framework ; ii ) they allow testing the significance of the identified relationships ; iii ) their calculation is fast , which allows testing multiple alternative models easily ; and iv ) they belong to a family of parametric statistics most commonly used by other scientists , hence allowing easy replication of the results . however , one difficulty with this type of analysis is the multi - colinearity between predictors , as the level of significance of a given variable in a model may be affected by another correlated variable in such a way as to confuse epidemiological interpretation . in addition , given the limited number of spatial units in the final data set , it was difficult to simultaneously fit more than three variables in any model . hence , we first explored the model space by running all possible models one can build for combinations of three explanatory variables out of the set of 14 . hc and hcp were modeled using multiple logistic regressions , and we used two indicators to assess these models goodness of fit . the akaike s information criteria ( aic ) is a measure of the goodness of fit of an estimated statistical model , and is estimated as aic = 2 k 2.ln(l ) , where k is the number of parameters in the model , and l is the maximized value of the likelihood function for the estimated model . in addition , we also computed the area under curve ( auc ) or the receiver operating characteristic curve ( roc ) . the roc curve is a plot that present the fraction of true positives as a function of the fraction of false positives for changing values of a discrimination criterion . the auc quantifies the area under the roc curve , and represents the overall ability of the predictions to discriminate between positives and negatives . useless predictions ( no better at identifying positives than flipping a coin ) have an auc of 0.5 , while perfect predictions ( zero false - positives and zero false - negatives ) have an auc of 1.00 . the auc is increasingly used to quantify the goodness of fit of models with binary outcomes because it is relatively independent to the rate of positives ( fielding and bell , 1997 ) . concurrently , prs was modeled using standard multiple regression models , with goodness of fit estimated by aic and r. in total , we estimated the aic , auc , and r of 364 different models from a round robin of three explanatory variables . this analysis was repeated using two sets of data : all - countries ( n = 61 ) , with china , indonesia , and india each as a single unit , and all - countries plus the individual provinces of china , indonesia , and india ( n = 124 ) . second , we aimed to select a subset of explanatory variables adequate to discriminate high h5n1 persistence assumed by all three indicators ( hc , hcp , and prs ) . we computed the median auc and r of all three - variable models and ranked the variables by their capacity to generate a high median auc score in models predicting hc and hcp combined with a high median r in models predicting prs . this yielded a restricted number of variables with superior predictive capacity to model hpai h5n1 human cases and seasonal h5n1 persistence in poultry , wild birds , and/or humans . the above subset of variables was subject to partitioning around a medoids method ( kaufman and rousseeuw , 1990 ) to identify five agro - ecological clusters or niches . this method is a clustering algorithm which aims to break a multivariate data set into groups . the algorithm identifies a set of medoids , that can be defined as the point in cluster , whose average dissimilarity to all other points in the cluster is minimal . after finding the set of medoids , each point of the data set the method is comparable to the k - mean clustering algorithm but is considered to be more robust to outlying values . the euclidian distances to the medoid of the niche with the greatest number of human cases was estimated in the space defined by the subset of variables and mapped as an index of dissimilarity . the analyses included all countries and chinese , indonesian , and indian provinces where hpai h5n1 has been registered between january 2004 and december 2008 ( n = 61 for the all - countries set , n = 124 for the all - countries and chinese , indonesian , and indian provinces set ; full list in supporting information ) . china , indonesia , and india were included at the province - level because of the countries sizes and importance to hpai h5n1 epidemiology ( vijaykrishna et al . , 2008 ; we therefore had two sets of spatial units for our analyses : one with only national level data ( n = 61 ) , and another one with 58 countries and 66 provinces from china , india , and indonesia . in order to account for heterogeneities in reporting and epidemiological situation , first , the number of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season number since the recorded introduction of hpai h5n1 ( prs , numeric , from 0 to 1 ) . second , the presence of human cases for more than 1 year over the study period ( hcp , binary ) . third , although it may represent a transient epidemic , i.e. , not true persistence , the presence of human cases ( hc , binary ) was also considered as a complementary index . this was based on the assumption that , in most instances , human cases were indicative of a relatively extensive virus circulation , i.e. , agro - ecological conditions permitting an easy transmission.table 1list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variablescodedefinitionunithcpresence of human casesbinary ( 0 or 1)hcppresence of human cases for more than 1 year over the study periodbinary ( 0 or 1)prsno . of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season no . since the recorded introduction of hpai h5n1proportion ( between 0 and 1)agpopdnagricultural population densitypeople / kmagpopdnoiagricultural population density by output / input chickensqrt(agpopdn oich)chdnchicken densityheads / kmchmtdnchicken meat densitykg / kmduchdnduck density by chicken densitysqrt(dudn chdn)dudnduck densityheads / kmoichchicken production output / inputmeat kg / headpopdntotal human population densitypeople / kmpppcpurchasing power per capitausd / persontmaxmaximum temperaturectminminimum temperaturectrngrange of temperaturecurpopdnurban population densitypeople / kmurrtproportion of urban populationproportion list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variables data on human cases of hpai h5n1 were compiled from who ( 2009 ) . the presence of hpai h5n1 in poultry and wild birds between autumn 2003 and autumn 2008 was established by season from the oie wahid ( world organization for animal health , 2009 ) and fao empres - i databases ( fao , 2009a ) . the first series of indicators described the poultry setting : chicken density ( chdn , heads / km ) : although this variable was not found significantly related to hpai h5n1 persistence in previous work ( gilbert et al . , 2008 ; ward et al . , 2008 ) , chicken production and trade appear to drive the expansion phase of h5n1 invasion ( slingenbergh and gilbert , 2008);chicken production output / input ( oich , meat kg / head ) : chicken density alone is a necessary but insufficient marker of the environment in which poultry - associated hpai may spread . the more industrialized the production , the more kg of meat is produced per poultry head in stock on an annual basis;duck density ( dudn , heads / km ) : duck density was previously shown to be a significant risk factor for hpai h5n1 ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) and is a marker of potential disease reservoirs;duck density by chicken density ( duchdn ; estimated as sqrt[dudn chdn ] ) : the product identifies areas where synergistic interactions between chicken and duck populations may take place;chicken meat density ( chmtdn , kg / km ) : the variable is an indicator of the density of broiler industry plants . chicken density ( chdn , heads / km ) : although this variable was not found significantly related to hpai h5n1 persistence in previous work ( gilbert et al . , 2008 ; ward et al . , 2008 ) , chicken production and trade appear to drive the expansion phase of h5n1 invasion ( slingenbergh and gilbert , 2008 ) ; chicken production output / input ( oich , meat kg / head ) : chicken density alone is a necessary but insufficient marker of the environment in which poultry - associated hpai may spread . the more industrialized the production , the more kg of meat is produced per poultry head in stock on an annual basis ; duck density ( dudn , heads / km ) : duck density was previously shown to be a significant risk factor for hpai h5n1 ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) and is a marker of potential disease reservoirs ; duck density by chicken density ( duchdn ; estimated as sqrt[dudn chdn ] ) : the product identifies areas where synergistic interactions between chicken and duck populations may take place ; chicken meat density ( chmtdn , kg / km ) : the variable is an indicator of the density of broiler industry plants . in addition to the above , a number of socio - demographic indicators were included : agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . , 2007 ; gilbert et al . , 2008 ) , with agricultural population density indicative of the density of smallholders;urban population density ( urpopdn , people / km ) and proportion of urban population ( urrt ) : expansions in poultry production in periurban areas worldwide have been reported to play a role in hpai epidemiology ( kapan et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation;total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface;purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard;agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . coexistence of old and new forms of poultry production may enhance viral circulation by increasing the mix of host types and farming sectors across local landscapes ( wallace et al . an increase in such diversity increases the kinds of ecological refugia in which livestock influenza can avoid extirpation . agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . 2008 ) , with agricultural population density indicative of the density of smallholders ; urban population density ( urpopdn , people / km ) and proportion of urban population ( urrt ) : expansions in poultry production in periurban areas worldwide have been reported to play a role in hpai epidemiology ( kapan et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation ; total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface ; purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard ; agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . coexistence of old and new forms of poultry production may enhance viral circulation by increasing the mix of host types and farming sectors across local landscapes ( wallace et al . an increase in such diversity increases the kinds of ecological refugia in which livestock influenza can avoid extirpation . finally , we also included indicators of climate ( table 1 ) : i ) minimum , maximum , and range of temperature ( tmin , tmax , trng , c ) : temperature may be indicative of virus persistence in the environment ( brown et al . national level agricultural and human demography indicators were collected from faostat data from 2007 ( fao , 2009b ) , purchasing power per capita was collected from the international monetary fund ( 2009 ) , and temperature data were collected from the tyndall center ( mitchell et al . , 2004 ) . subnational data for china were collected from animal husbandry yearbook data for chinese provinces ( editorial department of china animal husbandry yearbook , 2005 ) , and subnational data for india and indonesia were obtained from glipha ( fao , 2009c ) , statistics indonesia of the republic of indonesia ( badan pusat statistik , 2009 ) , and office of the registrar general and census commissioner , india ( 2009 ) . countries worldwide were included in the database so that the persistence models developed could be extrapolated to areas where hpai h5n1 has not yet spread . duck data were not available for many countries . because of the importance of ducks in hpai h5n1 epidemiology ( gilbert et al . , 2006 ) , we developed a model to predict national level duck numbers for countries for which this variable was missing ( see supplementary information ) . for mapping purposes , we masked areas where poultry production is considered to be impossible , i.e. , desert regions , using a suitability mask derived from the gridded livestock of the world database ( wint and robinson , 2007 ) . exploratory data analysis consisted of testing all explanatory indicators for normality , log - transforming those variables requiring so ( chdn , dudn , duchdn , chmtdn , agpopdn , urpopdn , popdn , and agpopdnoi ) . we aimed to quantify the statistical associations between the 14 predictors and the three different measures of hpai h5n1 persistence ( hc , hcp , and prs ) . linear models ( standard multiple linear regression for prs , and multiple logistic regressions for hc and hcp ) were chosen because : i ) they allow testing the statistical relationship between a set of independent variables and a quantitative ( prs ) , or binary independent variable ( hc and hcp ) using the same modeling framework ; ii ) they allow testing the significance of the identified relationships ; iii ) their calculation is fast , which allows testing multiple alternative models easily ; and iv ) they belong to a family of parametric statistics most commonly used by other scientists , hence allowing easy replication of the results . however , one difficulty with this type of analysis is the multi - colinearity between predictors , as the level of significance of a given variable in a model may be affected by another correlated variable in such a way as to confuse epidemiological interpretation . in addition , given the limited number of spatial units in the final data set , it was difficult to simultaneously fit more than three variables in any model . hence , we first explored the model space by running all possible models one can build for combinations of three explanatory variables out of the set of 14 . hc and hcp were modeled using multiple logistic regressions , and we used two indicators to assess these models goodness of fit . the akaike s information criteria ( aic ) is a measure of the goodness of fit of an estimated statistical model , and is estimated as aic = 2 k 2.ln(l ) , where k is the number of parameters in the model , and l is the maximized value of the likelihood function for the estimated model . in addition , we also computed the area under curve ( auc ) or the receiver operating characteristic curve ( roc ) . the roc curve is a plot that present the fraction of true positives as a function of the fraction of false positives for changing values of a discrimination criterion . the auc quantifies the area under the roc curve , and represents the overall ability of the predictions to discriminate between positives and negatives . useless predictions ( no better at identifying positives than flipping a coin ) have an auc of 0.5 , while perfect predictions ( zero false - positives and zero false - negatives ) have an auc of 1.00 . the auc is increasingly used to quantify the goodness of fit of models with binary outcomes because it is relatively independent to the rate of positives ( fielding and bell , 1997 ) . concurrently , prs was modeled using standard multiple regression models , with goodness of fit estimated by aic and r. in total , we estimated the aic , auc , and r of 364 different models from a round robin of three explanatory variables . this analysis was repeated using two sets of data : all - countries ( n = 61 ) , with china , indonesia , and india each as a single unit , and all - countries plus the individual provinces of china , indonesia , and india ( n = 124 ) . second , we aimed to select a subset of explanatory variables adequate to discriminate high h5n1 persistence assumed by all three indicators ( hc , hcp , and prs ) . we computed the median auc and r of all three - variable models and ranked the variables by their capacity to generate a high median auc score in models predicting hc and hcp combined with a high median r in models predicting prs . this yielded a restricted number of variables with superior predictive capacity to model hpai h5n1 human cases and seasonal h5n1 persistence in poultry , wild birds , and/or humans . the above subset of variables was subject to partitioning around a medoids method ( kaufman and rousseeuw , 1990 ) to identify five agro - ecological clusters or niches . this method is a clustering algorithm which aims to break a multivariate data set into groups . the algorithm identifies a set of medoids , that can be defined as the point in cluster , whose average dissimilarity to all other points in the cluster is minimal . after finding the set of medoids , each point of the data set the method is comparable to the k - mean clustering algorithm but is considered to be more robust to outlying values . the euclidian distances to the medoid of the niche with the greatest number of human cases was estimated in the space defined by the subset of variables and mapped as an index of dissimilarity . the outcome of the 364 models differed depending on the persistence variable modeled and the set of areas incorporated ( fig . 1 ) . a broad coherence was obtained when plotting the 364 data pairs representing the akaike s information criteria ( aic ) for the hc and prs models , and using the all - countries set ( fig . 1 , top left ) . the coherence disappeared when the province level data from china , indonesia , and india were included ( fig . 1 , top right ) . in contrast , aic values for hc and hcp models yielded a coherent cloud of values for the two sets of territories ( fig . 1 , bottom left and right ) . on the basis of these results , we used the set with all - countries in the follow - up analyses to quantify prs versus hc models , and the set of all - countries plus china , indonesia , and india provinces to quantify hcp versus hc models.figure 1akaike s information criteria of hpai h5n1 model groups . standard regression model aic of hpai h5n1 seasonal persistence ( prs ) as function of logistic regression model aic of hpai h5n1 human case presence ( hc ) for all countries ( top left ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( top right ) . logistic regression model aic of hpai h5n1 human cases persistence ( hcp ) as function of logistic regression model aic of hpai h5n1 human case presence ( hc ) for all countries ( bottom left ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( bottom right ) . standard regression model aic of hpai h5n1 seasonal persistence ( prs ) as function of logistic regression model aic of hpai h5n1 human case presence ( hc ) for all countries ( top left ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( top right ) . logistic regression model aic of hpai h5n1 human cases persistence ( hcp ) as function of logistic regression model aic of hpai h5n1 human case presence ( hc ) for all countries ( bottom left ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( bottom right ) . some of the explanatory variables provided consistently better results than others , but these variables were not the same across persistence measures ( fig . 2 and variable full names therein ) . using the set of all - countries , variables with the greatest median r for prs models were agpopdn , agpopdnoi , topopdn , while variables with the greatest median auc for hc models were pppc , agpopdn , agpopdnoi , and tmax . for all countries plus china , indonesia , and india provinces , the variables with the greatest median auc for hcp models comprised duchdn , chdn , tmin , pppc , and variables with greatest median auc for hc models were duchdn , pppc , agpopdn , dudn , and chdn . based on these results , we selected a subset of six variables that provided best models along the two sets of spatial units and measures of persistence , i.e. , located in the upper right quadrant of each plot : agpopdn ( agricultural population density ; log - scale ) , agpopdnoi ( product of agricultural population density and output / input chicken , log - scale ) , duchdn ( duck by chicken density , log - scale ) , pppc ( purchasing power per capita ) , chdn ( chicken density , log - scale ) , and dudn ( duck density , log - scale ) . with this subset , we identified five agro - ecological niches by partitioning around medoids ( see cluster plot , fig . 3 ) . niches 4 and 1 included the greatest number of human cases per million people ( fig . 4 , top left ) . in absolute numbers , niches 15 hosted 169 , 0 , 46 , 164 , and 0 human cases , and included 14 , 0 , 10 , 12 , and 0 spatial units with human cases , respectively . the medoid of the niche with the greatest number of human cases ( niche 4 ) was the southern chinese province of henan.figure 2goodness of fit indicators of hpai h5n1 model groups for all countries ( top left and right ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( bottom left and right ) . all - countries models ( top left and right ) are represented by the r of the seasonal persistence standard regression model ( prs ) as a function of the area under curve ( auc ) of the human case presence logistic regression model ( hc ) . all - countries and province models ( bottom left and right ) are presented by the auc of the human case persistence logistic regression model ( hcp ) as a function of the auc of the human case presence logistic regression model ( hc ) . left plots show all individual models ( gray dots ) and the median r or auc for each collection of models containing a variable ( black dots ) . the right plots show only r or auc median values for collection of models containing each variable ( the dotted lines are the median r or auc estimated over all models ) . variable abbreviations are as follows : agpopdn agricultural population density , agpopdnoi agricultural population density by output / input chicken , chdn chicken density , chmtdn chicken meat density , dudn duck density , duchdn duck by chicken density , oich output / input chicken , pppc purchasing power per capita , topopdn total population density , tmin , tmax , trng temperature min , max , and range , urpopdn urban population density , urrt urban to total population ratio.figure 3cluster plot of the partitioning around medoids presented in the two principal component space ( top ) , and map of the five agro - ecological clusters or niche in countries and chinese , indonesian , and indian provinces ( bottom ) where h5n1 was reported ( areas unsuitable to poultry production are masked in white).figure 4characterization of each niche by the number of h5n1 human cases per million people ( top left ) , the product of duck by chicken density ( duchdn , log scale [ top right ] ) , agricultural population density ( agpopdn , log - scale [ bottom left ] ) , and purchasing power per capita ( pppc [ bottom right ] ) . goodness of fit indicators of hpai h5n1 model groups for all countries ( top left and right ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( bottom left and right ) . all - countries models ( top left and right ) are represented by the r of the seasonal persistence standard regression model ( prs ) as a function of the area under curve ( auc ) of the human case presence logistic regression model ( hc ) . all - countries and province models ( bottom left and right ) are presented by the auc of the human case persistence logistic regression model ( hcp ) as a function of the auc of the human case presence logistic regression model ( hc ) . left plots show all individual models ( gray dots ) and the median r or auc for each collection of models containing a variable ( black dots ) . the right plots show only r or auc median values for collection of models containing each variable ( the dotted lines are the median r or auc estimated over all models ) . variable abbreviations are as follows : agpopdn agricultural population density , agpopdnoi agricultural population density by output / input chicken , chdn chicken density , chmtdn chicken meat density , dudn duck density , duchdn duck by chicken density , oich output / input chicken , pppc purchasing power per capita , topopdn total population density , tmin , tmax , trng temperature min , max , and range , urpopdn urban population density , urrt urban to total population ratio . cluster plot of the partitioning around medoids presented in the two principal component space ( top ) , and map of the five agro - ecological clusters or niche in countries and chinese , indonesian , and indian provinces ( bottom ) where h5n1 was reported ( areas unsuitable to poultry production are masked in white ) . characterization of each niche by the number of h5n1 human cases per million people ( top left ) , the product of duck by chicken density ( duchdn , log scale [ top right ] ) , agricultural population density ( agpopdn , log - scale [ bottom left ] ) , and purchasing power per capita ( pppc [ bottom right ] ) . the five niches differed in the patterns of main variables defining their structure ( figs . 3 and 4 ) . niche 5 was comprised largely of western european countries , japan , and south korea , and characterized by the highest purchasing power per capita and chicken production productivity , the lowest density of agricultural population , and an intermediate density of domestic ducks and chickens . niche 2 , with african countries , russia , western india provinces , and a low number of chinese and indonesia provinces , included areas with the lowest duck and chicken densities , a low purchasing power per capita and chicken production productivity , and an intermediate density of agricultural population . niche 3 , with laos , cambodia , nigeria , turkey , ukraine , and romania , was defined by slightly higher purchasing power per capita , and intermediate densities of ducks , chickens , and agricultural population . niche 4 ( egypt , the provinces of java , and several chinese provinces including guangdong and henan ) had the greatest density of domestic ducks , chickens , and agricultural population density , and at the same time an intermediate productivity of chicken production and purchasing power , higher than niches 1 , 2 , and 3 . finally , niche 1 supported a high density of agricultural population , ducks , and chickens , but lower than in niche 4 , and a low - to - intermediate chicken productivity and purchasing power per capita . niche 1 included countries from asia , such as thailand , vietnam , and several provinces of china and eastern india . we mapped the environmental distance to henan in the space defined by our set of six agro - ecological variables for all countries worldwide with the relevant data available ( fig . the map highlights that some countries geographically distant from henan share agro - ecological conditions : several china provinces , java province , bangladesh and some eastern india provinces , and egypt in africa . several spatial units with agro - ecologies somewhat close to that of henan included montenegro , poland , and hungary in europe ; nigeria and tunisia in africa ; haiti , guatemala . and el salvador in the americas ; and yemen and pakistan in western and southern asia . one should stress that the environmental distance to henan is thought to be an indicator of persistence upon introduction , i.e. , not the risk of introduction itself.figure 5distance to henan ( china ) in the space defined by the variables agricultural population density ( log - scale ) , duck density ( log - scale ) , duck by chicken density ( log - scale ) , chicken density ( log - scale ) , the product of agricultural population density and chicken output / input ratio ( log - scale ) , and purchasing power per capita ( areas unsuitable to poultry production are masked in white ) . distance to henan ( china ) in the space defined by the variables agricultural population density ( log - scale ) , duck density ( log - scale ) , duck by chicken density ( log - scale ) , chicken density ( log - scale ) , the product of agricultural population density and chicken output / input ratio ( log - scale ) , and purchasing power per capita ( areas unsuitable to poultry production are masked in white ) . the epidemiology of hpai h5n1 is characterized by a fundamental contradiction . on the one hand , hpai in poultry is highly infectious , spans a short generation time , tends toward boom - and - bust population dynamics ( villarreal et al . , 2000 ) , and would be expected to burn faster through the supply of susceptible hosts than low pathogenic variants ( dieckmann et al . , 2002 ; the hpai h5n1 epizootic , now in its 12th year , remains the longest recorded highly pathogenic influenza outbreak in poultry . first , the virus rotates across multiple host types ( olsen et al . , 2006 ; barrett et al . , 2008 ; wallace and fitch , 2008 ) , providing alternate hosts when one host species becomes too small to maintain transmission . second , the virus experiences shifts in life history across host types , including a reduced virulence ( and increased persistence ) in duck reservoirs ( keawcharoen et al . , 2008 ) . third , virulence can be an evolutionary stable strategy in situations where an ample supply of susceptibles is available , especially if such populations are distributed over wide geographical areas , as is the case with hpai h5n1 . fourth , persistence in the physical environment may contribute to sapronotic transmission ( brown et al . the results presented here suggest that some agro - ecologies are more capable of supporting hpai h5n1 persistence than others , perhaps by better integrating the aforementioned mechanisms . the multivariate analysis discriminated localities with human cases and high persistence , and identified five agro - ecological niches , which , although derived independently of hpai h5n1 data , vary by hpai h5n1 persistence ( fig . 3 ) . the models developed here , using human cases as the indicator of persistence , provided fairly good predictive power ( with auc values of 0.88 and 0.89 ) and highlight a series of variables that , although correlated among themselves , differentiated hpai s epidemiological environments . two of these variables were previously identified as risk factors in country - level studies ( gilbert et al . , 2008 ) : the density of ducks , known to play a critical role in hpai h5n1 epidemiology and evolution ( hulse - post et al . 2005 ; gilbert et al . , 2006 ) , and the density of agricultural population . the density of chickens influences disease flare up ( slingenbergh and gilbert , 2008 ) . the product of chicken by duck density appears most important in areas where both hosts act synergistically . importantly , the current study also revealed an as yet little - addressed third dimension : purchasing power per capita and associated poultry production intensification ( combined with agricultural population density ) . a number of underlying processes may contribute to this result . output / input chicken is a measure of poultry productivity and corresponding investment in biosecurity , shielding off invasive pathogens . similarly , higher purchasing power per capita generally implies a restricted interface between people and their poultry , with reduced exposure to any sick or dead birds . biosecurity , hygiene standards , and vaccination are common practice in modern poultry food chains , with a progressively diminishing reliance on live bird marketing . however , it is important to note that the niches with the greatest number of human cases were not those with the lowest levels of poultry productivity . niches 1 and 4 , with the greater number of human cases , were characterized by the largest duck and agricultural population densities , but not by the lowest output / input level , nor by the lowest purchasing power per capita . in particular , niche 4 , with the highest number of human cases per capita , mainly included countries or provinces with intermediate levels of productivity and low - to - medium purchasing power per capita ( e.g. , egypt , guangdong and anhui provinces ) . these regions , in the midst of an economic transition , with intermediate levels of poultry production and purchasing power , and a geographic mosaic of old and new modes of production , may offer the virus the array of micro - niches needed to spread and evolutionarily radiate . rice cultivation marked the transition between mesolithic foragers and the surplus food - producing economies of the neolithic ( zong et al . ducks were domesticated at least 3000 to 5000 years ago ( simoons , 1991 ; cherry and morris , 2008 ) and , by the middle of the ming dynasty , were deployed for pest control in the rice paddies of the pearl river delta ( peng , 1994 ) . the intensification of chicken and duck production , which appears to have driven the evolution of virulent influenza strains worldwide ( li et al . , 2004 ; vijaykrishna et al . , 2008 ) , was instituted in asia mostly during the economic liberalization of the past 30 years ( wallace , 2009 ) . the h5n1 duck poultry niche in asia has resulted from a series of agro - ecological changes , with changes in agricultural practices , ancient ( rice ) , modern ( ducks ) , and present - day ( poultry intensification ) , melding in a way that supports the present evolution of multiple virulent influenzas . at the global level , the h5n1 niches defined here are clearly structured by geography , with the most vulnerable niches arrayed across south and east asia , including along the chinese lowlands and coastline into the river basins of indochina and , further south , indonesia . at the same time , other important regions are scattered across h5n1 s range . for instance , egypt and wetland areas in northern nigeria support agro - ecologies similar to those that allow h5n1 to persist so well in china . the niches geographic distribution , then , may offer another mechanism by which hpai h5n1 persists . in acting as something of a western persistence hub , egypt may assist seeding outbreaks into sub - saharan africa . as is often the case , first , understanding the distribution of hpai h5n1 in relation to agro - ecological conditions depends heavily on the epidemiological records and conditions underlying the analyses . one way to circumvent the heterogeneity in disease report and epidemiological conditions was here to separately test multiple indicators of virus circulation , analyze these in relation to agro - ecological conditions , and assess whether they converge on a risk prediction . we found broadly coherent results at the global scale and country level , a coherence that disappeared when sub - national data on china , indonesia , and india were introduced ( fig . the disparity arises to a large extent because the distribution of human cases in china s provinces does not match that of outbreaks reported in domestic poultry . at least two possibilities may explain this pattern : human cases may truly be geographically disconnected from hpai h5n1 circulation in poultry , or they are geographically correlated but hpai h5n1 circulation remains undetected . when we consider the models based on human cases and human case persistence , we find a comparable scatter of models when comparing both ( i ) countries and ( ii ) all countries plus chinese , indonesian , and indian provinces ( fig . 1 bottom ) . this indicates that the risk factors influencing the distribution of human cases outside china are broadly similar to those found within china . given that the human cases outside china correlate also with the highest levels of seasonal persistence ( e.g. , indonesia , egypt , vietnam ) , this suggests that the same risk factors should underpin distribution of seasonal persistence in china . hence , there is a real possibility that hpai h5n1 virus circulation in china s poultry remains , in part , undetected . in concordance with this possibility , the virus is detected at high prevalence for poultry sampled in live bird markets across southern chinese provinces ( smith et al . , 2006 ) . second , in contrast to the models using human cases as a marker for persistent human infections , the models of seasonal persistence ( all - countries set ) offered a low level of predictability . the disparity is likely to stem from the difficulty to disentangle new introductions from local persistence and from the very high variability in disease surveillance in poultry and wild birds . for instance , germany , a high - income country with a high chicken productivity , and comparatively abundant resources devoted to disease surveillance and biosecurity , repeatedly reported hpai h5n1 cases several consecutive years , making it a country of intermediate persistence , comparable in our data set to myanmar , where disease surveillance and veterinary resources are sparse . the epidemiological situations of the two countries , however , are completely different . germany was exposed to repeated introductions , most likely by wild birds on winter escape ( starick et al . clinically silent infections did apparently occur in industrial duck - fattening holdings in germany ( harder et al . , 2009 ) , and small pockets of persistence in resident wild birds can not be fully ruled out ( globig et al . , 2009 ) . however , it can be realistically assumed that continuous viral circulation could hardly have gone undetected in germany , while the lack of veterinary infrastructure in myanmar is such that the true level of virus circulation is difficult to assess . first , it is important to avoid mistaking the agro - ecological niches identified here as deterministic in their effects on hpai h5n1 . the virus evolved during the study period , undergoing shifts in its transmission mode , virulence level , and host range . one may assume these changes are the means by which the virus adapts to the opportunities offered within each niche . a key test of the assumptions underlying niche modeling , and by extension a step toward modeling the pathway or trajectory of hpai emergence , would be to establish if these evolutionary changes exhibit repeated patterns . do localities with similar agro - ecological conditions select for hpai h5n1 variants with similar phenotypes ? the identification of different agro - ecological niches for hpai h5n1 persistence is a first and necessary step toward identifying what could be niche - specific evolutionary signatures . there are likely spatial signatures as well . in a situation where the training set consists of data pooled from heterogeneous sources representing different levels of detection capacity , a prediction of risk less dependent on the spatial distribution of the positive cases is helpful as it may highlight regions where agro - ecological conditions are more suitable ; that is , where the disease has not spread but may have the potential to persist , or regions to where it may have spread but where the introduction events may have gone undetected . that is why distance - based metrics from a region of assumed risk were estimated here , and were preferred to the probabilities for hpai presence arising from the more classically defined predictive models . first , with more phylogenetic data becoming available , it should be possible to separate true persistence in a country from re - introduction , e.g. , chaichoune et al . ( 2009 ) , which would enhance our capacity to identify the areas most susceptible to persistence . second , we should more finely estimate risk indices at sub - national levels , as country - level data often hide considerable within - country variation . for example , duck production in nigeria is concentrated in the central - northern part of the country , one of two sub - national sources fusaro et al . ( 2010 ) identify , where outbreaks persisted more so than elsewhere in the country . with sub - national data , one may find nigerian regions of considerable agro - ecological distance from niche 1 or 4 , while the central - northern region may be closer than suggested by the national level data ( fig . it is anticipated that many possible hotspots may be better identified with sub - national data , in particular in large countries that include considerable variation in agro - ecological conditions . all of java s comparatively small provinces are estimated here to be members of the niche of greatest risk for persistence . . expanding temporal coverage may help identify trajectories over which some countries , undergoing economic transitions , develop greater risk for hpai persistence . integrating such data sets with hpai genetic sequences will help to more fully describe the agro - ecological space in which influenza evolves . below is the link to the electronic supplementary material . supplementary material 1 ( doc 863 kb )

the highly pathogenic avian influenza ( hpai ) h5n1 virus has spread across eurasia and into africa . its persistence in a number of countries continues to disrupt poultry production , impairs smallholder livelihoods , and raises the risk a genotype adapted to human - to - human transmission may emerge . while previous studies identified domestic duck reservoirs as a primary risk factor associated with hpai h5n1 persistence in poultry in southeast asia , little is known of such factors in countries with different agro - ecological conditions , and no study has investigated the impact of such conditions on hpai h5n1 epidemiology at the global scale . this study explores the patterns of hpai h5n1 persistence worldwide , and for china , indonesia , and india includes individual provinces that have reported hpai h5n1 presence during the 20042008 period . multivariate analysis of a set of 14 agricultural , environmental , climatic , and socio - economic factors demonstrates in quantitative terms that a combination of six variables discriminates the areas with human cases and persistence : agricultural population density , duck density , duck by chicken density , chicken density , the product of agricultural population density and chicken output / input ratio , and purchasing power per capita . the analysis identifies five agro - ecological clusters , or niches , representing varying degrees of disease persistence . the agro - ecological distances of all study areas to the medoid of the niche with the greatest number of human cases are used to map hpai h5n1 risk globally . the results indicate that few countries remain where hpai h5n1 would likely persist should it be introduced.electronic supplementary materialthe online version of this article ( doi:10.1007/s10393 - 010 - 0324-z ) contains supplementary material , which is available to authorized users .

Electronic supplementary material I None Data Analysis R D Electronic supplementary material

the online version of this article ( doi:10.1007/s10393 - 010 - 0324-z ) contains supplementary material , which is available to authorized users . each new introduction of the highly pathogenic avian influenza h5n1 virus ( hpai h5n1 hereafter ) is a novel natural experiment providing clues about conditions that promote the virus s establishment and persistence . the approach requires quantitatively testing for matches between hpai h5n1 persistence and the agro - ecological conditions that the virus experienced during each new invasion . this article attempts to overcome these obstacles to analyze the agro - ecological conditions associated with hpai h5n1 globally . finally , the agro - ecological distances between countries worldwide and the niche with the greatest human h5n1 risk are mapped . china , indonesia , and india were included at the province - level because of the countries sizes and importance to hpai h5n1 epidemiology ( vijaykrishna et al . in order to account for heterogeneities in reporting and epidemiological situation , first , the number of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season number since the recorded introduction of hpai h5n1 ( prs , numeric , from 0 to 1 ) . , agro - ecological conditions permitting an easy transmission.table 1list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variablescodedefinitionunithcpresence of human casesbinary ( 0 or 1)hcppresence of human cases for more than 1 year over the study periodbinary ( 0 or 1)prsno . of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season no . since the recorded introduction of hpai h5n1proportion ( between 0 and 1)agpopdnagricultural population densitypeople / kmagpopdnoiagricultural population density by output / input chickensqrt(agpopdn oich)chdnchicken densityheads / kmchmtdnchicken meat densitykg / kmduchdnduck density by chicken densitysqrt(dudn chdn)dudnduck densityheads / kmoichchicken production output / inputmeat kg / headpopdntotal human population densitypeople / kmpppcpurchasing power per capitausd / persontmaxmaximum temperaturectminminimum temperaturectrngrange of temperaturecurpopdnurban population densitypeople / kmurrtproportion of urban populationproportion list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variables data on human cases of hpai h5n1 were compiled from who ( 2009 ) . , 2008 ) and is a marker of potential disease reservoirs ; duck density by chicken density ( duchdn ; estimated as sqrt[dudn chdn ] ) : the product identifies areas where synergistic interactions between chicken and duck populations may take place ; chicken meat density ( chmtdn , kg / km ) : the variable is an indicator of the density of broiler industry plants . in addition to the above , a number of socio - demographic indicators were included : agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation;total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface;purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard;agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation ; total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface ; purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard ; agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . linear models ( standard multiple linear regression for prs , and multiple logistic regressions for hc and hcp ) were chosen because : i ) they allow testing the statistical relationship between a set of independent variables and a quantitative ( prs ) , or binary independent variable ( hc and hcp ) using the same modeling framework ; ii ) they allow testing the significance of the identified relationships ; iii ) their calculation is fast , which allows testing multiple alternative models easily ; and iv ) they belong to a family of parametric statistics most commonly used by other scientists , hence allowing easy replication of the results . this analysis was repeated using two sets of data : all - countries ( n = 61 ) , with china , indonesia , and india each as a single unit , and all - countries plus the individual provinces of china , indonesia , and india ( n = 124 ) . this yielded a restricted number of variables with superior predictive capacity to model hpai h5n1 human cases and seasonal h5n1 persistence in poultry , wild birds , and/or humans . the euclidian distances to the medoid of the niche with the greatest number of human cases was estimated in the space defined by the subset of variables and mapped as an index of dissimilarity . china , indonesia , and india were included at the province - level because of the countries sizes and importance to hpai h5n1 epidemiology ( vijaykrishna et al . in order to account for heterogeneities in reporting and epidemiological situation , first , the number of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season number since the recorded introduction of hpai h5n1 ( prs , numeric , from 0 to 1 ) . , agro - ecological conditions permitting an easy transmission.table 1list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variablescodedefinitionunithcpresence of human casesbinary ( 0 or 1)hcppresence of human cases for more than 1 year over the study periodbinary ( 0 or 1)prsno . of seasons ( winter , spring , summer , and autumn ) with hpai h5n1 reports either in poultry , wild birds , or mammals , including humans , expressed as a proportion of total season no . since the recorded introduction of hpai h5n1proportion ( between 0 and 1)agpopdnagricultural population densitypeople / kmagpopdnoiagricultural population density by output / input chickensqrt(agpopdn oich)chdnchicken densityheads / kmchmtdnchicken meat densitykg / kmduchdnduck density by chicken densitysqrt(dudn chdn)dudnduck densityheads / kmoichchicken production output / inputmeat kg / headpopdntotal human population densitypeople / kmpppcpurchasing power per capitausd / persontmaxmaximum temperaturectminminimum temperaturectrngrange of temperaturecurpopdnurban population densitypeople / kmurrtproportion of urban populationproportion list of abbreviations for three indices of hpai h5n1 persistence and 14 explanatory variables data on human cases of hpai h5n1 were compiled from who ( 2009 ) . in addition to the above , a number of socio - demographic indicators were included : agricultural population density ( agpopdn , people / km ) : human population density was previously found to be a significant risk factor ( pfeiffer et al . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation;total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface;purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard;agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . urban centers with live bird markets attract viruses from different poultry settings , acting as both sink and source for viral infestation ; total human population density ( popdn , people / km ) : total density was included to make allowance for the possibility that total human density alone may inform us about the relative importance of the avian human interface ; purchasing power per capita ( pppc , usd / person ) : purchasing power is a general indicator of poverty / wealth , and thereby the degree to which poultry production practices and hygiene measures are up to standard ; agricultural population density by output / input chicken ( agpopdnoi ; estimated as sqrt[agpopdn oich ] ) : this product provides a measure for the extent to which poultry industrialization has replaced smallholder production . linear models ( standard multiple linear regression for prs , and multiple logistic regressions for hc and hcp ) were chosen because : i ) they allow testing the statistical relationship between a set of independent variables and a quantitative ( prs ) , or binary independent variable ( hc and hcp ) using the same modeling framework ; ii ) they allow testing the significance of the identified relationships ; iii ) their calculation is fast , which allows testing multiple alternative models easily ; and iv ) they belong to a family of parametric statistics most commonly used by other scientists , hence allowing easy replication of the results . this analysis was repeated using two sets of data : all - countries ( n = 61 ) , with china , indonesia , and india each as a single unit , and all - countries plus the individual provinces of china , indonesia , and india ( n = 124 ) . this yielded a restricted number of variables with superior predictive capacity to model hpai h5n1 human cases and seasonal h5n1 persistence in poultry , wild birds , and/or humans . the euclidian distances to the medoid of the niche with the greatest number of human cases was estimated in the space defined by the subset of variables and mapped as an index of dissimilarity . on the basis of these results , we used the set with all - countries in the follow - up analyses to quantify prs versus hc models , and the set of all - countries plus china , indonesia , and india provinces to quantify hcp versus hc models.figure 1akaike s information criteria of hpai h5n1 model groups . for all countries plus china , indonesia , and india provinces , the variables with the greatest median auc for hcp models comprised duchdn , chdn , tmin , pppc , and variables with greatest median auc for hc models were duchdn , pppc , agpopdn , dudn , and chdn . , located in the upper right quadrant of each plot : agpopdn ( agricultural population density ; log - scale ) , agpopdnoi ( product of agricultural population density and output / input chicken , log - scale ) , duchdn ( duck by chicken density , log - scale ) , pppc ( purchasing power per capita ) , chdn ( chicken density , log - scale ) , and dudn ( duck density , log - scale ) . the medoid of the niche with the greatest number of human cases ( niche 4 ) was the southern chinese province of henan.figure 2goodness of fit indicators of hpai h5n1 model groups for all countries ( top left and right ) , and all countries and chinese ( chn ) , indonesian ( idn ) , and indian ( ind ) provinces ( bottom left and right ) . variable abbreviations are as follows : agpopdn agricultural population density , agpopdnoi agricultural population density by output / input chicken , chdn chicken density , chmtdn chicken meat density , dudn duck density , duchdn duck by chicken density , oich output / input chicken , pppc purchasing power per capita , topopdn total population density , tmin , tmax , trng temperature min , max , and range , urpopdn urban population density , urrt urban to total population ratio.figure 3cluster plot of the partitioning around medoids presented in the two principal component space ( top ) , and map of the five agro - ecological clusters or niche in countries and chinese , indonesian , and indian provinces ( bottom ) where h5n1 was reported ( areas unsuitable to poultry production are masked in white).figure 4characterization of each niche by the number of h5n1 human cases per million people ( top left ) , the product of duck by chicken density ( duchdn , log scale [ top right ] ) , agricultural population density ( agpopdn , log - scale [ bottom left ] ) , and purchasing power per capita ( pppc [ bottom right ] ) . variable abbreviations are as follows : agpopdn agricultural population density , agpopdnoi agricultural population density by output / input chicken , chdn chicken density , chmtdn chicken meat density , dudn duck density , duchdn duck by chicken density , oich output / input chicken , pppc purchasing power per capita , topopdn total population density , tmin , tmax , trng temperature min , max , and range , urpopdn urban population density , urrt urban to total population ratio . cluster plot of the partitioning around medoids presented in the two principal component space ( top ) , and map of the five agro - ecological clusters or niche in countries and chinese , indonesian , and indian provinces ( bottom ) where h5n1 was reported ( areas unsuitable to poultry production are masked in white ) . characterization of each niche by the number of h5n1 human cases per million people ( top left ) , the product of duck by chicken density ( duchdn , log scale [ top right ] ) , agricultural population density ( agpopdn , log - scale [ bottom left ] ) , and purchasing power per capita ( pppc [ bottom right ] ) . niche 5 was comprised largely of western european countries , japan , and south korea , and characterized by the highest purchasing power per capita and chicken production productivity , the lowest density of agricultural population , and an intermediate density of domestic ducks and chickens . niche 2 , with african countries , russia , western india provinces , and a low number of chinese and indonesia provinces , included areas with the lowest duck and chicken densities , a low purchasing power per capita and chicken production productivity , and an intermediate density of agricultural population . niche 4 ( egypt , the provinces of java , and several chinese provinces including guangdong and henan ) had the greatest density of domestic ducks , chickens , and agricultural population density , and at the same time an intermediate productivity of chicken production and purchasing power , higher than niches 1 , 2 , and 3 . finally , niche 1 supported a high density of agricultural population , ducks , and chickens , but lower than in niche 4 , and a low - to - intermediate chicken productivity and purchasing power per capita . , not the risk of introduction itself.figure 5distance to henan ( china ) in the space defined by the variables agricultural population density ( log - scale ) , duck density ( log - scale ) , duck by chicken density ( log - scale ) , chicken density ( log - scale ) , the product of agricultural population density and chicken output / input ratio ( log - scale ) , and purchasing power per capita ( areas unsuitable to poultry production are masked in white ) . distance to henan ( china ) in the space defined by the variables agricultural population density ( log - scale ) , duck density ( log - scale ) , duck by chicken density ( log - scale ) , chicken density ( log - scale ) , the product of agricultural population density and chicken output / input ratio ( log - scale ) , and purchasing power per capita ( areas unsuitable to poultry production are masked in white ) . the multivariate analysis discriminated localities with human cases and high persistence , and identified five agro - ecological niches , which , although derived independently of hpai h5n1 data , vary by hpai h5n1 persistence ( fig . importantly , the current study also revealed an as yet little - addressed third dimension : purchasing power per capita and associated poultry production intensification ( combined with agricultural population density ) . however , it is important to note that the niches with the greatest number of human cases were not those with the lowest levels of poultry productivity . niches 1 and 4 , with the greater number of human cases , were characterized by the largest duck and agricultural population densities , but not by the lowest output / input level , nor by the lowest purchasing power per capita . in particular , niche 4 , with the highest number of human cases per capita , mainly included countries or provinces with intermediate levels of productivity and low - to - medium purchasing power per capita ( e.g. at the global level , the h5n1 niches defined here are clearly structured by geography , with the most vulnerable niches arrayed across south and east asia , including along the chinese lowlands and coastline into the river basins of indochina and , further south , indonesia . as is often the case , first , understanding the distribution of hpai h5n1 in relation to agro - ecological conditions depends heavily on the epidemiological records and conditions underlying the analyses . we found broadly coherent results at the global scale and country level , a coherence that disappeared when sub - national data on china , indonesia , and india were introduced ( fig . in a situation where the training set consists of data pooled from heterogeneous sources representing different levels of detection capacity , a prediction of risk less dependent on the spatial distribution of the positive cases is helpful as it may highlight regions where agro - ecological conditions are more suitable ; that is , where the disease has not spread but may have the potential to persist , or regions to where it may have spread but where the introduction events may have gone undetected .

loss of sight is strongly associated with chronological age and frequently used as an important marker of the awareness of getting old . severe vision impairment has been found in 20% of those 65 years and older and 25% of those 75 years and older [ 2 , 3 ] . age - related macular degeneration ( amd ) is the leading cause of poor vision in the industrialized countries , affecting one in five people between 65 and 74 years of age and one in three people 75 years of age or older . although amd treatment has significantly improved during recent years , currently available medical interventions can halt disease progression only in about 10 to 15% of cases . thus , the majority of older adults who are faced with amd are psychologically challenged to adapt to ongoing and progressive visual loss , which typically occurs as deterioration of the central visual field ( i.e. , scotoma ) , affecting reading ability , overall daily functioning , and mental health [ 6 , 7 ] . furthermore , other chronic conditions , such as glaucoma , diabetic retinopathy , or diseases of the optic nerve come with the need for psychological adaptation , because treatment options may be limited . in this paper , it is my goal to highlight and synthesize key research speaking to the psychological consequences of age - related vision impairment and how such consequences may become a significant component of efficient treatment and rehabilitation . evidence on the psychological situation of older adults with vision impairment is an important addition to ophthalmological research and treatment , and i suggest the term age - related psychoophthalmology ( apo ) to address this needed field . the bottom line is that demographic change and the constantly increasing life expectancy have reached the center of ophthalmology and optometric day - to - day practice . hence , the older patient is on the way to become the standard , eye care specialists are increasingly confronted with the psychological challenges related to aging and covarying conditions , loss of sight and respective quality - of - life implications in particular , but also frequently in conjunction with other occurrences , such as loss in hearing , mobility , and cognitive function . thus , i can hardly imagine ophthalmological treatment of the older patient only driven by an understanding of vision impairment as a circumscribed disease of the retina / fovea or another part of the optical apparatus . instead , knowledge of the psychological situation of the older patient is also needed and may significantly help further optimize treatment and rehabilitation outcomes , as well as the efficient involvement of other disciplines , such as psychology , social work , and occupational therapy . apo adds to a long tradition of strongly psychologically framed research addressing the role and consequences of severe vision impairment in infancy . in some contrast , while it is true that aging and change in normal vision function have long been a target for geropsychological research ( e.g. , ; see also ) , psychological research on older individuals ' efforts and experiences to cope with severe vision loss and needed psychosocial interventions is fairly new , having only been in existence since the end of the 1970s / beginning of the 1980s ( e.g. , as an early study in the area ) . more concretely , what are the primary goals of apo and how can this field add to the more medical part of ophthalmological treatment addressing older adults ? first , apo provides findings on the full range of psychological issues involved in the experience and behavioral and emotional outcome of age - related vision impairment . major areas include ( 1 ) everyday competence , ( 2 ) cognitive functioning , ( 3 ) social functioning , and ( 4 ) subjective well - being - related outcomes , depression , and adaptational processes . second , apo also translates its more fundamental research insights to psychosocially framed intervention programs with visually impaired older adults with the aim to improve their quality of life in terms of behavioral and emotional functioning . third , apo may also serve as a bridge builder between the aging - oriented areas of gerontology and geriatric medicine and ophthalmology . this includes , for example , recent concepts and theories developed in geropsychology speaking to how aging and the occurrence of chronic conditions are experienced , where psychological resilience may reach its limits , and what can be offered in a crisis situation . at the more practical level , apo should enable eye care specialists to make better use of the existing body of evidence regarding psychosocially framed findings on age - related vision impairment in their day - to - day professional action . i see four primary reasons why such evidence emerging out of apo may make a difference in high - quality ophthalmological treatment . first , having a better understanding of everyday competence , the role of cognitive functioning , social resources , and well - being - related dynamics in visually impaired older adults may enrich the ophthalmologists ' professional background knowledge of the patient . this knowledge is important for diagnostic evaluation , treatment decisions , predictions of long - term outcomes , and general recommendations aimed to inform the patient , as well as her / his family environment . for example , for an older patient with rich psychological and social resources to deal successfully with occurring vision impairment in day - to - day life , available but possibly risky treatment options promising a limited improvement of objective vision functioning may not be a good option . second , it is also critical to have an understanding of the more fundamental mechanisms and systemic interrelations in older patients ( e.g. , among visual , hearing , mobility , and cognitive impairment ) , because such evidence helps to evaluate overall vulnerability and likely future trajectories of the patient . for example , cognitive status plays a critical role for visual functioning , and the remaining useful field of vision depends on remaining information - processing speed and not only on high functioning of the end organ . third , psychoophthalmological concepts and findings may also help to enhance the professional relationship with the visually impaired older patient ; this can be a critical issue , when problematic and threatening diagnostic findings ( e.g. , the diagnosis of amd ) need communication and acceptance by the patient . for example , communicating the diagnosis of amd to a very old individual already confronted with other major chronic diseases , cognitive impairment or clear indications of loneliness should be linked with recommendations of psychosocial support services , and such recommendations should be as concrete as possible . high - quality ophthalmological treatment may profit from detailed knowledge of available psychosocial intervention programs ( to be addressed in more detail below ) and their efficiency , availability , and financial implications . that is , knowing about the full scale of interventions and rehabilitation efforts evolving from psychological research with visually impaired older individuals may enhance ophthalmologists ' repertoire of goal - directed consultations and recommendations for patients and caregivers , particularly in instances in which the boundary of pure ophthalmological treatment has been reached . here , i see a huge potential of high - level multiprofessionalism that includes psychologists , occupational therapists , and social workers . unfortunately , such highly synergetic professional collaboration is still in development but deserves much more attention in the future . finally , an important future task of apo is to incorporate medical - ophthalmological education and training situation for medical students and early - stage eye care specialists . here , the major issue is to be sensitive to the various needs and challenges of seeing the demographic revolution in a very concrete way in one 's own day - to - day practice . in terms of concepts important to understand visual impairment in later life , a lifespan developmental view is a key approach . as baltes argued , lifespan developmental trajectories reveal a complex intertwining of aging , age - normative occurrences ( such as retirement ) , and nonnormative events . vision impairment occurring in late life reflects , in a sense , all of these elements ; the likelihood of vision impairment clearly increases with age , and aging individuals expect in their late - life phase a more frequent occurrence of health instances . at the same time , severe and enduring vision loss , comes as a surprise for many older adults ( e.g. , ) , and there is thus also a nonnormative and critical life - event characteristic inherent in the experience of this functional - loss situation . while normative vision decrease is normally tolerated by most aging individuals as a condition affecting practically all older adults , pathological and hard - to - treat vision impairment is not ; furthermore , it typically requires serious and effortful strategies of psychological adaptation . adding to a lifespan view , research also revealed that the impact of vision loss is partially different in old age as compared to middle age . for example , change in career is not an issue for visually impaired older adults but high on the agenda of visually impaired middle - aged individuals . a core issue of understanding adaptational dynamics is related with the assumption that developing individuals have access to a toolbox of self - regulatory strategies enabling them to adjust to critical life situations . heckhausen , and schulz argued that human kind strives from birth to death for what they have labeled primary control , that is , ways and means to impact one 's own environment to achieve important life goals . however , aging and particularly pathological occurrences that accompany aging such as vision impairment challenge the fundamental motivation toward primary control and demand additional strategies to support primary - control goals . that is where secondary control strategies , such as disengagement from no - longer achievable goals and the reinterpretation of life priorities ( e.g. , secondary control should not be seen as something negative but as a means to enhance goal engagement based on remaining competencies and driven by personal needs and priorities . therefore , the lifespan theory of control suggested by heckhausen and colleagues is a helpful conceptual tool to understand better how visually impaired older adults cope with their situation ( e.g. , [ 17 , 18 ] ) . brandstdter and renner have developed a similar argument in their differentiation between assimilative and accommodative coping strategies . while assimilative strategies indicate operating in a state of ongoing goal pursuit , the accommodative mode allows for flexible goal adjustment . translated to chronic vision impairment , it seems critical for older adults with vision impairment not to psychologically give up but to keep the pursuit of goals such as autonomy and self - efficacy ; at the same time , flexibly adjusting for goals no longer achievable or only achievable with high costs or failure risks seems to be highly adaptive [ 2022 ] . vision loss brings serious challenges to one 's interaction with the physical - spatial environment and , therefore , person - environment fit models are important in understanding the day - to - day consequences of vision impairment . such models assume that environmental hazards , for example , bad light or floor conditions , may reveal a particularly strong negative impact when competence is low [ 23 , 24 ] . regarding the social environment , socioemotional selectivity theory argues that older adults invest much in the maintenance of their close and intimate relationships because of a more limited future - time perspective in old age , while less intimate relationships tend to lose importance and may decrease in importance . this social environment - oriented optimization strategy may also be very helpful for visually impaired older adults . the downgrading of less significant social relationships may even be stronger , because all social energy much of what we see in terms of adaptational dynamics in visually impaired older adults is driven by the human potential for habituation and resilience . as research on the well - being paradox of old age ( e.g. , ) shows , all of the previouslymentioned strategies contribute to some extent to adaptation in terms of long - term restoration of well - being (; see also , with an application to age - related vision impairment ) . in parallel , however , aging is strongly affected by cognitive downward trajectories in information - processing speed , key memory functions ( e.g. , working memory , episodic memory ) , and executive control functions , which brings additional stress to the stability of aging trajectories under the confining condition of severe vision loss . according to birren 's cascade hypothesis , age - related performance deficits in the sensory system may show a domino effect on impairment in other areas of functioning , such as cognitive abilities . the assumption is that the slackened rate of information flow to the brain due to sensory impairment produces a state of insufficient stimulation and deprivation ; this , in turn , has a negative effect on cognitive performance . the common cause hypothesis (; see also ) maintains that the cognitive , sensory , and motor systems increasingly dedifferentiate at the neocortex as well as at the behavioral level in later life . therefore , all of these changes may echo a common cause , that is , the common aging of central brain areas , such as the frontal lobe and the visual and motor cortex . generalizing further from such insights , it is important to acknowledge that , particularly in old - age , vision - related processes ( such as speed of visual information processing , selective visual attention processes , reading capacity , or using the remaining visual field most effectively ) are strongly linked with central nervous system function and are not solely rooted in the end organ , that is , the eye [ 10 , 31 ] . in conclusion , adaptation to visual impairment seems to reflect in particular clarity and sharpness , what lifespan researchers have termed the gain - loss dynamics of human development . on the one hand , a well - filled tool box of strategies is at hand to help the old individual affected by severe vision impairment to make the best out of her / his situation in terms of autonomy and well - being , because many older adults have optimized the world in which they live over the years as a highly predictable and socially stable one , they are able to counteract threats to their cognitive - emotional well - being to a large extent . however , if chronic stressors such as severe vision impairment can no longer be avoided or psychologically reinterpreted , high negative emotional arousal may occur , which is likely to require psychosocial intervention and wide - scale support . that said , the experience of visual impairment in old age may be seen as a prime example of reasons to learn about the interplay of strengths and vulnerabilities as people age . everyday competence is a concept frequently used in geropsychology to address the day - to - day functioning of aging individuals . everyday competence includes basic and instrumental activities of daily living ( adl / iadl ) but also activities covering socializing and leisure life . age - related vision impairment has robustly been found to be closely associated with significantly lower everyday competence , because visual capacity is a critical prerequisite for such behaviors . in contrast , hearing loss has been found not to have a major impact in particular on adl / iadl . indeed , lowered everyday competence appeared as the best of a range of variables ( including cognitive function and well - being - related measures ) used to differentiate between visually impaired and visually unimpaired older adults . found in a sample of community - dwelling older adults ( aged 5598 years ) that vision status mediated the connection between age and out - of - home competence ; therefore , the direct effect of age on everyday functions is clearly reduced after vision function is taken into consideration . furthermore , vision impairment affects more strongly iadl and leisure activities as compared to adl , because the execution of iadl is more complex and depends more strongly on environmental enhancing or hindering factors , such as unexpected construction areas or the availability of low - floor bus transportation [ 38 , 39 ] . hence , shrinkage in iadl competence reflects a kind of early behavioral marker of severe vision impairment , whereas significant adl decrease only happens later in the process of chronic vision loss . additional support for the critical role of iadl comes from at least two research directions . for one , . found , when comparing severely visually impaired and visually unimpaired older adults , that those being blind and severely visually impaired ( and living in apartments / houses with relatively more environmental hazards ) also were lower in iadl functioning , but not in basic adl . wahl et al . interpreted this finding as support for the assumption that visually impaired older adults invest as much as possible in the maintenance of basic adl , even in challenging physical environments , whereas the combination of being visually impaired and living in barrier - rich environments inevitably forfeits iadl . in contrast , in visually unimpaired older adults , environmental hazards were unimportant for autonomy outcomes , which supports the core assumption of person - environment fit models , that is , the physical environments gain importance only when competence has fallen below a certain threshold ( e.g. , due to vision loss ) . second , wahl et al . found that iadl ( but not adl ) change in visually impaired older adults is a critical driver for change in adaptational strategies , particularly in control - strategy regulation . it has been found that as long as iadl remains relatively high , visually impaired older individuals invest in maintaining goal - directed resources , whereas starting from a certain threshold of iadl competence loss , these individuals switch to secondary control strategies and disengage from no - longer - achievable goals ( see also ) . in addition to a more general notion of adl and iadl , it is obvious that there are two everyday competencies particularly critical for adapting to severe vision impairment . on the one hand , reading is of utmost importance for visually impaired older adults and therefore a major goal in rehabilitation . becker et al . found that control - strategy use is of importance for assistive - device use such as magnifying glasses and that those being higher in primary control use assistive devices more frequently and efficiently . that is , control regulation directly or at least indirectly ( via assistive devices ) also contributes to the maintenance of reading . on the other hand the need to give up driving has generally been found to be a major psychological loss experience , intensively symbolizing decrease in autonomy and participation . it is also obviously a threat to day - to - day function , particularly in countries with an underdeveloped public transport system , such as the u.s . . giving up driving has indeed been found to be a significant predictor of depression onset in older adults in the u.s . . linking sensory and cognitive decline as people age is an old research issue and dates back to the work of galton . at the same time , cognitive performance is also , as visual capacity , a major platform for exerting everyday competence . previous research has shown that reduced sensory function is accompanied by a decrease in cognitive performance in older adults . no clear difference between the sensory modalities of vision and hearing has been identified regarding their relationship with cognitive performance , although some evidence supports that the linkage may be stronger with vision loss . the work of lindenberger and baltes , based on the berlin aging study ( base)but also including additional studies with a wider age range is central in supporting a strong connection among vision , hearing , balance , and cognitive functioning in later life . the base has been particularly noteworthy in this context , because it assessed sensory functioning in a heterogeneous sample of old and very - old participants ( 70103 years of age ) by means of standardized ( objective ) procedures covering near- and far - vision functioning , while many geropsychological studies only rely on subjective assessments . in addition , a state - of - the - art cognitive assessment battery has been applied in base , which means that measurement went far beyond cognitive screening measures such as the mini - mental state examination , which are of limited sensitivity for detecting age - normative change in cognitive functioning . lindenberger and baltes found with a subset of participants of base that general intelligence correlated just as strongly with visual as with auditory ability . in a model containing age , sensory function as well as intelligence , visual and auditory function , predicted a large portion of interindividual differences in intelligence and indeed fully mediated the negative correlation between age and intelligence . this finding has meanwhile been replicated by a number of other research groups and may be regarded as rather robust ( e.g. , [ 52 , 53 ] ) . in addition , baltes and lindenberger observed that sensory measures were better predictors of intelligence than sociostructural variables such as education or social class . they also showed that the connection among sensory functioning and intelligence was much closer in older adults as compared to adults in early and middle adulthood ( see also ) . longitudinal data support the view that the causal dynamics involved in the vision and intelligence change connection are complex and that each of these variables can drive change in the others across longer periods of later life . henderson et al . were able to show based on their long - term data that the significant correlation between vision and intelligence in a sample aged 83 years remained after controlling for child intelligence at the age of 11 years . this supports as do many of the aforementioned findings the common cause hypothesis . however , henderson et al . also found that childhood intelligence is able to predict visual function 72 years later , at age 83 . a possible interpretation is that higher intelligence early in life leads to more investments into visual health across the life course ( searching for better treatment , more preventive efforts , etc . ) and therefore adds to maintaining visual status and provoking the significant correlation across an amazing time span . vision status also plays a role when it comes to the connection between cognitive function and everyday competence , a linkage that is generally challenged as people age and that may lead to endpoints such as dependence from others and transition to long - term care . observed that the link between vision status and out - of - home everyday competence is moderated by cognitive status . in a more recent study , being able to add to the understanding of such a moderation process , heyl and wahl [ 57 , 58 ] showed that the connection between cognitive function and everyday function is much closer in visually impaired older adults as compared to visually unimpaired older adults , which possibly means that visually impaired elders rely more intensely on their cognitive resources . reversely , ongoing age - related cognitive decrease may bring a particularly dramatic impact in visually impaired older adults , who are already challenged in their everyday competence due to their vision loss . as rovner et al . observed in a study with amd patients over 64 years of age covering a three - year observation period and two measurement occasions , that activity loss over time due to the visual loss led to cognitive decline happening between t1 and t2 . this finding fits well with the more general finding in the cognitive aging literature that the exertion of social and leisure activities is important for maintaining cognitive functioning . social relations , as well as social support , have generally been found to be of key importance for older adults . we concentrate in this section on issues that may accompany the experience of age - related vision impairment and , in the next section , on linkages between social relations and adaptational outcomes . existing evidence supports the view that social relations of older adults with severe vision impairment are not fundamentally different from sensory - unimpaired older individuals . reinhardt found that visually impaired older adults nominated , on average , 5.4 persons with intimate relations within their family network , and 3.5 persons within their friendship network , which is similar to sensory - unimpaired older adults , such as those assessed in the base . study , visually impaired older adults nominated 4.7 persons as being in the most intimate circle of their social network ; the respective number in a comparison group of visually unimpaired older adults amounted to 5.2 persons , which was not statistically significantly different from the visually impaired group 's mean . as has also been found , the differentiation between friendship and family support is also an important one for visually impaired older adults . additionally , the closest family members of visually impaired older adults are children ( 38% ) , spouses ( 33% ) , and other relatives ( 29% ) . in contrast to family relations , friendship is of voluntary nature and characterized by a strong affective base and mutuality . research has found that because of its affective as well as instrumental support , friendship becomes increasingly important for older adults with disability , but friends of older adults tend to be old as well ( the average age of visually impaired older adults ' closest friend was 70 years in the reinhardt and blieszner study ) ; thus , friendship networks and their respective support are vulnerable ( see also ) . from the perspective of socioemotional selectivity theory , it can generally be expected that older adults including sensory - impaired individuals invest much in the maintenance of their close relationships , because of a more limited - future time perspective in old age . wahl et al . found that data among visually impaired older adults revealed even lower numbers of persons in less intimate social domains as compared to visually unimpaired older adults ; this suggests that socioemotional selectivity toward more intimate social partners may even be more pronounced in visually impaired older adults . furthermore , although visual impairment may further increase the likelihood of experiencing loneliness , the majority of studies found visually impaired older adults not to be dramatically more affected by the experience of loneliness than visually unimpaired older adults [ 36 , 67 ] . visually impaired older adults are also particularly challenged to reestablish their social networks following vision loss , because social network members may have a tendency to sever social ties with visually impaired older adults , for example , because previous joint activities have become more difficult . as wang and boerner observed , two strategies seem prevalent in visually impaired older adults : namely , to readjust one 's behavior to maintain relationships or to let relationships go to some extent . 's finding of lower number of persons in the periphery of social networks of visually impaired older adults as compared to visually unimpaired individuals . particularly critical features of social partners are dependence - inducing behavior , the lack of an independence - supporting attitude , and overprotection . perceived overprotection in visually impaired older adults is on the rise , wherein functional disability and received instrumental support increases . also , perceived overprotection increases over time and has been found to be unaffected by rehabilitation service use . in general , it seems a challenge to provide visually impaired older adults with the needed instrumental support while at the same time avoiding to underestimating remaining capabilities . overprotection may put constraints on the visually impaired older adults ' true functional capacity and thereby contribute to loss in competence in the longer run due to disuse [ 69 , 73 ] . subjective well - being ( swb ) is frequently defined via its cognitive component as degree of satisfaction with one 's current life . many studies with visually impaired older adults have found that with age - related vision loss comes a significant decrease regarding the cognitive component of well - being [ 39 , 67 ] . as has also been observed , the discrepancy in swb between visually impaired and visually unimpaired older adults tends to persist over time [ 38 , 74 ] . however , pinquart and pfeiffer found in their meta - analysis of 198 studies that the effect size regarding differences in vision - unspecific measures ( such as general well - being or positive / negative affect scales ) was small , whereas larger negative effects regarding well - being appeared in vision - specific measures of quality of life ( such as the nei vfq-25 ; ) . in general , widening the picture of adaptation to visual impairment with the consideration of everyday competence , robust evidence underscores that differences in well - being - related indicators including constructs such as self - acceptance , positive relations with others , and tone of future - time perspective between visually impaired and visually unimpaired older adults are clearly less pronounced than differences in impairment in adl - iadl status and leisure activity level [ 36 , 77 ] . this suggests that although vision impairment is a pronounced psychological challenge in late life , many visually impaired older adults seem to adapt rather well at the level of cognitive well - being , thus also supporting what has been titled the paradox of well - being in later life . at the same time , it is critical to acknowledge that visually impaired older adults represent an at - risk population , in which the positive impact of human adaptation and the drawback of reaching the limits of psychological resilience go hand in hand . affect balance ( ratio of positive and negative effect ) has been found to be more toward the negative pole in visually impaired older adults and depression has consistently been found to be significantly increased in visually impaired older adults [ 7880 ] . rates identified in epidemiological studies roughly vary between 15% and 30% and are particularly high in amd patients . this is also important , because depressive symptoms may accelerate both cognitive decline and decline in everyday competence in amd patients . in addition to depressive mood , maintenance and decline in positive effect are important for adaptation . schilling and wahl found that positive effect goes down in the period after having received the diagnosis of amd , but then also increases again after 24 years . thereafter , rather constant decrease has been observed , probably due to the progressive course of amd in combination with growing comorbidity and other loss occurrences ( such as widowhood ) as people age further . control regulation , as argued by heckhausen et al . , also seems important for positive effect . wahl et al . observed in a respective analysis that compensatory control strategies ( called compensatory primary control by heckhausen et al . , ) , such as involving the help of others as well as strengthening one 's commitment to important life goals ( called selective secondary control by heckhausen et al . , ) coincides with positive effect , whereas the effect of exerting the impact on one 's environment ( called selective primary control by heckhausen et al . , ) on positive effect was fully mediated by adl - iadl and a vision - specific adaptation measure ( called adaptation to vision loss ; ) . the critical role of maintaining everyday competence ( and not objective vision impairment ) for well - being and adaptation to vision loss in visually impaired older adults has also been confirmed in other research , including the transition from assimilative to accommodative coping [ 19 , 20 ] . decrease in iadl competence also seems to drive the transition to modes of control regulation connected with goal disengagement and flexible goal adjustment , which heckhausen et al . in addition to change in self - regulation as a significant adaptational process , social resources have been found to play a critical role in adaptation to vision loss and other well - being - related outcomes cross - sectionally as well as longitudinally and family and friends provide distinct contributions to the maintenance of well - being [ 62 , 85 ] . perceived overprotection may also lead to negative consequences in terms of heightened depression and anxiety over time . dual sensory impairment affects one - fifth of those 70 years of age and older and therefore also deserves attention . previous research supports the notion that the overall psychosocial situation of those with dual sensory impairment is even worse as compared to those with sole vision impairment , particularly in the area of everyday functioning . in addition , higher rates of depression and lowered well - being have also been found in those affected by dual sensory loss . the research summary provided in the previous section underscores that the experience of age - related visual impairment comes with pronounced loss , although the capacity to maintain subjective well - being and positive effect has to be acknowledged at least in a considerable portion of visually impaired older adults . overall , i come to the conclusion that such naturally occurring psychological resilience and reserve capacity to counteract the threat elicited by the experience of chronic vision impairment are surprisingly high ; on the other hand , given the vulnerability of visually impaired older adults particularly , the alarmingly high ( [ 78 , p. 591 ] ) rate of depression qualified professional support of visually impaired older adults in the psychosocial domain with the potential to significantly add to the best available ophthalmological treatment seems to be a must . wahl et al . analyzed 15 intervention studies , with a considerable subportion of randomized , controlled intervention trials . most interventions could be characterized as self - management and disease management - like efforts and are promising for visually impaired older adults . major elements of such programs include stress - reducing strategies ( e.g. , muscle - relaxation exercises ) , goal - directed problem - solving strategies to evoke positive affect , activating available resources , and information and consultation . typically , such programs are conducted in a group format in an eye clinic , bringing together 68 visually impaired older adults , for weekly sessions of 2 - 3 hours , over 68 weeks . group sessions are moderated by a clinical psychologist with particularly professional experience with older adults . a case example emerging from our work in this area [ 89 , 90 ] may illustrate what can be achieved . singlewithout many family ties had been a traveling person throughout her life . after retirement from her job as an assistant to the director of a university library , her plan was to intensify her a. became , to her own surprise , very depressed and , to a large extent , unable to manage her daily affairs . in the group sessions of the self - management program , of which she learned in the practice of her eye doctor , she remained rather quiet and obviously in a depressed mood throughout the first three sessions . in the fourth week 's session , she reported having learned muscle relaxation and also felt somewhat better . the session was then also used to plan visiting a restaurant an activity she had not done in a year although she always found having lunch in a restaurant very enjoyable . in particular , all the steps to successfully conduct this activity were discussed and preplanned . later in the flow of the program , mrs . a. reported of two successful lunch events and said that she had been very proud of herself . she had also begun to reinstate phone conversations with some of her friends and indeed started to plan a 7-day holiday together with her best friend . although she still reported being frequently in a depressed mood at the end of the 8-session program , she also felt an increasing feelings of mastery in her day - to - day life . positive effects have been reported in such self - management programs regarding depression , increased well - being , self - efficacy , and stress reduction . dose of intervention is important ( too - short and less - intensive programs may indeed elicit negative effects ; ) . more recent work provides additional support for the assumption that self - management programs are useful [ 91 , 92 ] and add to the need to seriously consider such programs as a standard health treatment . still , many of the available studies operate on small samples and include a number of other methodological limitations , which means that larger high - quality studies are an important need in the area . it is also obvious that such programs should find a strong liaison with classic high - caliber rehabilitation programs , including effective reading training . given the findings on the role of cognitive resources in visually impaired older adults ( see respective section above ) , cognitive training may be an important addition to psychosocial intervention and rehabilitation [ 58 , 94 ] . furthermore , physical training programs , which have proven efficient with old and very old individuals including those being cognitively vulnerable may also be of significant advantage for visually impaired older adults . as has been found , such programs not only increase posture , gait , and general physical fitness , but also they prevent falls and enhance well - being , self - efficacy , and cognitive function , especially executive control . finally , it may be important to consider personality traits of visually impaired older adults so as to achieve the best psychosocial outcomes possible . in summation , i would recommend multi - component interventions as the best available psychosocially framed interventions for visually impaired older adults . such multicomponent interventions that not only offer direct psychosocial guidance ( as described previously ) , but are also closely intertwined with cognitive and physical engagement and independence training in adl . lastly , emerging evidence supports the notion that psychosocially framed interventions may also contribute to saving health costs ( e.g. , via reduced psychopharmacy ) and may also enhance commitment toward secondary prevention , for example , avoiding later treatments of depression . severe vision loss and blindness have always been an issue going far beyond the mere functional loss and respective medical - ophthalmological treatment into culture , society , and fundamental questions concerning the human condition . the prophet tiresias in greek mythology was blind but also was able to offer wise recommendations and helpful forecasts . the secular trend version of this cultural history comes with the more recent significant expansion of life expectancy , increasingly making the older adult the standard patient of eye care specialists . apo now provides a rich portfolio of research supporting the view that many older adults continue , in a sense , in the tiresias tradition and are able to make the best out of their vision impairment . however , major subgroups of older adults with vision impairment also are in need of high - quality professional support to improve their psychosocial adaptation . although promising intervention research has also been generated within apo , such psychosocial support services currently seem to be more the exception than the usual or even standard professional offer in the usa , as well as in europe or other regions of the world . this is disappointing and requires that the multiprofessional treatment and rehabilitation of visually impaired older adults should find high priority in public health terms in the future .

the intention is to summarize the body of evidence speaking to the psychological challenges faced by visually impaired older adults , as well as their coping efforts . this evidence is substantiated by a rich set of concepts , theories , and empirical findings that have accumulated under the umbrella of age - related psychoophthalmology ( apo ) . i introduce the field of apo and continue with a discussion of important concepts and theories for a better understanding of adaptational processes in visually impaired older adults . i then summarize the most relevant and most recent data from four areas : ( 1 ) everyday competence , ( 2 ) cognitive functioning , ( 3 ) social functioning , and ( 4 ) subjective well - being - related outcomes , depression , and adaptational processes . thereafter , major insights related to the current state - of - the art psychosocial interventions with visuallyimpaired older adults are reviewed . i close with the need that the public health community should become more aware of and address the psychosocial needs of visually impaired older adults .

1. Introduction 2. Why Age-Related Psychoophthalmology Is a Needed Field 3. Major Concepts, Models, and Theories for Age-Related Psychoophthalmology 4. Major Findings of Age-Related Psychoophthalmology 5. Psychosocially Framed Intervention Research with Visually Impaired Older Adults: Promises and Unknowns 6. Outlook

age - related macular degeneration ( amd ) is the leading cause of poor vision in the industrialized countries , affecting one in five people between 65 and 74 years of age and one in three people 75 years of age or older . , scotoma ) , affecting reading ability , overall daily functioning , and mental health [ 6 , 7 ] . furthermore , other chronic conditions , such as glaucoma , diabetic retinopathy , or diseases of the optic nerve come with the need for psychological adaptation , because treatment options may be limited . in this paper , it is my goal to highlight and synthesize key research speaking to the psychological consequences of age - related vision impairment and how such consequences may become a significant component of efficient treatment and rehabilitation . evidence on the psychological situation of older adults with vision impairment is an important addition to ophthalmological research and treatment , and i suggest the term age - related psychoophthalmology ( apo ) to address this needed field . hence , the older patient is on the way to become the standard , eye care specialists are increasingly confronted with the psychological challenges related to aging and covarying conditions , loss of sight and respective quality - of - life implications in particular , but also frequently in conjunction with other occurrences , such as loss in hearing , mobility , and cognitive function . instead , knowledge of the psychological situation of the older patient is also needed and may significantly help further optimize treatment and rehabilitation outcomes , as well as the efficient involvement of other disciplines , such as psychology , social work , and occupational therapy . more concretely , what are the primary goals of apo and how can this field add to the more medical part of ophthalmological treatment addressing older adults ? first , apo provides findings on the full range of psychological issues involved in the experience and behavioral and emotional outcome of age - related vision impairment . major areas include ( 1 ) everyday competence , ( 2 ) cognitive functioning , ( 3 ) social functioning , and ( 4 ) subjective well - being - related outcomes , depression , and adaptational processes . second , apo also translates its more fundamental research insights to psychosocially framed intervention programs with visually impaired older adults with the aim to improve their quality of life in terms of behavioral and emotional functioning . this includes , for example , recent concepts and theories developed in geropsychology speaking to how aging and the occurrence of chronic conditions are experienced , where psychological resilience may reach its limits , and what can be offered in a crisis situation . at the more practical level , apo should enable eye care specialists to make better use of the existing body of evidence regarding psychosocially framed findings on age - related vision impairment in their day - to - day professional action . first , having a better understanding of everyday competence , the role of cognitive functioning , social resources , and well - being - related dynamics in visually impaired older adults may enrich the ophthalmologists ' professional background knowledge of the patient . this knowledge is important for diagnostic evaluation , treatment decisions , predictions of long - term outcomes , and general recommendations aimed to inform the patient , as well as her / his family environment . for example , cognitive status plays a critical role for visual functioning , and the remaining useful field of vision depends on remaining information - processing speed and not only on high functioning of the end organ . third , psychoophthalmological concepts and findings may also help to enhance the professional relationship with the visually impaired older patient ; this can be a critical issue , when problematic and threatening diagnostic findings ( e.g. that is , knowing about the full scale of interventions and rehabilitation efforts evolving from psychological research with visually impaired older individuals may enhance ophthalmologists ' repertoire of goal - directed consultations and recommendations for patients and caregivers , particularly in instances in which the boundary of pure ophthalmological treatment has been reached . as baltes argued , lifespan developmental trajectories reveal a complex intertwining of aging , age - normative occurrences ( such as retirement ) , and nonnormative events . while normative vision decrease is normally tolerated by most aging individuals as a condition affecting practically all older adults , pathological and hard - to - treat vision impairment is not ; furthermore , it typically requires serious and effortful strategies of psychological adaptation . for example , change in career is not an issue for visually impaired older adults but high on the agenda of visually impaired middle - aged individuals . therefore , the lifespan theory of control suggested by heckhausen and colleagues is a helpful conceptual tool to understand better how visually impaired older adults cope with their situation ( e.g. this social environment - oriented optimization strategy may also be very helpful for visually impaired older adults . the downgrading of less significant social relationships may even be stronger , because all social energy much of what we see in terms of adaptational dynamics in visually impaired older adults is driven by the human potential for habituation and resilience . as research on the well - being paradox of old age ( e.g. , ) shows , all of the previouslymentioned strategies contribute to some extent to adaptation in terms of long - term restoration of well - being (; see also , with an application to age - related vision impairment ) . , working memory , episodic memory ) , and executive control functions , which brings additional stress to the stability of aging trajectories under the confining condition of severe vision loss . according to birren 's cascade hypothesis , age - related performance deficits in the sensory system may show a domino effect on impairment in other areas of functioning , such as cognitive abilities . the assumption is that the slackened rate of information flow to the brain due to sensory impairment produces a state of insufficient stimulation and deprivation ; this , in turn , has a negative effect on cognitive performance . the common cause hypothesis (; see also ) maintains that the cognitive , sensory , and motor systems increasingly dedifferentiate at the neocortex as well as at the behavioral level in later life . on the one hand , a well - filled tool box of strategies is at hand to help the old individual affected by severe vision impairment to make the best out of her / his situation in terms of autonomy and well - being , because many older adults have optimized the world in which they live over the years as a highly predictable and socially stable one , they are able to counteract threats to their cognitive - emotional well - being to a large extent . everyday competence is a concept frequently used in geropsychology to address the day - to - day functioning of aging individuals . age - related vision impairment has robustly been found to be closely associated with significantly lower everyday competence , because visual capacity is a critical prerequisite for such behaviors . indeed , lowered everyday competence appeared as the best of a range of variables ( including cognitive function and well - being - related measures ) used to differentiate between visually impaired and visually unimpaired older adults . found in a sample of community - dwelling older adults ( aged 5598 years ) that vision status mediated the connection between age and out - of - home competence ; therefore , the direct effect of age on everyday functions is clearly reduced after vision function is taken into consideration . found , when comparing severely visually impaired and visually unimpaired older adults , that those being blind and severely visually impaired ( and living in apartments / houses with relatively more environmental hazards ) also were lower in iadl functioning , but not in basic adl . interpreted this finding as support for the assumption that visually impaired older adults invest as much as possible in the maintenance of basic adl , even in challenging physical environments , whereas the combination of being visually impaired and living in barrier - rich environments inevitably forfeits iadl . in contrast , in visually unimpaired older adults , environmental hazards were unimportant for autonomy outcomes , which supports the core assumption of person - environment fit models , that is , the physical environments gain importance only when competence has fallen below a certain threshold ( e.g. found that iadl ( but not adl ) change in visually impaired older adults is a critical driver for change in adaptational strategies , particularly in control - strategy regulation . it has been found that as long as iadl remains relatively high , visually impaired older individuals invest in maintaining goal - directed resources , whereas starting from a certain threshold of iadl competence loss , these individuals switch to secondary control strategies and disengage from no - longer - achievable goals ( see also ) . on the one hand , reading is of utmost importance for visually impaired older adults and therefore a major goal in rehabilitation . at the same time , cognitive performance is also , as visual capacity , a major platform for exerting everyday competence . previous research has shown that reduced sensory function is accompanied by a decrease in cognitive performance in older adults . the work of lindenberger and baltes , based on the berlin aging study ( base)but also including additional studies with a wider age range is central in supporting a strong connection among vision , hearing , balance , and cognitive functioning in later life . the base has been particularly noteworthy in this context , because it assessed sensory functioning in a heterogeneous sample of old and very - old participants ( 70103 years of age ) by means of standardized ( objective ) procedures covering near- and far - vision functioning , while many geropsychological studies only rely on subjective assessments . in addition , a state - of - the - art cognitive assessment battery has been applied in base , which means that measurement went far beyond cognitive screening measures such as the mini - mental state examination , which are of limited sensitivity for detecting age - normative change in cognitive functioning . in a model containing age , sensory function as well as intelligence , visual and auditory function , predicted a large portion of interindividual differences in intelligence and indeed fully mediated the negative correlation between age and intelligence . they also showed that the connection among sensory functioning and intelligence was much closer in older adults as compared to adults in early and middle adulthood ( see also ) . vision status also plays a role when it comes to the connection between cognitive function and everyday competence , a linkage that is generally challenged as people age and that may lead to endpoints such as dependence from others and transition to long - term care . observed that the link between vision status and out - of - home everyday competence is moderated by cognitive status . in a more recent study , being able to add to the understanding of such a moderation process , heyl and wahl [ 57 , 58 ] showed that the connection between cognitive function and everyday function is much closer in visually impaired older adults as compared to visually unimpaired older adults , which possibly means that visually impaired elders rely more intensely on their cognitive resources . reversely , ongoing age - related cognitive decrease may bring a particularly dramatic impact in visually impaired older adults , who are already challenged in their everyday competence due to their vision loss . observed in a study with amd patients over 64 years of age covering a three - year observation period and two measurement occasions , that activity loss over time due to the visual loss led to cognitive decline happening between t1 and t2 . this finding fits well with the more general finding in the cognitive aging literature that the exertion of social and leisure activities is important for maintaining cognitive functioning . social relations , as well as social support , have generally been found to be of key importance for older adults . we concentrate in this section on issues that may accompany the experience of age - related vision impairment and , in the next section , on linkages between social relations and adaptational outcomes . reinhardt found that visually impaired older adults nominated , on average , 5.4 persons with intimate relations within their family network , and 3.5 persons within their friendship network , which is similar to sensory - unimpaired older adults , such as those assessed in the base . study , visually impaired older adults nominated 4.7 persons as being in the most intimate circle of their social network ; the respective number in a comparison group of visually unimpaired older adults amounted to 5.2 persons , which was not statistically significantly different from the visually impaired group 's mean . as has also been found , the differentiation between friendship and family support is also an important one for visually impaired older adults . additionally , the closest family members of visually impaired older adults are children ( 38% ) , spouses ( 33% ) , and other relatives ( 29% ) . research has found that because of its affective as well as instrumental support , friendship becomes increasingly important for older adults with disability , but friends of older adults tend to be old as well ( the average age of visually impaired older adults ' closest friend was 70 years in the reinhardt and blieszner study ) ; thus , friendship networks and their respective support are vulnerable ( see also ) . found that data among visually impaired older adults revealed even lower numbers of persons in less intimate social domains as compared to visually unimpaired older adults ; this suggests that socioemotional selectivity toward more intimate social partners may even be more pronounced in visually impaired older adults . furthermore , although visual impairment may further increase the likelihood of experiencing loneliness , the majority of studies found visually impaired older adults not to be dramatically more affected by the experience of loneliness than visually unimpaired older adults [ 36 , 67 ] . visually impaired older adults are also particularly challenged to reestablish their social networks following vision loss , because social network members may have a tendency to sever social ties with visually impaired older adults , for example , because previous joint activities have become more difficult . as wang and boerner observed , two strategies seem prevalent in visually impaired older adults : namely , to readjust one 's behavior to maintain relationships or to let relationships go to some extent . 's finding of lower number of persons in the periphery of social networks of visually impaired older adults as compared to visually unimpaired individuals . perceived overprotection in visually impaired older adults is on the rise , wherein functional disability and received instrumental support increases . in general , it seems a challenge to provide visually impaired older adults with the needed instrumental support while at the same time avoiding to underestimating remaining capabilities . overprotection may put constraints on the visually impaired older adults ' true functional capacity and thereby contribute to loss in competence in the longer run due to disuse [ 69 , 73 ] . subjective well - being ( swb ) is frequently defined via its cognitive component as degree of satisfaction with one 's current life . many studies with visually impaired older adults have found that with age - related vision loss comes a significant decrease regarding the cognitive component of well - being [ 39 , 67 ] . as has also been observed , the discrepancy in swb between visually impaired and visually unimpaired older adults tends to persist over time [ 38 , 74 ] . however , pinquart and pfeiffer found in their meta - analysis of 198 studies that the effect size regarding differences in vision - unspecific measures ( such as general well - being or positive / negative affect scales ) was small , whereas larger negative effects regarding well - being appeared in vision - specific measures of quality of life ( such as the nei vfq-25 ; ) . in general , widening the picture of adaptation to visual impairment with the consideration of everyday competence , robust evidence underscores that differences in well - being - related indicators including constructs such as self - acceptance , positive relations with others , and tone of future - time perspective between visually impaired and visually unimpaired older adults are clearly less pronounced than differences in impairment in adl - iadl status and leisure activity level [ 36 , 77 ] . this suggests that although vision impairment is a pronounced psychological challenge in late life , many visually impaired older adults seem to adapt rather well at the level of cognitive well - being , thus also supporting what has been titled the paradox of well - being in later life . at the same time , it is critical to acknowledge that visually impaired older adults represent an at - risk population , in which the positive impact of human adaptation and the drawback of reaching the limits of psychological resilience go hand in hand . affect balance ( ratio of positive and negative effect ) has been found to be more toward the negative pole in visually impaired older adults and depression has consistently been found to be significantly increased in visually impaired older adults [ 7880 ] . thereafter , rather constant decrease has been observed , probably due to the progressive course of amd in combination with growing comorbidity and other loss occurrences ( such as widowhood ) as people age further . , ) , such as involving the help of others as well as strengthening one 's commitment to important life goals ( called selective secondary control by heckhausen et al . the critical role of maintaining everyday competence ( and not objective vision impairment ) for well - being and adaptation to vision loss in visually impaired older adults has also been confirmed in other research , including the transition from assimilative to accommodative coping [ 19 , 20 ] . in addition to change in self - regulation as a significant adaptational process , social resources have been found to play a critical role in adaptation to vision loss and other well - being - related outcomes cross - sectionally as well as longitudinally and family and friends provide distinct contributions to the maintenance of well - being [ 62 , 85 ] . in addition , higher rates of depression and lowered well - being have also been found in those affected by dual sensory loss . the research summary provided in the previous section underscores that the experience of age - related visual impairment comes with pronounced loss , although the capacity to maintain subjective well - being and positive effect has to be acknowledged at least in a considerable portion of visually impaired older adults . overall , i come to the conclusion that such naturally occurring psychological resilience and reserve capacity to counteract the threat elicited by the experience of chronic vision impairment are surprisingly high ; on the other hand , given the vulnerability of visually impaired older adults particularly , the alarmingly high ( [ 78 , p. 591 ] ) rate of depression qualified professional support of visually impaired older adults in the psychosocial domain with the potential to significantly add to the best available ophthalmological treatment seems to be a must . most interventions could be characterized as self - management and disease management - like efforts and are promising for visually impaired older adults . typically , such programs are conducted in a group format in an eye clinic , bringing together 68 visually impaired older adults , for weekly sessions of 2 - 3 hours , over 68 weeks . group sessions are moderated by a clinical psychologist with particularly professional experience with older adults . positive effects have been reported in such self - management programs regarding depression , increased well - being , self - efficacy , and stress reduction . more recent work provides additional support for the assumption that self - management programs are useful [ 91 , 92 ] and add to the need to seriously consider such programs as a standard health treatment . given the findings on the role of cognitive resources in visually impaired older adults ( see respective section above ) , cognitive training may be an important addition to psychosocial intervention and rehabilitation [ 58 , 94 ] . furthermore , physical training programs , which have proven efficient with old and very old individuals including those being cognitively vulnerable may also be of significant advantage for visually impaired older adults . as has been found , such programs not only increase posture , gait , and general physical fitness , but also they prevent falls and enhance well - being , self - efficacy , and cognitive function , especially executive control . finally , it may be important to consider personality traits of visually impaired older adults so as to achieve the best psychosocial outcomes possible . in summation , i would recommend multi - component interventions as the best available psychosocially framed interventions for visually impaired older adults . apo now provides a rich portfolio of research supporting the view that many older adults continue , in a sense , in the tiresias tradition and are able to make the best out of their vision impairment . although promising intervention research has also been generated within apo , such psychosocial support services currently seem to be more the exception than the usual or even standard professional offer in the usa , as well as in europe or other regions of the world . this is disappointing and requires that the multiprofessional treatment and rehabilitation of visually impaired older adults should find high priority in public health terms in the future .

the association between copd and coronary artery disease ( cad ) is well known.1 in studies of the general population , it has been shown an increased all - cause mortality as well as cardiovascular mortality in patients with copd.2,3 patients with mild copd seem to have even a higher risk of dying from cardiovascular causes than from respiratory insufficiency.4 copd is highly prevalent among patients with cad.5 the causal connection between both diseases has historically been cigarette smoking.6 however , a recent epidemiological study has shown that systemic inflammation plays a significant role in both atherogenesis and copd.7 other evidence that highlights the close relationship between copd and cad is the fact that this pulmonary disease is an independent factor of poor outcome and mortality after coronary revascularization procedures.8 the diagnosis of copd was , together with other important clinical variables such as age , sex , and left ventricular ejection fraction , a predictor of mortality at 4 years of revascularization in the syntax ii score ( index that punctuates the complexity of cad by angiography and it helps to decide the optimal method of revascularization in patients with cad complex).9 as a result , treatment of cad in patients with copd can present therapeutic challenges . given that copd is associated with higher risk of adverse events after coronary artery bypass graft ( cabg ) surgery,10,11 percutaneous coronary intervention ( pci ) is now the most commonly used reperfusion procedure for copd patients undergoing revascularization.6 in fact , patients with copd currently make up almost 10% of patients undergoing pci.12 nonetheless , several studies have reported and elevated the risk of adverse outcomes among patients with copd after pci.5,1214 secular trends in the use of coronary revascularization procedures have been examined.15 culler et al16 have identified , in a recent retrospective study , all medicare beneficiaries undergoing a coronary revascularization procedure between 2008 and 2012 . they have observed , on the one hand , that the total number of revascularization procedures peaked in 2010 and declined by > 4% per year in 2011 and 2012 , and second , that mortality rates remained between 2.1% and 2.2% annually during the study period . however , despite the poor prognosis of patients with copd after pci and cabg , no previous studies have evaluated the changes in utilization and outcomes of coronary revascularization procedures over time in these patients . it is possible that may be there have been advances in recent years , not only in surgical techniques , but also in monitoring and anesthesia , which they could have helped to improve results.17 in this study , we used national hospital discharge data to describe and compare trends in the use of coronary revascularization procedures in copd and non - copd patients between 2001 and 2011 in spain . in particular , we analyzed trends in the use of cabg and pci , patient comorbidities , and in - hospital outcomes such as length of stay ( los ) and in - hospital mortality ( ihm ) . a retrospective , descriptive , epidemiological study was conducted using the spanish national hospital database ( cmbd , conjunto minimo bsico de datos ) , which compiles all public and private hospital data , hence covering more than 95% of hospital discharges.18 the cmbd database is managed by the spanish ministry of health , social services and equality and includes patient variables ( sex and date of birth ) , date of admittance , date of discharge , up to 14 discharge diagnoses , and up to 20 procedures performed during hospitalization . the spanish ministry of health , social services and equality sets recording standards and performs periodic audits.18 we selected all surgical admissions of patients who underwent coronary revascularization procedures using the international classification of diseases ninth revision , clinical modification ( icd-9-cm ) . the procedure codes used were 36.1036.19 for cabg and 36.06 , 36.07 , 36.09 , and 0.66 for pci . discharges were grouped by copd status as follows : no copd and copd ( icd-9-cm codes : 490492 and 495496 , respectively ) . the outcomes of interest included the percentage of patients who died during hospitalization , defined as ihm and the mean los . clinical characteristics included information on overall comorbidity at the time of surgery , which was assessed by computing the charlson comorbidity index ( cci ) . the index applies to 17 disease categories whose scores are totaled to obtain an overall score for each patient.19 the 17 diseases included in the cci are myocardial infarction , congestive heart failure , peripheral vascular disease , cerebrovascular disease , dementia , copd , connective tissue disease , ulcer disease , diabetes , renal disease , hemiplegia , any tumor , leukemia , lymphoma , liver disease , metastatic solid tumor , and aids . the index is subsequently categorized into three levels : 0 , no disease ; 1 , one or two diseases ; and 2 , three or more diseases.20 to calculate the cci , we used 17 disease categories , excluding copd and acute myocardial infarction , as described by thomsen et al.20 a descriptive statistical analysis was performed . rates for copd and non - copd patients for each coronary revascularization procedure were calculated in terms of 100,000 inhabitants . we also calculated the yearly age- and sex - specific incidence rates for copd and non - copd patients , dividing the number of cases per year , sex , and age group by the corresponding number of people in that population group , according to data from the spanish national institute of statistics , as reported on december 31 of each year.21 in our study , we used joinpoint log - linear regression to identify the years in which changes in tendency occurred in the use of pci and cabg in patients with and without copd , as well as to estimate the annual percent change ( apc ) in each of the periods delimited by the points of change . the analysis started with the minimum number of joinpoints and tested whether the inclusion of one or more joinpoints was statistically significant.22 in the final model , each joinpoint indicated a significant change in the tendency , and the apc was obtained in each of the segments delimited by the joinpoints , using the weighted least squares technique . the joinpoint regression program version 4.0.4 was used for the analysis.23 in order to test the time trend for ihm , logistic regression analyses were performed with mortality as a binary outcome , using year of discharge , sex , age , and cci as independent variables . models were generated for copd and non - copd subjects and for the entire population , in order to compare the ihm of those who have the disease and those who do not . statistical analyses were performed using stata version 10.1 ( statacorp lp , college station , tx , usa ) . patient identifiers were deleted before the database was provided to the authors , in order to maintain patient anonymity . it is not possible to identify patients at individual levels , either in this article or in the database . given the anonymous and mandatory nature of the dataset , it was not necessary to obtain informed consent . the study protocol was approved by the ethics committee of the rey juan carlos university . rates for copd and non - copd patients for each coronary revascularization procedure were calculated in terms of 100,000 inhabitants . we also calculated the yearly age- and sex - specific incidence rates for copd and non - copd patients , dividing the number of cases per year , sex , and age group by the corresponding number of people in that population group , according to data from the spanish national institute of statistics , as reported on december 31 of each year.21 in our study , we used joinpoint log - linear regression to identify the years in which changes in tendency occurred in the use of pci and cabg in patients with and without copd , as well as to estimate the annual percent change ( apc ) in each of the periods delimited by the points of change . the analysis started with the minimum number of joinpoints and tested whether the inclusion of one or more joinpoints was statistically significant.22 in the final model , each joinpoint indicated a significant change in the tendency , and the apc was obtained in each of the segments delimited by the joinpoints , using the weighted least squares technique . the joinpoint regression program version 4.0.4 was used for the analysis.23 in order to test the time trend for ihm , logistic regression analyses were performed with mortality as a binary outcome , using year of discharge , sex , age , and cci as independent variables . models were generated for copd and non - copd subjects and for the entire population , in order to compare the ihm of those who have the disease and those who do not . statistical analyses were performed using stata version 10.1 ( statacorp lp , college station , tx , usa ) . patient identifiers were deleted before the database was provided to the authors , in order to maintain patient anonymity . it is not possible to identify patients at individual levels , either in this article or in the database . given the anonymous and mandatory nature of the dataset , it was not necessary to obtain informed consent . the study protocol was approved by the ethics committee of the rey juan carlos university . from 2001 to 2011 , a total of 508,135 admissions of patients who underwent scheduled or unscheduled coronary revascularization procedures were recorded in spain . over the study period , 7.17% ( n=36,478 ) of all patients who underwent coronary revascularization procedures had copd . there were a total of 428,516 pcis ( 6.91% [ n=29,630 ] in patients with copd ) and 79,619 cabgs ( 8.6% [ n=6,848 ] in patients with copd ) . in patients who underwent a pci , there was a significant male predominance in patients both with and without copd ( 92.82% and 75.54% , respectively ) . mean age was 69.78 years ( standard deviation [ sd ] , 9.55 years ) in patients with copd and 65.02 years ( sd , 11.37 years ) in those without copd ( p<0.01 ) . patients with copd who underwent pcis had higher cci values compared with those without copd ( 56.79% vs 43.75% with one or more coexisting conditions , respectively ) . among those who received a pci , the median los was significantly higher in patients with copd ( 7.00 days [ interquartile range ( iqr ) 8.00 days ] ) compared with those without copd ( 6.00 days [ iqr 7.00 days ] ) . also , ihm was significantly higher in patients with copd ( 2.97% ) compared with patients without copd ( 2.12% ) . according to the results of the joinpoint analysis , we found that the sex and age - adjusted use of pci increased by 21.27% per year from 2001 to 2004 and by 5.47% per year from 2004 to 2011 in patients with copd ( figure 1a ) . in patients without copd , the use of pci increased by 14.7% per year from 2001 to 2005 . from 2005 to 2008 , it increased by 5.21% per year , and from 2008 to 2011 it also increased by 0.48% per year , but not significantly in both cases ( figure 1b ) . table 1 shows time trend outcomes in annual pcis in patients with and without copd in spain , from 2001 to 2011 . we found that the mean age of patients with copd who underwent a pci was 68.438.95 years in 2001 and increased to 70.6119.85 years in 2011 ( p<0.05 ) . the prevalence of those with a cci of one or more increased from 46.95% in 2001 to 62.10% ( p<0.05 ) . los after pci decreased significantly over the study period in both groups of patients ( p<0.001 ) , showing higher values among those with copd in all years analyzed ( table 1 ) . the ihm among those who underwent a pci increased for those without copd ( from 1.94% in 2001 to 2.42% in 2011 , p<0.001 ) . this parameter also increased in patients with copd ( from 2.37% in 2001 to 2.83% in 2011 , p<0.01 ) ( table 1 ) . as can been seen in table 2 , after multivariate adjustment , the ihm among patients with copd who underwent a pci increased significantly from 2001 to 2011 ( odds ratio [ or ] 1.11 ; 95% confidence interval [ ci ] 1.031.20 ) . by contrast , the ihm did not change significantly according to sex . on the other hand , the ihm was significantly higher in those with one or two ( or 1.93 ; 95% ci 1.652.26 ) or three or more ( or 3.26 ; 95% ci 2.544.18 ) comorbidities associated with copd . we found that , in patients who underwent a cabg , the mean age was significantly higher in patients with copd than in those without copd ( 68.48 years [ sd 8.42 years ] vs 66.72 years [ sd 9.55 years ] ) and there was higher proportion of males undergoing cabg procedures in both groups ( 93.17% in patients with copd vs 76.49% in patients without copd , p<0.05 ) . in our study , patients with copd who underwent a cabg had higher cci values than those without copd ( 58.15% vs 51.84% with one or more comorbidities ) . in patients with copd , the ihm was significantly higher than in those without copd ( 7.75% vs 7.17% ) ; however , the median los was significantly higher in patients with copd ( 17.00 days [ iqr 17.00 days ] compared with those without copd ( 15.00 days [ iqr 14.00 days ] ) . among patients with copd who underwent cabg , the sex and age - adjusted cabg incidence rate increased by 9.77% per year from 2001 to 2003 , then decreased by 3.15% through 2011 ( figure 1c ) . in patients without copd , the incidence rate decreased at a constant rate of 2.69% per year over the entire period of study ( figure 1d ) . as can be seen in table 3 , over the 11-year study period , the mean age of patients with copd who underwent cabg was 66.828.34 years in 2001 and 69.38.45 years in 2011 . the proportion of men was 94.24% in 2001 and 91.68% in 2011 ( no significant differences ) . in our study , los in patients with copd decreased significantly from 18 days ( iqr , 19 days ) in 2001 to 16 days ( iqr , 16 days ) in 2011 ( p<0.01 ) . in patients without copd , los also decreased significantly over the period of study ( table 3 ) . the ihm decreased significantly for those with and without copd during the 11-year study period ( 10.59% in 2001 vs 5.99% in 2011 and 8.85% vs 5.92% , respectively ) ( table 3 ) . after multivariate adjustment , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period ( or 0.93 ; 95% ci 0.900.96 ) . on the other hand , it did not change significantly according to sex . finally , the ihm was significantly higher in patients with copd with one or two ( or 1.47 ; 95% ci 1.211.78 ) and with three or more ( or 2.58 ; 95% ci 1.783.72 ) comorbidities . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd ( table 2 ) . in patients who underwent a pci , there was a significant male predominance in patients both with and without copd ( 92.82% and 75.54% , respectively ) . mean age was 69.78 years ( standard deviation [ sd ] , 9.55 years ) in patients with copd and 65.02 years ( sd , 11.37 years ) in those without copd ( p<0.01 ) . patients with copd who underwent pcis had higher cci values compared with those without copd ( 56.79% vs 43.75% with one or more coexisting conditions , respectively ) . among those who received a pci , the median los was significantly higher in patients with copd ( 7.00 days [ interquartile range ( iqr ) 8.00 days ] ) compared with those without copd ( 6.00 days [ iqr 7.00 days ] ) . also , ihm was significantly higher in patients with copd ( 2.97% ) compared with patients without copd ( 2.12% ) . according to the results of the joinpoint analysis , we found that the sex and age - adjusted use of pci increased by 21.27% per year from 2001 to 2004 and by 5.47% per year from 2004 to 2011 in patients with copd ( figure 1a ) . in patients without copd , the use of pci increased by 14.7% per year from 2001 to 2005 . from 2005 to 2008 , it increased by 5.21% per year , and from 2008 to 2011 it also increased by 0.48% per year , but not significantly in both cases ( figure 1b ) . table 1 shows time trend outcomes in annual pcis in patients with and without copd in spain , from 2001 to 2011 . we found that the mean age of patients with copd who underwent a pci was 68.438.95 years in 2001 and increased to 70.6119.85 years in 2011 ( p<0.05 ) . the prevalence of those with a cci of one or more increased from 46.95% in 2001 to 62.10% ( p<0.05 ) . los after pci decreased significantly over the study period in both groups of patients ( p<0.001 ) , showing higher values among those with copd in all years analyzed ( table 1 ) . the ihm among those who underwent a pci increased for those without copd ( from 1.94% in 2001 to 2.42% in 2011 , p<0.001 ) . this parameter also increased in patients with copd ( from 2.37% in 2001 to 2.83% in 2011 , p<0.01 ) ( table 1 ) . as can been seen in table 2 , after multivariate adjustment , the ihm among patients with copd who underwent a pci increased significantly from 2001 to 2011 ( odds ratio [ or ] 1.11 ; 95% confidence interval [ ci ] 1.031.20 ) . by contrast , the ihm did not change significantly according to sex . on the other hand , the ihm was significantly higher in those with one or two ( or 1.93 ; 95% ci 1.652.26 ) or three or more ( or 3.26 ; 95% ci 2.544.18 ) comorbidities associated with copd . we found that , in patients who underwent a cabg , the mean age was significantly higher in patients with copd than in those without copd ( 68.48 years [ sd 8.42 years ] vs 66.72 years [ sd 9.55 years ] ) and there was higher proportion of males undergoing cabg procedures in both groups ( 93.17% in patients with copd vs 76.49% in patients without copd , p<0.05 ) . in our study , patients with copd who underwent a cabg had higher cci values than those without copd ( 58.15% vs 51.84% with one or more comorbidities ) . in patients with copd , the ihm was significantly higher than in those without copd ( 7.75% vs 7.17% ) ; however , the median los was significantly higher in patients with copd ( 17.00 days [ iqr 17.00 days ] compared with those without copd ( 15.00 days [ iqr 14.00 days ] ) . among patients with copd who underwent cabg , the sex and age - adjusted cabg incidence rate increased by 9.77% per year from 2001 to 2003 , then decreased by 3.15% through 2011 ( figure 1c ) . in patients without copd , the incidence rate decreased at a constant rate of 2.69% per year over the entire period of study ( figure 1d ) . as can be seen in table 3 , over the 11-year study period , the mean age of patients with copd who underwent cabg was 66.828.34 years in 2001 and 69.38.45 years in 2011 . the proportion of men was 94.24% in 2001 and 91.68% in 2011 ( no significant differences ) . in our study , los in patients with copd decreased significantly from 18 days ( iqr , 19 days ) in 2001 to 16 days ( iqr , 16 days ) in 2011 ( p<0.01 ) . in patients without copd , the ihm decreased significantly for those with and without copd during the 11-year study period ( 10.59% in 2001 vs 5.99% in 2011 and 8.85% vs 5.92% , respectively ) ( table 3 ) . after multivariate adjustment , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period ( or 0.93 ; 95% ci 0.900.96 ) . on the other hand , it did not change significantly according to sex . finally , the ihm was significantly higher in patients with copd with one or two ( or 1.47 ; 95% ci 1.211.78 ) and with three or more ( or 2.58 ; 95% ci 1.783.72 ) comorbidities . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd ( table 2 ) . in the present study , we found different trends over the last 11 years in the hospitalizations of subjects with and without copd who underwent coronary revascularization procedures . our results reveal that patients with copd account for 7.17% of all revascularization procedures in spain . we found an increase in pci procedure rates from 2001 to 2011 ; and a decline in hospital admissions for cabg in patients with copd from 2003 to 2011 . although there are no previous studies that have assessed the trends in coronary revascularization procedure for copd over time , our results are consistent with studies conducted in patients with other chronic diseases . so , for example , in patients with diabetes , it has been observed that pci rates have increased significantly due to advances in stent device technology and adjunctive pharmacology . by contrast , cabg rates have declined due to the fact that this procedure is more invasive than pci procedures.24,25 in spain , as other countries , the indications for pci have become wider over the last years ; more hospitals have included units where pci procedures are conducted ; pci have improved in effectiveness in the last years and have very low morbidity , mortality , and cost compared with cabg . these tendencies have been found in studies conducted elsewhere.2628 we found that ihm increased significantly over time among copd patients after pci . in addition , the ihm was significantly higher in those with one or more comorbidities associated with copd . previous studies that focused on mortality in patients undergoing pci suggested worse prognosis in patients with copd.12,13 more recently , konecny et al5 have shown that there is a clearly increased mortality rate with each additional level of copd severity ; namely , patients with mild or moderate copd did better than those with severe copd who , in turn , did better than those with very severe copd . patients without copd had better survival than those with any level of copd . in another study in which it was assessed the incidence of copd in patients with acute st - segment elevation myocardial infarction undergoing primary pci and the impact of copd on the prognostic outcome of this patient population , it was demonstrated that copd was not an independent predictor of short - term and medium - term mortality.29 although the inconsistent findings between this study and previous reports remain poorly explained , there are some reasons that may explain the discrepancy . first , the enrollment and exclusion criteria and the diagnosis of copd may not be identical between the different studies . second , primary pci , which was not a criterion for study enrollment in the majority of the recent studies , was performed in all patients in the last study.29 in any case , there are no previous studies that have evaluated the evolution of mortality in these patients over time . incomplete revascularization is the frequent final outcome in patients with multivessel coronary disease who undergo pci . however , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period . leavit et al30 also showed , to assess the long - term survival of patients with copd undergoing cabg , that the presence of copd and $ 1 other comorbidity significantly reduced the 10-year survival . in copd patients with 1 other comorbidity , the 10-year survival was 30% , and the incidence rate of death was 9.4 deaths per 100 person years of follow - up . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd in our study . several studies have also demonstrated similar ihm in cabg patients with and without copd.3134 manganas et al34 found no evidence of increased post - cabg mortality in patients with copd , although these patients demonstrated an increased incidence of postoperative chest infections and atrial fibrillation , and required longer hospital stay . in a large retrospective analysis , angouras et al35 did not find any difference ihm and major morbidities between patients with and without copd , although those with copd had a longer hospital stay and a higher long - term mortality rate . by contrast , other studies have reported an increased risk for long - term mortality and lower survival in copd patients.30,36,37 so , saleh et al38 found , in a large retrospective study , that the severity of copd proved to be a prognostic marker in patients undergoing cabg . more advanced stages of copd , rather than mild or moderate forms , were found to be significantly associated with early mortality following cabg . to improve the outcomes , it has been suggested that off - pump coronary artery bypass grafting may be more suitable for patients with pulmonary diseases with a lower mortality and morbidity risk as compared with conventional cabg . however , almassi et al39 did not find benefit to use an off - pump coronary artery bypass approach rather than conventional cabg in copd patients . in our investigation , the median los for pci decreased from 9 days in 2001 to 6 days in 2011 , among copd patients , and from 6 to 5 days among non - copd patients . with regard to cabg , the median was 18 days in 2001 and 16 days in 2011 for those with copd , with equivalent figures of 17 and 14 days for those without copd . postoperative respiratory complications can increase hospital los as well as midterm mortality of cabg patients with severe left ventricular dysfunction.40 these , together with prolonged mechanical ventilation time , have been also identified as independent risk factors of postoperative acute renal failure requiring renal replacement therapy after cardiac surgery in patients with preoperatively normal renal functions , a complication that increases hospital los and ihm.41 in patients without copd who had undergone either a pci or a cabg , women had worse outcomes than men . these results contrast with those obtained in other chronic diseases such as diabetes where it has been suggested that the worse effect of diabetes on outcomes in women might be related to the onset mechanism for cardiovascular disease , the success of the revascularization coronary procedure , and the higher burden of cardiovascular risk factors.42,43 we should recognize strength and limitations of the current study . the main strength lies in its large sample size , its 11-year follow - up period , and its standardized methodology maintained constant along the study period . first , a potential source of bias comes from the use of icd-9-cm diagnosis codes for identifying patients hospitalized for copd.44,45 it might generate underdiagnosis of copd , because all patients receiving pci or cabg did not perform pulmonary function tests . however , the icd-9-cm codes have been previously used for epidemiological investigations and proved to be a valid method to identify patients suffering from this disease.46,47 in relation to cad , we have not provided information of this diagnosis , such as st - elevation myocardial infarction , non - st - elevation acute coronary syndrome , or stable angina . by contrast if we stratify cad , we would lose power because some clinical categories would have a very small number of subjects . on the other hand , this study and its outcomes were limited to the variables coded which do not include relevant data such as the extent of the coronary disease , the severity of copd , or the pharmacologic treatment . in addition , the endpoint of this study is limited to short - term mortality and we can not provide information of long - term mortality . another limitation of this database is its anonymity ( no identifying items such as clinical history number ) , which makes it impossible to detect whether the same patient was admitted more than once during the same year . nevertheless the administrative database used in our study was introduced in spain in 1982 , is a mandatory register , and its coverage is estimated to be greater than 95%.18 concerns have been raised about the accuracy of routinely collected datasets ; however , these datasets are periodically audited . consequently , the quality and validity of our dataset have been assessed and shown to be useful for health research.48 another limitation of our study is the missing causal relationship between ihm and cabg / pci as patients admitted for cancer and non - cardiovascular were also included , the mortality of whom may not have been affected by the cabg / pci . our results show that the apc in pci procedures increased over study period in copd and non - copd patients . other outcome such as los was worse among individuals with copd than those without copd for pci although they improved over the entire study period for both groups . higher comorbidity was associated with higher ihm in pci procedures in both groups of patients . on the other hand , we found a decrease in the use of cabg procedures in patients with and without copd . the ihm did not change according to the existence , or not , of copd among patients who underwent a cabg . these findings emphasize the need for further improvement in the control of cardiovascular risk factors in people with copd .

backgroundpeople with copd suffering from coronary artery disease are frequently treated with revascularization procedures . we aim to compare trends in the use and outcomes of these procedures in copd and non - copd patients in spain between 2001 and 2011.methodswe identified all patients who had undergone percutaneous coronary interventions ( pcis ) and coronary artery bypass graft ( cabg ) surgeries , using national hospital discharge data . discharges were grouped into : copd and no copd.resultsfrom 2001 to 2011 , 428,516 pcis and 79,619 cabgs were performed . the sex and age - adjusted use of pci increased by 21.27% per year from 2001 to 2004 and by 5.47% per year from 2004 to 2011 in patients with copd . in - hospital mortality ( ihm ) among patients with copd who underwent a pci increased significantly from 2001 to 2011 ( odds ratio 1.11 ; 95% confidence interval 1.031.20 ) . among patients with copd who underwent a cabg , the sex and age - adjusted cabg incidence rate increased by 9.77% per year from 2001 to 2003 , and then decreased by 3.15% through 2011 . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd ( odds ratio , 1.06 ; 95% confidence interval 0.961.17).conclusionthe annual percent change in pci procedures increased in copd and non - copd patients . we found a decrease in the use of cabg procedures in both groups . ihm was higher in patients with copd who underwent a pci than in those without copd . however , copd did not increase the probability of dying during hospitalization in patients who underwent a cabg .

Background Methods Statistical analysis Ethical aspects Results Percutaneous coronary intervention Coronary artery bypass graft Discussion Conclusion

the association between copd and coronary artery disease ( cad ) is well known.1 in studies of the general population , it has been shown an increased all - cause mortality as well as cardiovascular mortality in patients with copd.2,3 patients with mild copd seem to have even a higher risk of dying from cardiovascular causes than from respiratory insufficiency.4 copd is highly prevalent among patients with cad.5 the causal connection between both diseases has historically been cigarette smoking.6 however , a recent epidemiological study has shown that systemic inflammation plays a significant role in both atherogenesis and copd.7 other evidence that highlights the close relationship between copd and cad is the fact that this pulmonary disease is an independent factor of poor outcome and mortality after coronary revascularization procedures.8 the diagnosis of copd was , together with other important clinical variables such as age , sex , and left ventricular ejection fraction , a predictor of mortality at 4 years of revascularization in the syntax ii score ( index that punctuates the complexity of cad by angiography and it helps to decide the optimal method of revascularization in patients with cad complex).9 as a result , treatment of cad in patients with copd can present therapeutic challenges . given that copd is associated with higher risk of adverse events after coronary artery bypass graft ( cabg ) surgery,10,11 percutaneous coronary intervention ( pci ) is now the most commonly used reperfusion procedure for copd patients undergoing revascularization.6 in fact , patients with copd currently make up almost 10% of patients undergoing pci.12 nonetheless , several studies have reported and elevated the risk of adverse outcomes among patients with copd after pci.5,1214 secular trends in the use of coronary revascularization procedures have been examined.15 culler et al16 have identified , in a recent retrospective study , all medicare beneficiaries undergoing a coronary revascularization procedure between 2008 and 2012 . however , despite the poor prognosis of patients with copd after pci and cabg , no previous studies have evaluated the changes in utilization and outcomes of coronary revascularization procedures over time in these patients . it is possible that may be there have been advances in recent years , not only in surgical techniques , but also in monitoring and anesthesia , which they could have helped to improve results.17 in this study , we used national hospital discharge data to describe and compare trends in the use of coronary revascularization procedures in copd and non - copd patients between 2001 and 2011 in spain . in particular , we analyzed trends in the use of cabg and pci , patient comorbidities , and in - hospital outcomes such as length of stay ( los ) and in - hospital mortality ( ihm ) . discharges were grouped by copd status as follows : no copd and copd ( icd-9-cm codes : 490492 and 495496 , respectively ) . rates for copd and non - copd patients for each coronary revascularization procedure were calculated in terms of 100,000 inhabitants . we also calculated the yearly age- and sex - specific incidence rates for copd and non - copd patients , dividing the number of cases per year , sex , and age group by the corresponding number of people in that population group , according to data from the spanish national institute of statistics , as reported on december 31 of each year.21 in our study , we used joinpoint log - linear regression to identify the years in which changes in tendency occurred in the use of pci and cabg in patients with and without copd , as well as to estimate the annual percent change ( apc ) in each of the periods delimited by the points of change . we also calculated the yearly age- and sex - specific incidence rates for copd and non - copd patients , dividing the number of cases per year , sex , and age group by the corresponding number of people in that population group , according to data from the spanish national institute of statistics , as reported on december 31 of each year.21 in our study , we used joinpoint log - linear regression to identify the years in which changes in tendency occurred in the use of pci and cabg in patients with and without copd , as well as to estimate the annual percent change ( apc ) in each of the periods delimited by the points of change . from 2001 to 2011 , a total of 508,135 admissions of patients who underwent scheduled or unscheduled coronary revascularization procedures were recorded in spain . there were a total of 428,516 pcis ( 6.91% [ n=29,630 ] in patients with copd ) and 79,619 cabgs ( 8.6% [ n=6,848 ] in patients with copd ) . in patients who underwent a pci , there was a significant male predominance in patients both with and without copd ( 92.82% and 75.54% , respectively ) . mean age was 69.78 years ( standard deviation [ sd ] , 9.55 years ) in patients with copd and 65.02 years ( sd , 11.37 years ) in those without copd ( p<0.01 ) . patients with copd who underwent pcis had higher cci values compared with those without copd ( 56.79% vs 43.75% with one or more coexisting conditions , respectively ) . among those who received a pci , the median los was significantly higher in patients with copd ( 7.00 days [ interquartile range ( iqr ) 8.00 days ] ) compared with those without copd ( 6.00 days [ iqr 7.00 days ] ) . also , ihm was significantly higher in patients with copd ( 2.97% ) compared with patients without copd ( 2.12% ) . according to the results of the joinpoint analysis , we found that the sex and age - adjusted use of pci increased by 21.27% per year from 2001 to 2004 and by 5.47% per year from 2004 to 2011 in patients with copd ( figure 1a ) . in patients without copd , the use of pci increased by 14.7% per year from 2001 to 2005 . from 2005 to 2008 , it increased by 5.21% per year , and from 2008 to 2011 it also increased by 0.48% per year , but not significantly in both cases ( figure 1b ) . table 1 shows time trend outcomes in annual pcis in patients with and without copd in spain , from 2001 to 2011 . we found that the mean age of patients with copd who underwent a pci was 68.438.95 years in 2001 and increased to 70.6119.85 years in 2011 ( p<0.05 ) . the ihm among those who underwent a pci increased for those without copd ( from 1.94% in 2001 to 2.42% in 2011 , p<0.001 ) . this parameter also increased in patients with copd ( from 2.37% in 2001 to 2.83% in 2011 , p<0.01 ) ( table 1 ) . as can been seen in table 2 , after multivariate adjustment , the ihm among patients with copd who underwent a pci increased significantly from 2001 to 2011 ( odds ratio [ or ] 1.11 ; 95% confidence interval [ ci ] 1.031.20 ) . on the other hand , the ihm was significantly higher in those with one or two ( or 1.93 ; 95% ci 1.652.26 ) or three or more ( or 3.26 ; 95% ci 2.544.18 ) comorbidities associated with copd . we found that , in patients who underwent a cabg , the mean age was significantly higher in patients with copd than in those without copd ( 68.48 years [ sd 8.42 years ] vs 66.72 years [ sd 9.55 years ] ) and there was higher proportion of males undergoing cabg procedures in both groups ( 93.17% in patients with copd vs 76.49% in patients without copd , p<0.05 ) . in our study , patients with copd who underwent a cabg had higher cci values than those without copd ( 58.15% vs 51.84% with one or more comorbidities ) . in patients with copd , the ihm was significantly higher than in those without copd ( 7.75% vs 7.17% ) ; however , the median los was significantly higher in patients with copd ( 17.00 days [ iqr 17.00 days ] compared with those without copd ( 15.00 days [ iqr 14.00 days ] ) . among patients with copd who underwent cabg , the sex and age - adjusted cabg incidence rate increased by 9.77% per year from 2001 to 2003 , then decreased by 3.15% through 2011 ( figure 1c ) . as can be seen in table 3 , over the 11-year study period , the mean age of patients with copd who underwent cabg was 66.828.34 years in 2001 and 69.38.45 years in 2011 . in our study , los in patients with copd decreased significantly from 18 days ( iqr , 19 days ) in 2001 to 16 days ( iqr , 16 days ) in 2011 ( p<0.01 ) . after multivariate adjustment , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period ( or 0.93 ; 95% ci 0.900.96 ) . finally , the ihm was significantly higher in patients with copd with one or two ( or 1.47 ; 95% ci 1.211.78 ) and with three or more ( or 2.58 ; 95% ci 1.783.72 ) comorbidities . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd ( table 2 ) . in patients who underwent a pci , there was a significant male predominance in patients both with and without copd ( 92.82% and 75.54% , respectively ) . mean age was 69.78 years ( standard deviation [ sd ] , 9.55 years ) in patients with copd and 65.02 years ( sd , 11.37 years ) in those without copd ( p<0.01 ) . patients with copd who underwent pcis had higher cci values compared with those without copd ( 56.79% vs 43.75% with one or more coexisting conditions , respectively ) . among those who received a pci , the median los was significantly higher in patients with copd ( 7.00 days [ interquartile range ( iqr ) 8.00 days ] ) compared with those without copd ( 6.00 days [ iqr 7.00 days ] ) . also , ihm was significantly higher in patients with copd ( 2.97% ) compared with patients without copd ( 2.12% ) . according to the results of the joinpoint analysis , we found that the sex and age - adjusted use of pci increased by 21.27% per year from 2001 to 2004 and by 5.47% per year from 2004 to 2011 in patients with copd ( figure 1a ) . in patients without copd , the use of pci increased by 14.7% per year from 2001 to 2005 . from 2005 to 2008 , it increased by 5.21% per year , and from 2008 to 2011 it also increased by 0.48% per year , but not significantly in both cases ( figure 1b ) . table 1 shows time trend outcomes in annual pcis in patients with and without copd in spain , from 2001 to 2011 . we found that the mean age of patients with copd who underwent a pci was 68.438.95 years in 2001 and increased to 70.6119.85 years in 2011 ( p<0.05 ) . the ihm among those who underwent a pci increased for those without copd ( from 1.94% in 2001 to 2.42% in 2011 , p<0.001 ) . this parameter also increased in patients with copd ( from 2.37% in 2001 to 2.83% in 2011 , p<0.01 ) ( table 1 ) . as can been seen in table 2 , after multivariate adjustment , the ihm among patients with copd who underwent a pci increased significantly from 2001 to 2011 ( odds ratio [ or ] 1.11 ; 95% confidence interval [ ci ] 1.031.20 ) . on the other hand , the ihm was significantly higher in those with one or two ( or 1.93 ; 95% ci 1.652.26 ) or three or more ( or 3.26 ; 95% ci 2.544.18 ) comorbidities associated with copd . we found that , in patients who underwent a cabg , the mean age was significantly higher in patients with copd than in those without copd ( 68.48 years [ sd 8.42 years ] vs 66.72 years [ sd 9.55 years ] ) and there was higher proportion of males undergoing cabg procedures in both groups ( 93.17% in patients with copd vs 76.49% in patients without copd , p<0.05 ) . in our study , patients with copd who underwent a cabg had higher cci values than those without copd ( 58.15% vs 51.84% with one or more comorbidities ) . in patients with copd , the ihm was significantly higher than in those without copd ( 7.75% vs 7.17% ) ; however , the median los was significantly higher in patients with copd ( 17.00 days [ iqr 17.00 days ] compared with those without copd ( 15.00 days [ iqr 14.00 days ] ) . among patients with copd who underwent cabg , the sex and age - adjusted cabg incidence rate increased by 9.77% per year from 2001 to 2003 , then decreased by 3.15% through 2011 ( figure 1c ) . in patients without copd , the incidence rate decreased at a constant rate of 2.69% per year over the entire period of study ( figure 1d ) . as can be seen in table 3 , over the 11-year study period , the mean age of patients with copd who underwent cabg was 66.828.34 years in 2001 and 69.38.45 years in 2011 . in our study , los in patients with copd decreased significantly from 18 days ( iqr , 19 days ) in 2001 to 16 days ( iqr , 16 days ) in 2011 ( p<0.01 ) . in patients without copd , the ihm decreased significantly for those with and without copd during the 11-year study period ( 10.59% in 2001 vs 5.99% in 2011 and 8.85% vs 5.92% , respectively ) ( table 3 ) . after multivariate adjustment , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period ( or 0.93 ; 95% ci 0.900.96 ) . finally , the ihm was significantly higher in patients with copd with one or two ( or 1.47 ; 95% ci 1.211.78 ) and with three or more ( or 2.58 ; 95% ci 1.783.72 ) comorbidities . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd ( table 2 ) . in the present study , we found different trends over the last 11 years in the hospitalizations of subjects with and without copd who underwent coronary revascularization procedures . our results reveal that patients with copd account for 7.17% of all revascularization procedures in spain . we found an increase in pci procedure rates from 2001 to 2011 ; and a decline in hospital admissions for cabg in patients with copd from 2003 to 2011 . by contrast , cabg rates have declined due to the fact that this procedure is more invasive than pci procedures.24,25 in spain , as other countries , the indications for pci have become wider over the last years ; more hospitals have included units where pci procedures are conducted ; pci have improved in effectiveness in the last years and have very low morbidity , mortality , and cost compared with cabg . previous studies that focused on mortality in patients undergoing pci suggested worse prognosis in patients with copd.12,13 more recently , konecny et al5 have shown that there is a clearly increased mortality rate with each additional level of copd severity ; namely , patients with mild or moderate copd did better than those with severe copd who , in turn , did better than those with very severe copd . however , the ihm among patients with copd who underwent a cabg decreased significantly over the entire study period . in copd patients with 1 other comorbidity , the 10-year survival was 30% , and the incidence rate of death was 9.4 deaths per 100 person years of follow - up . the probability of dying during hospitalization in patients who underwent a cabg did not change significantly in patients with and without copd in our study . several studies have also demonstrated similar ihm in cabg patients with and without copd.3134 manganas et al34 found no evidence of increased post - cabg mortality in patients with copd , although these patients demonstrated an increased incidence of postoperative chest infections and atrial fibrillation , and required longer hospital stay . in a large retrospective analysis , angouras et al35 did not find any difference ihm and major morbidities between patients with and without copd , although those with copd had a longer hospital stay and a higher long - term mortality rate . however , almassi et al39 did not find benefit to use an off - pump coronary artery bypass approach rather than conventional cabg in copd patients . in our investigation , the median los for pci decreased from 9 days in 2001 to 6 days in 2011 , among copd patients , and from 6 to 5 days among non - copd patients . with regard to cabg , the median was 18 days in 2001 and 16 days in 2011 for those with copd , with equivalent figures of 17 and 14 days for those without copd . postoperative respiratory complications can increase hospital los as well as midterm mortality of cabg patients with severe left ventricular dysfunction.40 these , together with prolonged mechanical ventilation time , have been also identified as independent risk factors of postoperative acute renal failure requiring renal replacement therapy after cardiac surgery in patients with preoperatively normal renal functions , a complication that increases hospital los and ihm.41 in patients without copd who had undergone either a pci or a cabg , women had worse outcomes than men . first , a potential source of bias comes from the use of icd-9-cm diagnosis codes for identifying patients hospitalized for copd.44,45 it might generate underdiagnosis of copd , because all patients receiving pci or cabg did not perform pulmonary function tests . our results show that the apc in pci procedures increased over study period in copd and non - copd patients . higher comorbidity was associated with higher ihm in pci procedures in both groups of patients . on the other hand , we found a decrease in the use of cabg procedures in patients with and without copd . the ihm did not change according to the existence , or not , of copd among patients who underwent a cabg .

gold nanoparticles ( aunps ) belong to a class of nanometer - sized gold structures that display unique photothermal and optical properties due to their surface plasmon resonance ( spr ) . shell hybrid nanostructure allows applications of such properties in the design of catalysts , sensors , imaging devices , photothermal agents , and delivery systems of genes and therapeutic agents for targeted therapy . a large fraction of these studies have focused on exploring new functional applications of the aunp - based nanoparticles based on their photophysical properties . however , three - dimensional features that vary in response to the interaction between aunp - based nanoparticles and their biological targets such as particle size distribution can provide physical insights on their interaction although they remain much less characterized in most aunp - based delivery systems . here , we studied the interaction of a dendrimer chemisorbed aunp with its protein target by atomic force microscopy ( afm ) as a biophysical method to correlate protein binding with the geometrical alteration of the aunp nanocomposite . this study demonstrates that afm serves as an effective technique for characterizing the interaction of aunp - based nanoparticles and target proteins in vitro by quantitative measurement of size distribution . dendrimer nps used for surface modification of aunps are based on a fifth generation ( g5 ) poly(amido amine ) ( pamam ) dendrimer conjugated with a riboflavin ( rf ) molecule . as a vitamin molecule ( b2 ) essential for the biosynthesis of flavin - based redox cofactors , rf is taken up by riboflavin receptors , also referred to as rf carriers , which are expressed as both soluble and membrane - bound isoforms . moreover , rf receptors constitute one type of potential tumor biomarker due to their overexpression in certain malignant cells from human breast and prostate cancers . this biomarker is relatively new as a receptor considered for cancer targeting compared to other biomarker proteins that include folic acid receptor ( far ) , v3 integrin , prostate - specific membrane antigen , her2 receptor , and epidermal growth factor receptor . recently , we applied the concept of targeted drug delivery to the rf receptor by designing rf - conjugated pamam dendrimer nanoparticles and demonstrated their effectiveness in vitro for rf receptor targeted delivery of methotrexate ( mtx ) in kb cancer cells that overexpress the riboflavin receptors . in a follow - up study , we employed isothermal titration calorimetry ( itc ) and differential scanning calorimetry ( dsc ) to address the thermodynamic aspects of the rf receptor ligand interactions for rf - conjugated dendrimers . combination of these two methods provided biophysical information important for the correlation of binding affinity to design factors such as ligand valency and the rf attachment position of the dendrimer conjugates . these studies allowed characterization of the structural features dictating the thermodynamic aspects of the interaction between the dendrimer and the receptor protein . in the current study , we employed afm for structural characterization of rf receptor targeted aunp systems . afm has been used for the imaging of pamam dendrimer nps of various dendrimer sizes . the rigidity of generation 7 or higher dendrimers makes it possible to image them by afm , whereas the softer g5 dendrimers used for rf conjugation ( d = 5.4 nm ) in this study tend to flatten on mica surfaces , providing fuzzy afm images . first , aunp brings a number of photophysical and photothermal properties , and its surface functionalization with the dendrimer platform creates a hybrid nanosystem that enables both imaging and specific cancer targeting . second , in contrast to the soft dendrimer , the aunp provides advantages for performing the afm study primarily because it can serve as a marker , due to its metallic core and can be obtained in sizes greater than the dendrimer np . hence afm is ideally suited for the determination of the size alteration as a quantifiable physical property that follows binding of the target protein to the dendrimer - conjugated aunp . recently , structural characterization of aunp hybrids by afm has been demonstrated for a number of aunp hybrid systems , each aunp conjugated with oligonucleotide ( dna ) probes , polyphenylene dendrimer , peg , poly(4-vinylpyridine ) , and cyclodextrin . finally , afm can image surfaces with nanoscale resolution , and unlike other imaging techniques such as transmission electron microscopy ( tem ) , it can image a variety of biomacromolecular complexes and biological events under nearly physiological conditions . as a result , afm has been widely applied to numerous applications in biology and drug discovery . the present report focuses on afm characterization of aunps functionalized on the surface with dendrimer nps as a new class of rf receptor targeted delivery systems . this afm study was performed in combination with complementary techniques including uv vis spectrometry and in vitro confocal microscopy to image binding of the dendrimer - functionalized aunps to riboflavin receptor in a cancer cell . this study illustrates how physical measurement of particle size distribution of aunps by afm enables the characterization and precise monitoring of the biological interaction between a dendrimer conjugated aunp and its target protein in a physiological solution . unless noted otherwise , all reagents and solvents were purchased from sigma - aldrich including ( )--lipoic acid ( 98% ) , ( )-riboflavin ( 98% ) , riboflavin binding protein ( apo - form ) , 3-(aminopropyl)triethoxysilane , and gold(iii ) chloride trihydrate ( 99.9% ) . a fifth generation ( g5 ) pamam dendrimer ( g5-nh2 ) was purchased from dendritech , inc . ( midland , mi ) as a solution in methanol ( 17.5% by wt / wt ) . it was purified by dialysis against deionized water prior to use as described elsewhere . the mean number ( n = 114 ) of primary amines per g5-(nh2)n was determined by potentiometric titration using a mettler toledo mp230 ph meter equipped with an inlab micro electrode . each modified dendrimer was purified by dialysis using cellulose membrane tubing from spectrum laboratories ( mwco 10 000 ) . each dendrimer , including the parent and surface - modified g5 dendrimers , was fully characterized by a number of standard analytical methods as described below . nmr characterization of the dendrimers was performed at 500 mhz for h nucleus using a varian nmr spectrometer.h nmr spectra are reported in ppm relative to an internal standard 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt ( dss ; = 0.00 ppm ) . molecular weights of dendrimers conjugated with lipoic acid and riboflavin were determined by matrix - assisted laser desorption ionization time - of - flight ( maldi tof ) mass spectrometry with a waters tofspec-2e spectrometer ( mw for a parent g5 dendrimer = 27 600 g / mol ) . uv vis spectrometry was performed on a perkin - elmer lambda 20 spectrophotometer . the purity of each dendrimer was determined by ultraperformance liquid chromatography ( uplc ) on a waters acquity peptide mapping system equipped with a waters photodiode array detector . each sample was run on a c4 beh column ( 150 2.1 mm , 300 ) with a linear gradient method beginning with 98:2 ( v / v ) water / acetonitrile ( with trifluoroacetic acid at 0.14 wt % in each eluent ) at a flow rate of 1 ml / min . molecular weights ( mw , mn ) and polydispersity index values ( pdi = mw / mn ) of g5 pamam dendrimer were determined by gel permeation chromatography ( gpc ) , and details for the experimental method are already described earlier . the pdi value determined for the purified g5 dendrimer was 1.010 ( mn = 26 270 g / mol ) . the size distribution of gold nanoparticles ( aunps ) was determined by dynamic light scattering measured at 25 c on a zetasizer nano zs ( malvern ) . to a solution of ( )-lipoic acid ( 29 mg , 0.14 mmol ) in dmf ( 5 ml ) was added n - hydroxybenzotriazole ( hobt ; 22 mg , 0.14 mmol ) , n , n-diisopropylethylamine ( dipea ; 49 l , 0.28 mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edc ; 56 mg , 0.29 mmol ) . the reaction mixture was stirred at room temperature ( rt ) for 4 h. g5 pamam dendrimer ( 250 mg , 9.4 mol ) was dissolved in meoh ( 100 ml ) in a separate reaction flask . to this dendrimer solution was then added the activated ester of lipoic acid prepared above while the mixture was stirred . the final mixture was stirred for 15 h , and excess unreacted amine groups of the dendrimer were fully modified by reacting with glutaric anhydride ( 210 mg , 1.84 mmol ) and dipea ( 310 l , 1.8 mmol ) . after stirring for 6 h , the mixture was concentrated in vacuo , and the residue was dissolved in 10 ml of phosphate buffered saline ( pbs , ph 7.4 ) . the solution was loaded into a membrane dialysis bag ( mwco 10 kda ) , and dialyzed against pbs ( 2 2l ) and deionized water ( 3 2l ) over 2 days until its dendrimer purity was greater than 95% as determined by analytical uplc . the dialyzed solution was collected and freeze - dried to afford 3 g5(la)10 as a pale beige solid ( 378 mg ) . maldi tof determined molecular weight ( m / z , g mol ) : 40 700 . h nmr ( 500 mhz , d2o ) : 3.443.36 ( broad ) , 3.32 ( br s ) , 3.062.9 ( br ) , 2.862.72 ( br ) , 2.562.46 ( br ) , 2.282.23 ( br t ) , 2.22.17 ( br t ) , 1.861.79 ( br quin ) , 1.6 ( br m ) , 1.4 ( br m ) ppm ( abbreviation : br = broad ; s = singlet ; t = triplet ; quin = quintet ; m = multiplet ) . the theoretical number of terminal branches per g5 dendrimer is 128 ( experimentally , 114 based on potentiometric titration ) , but only a fraction of the branches are shown for clarity . in conjugates 3 and 4 , each branch is terminated with a glutaric acid group unless its carboxylic acid is further conjugated with la or rf . reagents and conditions : ( i ) ethyl bromoacetate , k2co3 , dmf , 85 c ; ( ii ) 6 m hcl , 90 c ; ( iii ) 2,2-(ethylenedioxy)bis(ethylamine ) , pybop , hobt , n , n-diisopropylethylamine , dmf , rt ; ( iv ) ( a ) edchcl , hobt , n , n-diisopropylethylamine , ( )-lipoic acid , rt ; ( b ) glutaric anhydride ( 180 equiv to dendrimer ) , n , n-diisopropylethylamine ( 180 equiv ) ; ( v ) ( a ) pybop , hobt , n , n diisopropylethylamine , dmf , rt ; ( b ) 2 ( 8 equiv ) , 24 h , rt . n3-carboxymethylriboflavin 1(31,54,55 ) was first converted to its linker - extended form 2 prior to its conjugation with 3 g5(la)10 . to a stirred solution of n3-carboxymethylriboflavin ( 15 mg , 35 mol ) dissolved in dmf ( 9 ml ) was added hobt ( 6.4 mg , 42 mol ) , dipea ( 12 l , 69 mol ) and pybop ( 22 mg , 42 mol ) sequentially . the reaction mixture was stirred at rt for 17 h , followed by addition of 2,2-(ethylenedioxy)bis(ethylamine ) ( 5.1 l , 34 mol ) . after stirring for an additional 7 h , product 2 in the reaction mixture was used for dendrimer conjugation in the next step without further treatment as described below . to a mixture of g5(la)10 ( 50 mg , 1.2 mol ) , hobt ( 33 mg , 216 mol ) , and dipea ( 50 l , 287 mol ) in dmf ( 5 ml ) was added pybop ( 111 mg , 213 mol ) . the mixture was stirred for 20 h at rt prior to the addition of 2 in dmf ( 2.4 ml ; prepared above ) . after stirring for an additional 24 h , the reaction mixture was concentrated in vacuo , and the residue was dissolved in 10 ml of phosphate buffered saline ( pbs , ph 7.4 ) . the product conjugate was purified by membrane dialysis ( mwco 10 kda ) against pbs ( 2 2 l ) , and deionized water ( 3 2 l ) over 2 days until impurities were lower than 5% , as determined by analytical uplc . the dialyzed solution was collected and freeze - dried to afford 4 g5(la)10(rf)5 as a pale yellow solid . the number ( n = 5 1 ) of riboflavin molecules attached per dendrimer was estimated on a mean basis by uv vis spectrometry . maldi tof mass spectrometry ( m / z , gmol ) : 41 000 . h nmr ( 500 mhz , d2o ) : 3.9 ( br ) , 3.73.4 ( m ) , 3.53.3 ( br ) , 3.33.1 ( br m ) , 2.952.75 ( br ) , 2.752.6 ( br m ) , 2.552.35 ( br m ) , 2.32.15 ( br m ) , 1.95 ( m ) , 1.85 ( m ) , 1.6 ( br m ) , 1.4 ( br ) ppm . uv vis ( pbs , ph 7.4 ) : max = 448 nm ( = 4045 m cm ; rf ) , 350 nm ( = 4847 m cm ; rf ) , 274 nm ( = 28 800 m cm ) . gold nanoparticles ( aunps , mean diameter = 16 nm ) were synthesized by an established method . an aqueous solution of sodium citrate was added to a boiling solution of haucl4 in water , each mixed at a ratio of [ citrate]/[au(iii ) ] = 2.72 ( [ citrate ] = 0.68 mm ; [ au ] = 0.25 mm ) . uv vis ( pbs , ph 7.4 ) : max = 520 ( 522 ) nm . distribution of the size of aunps was measured by dynamic light scattering measured at 25 c ( z - average hydrodynamic diameter = 30.27 nm , pdi = 0.433 ; figure s4 , supporting information ) , and also by afm ( mean core diameter = 13.5 2.2 nm vs 13 nm ( tem ) ) . for dendrimer 3aunp , 3 g5(la)10 ( 2.5 m in water as a stock solution ) was titrated into a solution of aunp ( 2 ml ; [ au ] = 0.125 mm ) in 10 l increments . after each addition , the solution was mixed thoroughly , and its uv vis spectrum was measured as summarized in figure 1 . proposed models for chemisorption of aunps by dendrimer nanoparticles 3 and 4 g5(la)10(rf)n ( n = 0 , 5 ) . each acquired during the titration of 3 g5(la)10 or 4 g5(la)10(rf)5 ( 2.5 m ) into an aunp solution in water ( [ aunp ] = 10 nm ; 2 ml ) . arrays of uv vis spectra acquired during the titration of riboflavin binding protein ( rfbp ) to the surface - modified aunps ( 10 nm ) , each aunp coated with 3 g5(la)10 ( c ) or 4 g5(la)10(rf)5 ( d ) . the amount of dendrimer or rfbp indicated in the legend is expressed as a final concentration ( nm ) of the dendrimer or protein solution added . the plot in ( e ) summarizes the change ( % ) of absorption at the wavelength of max as a function of [ rfbp]added/[dendrimer ] . a solution of dendrimer 4aunp was prepared as described above by reacting 4 ( 25 m , 70 l ) with aunp ( 2 ml ) . rfbp ( 3.3 m ) was titrated into this solution in 10 l increments ( each 0.5% increase in volume ) until a saturation point was reached ( 70 l ; 3% increase relative to an initial volume ) . after each addition , the uv vis spectrum was recorded as summarized in figure 1 . aunp and its dendrimer nanoconjugates were imaged using a multimode atomic force microscope ( bruker , santa barbara , ca ) . n / m spring constant and 120180 khz resonance frequency were used for afm imaging in pbs buffer . prior to imaging , the vertically engaging e - scanner was calibrated for accuracy in the x , y , z direction by using a 1 m grid with a depth of 20 nm . freshly cleaved mica was modified with a 100 l deposit of 0.01% 3-(aminopropyl)triethoxysilane ( aptes ) solution . after a 20 min incubation period , the mica surface was rinsed six times with 1 ml aliquots of nanopure water and dried with compressed nitrogen . the modified mica substrate was incubated for 5 min with 10 l of conjugate aunp and placed in the afm fluid cell holder for imaging . all images were collected in fluid scanasyst mode under pbs buffer ( ph 7.4 ) with a scan rate of 1 hz all images were corrected for tip artifacts and surface bowing only , and analyzed using a scanning probe image processor ( spip ) software ( version 6.0.2 , image metrology a / s , lyngby , denmark ) . the particle size analysis was conducted by the particle and pore analysis module available in the spip software . an automatic threshold was applied for each image with particle sizes less than 7 nm excluded from the analysis . for the height analysis of aunps ( a representative image shown in figure 3a ) , seventeen 5 m 5 m images were analyzed with 700 np per image ( n = 12 063 ) . for the height analysis of dendrimer 4aunp ( a representative image shown in figure 3b ) , nine 10 m 10 m images were analyzed with 250 np per image ( n = 2281 ) . for the height analysis of dendrimer 4aunp + rfbp ( a representatitve image shown in figure 3c ) , nine 10 m 10 m images were analyzed with 280 np per image ( n = 2476 ) . for the height analysis of dendrimer 4aunp + rfbp + rf ( a representatitve image shown in figure 3d ) , twelve 10 m 10 m images were analyzed with 210 np per image ( n = 2551 ) . histograms and kernel density function plots were generated to examine the height distribution of nanoparticles . all density curves reported were generated using 512 points and the gaussian kernel function . peak heights were obtained by fitting the density curve to the minimum number of gaussians and taking the peak value and the width of the gaussian using the commercially available peakfit software . kb cells were grown as a monolayer culture in a riboflavin - deficient rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) . kb cells were seeded overnight on eight - well chambered cover glass slides at a density of 5 10 cells / well ( lab - tek ) . cells were treated with 3aunp or 4aunp at different concentrations ( 50 or 80 nm ) in media for 2 h or 4 h at 37 c followed by washing with pbs ( three times ) . the cells were fixed with 4% paraformaldehyde and mounted in prolong gold with 4,6-diamidino-2-phenylindole ( dapi ) ( life technologies ) . the signal from the spr scattering of aunps was collected on a leica inverted sp5x confocal microscope ( leica microsystems ) using a 63 oil immersion objective . spr of the aunps was measured by excitation with the white light laser at 514 nm , with sequential scanning of emission at 474506 and 522570 nm , similar to what was described previously . emissions from these two channels were overlaid to yield the total spr scattering signal which was merged with a differential interference contrast ( dic ) image . conjugates 3 and 4 used in this study are based on a fifth generation ( g5 ) poly(amido amine ) ( pamam ) dendrimer g5-(nh2 ) ( diameter 5.4 nm ) . we prepared each by conjugating g5-(nh2 ) with ( )--lipoic acid ( la ) alone ( 3 ) or in combination with the rf ligand ( 4 ) as summarized in scheme 1 . in the design of these dendrimer conjugates , we introduced ( )--lipoic acid as a bifunctional molecule that provides both a carboxylic acid for dendrimer conjugation and a dithiolanyl moiety to serve as a disulfide handle for dendrimer chemisorption to the surface of aunp through au s bond formation . first , we prepared the 3 g5(la)m=10 conjugate ( scheme 1 ) by reaction of g5-(nh2)114 with freshly prepared lipoic hobt ester added at 15 molar equivalency to the dendrimer . after this amide coupling step , each of the unreacted surface amines was modified to n - glutarylamide . the number ( m ) of lipoic acid molecules attached per dendrimer was estimated on a mean basis by the h nmr integration method . in this method , the integration area ( aucga ) for those ch2 signals of the glutaric amide residue ( each at 2.25 , 2.20 , 1.83 ppm ; figure s3 , supporting information ) was compared to the area ( aucla ) of those ch2 signals of the lipoic amide residue ( selected , each at 1.4 , 1.6 ppm ) : m + p = 114 , and m / p = aucla / aucga where m and p refer to the number of la and ga residues , respectively . second , we prepared the conjugate 4 g5(la)10(rf)5 by amide conjugation of 3 g5(la)10 with a rf ligand 1 through an ethylenedioxybis(ethylamine ) spacer . here , 3-(carboxymethyl)riboflavin ( 1 ) was selected as the rf ligand because of its carboxylic acid located at its n-3 position of an isoalloxazine head and the retained binding affinity of n-3 derivatives ( e.g. , kd = 2.5 nm for n-3 me ) to riboflavin binding protein ( rfbp ) . thus each rf molecule was attached to the dendrimer through the two carbon spacers provided by 1 extended with the additional ethylene oxide - based spacer . this linker design aimed to provide a linker long enough for the dendrimer - attached rf to bind the targeted receptor protein without any steric constraint as supported by our prior studies based on cellular binding , and our spr studies . the synthetic method used for the conjugate 4 was based on a pybop - based activation of 3 prior to the reaction with 2 . each conjugate was purified by exhaustive dialysis using a membrane tubing ( mwco 10 kda ) until its purity was greater than 95% without detectable amounts of free la and/or the rf ligand as determined by hplc ( figure s1 , supporting information ) . each of these conjugates 3 and 4 was fully characterized by employing standard analytical techniques including uv vis spectrometry , h nmr spectroscopy , maldi tof mass spectrometry , and gpc ( table 1 , supporting information ) . the uv vis spectrum of 4 provides strong absorption at long wavelengths assigned for the isoalloxazine chromophore of rf ( figure s2 , supporting information ; max = 449 nm , = 12 100 m cm ) . the analysis of such uv vis spectral data allowed determination of the number ( n = 5 ) of rf ligands attached to the dendrimer particle on a mean basis . use of another complementary method based on h nmr integration was inapplicable for this analysis due to the strong line broadening of rf aromatic proton signals . the efficiency for small molecule conjugation to the dendrimer ( [ ligand]attached vs [ ligand]added ) was approximately 70% for la and 20% for the rf ligand . this is possibly attributable to steric congestion by preexisting la presented on the dendrimer surface , as well as the in situ method used for linker extension . mean molecular weight ( = [ mn + mw ] 2 ) determined by gel permeation chromatography ( gpc ) . aunps were prepared as citrate - stabilized particles by the gold(iii ) reduction method performed at a ratio of ( [ citrate]/[au(iii ) ] = 2.72 ) as described elsewhere . the surface plasmon resonance absorption spectrum of aunps was measured by uv vis spectrometry ( figure 1 ) . the maximal absorption band ( max ) at 520 nm and lack of longer max absorption at 600700 nm by gold ellipsoids and nanorods ( figure 1 ) suggest that these aunps are spherical and in the nonaggregated state , in agreement with dynamic light scattering measurements which gave a hydrodynamic diameter of 30 nm ( z - average ) . this hydrodynamic size ( the stokes diameter calculated from the diffusion properties of tumbling , solvated particles ) includes the contribution from the hydrated diffusion layer surrounding the aunp core and is apparently much greater than the size determined by current afm analysis ( discussed below ) showing that these aunps are spherical particles with a mean core diameter of 13.5 2.2 nm ( 1 error ) in buffered solution . this size analysis is in good agreement with the value published in the literature ( 13 nm ) determined by transmission electron microscopy ( tem ) . surface functionalization of aunps with dendrimer conjugates 3 and 4 was performed by au s chemisorption through a mechanism that involves the reaction of the cyclic disulfide moiety at the terminus of the la with au surface . dendrimer chemisorption was performed by incubation of each conjugate solution with aunps in water , and its progress was monitored by uv vis spectroscopy ( figure 1 ) . upon dendrimer incubation , a large red shift was observed in the uv vis spectra for dendrimer 3aunp ( max = 615 nm ) , and for dendrimer 4aunp ( max = 653 nm ) . such a red shift has previously been reported in the chemisorption of polymer molecules on gold surfaces though the spectral shifts observed with our dendrimer nanoparticles were much greater . we believe that such shifts are not due to the formation of large dendrimer - mediated aggregates of aunps as supported by an afm analysis to be presented later showing the size distribution of dendrimer 4aunp with a peak diameter of 20.5 4.0 nm . in each aunp functionalization , the amount of the dendrimer particles required for maximal coverage of the aunp surface was determined by the titration method as shown in figure 1 . we investigated the interaction of rfbp with aunps modified with dendrimer 3 or 4 by uv vis spectrometry ( figure 1 ) . with incremental addition of rfbp to dendrimer 3aunp , the absorption bands decreased significantly at first but insignificantly later ( 5% ) with a small shift in the wavelength of max to 622 nm at saturation ( wavelength = 7 nm ; figure 1d , e ) . such spectral changes are quite distinct with those observed from dendrimer 4aunp , which showed a concentration - dependent decrease ( 16% ) in absorption with a small red shift to 659 nm ( wavelength = 6 nm ; figure 1d , e ) . this absorption decrease is significant and not due to increase ( 3% ) in the volume of the solution that occurred during the titration . we attribute this spectral trend for dendrimer 4aunp to specific binding of rfbp to the rf ligand presented on the surface of aunp - attached dendrimer . in contrast , there is no specific spectral trend indicative of specific binding of rfbp to dendrimer 3aunp that does not present rf ligands on the dendrimer surface though small extent of nonspecific binding or aggregation might occur with dendrimer 3aunp as suggested by the initial decrease and shift in the absorption . thus uv vis absorption spectral traces provide evidence for specific interaction of rfbp with rf - presenting dendrimer 4aunp . in addition to the uv titration study in solution , we performed a confocal imaging study in vitro for demonstrating the binding and uptake ( collectively termed as association ) of dendrimer 4aunp in kb cancer cells that express the rf receptors as described elsewhere . in terms of imaging modality , aunps display several optophysical properties such as surface plasmon resonance ( spr ) absorption in the visible region of the light spectrum , visible luminescence , and spr scattering effects . due to these properties , aunps have recently attracted significant attention as visible probes for biological sensing and imaging studies for in vitro and in vivo applications . in this study , we were interested in their spr scattering properties because detection of aunps in biological samples can occur without additional fluorescent labels or modifications . following the imaging methods as demonstrated in previous studies using dark field light microscopy and confocal microscopy , we investigated whether aunp or its dendrimer nanocomposites undergo cellular association in cancer cells in vitro . we first verified the spr scattering of aunp as its detection modality using unmodified citrate - adsorbed aunp ( no fluorescent dye or chemisorbed dendrimer ) in kb cancer cells . after a 2 h incubation with aunp ( 50 nm ) , the treated kb cells were imaged by confocal microscopy with two different detection configurations : ( i ) scattered spr ( excitation 514 nm ; emission 474506 and 522570 nm ) and ( ii ) luminescence ( excitation at 514 nm ; emission at 550650 nm ) as shown in figure 2a c . aunps associated with the cells are clearly detectable both by spr scattering and by luminescence , although their detection intensities are weak indicative of the low level of cellular association . thus , despite the lack of fluorescent dye molecules , it was possible to detect aunps associated with and internalized in the cells by two different imaging methods . cellular uptake of aunp derived constructs in kb cells . ( a)(c ) kb cells treated with 50 nm of unmodified aunp for 2 h were imaged by ( a ) spr scattering or ( b ) luminescence . ( c ) co - localization of the signals was confirmed by the overlay . spr scattering imaging was taken for kb cells treated for 4 h with 80 nm of ( d ) aunp , ( e ) dendrimer 3aunp , or ( f ) dendrimer 4aunp ( inset is a magnified view of the adjacent cells ) . confocal microscopic images ( 63 magnification ) were taken as described in the experimental section . signal from the spr scattering was overlaid with differential interference contrast ( dic ) images . we next treated kb cells with aunp , dendrimer 3aunp or dendrimer 4aunp , each at 80 nm , for 4 h , and imaged the treated cells in the spr scattering mode as shown in figure 2d f . from these scattering images , qualitative differences between the aunp and dendrimer cells treated with the riboflavin targeted dendrimer 4aunp showed more punctate and localized areas of signal which were distinctly more intense than those found in the bare aunp and dendrimer 3aunp treated cells at this time point ( figure 2f ) . this scattering is attributable to aggregated aunps either on the cell surface or inside the cell . in summary , dendrimer chemisorbed 4aunps were detectable by the spr scattering method using confocal microscopy , providing evidence for the cellular localization of these aunps . our confocal microscopy studies demonstrated the utility of the dual detection modes of aunps based on spr scattering and luminescence , both of which are inherent properties of aunps , for determining the cellular localization of aunps without the need for additional particle labeling . we employed mica as a substrate in afm imaging due to its atomic flatness . however , the negative surface charge of mica makes immobilization of previously described nanoparticles unfavorable due to the presence of the negatively charged citrate groups on the surface of aunps ( figure s6a , supporting information ) . similarly , the dendrimers conjugated to aunps have an overall negative charge due to excess linker branches containing glutaric acid ( pka = 4.31 , 5.41 ) . to obtain an adequate surface coverage of conjugated and unconjugated gold nanoparticles , the mica surface needs to be pretreated to generate a positive surface charge . common approaches used for functionalizing mica substrates are to treat the mica surface with mgcl2 , nicl2 , or 3-(aminopropyl)triethoxysilane . the mgcl2 treated mica surface did not , however , yield substantial coverage of aunps on the surface , resulting in only very few observable nanoparticles when imaged by afm in pbs buffer ( figure s6b , supporting information ) . similar observations have been noted with mg treated mica surfaces , and the immobilization appears to depend on the size of the nanoparticles with larger aunps not adhering to the mgcl2 treated mica surface . 3-(aminopropyl)triethoxysilane ( aptes ) treated mica is also commonly used for immobilizing biological material and has been used extensively in imaging dna on mica substrates . mica substrates treated with 0.01% aptes solution were found to be favorably disposed toward achieving electrostatic immobilization of unconjugated aunps ( figure 3a ) . the immobilization of conjugated aunps is also likely aided by the hydrophobic surface of the aptes mica as opposed to the hydrophilic surface of a freshly cleaved mica surface . the height of the unconjugated aunps from figure 3a was found to be 13.5 2.2 nm ( obtained from seventeen 5 m 5 m images , each with 700 nps ) . afm images of aunps and dendrimer chemisorbed aunps on 0.01% aptes treated mica in pbs buffer ( ph 7.4 ) : ( a ) 2 nm aunps ( z - scale 25 nm ) ; ( b ) 7.5 ( 2.5 ) nm dendrimer 4aunp ( z - scale 50 nm ) ; ( c ) 7.5 ( 2.5 ) nm dendrimer 4aunp after the addition of 0.5 m rfbp ( z - scale 60 nm ) ; ( d ) 7.5 ( 2.5 ) nm dendrimer 4aunp with 0.5 m rfbp after the addition of 5 m rf ( z - scale 30 nm ) . inset for ( a ) and ( b ) : close - up view of the nps imaged in air ( 1 m 1 m ) . afm was used to determine the height distribution of dendrimer 4aunps for comparison with the height distribution of bare aunps ( 13.5 2.2 nm ) covered with citrate anions ( figure 3a , b ) . all afm imaging work was carried out on 0.01% aptes modified mica substrates , as per the results of immobilization work presented above . analysis of figure 3a , b afm images suggests an 83% decrease in surface density of the adsorbed 4aunps ( 3b ) in comparison to the bare aunps ( 3a ) . this is likely due to the overall charge on the bare aunp being much more negative in comparison to the dendrimer 4aunp , resulting in less attraction of the conjugated aunps to the aptes modified mica surface . in addition , the conjugation of dendrimers to the aunp via the longer lipoic amide - based linkers shields most of the negative charges otherwise conferred by citrate anions on the bare aunp . it is also plausible that certain groups in the dendrimer 4 including the rf ligand attached ( pka of pteridine head = 9.69 ) and a minor fraction of internal tertiary amines ( theoretically 126 per particle ; pka = 6.36.85 for its conjugate acid ) might be protonated in pbs ( ph 7.4 ) . the presence of such positively charged groups could contribute to partial neutralization of the dendrimer surface or repulsive dendrimer interaction with the aptes mica surface , leading to the decreased surface adsorption of 4aunp . the height distribution of dendrimer 4aunps shows predominantly two peaks , with the first larger peak at 12.0 2.9 nm and a second smaller peak at 20.5 4.0 nm , each from the simulated curve ( figure 4a ) . the shoulder observed at 8 nm is due to the 7 nm threshold applied in the data analysis and appears in all of the distributions presented in figure 4 ; it will not be further discussed . the larger peak in height distribution ( at 20.5 nm ) is estimated in the range of + 7 nm of the peak height in the dendrimer 3aunp height distribution . this increase in size appears to be comparable to the size of individual dendrimer conjugates ( mean diameter of a parent g5 dendrimer = 5.4 nm ) and suggests the formation of a dendrimer monolayer on the gold surface of the aunp as proposed from the uv vis titration experiments ( figure 1a , b ) . we believe that this heterogeneous distribution composed of the smaller and larger particles of dendrimer 4aunp might be attributable to the distribution in the number of dendrimer particles adsorbed on each aunp with the larger size corresponding to aunps with higher numbers of adsorbed dendrimers . it is notable that this distribution was detectable by afm , but not by standard uv vis spectrometric titration ( figure 1 ) . nanoparticle height histogram plots : ( a ) 7.5 ( 2.5 ) nm dendrimer 4aunp ; ( b ) 7.5 ( 2.5 ) nm dendrimer 4aunp after the addition of 0.5 m rfbp ; ( c ) 7.5 ( 2.5 ) nm dendrimer 4aunp with 0.5 m rfbp after the addition of 5 m rf . in each plot we next investigated the interaction of rfbp with dendrimer 4 adsorbed on aunp by afm . first , the dendrimer 4aunp was imaged in the presence of 0.5 m rfbp ( figure 3c ) , and then binding of the rfbp to dendrimer surface was selectively blocked by addition of 5.0 m rf as a competitive ligand ( figure 3d ) . the height distributions of dendrimer 4aunps under each of these conditions are represented as histograms in figure 4b , c and as kernel density function plots in figure 5 . kernel density plot showing height distribution analysis of each step of a three - step competitive binding experiment . step 1 : 7.5 ( 2.5 ) nm dendrimer 4aunp imaged on modified mica under pbs buffer ( red line ) . step 2 : 0.5 m rf - targeting rfbp was added to dendrimer 4aunp ( green line ) . step 3 : 5.0 m rf was added to dendrimer 4aunp with 0.5 m rfbp ( blue line ) . binding of rfbp to the dendrimer 4aunps is indicated by peak height shifts to 16.3 5.0 nm with a broad shoulder centered at 26.7 4.5 nm ( figure 4b ) . these shifts correspond to approximately 4 and 6 nm increases in each of the peak heights , respectively , relative to untreated dendrimer 4aunp alone . we hypothesize that these observed height increases upon rfbp addition could reflect the number of rfbp molecules bound , and the heterogeneity in the rf ligand density on the dendrimer 4aunp . recently , a number of reports have shown evidence that ligand conjugation to dendrimers through conventional methods such as amide coupling is unable to produce dendrimers with precisely defined ligand valency but rather yields stochastic distributions of ligand valencies . in this study , we assigned a valency of five rf ligands attached per dendrimer for dendrimer 4 . however , this ligand valency is calculated on an average basis , as dendrimer 4 is composed of mixed ligand valencies with a theoretical poisson distribution ( figure s5 , supporting information ) . therefore , we believe that fewer rfbp molecules bind to those dendimer 4aunps coated with lower - valent dendrimer species compared to those with higher - valent species . second , we analyzed the number density of nanoparticles in the broad 2040 nm range , which was significantly greater than the particle height at the main peak . this density comparison suggests that rfbp binding to the dendrimer 4aunps resulted in an increase in this population ( from 17.0% to 28.9% ; table s1 , supporting information ) . a binomial proportions test showed this increase to be significant ( p < 0.05 ) ( figure s7 , supporting information ) . to confirm that this increase was due to the binding of rfbp to dendrimer 4 , free rf ( 5 m ) was added to competitively bind rfbp in the medium and thereby prevent rfbp from binding to dendrimer 4aunp . the resulting peak height distributions show peaks centered at 14.4 2.7 and 22.8 3.9 nm ( figure 4c ) . in addition , a comparison of the height distribution of the nanoparticles after the addition of rf shows the density of nanoparticles in the 2040 nm range decreased from 28.9% to 17.4% , a statistically significant change ( p < 0.05 ; figure s7 , supporting information ) . the kernel density ( figure 5 ) in the 2040 nm region for dendrimer 4aunp , prior to the addition of rfbp and after the competitive binding of rf with rfbp , shows a modest change in particle density from 17.0% to 17.4% , which , on the basis of the binomial proportions test , shows no statistical significance ( p = 0.45 ) . we probed its receptor binding in vitro by using an array of complementary methods including uv vis spectroscopy , confocal spr imaging and afm . first , we illustrated the capability of afm as a quantitative method for investigating receptor ligand interactions at the aunp specific binding of rfbp to rf - presenting dendrimer aunp was probed by detecting changes in particle height distribution as a metric . such afm - based quantitative analysis is not achievable by corresponding uv vis spectrometric titration experiments or confocal image analysis . second , our afm study suggests preexisting heterogeneity in the rf ligand distribution on dendrimers as the basis that leads to heterogeneous populations of rfbp bound to the dendrimer - adsorbed aunp . these results corroborate a model for the rf receptor ligand interaction suggested by uv vis spectrometry and confocal microscopic studies . this study clearly demonstrates that afm serves as an imaging tool for the determination of size distributions as well as for quantifying receptor

riboflavin receptors are overexpressed in malignant cells from certain human breast and prostate cancers , and they constitute a group of potential surface markers important for cancer targeted delivery of therapeutic agents and imaging molecules . here we report on the fabrication and atomic force microscopy ( afm ) characterization of a core shell nanocomposite consisting of a gold nanoparticle ( aunp ) coated with riboflavin receptor - targeting poly(amido amine ) dendrimer . we designed this nanocomposite for potential applications such as a cancer targeted imaging material based on its surface plasmon resonance properties conferred by aunp . we employed afm as a technique for probing the binding interaction between the nanocomposite and riboflavin binding protein ( rfbp ) in solution . afm enabled precise measurement of the aunp height distribution before ( 13.5 nm ) and after chemisorption of riboflavin - conjugated dendrimer ( aunp dendrimer ; 20.5 nm ) . binding of rfbp to the aunp dendrimer caused a height increase to 26.7 nm , which decreased to 22.8 nm when coincubated with riboflavin as a competitive ligand , supporting interaction of aunp dendrimer and its target protein . in summary , physical determination of size distribution by afm imaging can serve as a quantitative approach to monitor and characterize the nanoscale interaction between a dendrimer - covered aunp and target protein molecules in vitro .

Introduction Experimental Section Results and Discussion Conclusions

a large fraction of these studies have focused on exploring new functional applications of the aunp - based nanoparticles based on their photophysical properties . however , three - dimensional features that vary in response to the interaction between aunp - based nanoparticles and their biological targets such as particle size distribution can provide physical insights on their interaction although they remain much less characterized in most aunp - based delivery systems . here , we studied the interaction of a dendrimer chemisorbed aunp with its protein target by atomic force microscopy ( afm ) as a biophysical method to correlate protein binding with the geometrical alteration of the aunp nanocomposite . this study demonstrates that afm serves as an effective technique for characterizing the interaction of aunp - based nanoparticles and target proteins in vitro by quantitative measurement of size distribution . dendrimer nps used for surface modification of aunps are based on a fifth generation ( g5 ) poly(amido amine ) ( pamam ) dendrimer conjugated with a riboflavin ( rf ) molecule . as a vitamin molecule ( b2 ) essential for the biosynthesis of flavin - based redox cofactors , rf is taken up by riboflavin receptors , also referred to as rf carriers , which are expressed as both soluble and membrane - bound isoforms . moreover , rf receptors constitute one type of potential tumor biomarker due to their overexpression in certain malignant cells from human breast and prostate cancers . this biomarker is relatively new as a receptor considered for cancer targeting compared to other biomarker proteins that include folic acid receptor ( far ) , v3 integrin , prostate - specific membrane antigen , her2 receptor , and epidermal growth factor receptor . recently , we applied the concept of targeted drug delivery to the rf receptor by designing rf - conjugated pamam dendrimer nanoparticles and demonstrated their effectiveness in vitro for rf receptor targeted delivery of methotrexate ( mtx ) in kb cancer cells that overexpress the riboflavin receptors . in a follow - up study , we employed isothermal titration calorimetry ( itc ) and differential scanning calorimetry ( dsc ) to address the thermodynamic aspects of the rf receptor ligand interactions for rf - conjugated dendrimers . combination of these two methods provided biophysical information important for the correlation of binding affinity to design factors such as ligand valency and the rf attachment position of the dendrimer conjugates . these studies allowed characterization of the structural features dictating the thermodynamic aspects of the interaction between the dendrimer and the receptor protein . in the current study , we employed afm for structural characterization of rf receptor targeted aunp systems . the rigidity of generation 7 or higher dendrimers makes it possible to image them by afm , whereas the softer g5 dendrimers used for rf conjugation ( d = 5.4 nm ) in this study tend to flatten on mica surfaces , providing fuzzy afm images . first , aunp brings a number of photophysical and photothermal properties , and its surface functionalization with the dendrimer platform creates a hybrid nanosystem that enables both imaging and specific cancer targeting . second , in contrast to the soft dendrimer , the aunp provides advantages for performing the afm study primarily because it can serve as a marker , due to its metallic core and can be obtained in sizes greater than the dendrimer np . hence afm is ideally suited for the determination of the size alteration as a quantifiable physical property that follows binding of the target protein to the dendrimer - conjugated aunp . recently , structural characterization of aunp hybrids by afm has been demonstrated for a number of aunp hybrid systems , each aunp conjugated with oligonucleotide ( dna ) probes , polyphenylene dendrimer , peg , poly(4-vinylpyridine ) , and cyclodextrin . finally , afm can image surfaces with nanoscale resolution , and unlike other imaging techniques such as transmission electron microscopy ( tem ) , it can image a variety of biomacromolecular complexes and biological events under nearly physiological conditions . the present report focuses on afm characterization of aunps functionalized on the surface with dendrimer nps as a new class of rf receptor targeted delivery systems . this afm study was performed in combination with complementary techniques including uv vis spectrometry and in vitro confocal microscopy to image binding of the dendrimer - functionalized aunps to riboflavin receptor in a cancer cell . this study illustrates how physical measurement of particle size distribution of aunps by afm enables the characterization and precise monitoring of the biological interaction between a dendrimer conjugated aunp and its target protein in a physiological solution . unless noted otherwise , all reagents and solvents were purchased from sigma - aldrich including ( )--lipoic acid ( 98% ) , ( )-riboflavin ( 98% ) , riboflavin binding protein ( apo - form ) , 3-(aminopropyl)triethoxysilane , and gold(iii ) chloride trihydrate ( 99.9% ) . molecular weights ( mw , mn ) and polydispersity index values ( pdi = mw / mn ) of g5 pamam dendrimer were determined by gel permeation chromatography ( gpc ) , and details for the experimental method are already described earlier . the final mixture was stirred for 15 h , and excess unreacted amine groups of the dendrimer were fully modified by reacting with glutaric anhydride ( 210 mg , 1.84 mmol ) and dipea ( 310 l , 1.8 mmol ) . the theoretical number of terminal branches per g5 dendrimer is 128 ( experimentally , 114 based on potentiometric titration ) , but only a fraction of the branches are shown for clarity . distribution of the size of aunps was measured by dynamic light scattering measured at 25 c ( z - average hydrodynamic diameter = 30.27 nm , pdi = 0.433 ; figure s4 , supporting information ) , and also by afm ( mean core diameter = 13.5 2.2 nm vs 13 nm ( tem ) ) . for dendrimer 3aunp , 3 g5(la)10 ( 2.5 m in water as a stock solution ) was titrated into a solution of aunp ( 2 ml ; [ au ] = 0.125 mm ) in 10 l increments . arrays of uv vis spectra acquired during the titration of riboflavin binding protein ( rfbp ) to the surface - modified aunps ( 10 nm ) , each aunp coated with 3 g5(la)10 ( c ) or 4 g5(la)10(rf)5 ( d ) . the amount of dendrimer or rfbp indicated in the legend is expressed as a final concentration ( nm ) of the dendrimer or protein solution added . aunp and its dendrimer nanoconjugates were imaged using a multimode atomic force microscope ( bruker , santa barbara , ca ) . peak heights were obtained by fitting the density curve to the minimum number of gaussians and taking the peak value and the width of the gaussian using the commercially available peakfit software . kb cells were grown as a monolayer culture in a riboflavin - deficient rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) , penicillin ( 100 iu / ml ) , and streptomycin ( 100 g / ml ) . conjugates 3 and 4 used in this study are based on a fifth generation ( g5 ) poly(amido amine ) ( pamam ) dendrimer g5-(nh2 ) ( diameter 5.4 nm ) . in the design of these dendrimer conjugates , we introduced ( )--lipoic acid as a bifunctional molecule that provides both a carboxylic acid for dendrimer conjugation and a dithiolanyl moiety to serve as a disulfide handle for dendrimer chemisorption to the surface of aunp through au s bond formation . in this method , the integration area ( aucga ) for those ch2 signals of the glutaric amide residue ( each at 2.25 , 2.20 , 1.83 ppm ; figure s3 , supporting information ) was compared to the area ( aucla ) of those ch2 signals of the lipoic amide residue ( selected , each at 1.4 , 1.6 ppm ) : m + p = 114 , and m / p = aucla / aucga where m and p refer to the number of la and ga residues , respectively . , kd = 2.5 nm for n-3 me ) to riboflavin binding protein ( rfbp ) . this linker design aimed to provide a linker long enough for the dendrimer - attached rf to bind the targeted receptor protein without any steric constraint as supported by our prior studies based on cellular binding , and our spr studies . the analysis of such uv vis spectral data allowed determination of the number ( n = 5 ) of rf ligands attached to the dendrimer particle on a mean basis . this hydrodynamic size ( the stokes diameter calculated from the diffusion properties of tumbling , solvated particles ) includes the contribution from the hydrated diffusion layer surrounding the aunp core and is apparently much greater than the size determined by current afm analysis ( discussed below ) showing that these aunps are spherical particles with a mean core diameter of 13.5 2.2 nm ( 1 error ) in buffered solution . this size analysis is in good agreement with the value published in the literature ( 13 nm ) determined by transmission electron microscopy ( tem ) . we believe that such shifts are not due to the formation of large dendrimer - mediated aggregates of aunps as supported by an afm analysis to be presented later showing the size distribution of dendrimer 4aunp with a peak diameter of 20.5 4.0 nm . with incremental addition of rfbp to dendrimer 3aunp , the absorption bands decreased significantly at first but insignificantly later ( 5% ) with a small shift in the wavelength of max to 622 nm at saturation ( wavelength = 7 nm ; figure 1d , e ) . we attribute this spectral trend for dendrimer 4aunp to specific binding of rfbp to the rf ligand presented on the surface of aunp - attached dendrimer . in contrast , there is no specific spectral trend indicative of specific binding of rfbp to dendrimer 3aunp that does not present rf ligands on the dendrimer surface though small extent of nonspecific binding or aggregation might occur with dendrimer 3aunp as suggested by the initial decrease and shift in the absorption . in addition to the uv titration study in solution , we performed a confocal imaging study in vitro for demonstrating the binding and uptake ( collectively termed as association ) of dendrimer 4aunp in kb cancer cells that express the rf receptors as described elsewhere . in terms of imaging modality , aunps display several optophysical properties such as surface plasmon resonance ( spr ) absorption in the visible region of the light spectrum , visible luminescence , and spr scattering effects . from these scattering images , qualitative differences between the aunp and dendrimer cells treated with the riboflavin targeted dendrimer 4aunp showed more punctate and localized areas of signal which were distinctly more intense than those found in the bare aunp and dendrimer 3aunp treated cells at this time point ( figure 2f ) . we employed mica as a substrate in afm imaging due to its atomic flatness . however , the negative surface charge of mica makes immobilization of previously described nanoparticles unfavorable due to the presence of the negatively charged citrate groups on the surface of aunps ( figure s6a , supporting information ) . the mgcl2 treated mica surface did not , however , yield substantial coverage of aunps on the surface , resulting in only very few observable nanoparticles when imaged by afm in pbs buffer ( figure s6b , supporting information ) . similar observations have been noted with mg treated mica surfaces , and the immobilization appears to depend on the size of the nanoparticles with larger aunps not adhering to the mgcl2 treated mica surface . the immobilization of conjugated aunps is also likely aided by the hydrophobic surface of the aptes mica as opposed to the hydrophilic surface of a freshly cleaved mica surface . afm was used to determine the height distribution of dendrimer 4aunps for comparison with the height distribution of bare aunps ( 13.5 2.2 nm ) covered with citrate anions ( figure 3a , b ) . analysis of figure 3a , b afm images suggests an 83% decrease in surface density of the adsorbed 4aunps ( 3b ) in comparison to the bare aunps ( 3a ) . this is likely due to the overall charge on the bare aunp being much more negative in comparison to the dendrimer 4aunp , resulting in less attraction of the conjugated aunps to the aptes modified mica surface . in addition , the conjugation of dendrimers to the aunp via the longer lipoic amide - based linkers shields most of the negative charges otherwise conferred by citrate anions on the bare aunp . the height distribution of dendrimer 4aunps shows predominantly two peaks , with the first larger peak at 12.0 2.9 nm and a second smaller peak at 20.5 4.0 nm , each from the simulated curve ( figure 4a ) . the shoulder observed at 8 nm is due to the 7 nm threshold applied in the data analysis and appears in all of the distributions presented in figure 4 ; it will not be further discussed . the larger peak in height distribution ( at 20.5 nm ) is estimated in the range of + 7 nm of the peak height in the dendrimer 3aunp height distribution . this increase in size appears to be comparable to the size of individual dendrimer conjugates ( mean diameter of a parent g5 dendrimer = 5.4 nm ) and suggests the formation of a dendrimer monolayer on the gold surface of the aunp as proposed from the uv vis titration experiments ( figure 1a , b ) . we believe that this heterogeneous distribution composed of the smaller and larger particles of dendrimer 4aunp might be attributable to the distribution in the number of dendrimer particles adsorbed on each aunp with the larger size corresponding to aunps with higher numbers of adsorbed dendrimers . in each plot we next investigated the interaction of rfbp with dendrimer 4 adsorbed on aunp by afm . first , the dendrimer 4aunp was imaged in the presence of 0.5 m rfbp ( figure 3c ) , and then binding of the rfbp to dendrimer surface was selectively blocked by addition of 5.0 m rf as a competitive ligand ( figure 3d ) . binding of rfbp to the dendrimer 4aunps is indicated by peak height shifts to 16.3 5.0 nm with a broad shoulder centered at 26.7 4.5 nm ( figure 4b ) . we hypothesize that these observed height increases upon rfbp addition could reflect the number of rfbp molecules bound , and the heterogeneity in the rf ligand density on the dendrimer 4aunp . to confirm that this increase was due to the binding of rfbp to dendrimer 4 , free rf ( 5 m ) was added to competitively bind rfbp in the medium and thereby prevent rfbp from binding to dendrimer 4aunp . in addition , a comparison of the height distribution of the nanoparticles after the addition of rf shows the density of nanoparticles in the 2040 nm range decreased from 28.9% to 17.4% , a statistically significant change ( p < 0.05 ; figure s7 , supporting information ) . the kernel density ( figure 5 ) in the 2040 nm region for dendrimer 4aunp , prior to the addition of rfbp and after the competitive binding of rf with rfbp , shows a modest change in particle density from 17.0% to 17.4% , which , on the basis of the binomial proportions test , shows no statistical significance ( p = 0.45 ) . first , we illustrated the capability of afm as a quantitative method for investigating receptor ligand interactions at the aunp specific binding of rfbp to rf - presenting dendrimer aunp was probed by detecting changes in particle height distribution as a metric . second , our afm study suggests preexisting heterogeneity in the rf ligand distribution on dendrimers as the basis that leads to heterogeneous populations of rfbp bound to the dendrimer - adsorbed aunp .

cloning of a codon - optimized variant of the homo sapiens traak gene ( uniprot q9nyg8 - 2 ) and heterologous expression in pichia pastoris was previously described . the construct used in this study is c - terminally truncated by an additional ten amino acids compared to our previous reports and similarly incorporates two mutations to remove n - linked glycosylation sites ( n104q / n108q ) and is expressed as a c - terminal precission protease - cleavable egfp-10xhis fusion protein . traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 is referred to as traak in the text for simplicity . frozen pichia cells expressing traak were disrupted by milling ( retsch model mm301 ) 5 times for 3 minutes at 25 hz . all subsequent purification steps were carried out at 4 c. for crystallization in k , cell powder was added to lysis buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 60 mm decyl--d - maltoside ( dm , affymetrix ) , 0.1 mg / ml dnase 1 , 1 g / ml pepstatin , 1 g / ml leupeptin , 1 g / ml aprotinin , 10 g / ml soy trypsin inhibitor , 1 mm benzamidine , and 1 mm phenylmethysulfonyl fluoride added immediately before use ) at a ratio of 1 g cell pellet/4 ml lysis buffer . membranes were extracted for 3 hours with stirring followed by centrifugation at 35000g for 45 minutes . cobalt resin ( clontech ) was added to the supernatant ( 1 ml resin/5 g cell pellet ) and stirred gently for 3 hours . resin was collected on a column and serially washed and eluted in buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 6 mm dm ) with 10 mm , 30 mm , and 300 mm imidazole ph 8.0 . edta ph 8.0 ( 1 mm final ) and prescission protease ( ~1:50 wt : wt ) were added to the elution before incubation with gentle rocking overnight . cleaved protein was concentrated ( 50 kda mwco ) and applied to a superdex 200 column ( ge healthcare ) equilibrated in sec buffer ( 20 mm tris ph 8.0 , 150 mm kcl , 1 mm edta , 4 mm n - decyl--d - maltopyranoside ( dm ) ) . fab was prepared from monoclonal antibody 13e9 against traak as described and buffer exchanged on a hitrap desalting column ( ge healthcare ) into 20 mm tris 150 mm kcl ph 8.0 for crystallization in kcl or 20 mm tris 150 mm kno3 ph 8.0 for crystallization in tlno3 . traak - fab complexes were prepared by incubating purified channel concentrated ( 50 kda mwco ) to ~10 mg / ml with purified fab concentrated ( 10 kda mwco ) to ~30 mg / ml at a 1:2.5 molar ratio in sec buffer for 10 min at 4c . traak - fab complex was separated from excess free fab on a superdex 200 column ( ge healthcare ) equilibrated in sec buffer . traak - fab complexes were concentrated ( 10 kda mwco ) to 30 mg / ml for crystallization . for crystallization in tl , traak - fab complexes were purified identically except that 150 mm kno3 replaced the 150 mm kcl in all buffers except for the final sec buffer in which 150 mm tlno3 replaced the 150 mm kcl . crystals were grown in drops of 0.250.35 l protein added to an equal volume of reservoir in hanging drops over 100 l reservoir at 4c . reservoir for the nonconductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 . reservoir for the nonconductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 . reservoir for the conductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 , 4% ( vol / vol ) polypropylene glycol 400 . reservoir for the conductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 , 52 mm trichloroethanol . crystals grew to ~ 100 m 100 m 200 m in 46 weeks . for cryoprotection , an approximately equal volume of mother liquor supplemented to be 30% ( vol / vol ) peg 400 was added to one side of the drop and crystals were moved through this solution with a cryoloop before being plunged into liquid nitrogen . thallium - containing crystals were collected at the energy giving a maximum imaginary scattering component f determined by a fluorescent scan . data were processed with hkl2000 and structures were solved by molecular replacement with phaser using the previously solved traak - fab structure with ligands , tm2-tm3 and tm4 regions removed as the search model . consistent with recent analysis , incorporation of weak data at high resolution ( to cc1/2=3560% ) improved map quality . two - fold local ncs and jelly body restraints were used throughout refinement and three tls groups per protein chain were incorporated during the final rounds of refinement . 1350 of 1454 total amino acids ( 93% ) are modeled in the structures : the termini ( amino acids 126 and 285290 plus a nine amino acid linker ) and a loop connecting the helical cap to pore helix 1 ( amino acids a104-a109 and b106-b109 ) in each traak protomer and a loop in each fab heavy chain ( amino acids 130135 ) were not visible in the electron density . in the nonconductive structure in tl , residues 275284 from traak subunit the positions of seven tl ions in the nonconductive conformation ( two bound to the extracellular surface of traak , one above the cytoplasmic mouth of the filter , and four in the selectivity filter ) and eight tl ions in the conductive conformation ( with an additional tl in the cavity ) were identified in model phased anomalous difference fourier maps as the highest peaks above background . the presence of an acyl chain in the channel cavity was evaluated based on the shape of the electron density ( in omit maps ) and statistics after crystallographic refinement . the mean b - factor and real space correlation coefficient of the acyl chain ( 101 / 0.76 in the k structure and 132 / 0.74 in the tl structure ) are comparable to the values of nearby residues in tm2 and tm4 that project into the cavity ( amino acids 154,155,158,161,269,272,273 , and 276 have an average b - factor / real space correlation coefficient of 125 / 0.78 in the k structure and 142 / 0.83 in the tl structure ) . phases for omit maps were calculated from a ligand - free model with every atom randomly shifted by a maximum of 0.5 prior to 20 cycles of refinement in refmac to remove model bias . omit density in these maps was essentially indistinguishable from maps generated with nave models that never had ligands modeled or simulated annealing omit maps calculated with phenix . structures were compared after alignment of pore helices and selectivity filters ( 119133 and 228242 from chain a and b ) using lsqkab in ccp4 . the area calculation used a surface calculated with 1.4 added to van der waals radii of protein atoms and a probe radius of 3.5 to approximate the lipid accessible surface area . a water cylinder with a 6 radius was used to fill the cavity of the traak channels to exclude its contribution from the calculation . 3 and 4 were made with symmetric traak channels ( i.e. both subunits had the same conformation ) generated by rotation of a copy of one subunit onto the other . structure figures were made with pymol ( version 1.7.2 schrdinger , llc ) or the ucsf chimera package . chimera is developed by the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nigms p41-gm103311 ) . for cellular recording , cho - k1 cells ( atcc ) ( which do not express endogenous mechanosensitive channels ) were cultured in dmem - f12 ( gibco ) with 10% fbs , 0.2 mm l - alanyl - l - glutamine ( glutamax , gibco ) , 100 u / ml penicillin , and 100 ug / ml streptomycin . cells were plated in 35 mm polystyrene dishes ( bd biosciences ) or on polyester thermanox coverslips ( thermo scientific ) ~24 hrs before transfection with fugenehd ( promega ) following manufacturers protocol . after 48 hrs , dishes or coverslips dna encoding amino acids 1290 of homo sapiens traak was cloned into a pir es2-acgfp vector ( clontech ) to generate untagged traak1290 used in experiments shown in fig . dna encoding amino acids 1300 of homo sapiens traak was cloned into the ecor1/xho1 sites of a modified pceh vector to generate a c - terminally egfp tagged construct ( traak1300 - snsavdaglvprgsaaa - egfp - h10 ) . this construct and mutants generated with inverse pcr were used in experiments presented in fig . reconstitution of traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 and recording from proteoliposome blisters was performed as described for traak1300(n104q , n108q)-sns - levlfq / gp - egfp - h10 . azolectin ( egg l--phosphatidylcholine , 840051 ) and dphpc ( 1,2-diphytanoyl - sn - glycero-3-phosphocholine , 850356 ) lipids were from avanti polar lipids , inc . pipettes were pulled from borosilicate glass ( sutter instruments bf150 - 86 - 10 ) to 34 m resistance . recordings were made with an axopatch 200b amplifier ( molecular devices ) , filtered at 1 khz and digitized at 10100 khz ( digidata 1440a , molecular devices ) . pressure application through the patch pipette was accomplished with a high - speed pressure clamp ( hspc , ala scientific ) controlled through the clampex software . all recordings were performed in a ten - fold concentration gradient of k. for cellular recordings , pipette solution was 10 mm hepes , 150 mm kcl , 3 mm mgcl2 , 5 mm egta , ph 7.2 ( adjusted with koh ) and bath solution was 10 mm hepes , 15 mm kcl , 135 mm nacl , 3 mm mgcl2 , 1 mm cacl2 , ph 7.3 ( adjusted with naoh ) . for experiments in fig . 2 and extended data fig . 3 , reducing bath solution additionally contained 10 mm dtt added from a freshly thawed 1 m stock of dtt in water . reducing solution pipette solution was 5 mm hepes , 180 mm nacl , 20 mm kcl , ph 7.2 ( adjusted with naoh ) and bath solution was 5 mm hepes , 200 mm kcl , 40 mm mgcl2 , ph 7.2 ( adjusted with koh ) . perfusion was accomplished with a nitrogen pressurized micro - perfusion system ( vc-8xp , ala scientific ) . all recordings in the manuscript are presented such that positive currents indicate k flux from the high [ k ] to low [ k ] side . cloning of a codon - optimized variant of the homo sapiens traak gene ( uniprot q9nyg8 - 2 ) and heterologous expression in pichia pastoris was previously described . the construct used in this study is c - terminally truncated by an additional ten amino acids compared to our previous reports and similarly incorporates two mutations to remove n - linked glycosylation sites ( n104q / n108q ) and is expressed as a c - terminal precission protease - cleavable egfp-10xhis fusion protein . traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 is referred to as traak in the text for simplicity . frozen pichia cells expressing traak were disrupted by milling ( retsch model mm301 ) 5 times for 3 minutes at 25 hz . all subsequent purification steps were carried out at 4 c. for crystallization in k , cell powder was added to lysis buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 60 mm decyl--d - maltoside ( dm , affymetrix ) , 0.1 mg / ml dnase 1 , 1 g / ml pepstatin , 1 g / ml leupeptin , 1 g / ml aprotinin , 10 g / ml soy trypsin inhibitor , 1 mm benzamidine , and 1 mm phenylmethysulfonyl fluoride added immediately before use ) at a ratio of 1 g cell pellet/4 ml lysis buffer . membranes were extracted for 3 hours with stirring followed by centrifugation at 35000g for 45 minutes . cobalt resin ( clontech ) was added to the supernatant ( 1 ml resin/5 g cell pellet ) and stirred gently for 3 hours . resin was collected on a column and serially washed and eluted in buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 6 mm dm ) with 10 mm , 30 mm , and 300 mm imidazole ph 8.0 . edta ph 8.0 ( 1 mm final ) and prescission protease ( ~1:50 wt : wt ) were added to the elution before incubation with gentle rocking overnight . cleaved protein was concentrated ( 50 kda mwco ) and applied to a superdex 200 column ( ge healthcare ) equilibrated in sec buffer ( 20 mm tris ph 8.0 , 150 mm kcl , 1 mm edta , 4 mm n - decyl--d - maltopyranoside ( dm ) ) . fab was prepared from monoclonal antibody 13e9 against traak as described and buffer exchanged on a hitrap desalting column ( ge healthcare ) into 20 mm tris 150 mm kcl ph 8.0 for crystallization in kcl or 20 mm tris 150 mm kno3 ph 8.0 for crystallization in tlno3 . traak - fab complexes were prepared by incubating purified channel concentrated ( 50 kda mwco ) to ~10 mg / ml with purified fab concentrated ( 10 kda mwco ) to ~30 mg / ml at a 1:2.5 molar ratio in sec buffer for 10 min at 4c . traak - fab complex was separated from excess free fab on a superdex 200 column ( ge healthcare ) equilibrated in sec buffer . traak - fab complexes were concentrated ( 10 kda mwco ) to 30 mg / ml for crystallization . for crystallization in tl , traak - fab complexes were purified identically except that 150 mm kno3 replaced the 150 mm kcl in all buffers except for the final sec buffer in which 150 mm tlno3 replaced the 150 mm kcl . crystals were grown in drops of 0.250.35 l protein added to an equal volume of reservoir in hanging drops over 100 l reservoir at 4c . reservoir for the nonconductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 . reservoir for the nonconductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 . reservoir for the conductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 , 4% ( vol / vol ) polypropylene glycol 400 . reservoir for the conductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 , 52 mm trichloroethanol . crystals grew to ~ 100 m 100 m 200 m in 46 weeks . for cryoprotection , an approximately equal volume of mother liquor supplemented to be 30% ( vol / vol ) peg 400 was added to one side of the drop and crystals were moved through this solution with a cryoloop before being plunged into liquid nitrogen . thallium - containing crystals were collected at the energy giving a maximum imaginary scattering component f determined by a fluorescent scan . data were processed with hkl2000 and structures were solved by molecular replacement with phaser using the previously solved traak - fab structure with ligands , tm2-tm3 and tm4 regions removed as the search model . consistent with recent analysis , incorporation of weak data at high resolution ( to cc1/2=3560% ) improved map quality . two - fold local ncs and jelly body restraints were used throughout refinement and three tls groups per protein chain were incorporated during the final rounds of refinement . 1350 of 1454 total amino acids ( 93% ) are modeled in the structures : the termini ( amino acids 126 and 285290 plus a nine amino acid linker ) and a loop connecting the helical cap to pore helix 1 ( amino acids a104-a109 and b106-b109 ) in each traak protomer and a loop in each fab heavy chain ( amino acids 130135 ) were not visible in the electron density . in the nonconductive structure in tl , residues 275284 from traak subunit b were additionally not modeled . the positions of seven tl ions in the nonconductive conformation ( two bound to the extracellular surface of traak , one above the cytoplasmic mouth of the filter , and four in the selectivity filter ) and eight tl ions in the conductive conformation ( with an additional tl in the cavity ) were identified in model phased anomalous difference fourier maps as the highest peaks above background . the presence of an acyl chain in the channel cavity was evaluated based on the shape of the electron density ( in omit maps ) and statistics after crystallographic refinement . the mean b - factor and real space correlation coefficient of the acyl chain ( 101 / 0.76 in the k structure and 132 / 0.74 in the tl structure ) are comparable to the values of nearby residues in tm2 and tm4 that project into the cavity ( amino acids 154,155,158,161,269,272,273 , and 276 have an average b - factor / real space correlation coefficient of 125 / 0.78 in the k structure and 142 / 0.83 in the tl structure ) . phases for omit maps were calculated from a ligand - free model with every atom randomly shifted by a maximum of 0.5 prior to 20 cycles of refinement in refmac to remove model bias . omit density in these maps was essentially indistinguishable from maps generated with nave models that never had ligands modeled or simulated annealing omit maps calculated with phenix . structures were compared after alignment of pore helices and selectivity filters ( 119133 and 228242 from chain a and b ) using lsqkab in ccp4 . the area calculation used a surface calculated with 1.4 added to van der waals radii of protein atoms and a probe radius of 3.5 to approximate the lipid accessible surface area . a water cylinder with a 6 radius was used to fill the cavity of the traak channels to exclude its contribution from the calculation . all area calculations and structures in figs . 3 and 4 were made with symmetric traak channels ( i.e. both subunits had the same conformation ) generated by rotation of a copy of one subunit onto the other . structure figures were made with pymol ( version 1.7.2 schrdinger , llc ) or the ucsf chimera package . chimera is developed by the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nigms p41-gm103311 ) . for cellular recording , cho - k1 cells ( atcc ) ( which do not express endogenous mechanosensitive channels ) were cultured in dmem - f12 ( gibco ) with 10% fbs , 0.2 mm l - alanyl - l - glutamine ( glutamax , gibco ) , 100 u / ml penicillin , and 100 ug / ml streptomycin . cells were plated in 35 mm polystyrene dishes ( bd biosciences ) or on polyester thermanox coverslips ( thermo scientific ) ~24 hrs before transfection with fugenehd ( promega ) following manufacturers protocol . after 48 hrs , dishes or coverslips dna encoding amino acids 1290 of homo sapiens traak was cloned into a pir es2-acgfp vector ( clontech ) to generate untagged traak1290 used in experiments shown in fig . dna encoding amino acids 1300 of homo sapiens traak was cloned into the ecor1/xho1 sites of a modified pceh vector to generate a c - terminally egfp tagged construct ( traak1300 - snsavdaglvprgsaaa - egfp - h10 ) . this construct and mutants generated with inverse pcr were used in experiments presented in fig . reconstitution of traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 and recording from proteoliposome blisters was performed as described for traak1300(n104q , n108q)-sns - levlfq / gp - egfp - h10 . azolectin ( egg l--phosphatidylcholine , 840051 ) and dphpc ( 1,2-diphytanoyl - sn - glycero-3-phosphocholine , 850356 ) lipids were from avanti polar lipids , inc . pipettes were pulled from borosilicate glass ( sutter instruments bf150 - 86 - 10 ) to 34 m resistance . recordings were made with an axopatch 200b amplifier ( molecular devices ) , filtered at 1 khz and digitized at 10100 khz ( digidata 1440a , molecular devices ) . pressure application through the patch pipette was accomplished with a high - speed pressure clamp ( hspc , ala scientific ) controlled through the clampex software . pressure application velocity was set to the maximum rate of 8.3 mmhg / msec . all recordings were performed in a ten - fold concentration gradient of k. for cellular recordings , pipette solution was 10 mm hepes , 150 mm kcl , 3 mm mgcl2 , 5 mm egta , ph 7.2 ( adjusted with koh ) and bath solution was 10 mm hepes , 15 mm kcl , 135 mm nacl , 3 mm mgcl2 , 1 mm cacl2 , ph 7.3 ( adjusted with naoh ) . for experiments in fig . 2 and extended data fig . 3 , reducing bath solution additionally contained 10 mm dtt added from a freshly thawed 1 m stock of dtt in water . reducing solution pipette solution was 5 mm hepes , 180 mm nacl , 20 mm kcl , ph 7.2 ( adjusted with naoh ) and bath solution was 5 mm hepes , 200 mm kcl , 40 mm mgcl2 , ph 7.2 ( adjusted with koh ) . perfusion was accomplished with a nitrogen pressurized micro - perfusion system ( vc-8xp , ala scientific ) . all recordings in the manuscript are presented such that positive currents indicate k flux from the high [ k ] to low [ k ] side . ( a , b ) view from the membrane plane of the traak central cavity in the ( a ) nonconductive and ( b ) conductive conformations . the exposed surface of hydrophobic amino acids are colored white , arginine and lysine are blue , glutamate and aspartate are red , and polar residues are green . the positions of k ions in the filter are outlined and residue t277 in tm4 is indicated with an asterisk . ( c ) diameter of the ion conduction pathway as a function of distance through the membrane for nonconductive traak ( red ) , conductive traak ( blue ) and twik1 ( gray , pdb:3ukm ) . the green box indicates the position of the selectivity filter and dashed gray lines are the approximate boundaries of the lipid membrane . the pore diameter is larger in traak than in twik1 and expands below t277 in the conductive conformation . ( a ) current recorded from traak proteoliposome patches as a function of holding voltage ( mean sem , n=9 patches each ) . current through traak reconstituted in phosphatidylcholine lipids with branched acyl chains ( 1,2-diphytanoyl - sn - glycero-3-phosphocholine , dphpc ) was significantly higher than in non - branched acyl chains ( egg l--phosphatidylcholine , pc ) at each voltage measured ( 5.0 fold higher at 0 mv , p < 0.0001 , student s t - test ) . ( b , c ) representative recording of pressure ( lower trace ) activation of traak current ( upper trace ) in ( b ) pc or ( c ) dphpc lipids . ( d ) quantification of pressure activation of traak in pc and dphpc ( mean fold pressure activation at 0 mv sem , n=9 patches each , * * * p < 0.0001 , student s t - test ) . in these experiments and those in fig . 2 , inside - out patches from cells expressing wild - type or mutant traak channels were excised and perfused with reducing bath solution ( with 10 mm dtt ) . after stabilization of the patch ( traak channels exhibit a gradual run - up of current following excision to an equilibrium value , e.g. extended data fig . 4 ) , the perfusion solution was switched to oxidizing bath solution ( no dtt ) . ( a , b ) representative voltage family from a i159c r284c traak patch during perfusion of ( a ) reducing and ( b ) oxidizing solution . the voltage family protocol is illustrated . ( c , d ) same as ( a , b ) but from a wild - type traak patch . ( e , f ) current response ( upper ) to pressure application ( lower ) at 0 mv from the same i159c r284c traak patch during perfusion of ( e ) reducing or ( f ) oxidizing bath solution . ( g , h ) same as ( e , f ) , but from a wild - type traak patch . ( a ) whole cell current from a traak - expressing cell during a voltage step protocol in a ten - fold gradient of [ k ] ( ek+ = 59 mv , holding voltage = 80 mv , v = 10 mv , indicated steps shown ) . ( c ) currents ( upper traces ) recorded from an outside - out patch excised from the same cell as in ( a , b ) . the voltage protocol in ( a ) was used with an additional pressure step ( lower trace ) during each voltage step . ( d ) current - voltage relationship from data in ( b ) ( mean current 5 min after patch excision before pressure ( red ) and peak current during pressure step ( gray ) ) and a recording immediately after pulling the patch ( red dashes ) . the excised patch contains < 1% of the whole cell membrane area , but gives ~25% of the whole cell current before and similar current during a pressure step . this is explained by very low basal activity of traak with near - zero membrane tension ( whole cell ) and channel activation by increasing membrane tension over a broad range ( intermediate tension in an excised patch to high tension in a pressurized patch ) . stereo view from the cytoplasm of an overlay of nonconductive ( red ) and conductive tm2-tm3 rotated ( blue ) conformations . amino acids that sterically prevent tm2-tm3 rotation when tm4 is down are shown as sticks . this rotation can only occur if tm4 is up because amino acids l172 , f201 and g205 on tm2-tm3 shift ( 0.75 2.1 ) to a position that would sterically clash with amino acids y271 and v275 on tm4 in a down conformation . translation of y271 and v275 3.14.1 in tm4 up conformations creates space for the tm2-tm3 rotation . data collection and refinement statistics data from 1 , 1 , 3 and 2 crystals were merged for the conductive k , conductive tl , nonconductive k and nonconductive tl structures , respectively . pairwise root mean square deviation ( ) between conformationally static regions between traak structures conformationally static regions of the traak channel in these structures include the entire a chain and residues 1155 of chain b. summary of traak channel conformations in crystal structures structures shown in figure 3 are highlighted with the color used in the figure .

summaryactivation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch , hearing and pain15 . traak ion channels are neuronally expressed members of the two - pore domain k+ ( k2p ) channel family and are mechanosensitive6 . they are involved in controlling mechanical and temperature nociception in mice7 . mechanosensitivity of traak is mediated directly through the lipid bilayer : it is a membrane tension gated channel8 . however , the molecular mechanism of traak channel gating and mechanosensitivity is unknown . here we present crystal structures of traak in conductive and nonconductive conformations defined by the presence of permeant ions along the conduction pathway . in the nonconductive state , a lipid acyl chain accesses the channel cavity through a 5 - wide lateral opening in the membrane inner leaflet and physically blocks ion passage . in the conductive state , rotation of a transmembrane helix ( tm4 ) about a central hinge seals the intramembrane opening , preventing lipid block of the cavity and permitting ion entry . additional rotation of a membrane interacting tm2-tm3 segment , unique to mechanosensitive k2ps , against tm4 may further stabilize the conductive conformation . comparison of the structures reveals a biophysical explanation for traak mechanosensitivity : an expansion in cross sectional area up to 2.7 nm2 in the conductive state is expected to create a membrane tension - dependent energy difference between conformations that promotes force activation . our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel .

Methods Protein expression and purification Crystallization, data collection, and structure determination Electrophysiology Extended Data

cloning of a codon - optimized variant of the homo sapiens traak gene ( uniprot q9nyg8 - 2 ) and heterologous expression in pichia pastoris was previously described . traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 is referred to as traak in the text for simplicity . edta ph 8.0 ( 1 mm final ) and prescission protease ( ~1:50 wt : wt ) were added to the elution before incubation with gentle rocking overnight . traak - fab complexes were prepared by incubating purified channel concentrated ( 50 kda mwco ) to ~10 mg / ml with purified fab concentrated ( 10 kda mwco ) to ~30 mg / ml at a 1:2.5 molar ratio in sec buffer for 10 min at 4c . traak - fab complex was separated from excess free fab on a superdex 200 column ( ge healthcare ) equilibrated in sec buffer . for crystallization in tl , traak - fab complexes were purified identically except that 150 mm kno3 replaced the 150 mm kcl in all buffers except for the final sec buffer in which 150 mm tlno3 replaced the 150 mm kcl . crystals were grown in drops of 0.250.35 l protein added to an equal volume of reservoir in hanging drops over 100 l reservoir at 4c . reservoir for the nonconductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 . reservoir for the nonconductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 . reservoir for the conductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 , 4% ( vol / vol ) polypropylene glycol 400 . reservoir for the conductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 , 52 mm trichloroethanol . for cryoprotection , an approximately equal volume of mother liquor supplemented to be 30% ( vol / vol ) peg 400 was added to one side of the drop and crystals were moved through this solution with a cryoloop before being plunged into liquid nitrogen . thallium - containing crystals were collected at the energy giving a maximum imaginary scattering component f determined by a fluorescent scan . consistent with recent analysis , incorporation of weak data at high resolution ( to cc1/2=3560% ) improved map quality . two - fold local ncs and jelly body restraints were used throughout refinement and three tls groups per protein chain were incorporated during the final rounds of refinement . 1350 of 1454 total amino acids ( 93% ) are modeled in the structures : the termini ( amino acids 126 and 285290 plus a nine amino acid linker ) and a loop connecting the helical cap to pore helix 1 ( amino acids a104-a109 and b106-b109 ) in each traak protomer and a loop in each fab heavy chain ( amino acids 130135 ) were not visible in the electron density . in the nonconductive structure in tl , residues 275284 from traak subunit the positions of seven tl ions in the nonconductive conformation ( two bound to the extracellular surface of traak , one above the cytoplasmic mouth of the filter , and four in the selectivity filter ) and eight tl ions in the conductive conformation ( with an additional tl in the cavity ) were identified in model phased anomalous difference fourier maps as the highest peaks above background . the presence of an acyl chain in the channel cavity was evaluated based on the shape of the electron density ( in omit maps ) and statistics after crystallographic refinement . the mean b - factor and real space correlation coefficient of the acyl chain ( 101 / 0.76 in the k structure and 132 / 0.74 in the tl structure ) are comparable to the values of nearby residues in tm2 and tm4 that project into the cavity ( amino acids 154,155,158,161,269,272,273 , and 276 have an average b - factor / real space correlation coefficient of 125 / 0.78 in the k structure and 142 / 0.83 in the tl structure ) . the area calculation used a surface calculated with 1.4 added to van der waals radii of protein atoms and a probe radius of 3.5 to approximate the lipid accessible surface area . a water cylinder with a 6 radius was used to fill the cavity of the traak channels to exclude its contribution from the calculation . both subunits had the same conformation ) generated by rotation of a copy of one subunit onto the other . structure figures were made with pymol ( version 1.7.2 schrdinger , llc ) or the ucsf chimera package . chimera is developed by the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nigms p41-gm103311 ) . cells were plated in 35 mm polystyrene dishes ( bd biosciences ) or on polyester thermanox coverslips ( thermo scientific ) ~24 hrs before transfection with fugenehd ( promega ) following manufacturers protocol . dna encoding amino acids 1300 of homo sapiens traak was cloned into the ecor1/xho1 sites of a modified pceh vector to generate a c - terminally egfp tagged construct ( traak1300 - snsavdaglvprgsaaa - egfp - h10 ) . this construct and mutants generated with inverse pcr were used in experiments presented in fig . reconstitution of traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 and recording from proteoliposome blisters was performed as described for traak1300(n104q , n108q)-sns - levlfq / gp - egfp - h10 . pressure application through the patch pipette was accomplished with a high - speed pressure clamp ( hspc , ala scientific ) controlled through the clampex software . all recordings were performed in a ten - fold concentration gradient of k. for cellular recordings , pipette solution was 10 mm hepes , 150 mm kcl , 3 mm mgcl2 , 5 mm egta , ph 7.2 ( adjusted with koh ) and bath solution was 10 mm hepes , 15 mm kcl , 135 mm nacl , 3 mm mgcl2 , 1 mm cacl2 , ph 7.3 ( adjusted with naoh ) . for experiments in fig . 2 and extended data fig . reducing solution pipette solution was 5 mm hepes , 180 mm nacl , 20 mm kcl , ph 7.2 ( adjusted with naoh ) and bath solution was 5 mm hepes , 200 mm kcl , 40 mm mgcl2 , ph 7.2 ( adjusted with koh ) . perfusion was accomplished with a nitrogen pressurized micro - perfusion system ( vc-8xp , ala scientific ) . all recordings in the manuscript are presented such that positive currents indicate k flux from the high [ k ] to low [ k ] side . cloning of a codon - optimized variant of the homo sapiens traak gene ( uniprot q9nyg8 - 2 ) and heterologous expression in pichia pastoris was previously described . the construct used in this study is c - terminally truncated by an additional ten amino acids compared to our previous reports and similarly incorporates two mutations to remove n - linked glycosylation sites ( n104q / n108q ) and is expressed as a c - terminal precission protease - cleavable egfp-10xhis fusion protein . traak1290(n104q , n108q)-sns - levlfq / gp - egfp - h10 is referred to as traak in the text for simplicity . frozen pichia cells expressing traak were disrupted by milling ( retsch model mm301 ) 5 times for 3 minutes at 25 hz . membranes were extracted for 3 hours with stirring followed by centrifugation at 35000g for 45 minutes . resin was collected on a column and serially washed and eluted in buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 6 mm dm ) with 10 mm , 30 mm , and 300 mm imidazole ph 8.0 . for crystallization in tl , traak - fab complexes were purified identically except that 150 mm kno3 replaced the 150 mm kcl in all buffers except for the final sec buffer in which 150 mm tlno3 replaced the 150 mm kcl . crystals were grown in drops of 0.250.35 l protein added to an equal volume of reservoir in hanging drops over 100 l reservoir at 4c . reservoir for the nonconductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 . reservoir for the nonconductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 . reservoir for the conductive conformation in k was 50 mm tris ph 8.8 , 200 mm cacl2 , 2730% ( vol / vol ) peg400 , 4% ( vol / vol ) polypropylene glycol 400 . reservoir for the conductive conformation in tl was 50 mm tris ph 8.8 , 200 mm ca(no3)2 , 2730% ( vol / vol ) peg400 , 52 mm trichloroethanol . for cryoprotection , an approximately equal volume of mother liquor supplemented to be 30% ( vol / vol ) peg 400 was added to one side of the drop and crystals were moved through this solution with a cryoloop before being plunged into liquid nitrogen . data were processed with hkl2000 and structures were solved by molecular replacement with phaser using the previously solved traak - fab structure with ligands , tm2-tm3 and tm4 regions removed as the search model . consistent with recent analysis , incorporation of weak data at high resolution ( to cc1/2=3560% ) improved map quality . two - fold local ncs and jelly body restraints were used throughout refinement and three tls groups per protein chain were incorporated during the final rounds of refinement . 1350 of 1454 total amino acids ( 93% ) are modeled in the structures : the termini ( amino acids 126 and 285290 plus a nine amino acid linker ) and a loop connecting the helical cap to pore helix 1 ( amino acids a104-a109 and b106-b109 ) in each traak protomer and a loop in each fab heavy chain ( amino acids 130135 ) were not visible in the electron density . in the nonconductive structure in tl , residues 275284 from traak subunit b were additionally not modeled . the positions of seven tl ions in the nonconductive conformation ( two bound to the extracellular surface of traak , one above the cytoplasmic mouth of the filter , and four in the selectivity filter ) and eight tl ions in the conductive conformation ( with an additional tl in the cavity ) were identified in model phased anomalous difference fourier maps as the highest peaks above background . the presence of an acyl chain in the channel cavity was evaluated based on the shape of the electron density ( in omit maps ) and statistics after crystallographic refinement . the mean b - factor and real space correlation coefficient of the acyl chain ( 101 / 0.76 in the k structure and 132 / 0.74 in the tl structure ) are comparable to the values of nearby residues in tm2 and tm4 that project into the cavity ( amino acids 154,155,158,161,269,272,273 , and 276 have an average b - factor / real space correlation coefficient of 125 / 0.78 in the k structure and 142 / 0.83 in the tl structure ) . phases for omit maps were calculated from a ligand - free model with every atom randomly shifted by a maximum of 0.5 prior to 20 cycles of refinement in refmac to remove model bias . the area calculation used a surface calculated with 1.4 added to van der waals radii of protein atoms and a probe radius of 3.5 to approximate the lipid accessible surface area . a water cylinder with a 6 radius was used to fill the cavity of the traak channels to exclude its contribution from the calculation . 3 and 4 were made with symmetric traak channels ( i.e. both subunits had the same conformation ) generated by rotation of a copy of one subunit onto the other . chimera is developed by the resource for biocomputing , visualization , and informatics at the university of california , san francisco ( supported by nigms p41-gm103311 ) . cells were plated in 35 mm polystyrene dishes ( bd biosciences ) or on polyester thermanox coverslips ( thermo scientific ) ~24 hrs before transfection with fugenehd ( promega ) following manufacturers protocol . dna encoding amino acids 1300 of homo sapiens traak was cloned into the ecor1/xho1 sites of a modified pceh vector to generate a c - terminally egfp tagged construct ( traak1300 - snsavdaglvprgsaaa - egfp - h10 ) . azolectin ( egg l--phosphatidylcholine , 840051 ) and dphpc ( 1,2-diphytanoyl - sn - glycero-3-phosphocholine , 850356 ) lipids were from avanti polar lipids , inc . recordings were made with an axopatch 200b amplifier ( molecular devices ) , filtered at 1 khz and digitized at 10100 khz ( digidata 1440a , molecular devices ) . pressure application through the patch pipette was accomplished with a high - speed pressure clamp ( hspc , ala scientific ) controlled through the clampex software . pressure application velocity was set to the maximum rate of 8.3 mmhg / msec . 3 , reducing bath solution additionally contained 10 mm dtt added from a freshly thawed 1 m stock of dtt in water . reducing solution pipette solution was 5 mm hepes , 180 mm nacl , 20 mm kcl , ph 7.2 ( adjusted with naoh ) and bath solution was 5 mm hepes , 200 mm kcl , 40 mm mgcl2 , ph 7.2 ( adjusted with koh ) . all recordings in the manuscript are presented such that positive currents indicate k flux from the high [ k ] to low [ k ] side . ( a , b ) view from the membrane plane of the traak central cavity in the ( a ) nonconductive and ( b ) conductive conformations . the exposed surface of hydrophobic amino acids are colored white , arginine and lysine are blue , glutamate and aspartate are red , and polar residues are green . the positions of k ions in the filter are outlined and residue t277 in tm4 is indicated with an asterisk . ( c ) diameter of the ion conduction pathway as a function of distance through the membrane for nonconductive traak ( red ) , conductive traak ( blue ) and twik1 ( gray , pdb:3ukm ) . the green box indicates the position of the selectivity filter and dashed gray lines are the approximate boundaries of the lipid membrane . the pore diameter is larger in traak than in twik1 and expands below t277 in the conductive conformation . current through traak reconstituted in phosphatidylcholine lipids with branched acyl chains ( 1,2-diphytanoyl - sn - glycero-3-phosphocholine , dphpc ) was significantly higher than in non - branched acyl chains ( egg l--phosphatidylcholine , pc ) at each voltage measured ( 5.0 fold higher at 0 mv , p < 0.0001 , student s t - test ) . ( b , c ) representative recording of pressure ( lower trace ) activation of traak current ( upper trace ) in ( b ) pc or ( c ) dphpc lipids . ( d ) quantification of pressure activation of traak in pc and dphpc ( mean fold pressure activation at 0 mv sem , n=9 patches each , * * * p < 0.0001 , student s t - test ) . in these experiments and those in fig . after stabilization of the patch ( traak channels exhibit a gradual run - up of current following excision to an equilibrium value , e.g. extended data fig . 4 ) , the perfusion solution was switched to oxidizing bath solution ( no dtt ) . ( e , f ) current response ( upper ) to pressure application ( lower ) at 0 mv from the same i159c r284c traak patch during perfusion of ( e ) reducing or ( f ) oxidizing bath solution . ( g , h ) same as ( e , f ) , but from a wild - type traak patch . ( c ) currents ( upper traces ) recorded from an outside - out patch excised from the same cell as in ( a , b ) . ( d ) current - voltage relationship from data in ( b ) ( mean current 5 min after patch excision before pressure ( red ) and peak current during pressure step ( gray ) ) and a recording immediately after pulling the patch ( red dashes ) . the excised patch contains < 1% of the whole cell membrane area , but gives ~25% of the whole cell current before and similar current during a pressure step . this is explained by very low basal activity of traak with near - zero membrane tension ( whole cell ) and channel activation by increasing membrane tension over a broad range ( intermediate tension in an excised patch to high tension in a pressurized patch ) . this rotation can only occur if tm4 is up because amino acids l172 , f201 and g205 on tm2-tm3 shift ( 0.75 2.1 ) to a position that would sterically clash with amino acids y271 and v275 on tm4 in a down conformation . data collection and refinement statistics data from 1 , 1 , 3 and 2 crystals were merged for the conductive k , conductive tl , nonconductive k and nonconductive tl structures , respectively . pairwise root mean square deviation ( ) between conformationally static regions between traak structures conformationally static regions of the traak channel in these structures include the entire a chain and residues 1155 of chain b. summary of traak channel conformations in crystal structures structures shown in figure 3 are highlighted with the color used in the figure .

migraine is a common episodic neurovascular disorder characterized by recurrent attacks of typically throbbing , unilateral , and often severe headache with associated features such as nausea , vomiting , and hypersensitivity to light , sound , and smell . in one - third of migraines , the headache is preceded by transient neurological symptoms ( visual , motor , or somatosensory ) lasting for up to an hour or sometimes for several days and known as an aura [ 1 , 2 ] . migraine is clinically divided into two main subtypes based on the absence or presence of an aura : migraine without aura ( mwoa ) and migraine with aura ( mwa ) . migraine affects approximately 15% of the general population , making it the third most frequent disease worldwide in both genders . the neurovascular system is one of the most important mechanisms involved in the pathogenesis of migraine . migraine episodically leads to the activation and sensitization of the trigeminovascular system ( tgvs ) . this system is thought to play an integral role in regulating vascular tone and in the transmission of pain signals . the tgvs consists of the trigeminal nerve and nerve fibers that innervate the network of extra- and intracranial meningeal blood vessels and the brain stem . in detail , the peripheral terminations of the tgvs are also located in extracranial soft tissues such as structures in the eyes and arteries , as well as in intracranial structures . activation of the tgvs stimulates the release of vasoactive neurotransmitters from peripheral terminations of the trigeminal nerve which cause the vascular and inflammatory changes associated with migraine pain . it is possible that not only the hypoperfusion in migraineurs but also a hyperperfusion , which may even follow each other , may involve areas other than the brain , including the retina and choroid [ 47 ] . although the vasoconstriction of cerebral and retinal blood vessels is a transient phenomenon , the chronic nature of migraines might cause permanent structural abnormalities in the brain as well as in the retina and choroid . structurally and functionally normal retinal and choroidal vasculature is essential for the function of the retina , as abnormal choroidal blood volume and/or compromised flow can result in photoreceptor dysfunction and death . changes in the retinal and choroidal blood flow may lead to an alteration of the retinal and choroidal vasculature in migraine . despite this , few studies have evaluated whether the retina and choroid are involved in migraine patients . the aim of this study was to compare the thickness of the retinal nerve fiber layer ( rnfl ) , macula , and choroid in the eyes of migraine patients with age - matched healthy subjects using enhanced depth imaging ( edi ) optical coherence tomography ( oct ) . this prospective , observational , cross - sectional study was conducted at the eye and neurology clinics at the kayseri training and research hospital on 76 patients who had previously been diagnosed as having migraine , with or without aura , according to the criteria of the headache international society . control subjects were required to have had fewer than three headaches in the past year and to have never experienced a migraine . the study protocol was approved by our institutional review board and performed according to the declaration of helsinki . written informed consent for participation in the study all selected eyes had a best - corrected visual acuity ( bcva ) of 0.20 or better ( logarithm of the minimum angle resolution ( logmar ) ) . a normal optic disc was defined as having a cup / disc area ratio less than or equal to 0.4 and a neuroretinal rim with no glaucomatous changes such as localized rim loss , thinning of the rim , or peripapillary hemorrhages seen ophthalmoscopically . if both eyes met all the inclusion criteria , only one eye per patient was selected based on the laterality of migraine . the exclusion criteria were pregnancy , smoking , organic eye disease , previous ocular trauma , intraocular surgery , cataract , glaucoma , strabismus , eccentric fixation , laser treatment , neurological disorders , retinal or optic nerve disorders , and diabetic or hypertensive retinopathy . each participant underwent a comprehensive neurologic and ophthalmologic examination , which included the following tests : bcva ( logmar , early treatment diabetic retinopathy study ( etdrs ) chart ) , slit - lamp biomicroscopy , gonioscopy , dilated funduscopic examination with a 90-diopter lens , and intraocular pressure ( iop ) ( goldmann applanation tonometry ) , axial length ( al ) , and oct measurements . the iol master ( carl zeiss meditec , dublin , ca ) was used for the ocular biometry to measure the corneal curvature and al , repeating the measurements until five valid values were obtained . the al measurements were interpreted based on the signal - to - noise ratio ( snr ) above 2.0 and the appearance of the graphs . edi - oct software ( version 5.6.4.0 ; spectralis oct heidelberg engineering , dossenheim , germany ) was used to assess the thickness of the rnfl , macula , and choroid . the spectralis oct device was used to assess the peripapillary rnfl thickness and the macular thickness . scans for all participants were performed with pupillary dilatation under the same intensity of dim room lighting by the same experienced technician . an internal fixation target was also used in all scans with the real - time eye tracking system to adjust for eye motion . the macular thickness ( m ) the fast macular thickness map included a 25-line raster volume scan , 20 20 degrees , and was centered on the fovea . in the raster scans utilized for the macular measurements , the scans were obtained in high - speed mode with the automated real - time ( art ) feature enabled and set at 9 frames . the infrared scanning laser ophthalmoscope scan angle was set at 30 degrees for all acquired scans . we selected the macular map analysis protocol on the spectralis to display the numeric averages of the measurements for each of the nine subfields as defined by the etdrs circle grid . the diameters of the concentric circles were 1 , 3 , and 6 mm for the macular scan . scans were obtained in high - speed mode with the art feature enabled and set at 16 frames . this software provided a thickness profile across the temporal - superior - nasal - inferior temporal areas of the standard 12-degree circular scan . the software also calculated average thickness values ( m ) globally and for each of the 6 sectors centered on the optic disc ( temporal , temporal superior , temporal inferior , nasal , nasal inferior , and nasal superior ) . spectralis uses a signal - to - noise estimate ( snr in db ) for the quality score . after all exposures , the scans not centered or with snr < 20 db were excluded from the study . each section , consisting of 30 average scans , was obtained in a 15 30-degree rectangle centered at the macula . choroidal thickness was determined as the distance from the outer surface of the hyperreflective line , referred to as the retinal pigment epithelium layer , to the hyperreflective line of the inner scleral border . choroidal thickness was measured at the fovea , 1500 m nasal and 1500 m temporal to the fovea in a horizontal scan section . figures 1(a ) and 1(b ) show pictures of the scanning imaging obtained from two of our subjects . the axial resolution is 3.9 mm digital , which is the same as for routine oct images obtained by spectralis oct . measurements were evaluated by two independent ophthalmologists ( sleyman demircan and mustafa ata ) , and the mean value was generated for analysis . kruskal - wallis h test and one - way analysis of variance ( anova ) were used to compare the differences for continuous variables . one - way multivariate analysis of variance ( manova ) was used for multivariate analysis . this study included 76 eyes in the same number of patients in the migraine group , 36 in patients with aura and 40 in patients without aura , and 38 eyes in 38 participants in the control group . the mean ages were 38.8 11.6 , 37.6 15.7 , and 37.9 11.1 years in the mwa , mwoa , and control groups , respectively . the mean attack frequency was 3.1 1.6 attacks / month in the mwa group and 2.9 1.6 attacks / month in the mwoa group , respectively , and the mean duration of disease was 10.8 8.1 years and 11.1 7.9 years . there was no significant difference between the mean duration of disease ( p = 0.928 ) or the mean number of attacks ( p = 0.688 ) . there were also no statistically significant differences between the migraineurs and the healthy subjects in terms of sex , bcva , al , or iop ( table 1 ) . the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors were significantly less ( p 0.018 ) in the eyes of the migraine group than in those of the control group ( table 2 ) . the manova did not reveal any significant difference between the mwoa , mwa , and control groups ( p = 0.139 ) . the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . similarly , the mean choroid thickness ( m ) at the fovea , 1500 m nasal and 1500 m temporal to the fovea , was significantly less ( p < 0.0001 ) in the eyes of the migraine group than in those of the control group ( table 3 ) . however , there were no significant differences between the two groups in the mean macular thickness ( m ) in any sector ( table 4 ) . most current models of migraine pathogenesis claim that a condition of brain hyperresponsivity to several exogenous and endogenous stimuli may underlie the susceptibility to migraine attacks . however , the exact pathophysiological mechanisms leading to the onset of an attack remain under debate . cortical spreading depression ( csd ) is the underlying mechanism of the migraine aura characterized by brief neuronal excitation , which is followed by a prolonged inhibition of neuronal activity . it may also trigger the headache phase of migraine attacks by activating and sensitizing the trigeminovascular system ( tgvs ) , initiating a series of neural , vascular , and inflammatory events that result in pain [ 9 , 10 ] . the tgvs consists of the trigeminal nerve and nerve fibers that innervate the network of extra- and intracranial meningeal blood vessels and the brain stem . this system is thought to play an integral role in regulating vascular tone and in the transmission of pain signals . in detail , the peripheral terminations of the tgvs are located in extracranial soft tissues such as muscles , eyes , ears , skin , subcutaneous tissue , nasal cavities , arteries , and periosteum , as well as in intracranial structures or venous sinuses and the vagus and glossopharyngeal nerves . activation of the tgvs stimulates the release of neuropeptides from peripheral endings of the trigeminal nerve which cause the vascular and inflammatory changes associated with migraine pain . it is believed that these neuropeptides play a role in causing a sterile neurogenically driven inflammation of the meningeal blood vessels wall ( the dura mater ) , including mast cell degranulation and changes in postcapillary venules including extravasation of plasma proteins and platelet aggregation , and in maintaining the migraine pain . neurogenic plasma extravasation in both dura and the retina can be seen after electrical stimulation of the trigeminal ganglion in experimental animals [ 11 , 12 ] . this presumed neurogenic inflammation can also occur in the choroid , and dadaci et al . and karalezli et al . studies have detected an increased risk for ischemic stroke in patients who had migraine with aura [ 1518 ] . vasospasm emerging prior to or during the pain has been considered to occur concurrently in tissues located outside the brain , and , by extension , local infarctions have been considered to lead to histopathological and functional disorders at the tissue level . killer et al . demonstrated a reduction in blood flow in the lower temporal artery in a patient who had a visual area defect in the left eye during a migraine attack . using colored doppler ultrasonography , . found a reduction in blood flow at the level of the central retinal and posterior ciliary arteries in migraine patients without aura relative to healthy individuals during intercritical migraine periods . using scanning laser polarimetry in studies in migraine patients both with and without aura , tan et al . in contrast , martinez et al . reported that the mean rnfl average thickness parameter was significantly less in the migraine subjects than that in the control group , although there were no differences between the migraine subjects and controls in the mean rnfl thickness in the superior and inferior areas . in another study , martinez et al . found that the mean rnfl thickness in migraine patients was similar to that of healthy individuals , with only the thickness of the temporal quadrant . the rnfl thickness parameters correlate with the migraine disability assessment score ( midas ) , number of attacks , and length of migraine history . the average rnfl thicknesses for global and superior , inferior , and temporal quadrants were significantly thinner in the eyes of the length of migraine history at or above 15 years than in those of the length of migraine history below 15 years . this finding raises questions regarding whether migraine could be associated with glaucoma , although this is controversial . the functional vasospasm of brain and retrobulbar vessels is linked to the pathogenesis of migraine . attacks of migraine may be related to decreased blood flow in the retina and optic nerve . this in turn may lead to unstable ocular perfusion and thereby to ischemia and reperfusion damage in duration of migraine history . ocular blood flow changes are involved in both the pathogenesis of glaucoma and the progression of glaucomatous damage . reported that normal tension glaucoma patients with migraine had a faster course of visual - field deterioration than those without a history of migraine . in addition , a statistically significant correlation was found between the migraine severity and rnfl thickness . . found significant thinning in the rnfl thickness in the upper quadrant but found no differences in the foveal thickness and macular volume in female migraine patients relative to healthy women . we found that the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with the reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . . found no correlation between reduction in rnfl thickness and duration and frequency of migraine . they proposed that the reduction in rnfl thickness strictly related to the presence of migraine . found no statistically significant differences between the control group and the migraine patients for the retinal thickness in any of the quadrants ( p > 0.05 ) . in this study , the average rnfl thickness was significantly thinner in the migraineurs than in the control subjects , and the anova did not reveal any significant difference between the mwoa , mwa , and control groups . we found that the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors were significantly less ( p 0.018 ) in the eyes of the migraine group than in those of the control group . however , the average global rnfl thickness was no statistically significant difference between the control group and the migraine patients . the manova did not reveal any significant difference between the mwoa , mwa , and control groups ( p = 0.139 ) . evidences from the literature suggest that there is a stronger vascular risk associated with mwa than with mwoa which focuses attention on the importance of the aura in vascular disease [ 1518 ] . we speculated that a putative vascular mechanism linking migraine and modification of posterior ocular structures is specifically referred to mwa . however , we did not find any differences between rnfl , macula , and choroid thicknesses in mwa and mwoa subgroups . this result may be explained by the fact that there is a common putative pathogenic mechanism for both migraine types . our findings suggest that some patients , not only mwa but also mwoa , may be at increased risk for possible migraine - related ischemic events . it is likely that there may be a correlation between migraine - related structural brain changes and ocular changes . the structural brain studies indicated that female migraineurs , both with and without aura , are at increased risk of deep white matter and brainstem hyperintensities . these hyperintensities and posterior circulation territory infarct - like lesions are believed to be of ischemic origin . in a recent study palm - meinders et al . evaluated the pathogenesis and relevance of migraine - related structural brain changes and their possible relation with ischemic events . women in the migraine group had a higher prevalence and a greater increase of deep white matter hyperintensities than women in the control group . although migraine was associated with a higher prevalence of infratentorial hyperintensities at follow - up , there were no significant associations of migraine with progression of infratentorial hyperintensities or posterior circulation territory infarct - like lesions among women . in addition , the number of migraines , frequency of migraines , migraine severity , type of migraine ( mwa or mwoa ) , and migraine therapy were not associated with lesion progression . similarly , we found no correlation between the mean duration of migraine , the mean number of attacks of the migraine , and type of migraine and reduction in rnfl thickness . a limitation of our study is that most of the treatments used for migraine attack are vasoactive ( i.e. , triptans ) . although triptans are generally considered safe for use in migraine , schmetterer et al . found that ocular fundus pulsations were slightly reduced after sumatriptan . measurement of ocular fundus pulsations indicates that sumatriptan also has a small vasoconstrictor action on resistance vessels . at least theoretically , the migraine - ischemic insult link may be the consequence of the unfavourable effect of migraine - specific drugs ( i.e. , triptans or ergot alkaloids ) . therefore it may be speculated that their vasoactive properties are a confounding factor for our results of the study . an additional study limitation is that severity of migraine or midas disability score was not evaluated . martinez et al . showed that the average rnfl thickness was significantly correlated with midas . however , ylek et al . found that the visual analogue scale score of the migraine patients was not statistically significantly correlated with any of the parameters , while the length of migraine history was negatively correlated with the average rnfl thickness , supporting a possible association between these pathologies . in another study , ekinci et al . found that the thinning of the rnfl and ganglion cell layer was detected only in the mwa group when groups were analysed . . used sd - oct to investigate the rnfl thickness and macular changes in patients with chronic migraine ( cm ) without visual impairment in comparison to healthy controls . the average rnfl thickness was not significantly thinner in the patients with cm , but the mean superior quadrant rnfl thickness was significantly less in the cm patients than in healthy controls . we found that the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . similar to our findings , migraine duration and the frequency of attacks were unaffected by the rnfl thickness , and the macular changes were not significantly different between the cm patients and healthy controls . similar to our results , sorkhabi et al . found that rnfl thickness was significantly thinner only in the nasal quadrant in migraineurs relative to a control group , and there was no statistical difference between the two migraine subgroups . in addition , the reduction in the average rnfl thicknesses for nasal quadrant was not significantly correlated with the mean duration of migraine from diagnosis , the mean number , or the severity of attacks of the migraine patients . the discrepancy between the results of these studies may be explained by the difference in methods used and the migraine severity [ 2022 ] . [ 21 , 22 ] was less than that in the study by tan et al . . al could influence the measured thickness of the retinal structures due to the reflectance directionality , which can degrade the accuracy of technologies assessing rnfl thickness . the choroid is responsible for most of the ocular blood flow , which is the highest of any tissue in the body , to supply the normal metabolic demands of the outer retina . migraine is known to reduce blood flow at the level of the central retinal and posterior ciliary arteries ; a thinning of the choroid layer is an expected clinical outcome in migraine patients . noted that an increased choroidal thickness during the migraine attack period could reflect an alteration of ocular circulation . however , the other studies reported a reduction in choroidal thickness during the migraine attack period in mwa and mwoa groups [ 33 , 34 ] . the discrepancy between the results of these studies may be explained by the fact that both groups of migraine evolve from hypoperfusion to hyperperfusion during their time course , although perhaps with a difference in intensity . like ekinci , we found that the choroidal thickness was significantly decreased in migraine patients , with or without aura , in comparison to healthy participants . this can be explained by the detrimental effect of migraine on the vascular system in chronic nature of the disease . a reduction in the rnfl thickness has been reported in migraine patients and is most likely a consequence of retinal ischemia . in contrast to the choroidal vessels , which have an intense autonomic innervation , the intraocular portion of the retinal vessels has no autonomic innervation . the superficial layer of the optic nerve head is supplied by small branches originating from the central retinal artery , while the anterior part of the optic nerve is supplied primarily by the short posterior ciliary arteries and choroidal vessels . an alteration in the quality of the blood supply in the optic nerve or rnfl could be indirectly triggered to a greater or lesser extent by altered rheological characteristics of the blood , local vasospasm , or changes in the physiological and/or physical characteristics of the blood vessels implicated in the process . it could be hypothesized that an alteration in the quality of blood supply in the anterior optic nerve head leads to an oligemic - hypoxic insult , which then contributes to ganglion cell death . thus , alterations in these structures may be associated with diseases of the optic nerve . in fact , decreased blood flow in the ophthalmic and posterior ciliary arteries has been shown to be associated with the progression of glaucoma . the chronic ischemic insult related to migraine pathogenic mechanisms may cause structural changes in the lamina cribrosa which have been implicated in the pathogenesis of glaucomatous optic neuropathy . the laminar region of the optic nerve head is the principal site of the retinal ganglion cell axonal insult in glaucoma . according to our oct findings , seong et al . found that patients with normal tension glaucoma had reduction in superior and inferior quadrants , similar to our patients with mwa or mwoa . further studies are needed to determine the alterations in the lamina cribrosa in patients with migraine . in addition , alterations in the ocular and systemic circulation have been demonstrated in patients with glaucoma . the oct results in this study suggest that the migraine pathological process could have a disruptive impact on the posterior ocular structures in chronic nature or severity of the disease and support the theory that mwa and mwoa may be not distinct disorders because they lead to the same results [ 3941 ] . in conclusion , the peripapillary rnfl and choroid were significantly thinner in the migraine patients , with or without aura , than in the age- and sex - matched healthy control subjects . our study suggests that migraine leads to a reduction in the peripapillary rnfl thickness and to thinning in choroidal structures in presence of the migraine as a result of the chronic nature and severity of disease . these findings can be explained by a chronic ischemic insult related to migraine pathogenic mechanisms and these findings are considered supportive of the relationship between glaucoma and migraine .

purpose . to investigate the thickness of the retinal nerve fiber layer ( rnfl ) and choroid in patients who have migraines in comparison to healthy controls . methods . this study included 76 eyes and patients in the migraine group , 36 with aura ( mwa group ) and 40 without ( mwoa group ) , and 38 eyes as control subjects . the rnfl and macular thicknesses were analysed with standard oct protocol while choroidal thickness was analysed with edi protocol in all subjects . choroidal thickness was measured at the fovea , 1500 m nasal and 1500 m temporal to the fovea in a horizontal section . results . the mean rnfl thickness for nasal and nasal inferior sectors was significantly thinner ( p 0.018 ) in the migraineurs ' eyes than in those of the controls , as was the mean choroid thickness at the fovea and measured points ( p < 0.0001 ) . however , the mean macular thickness was not significantly different between the groups . conclusions . this study suggests that migraine leads to a reduction in the peripapillary rnfl thickness and to thinning in choroidal structures . these findings can be explained by a chronic ischemic insult related to migraine pathogenic mechanisms and these findings are considered supportive of the relationship between glaucoma and migraine .

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

migraine is a common episodic neurovascular disorder characterized by recurrent attacks of typically throbbing , unilateral , and often severe headache with associated features such as nausea , vomiting , and hypersensitivity to light , sound , and smell . migraine is clinically divided into two main subtypes based on the absence or presence of an aura : migraine without aura ( mwoa ) and migraine with aura ( mwa ) . migraine affects approximately 15% of the general population , making it the third most frequent disease worldwide in both genders . the neurovascular system is one of the most important mechanisms involved in the pathogenesis of migraine . migraine episodically leads to the activation and sensitization of the trigeminovascular system ( tgvs ) . in detail , the peripheral terminations of the tgvs are also located in extracranial soft tissues such as structures in the eyes and arteries , as well as in intracranial structures . activation of the tgvs stimulates the release of vasoactive neurotransmitters from peripheral terminations of the trigeminal nerve which cause the vascular and inflammatory changes associated with migraine pain . although the vasoconstriction of cerebral and retinal blood vessels is a transient phenomenon , the chronic nature of migraines might cause permanent structural abnormalities in the brain as well as in the retina and choroid . structurally and functionally normal retinal and choroidal vasculature is essential for the function of the retina , as abnormal choroidal blood volume and/or compromised flow can result in photoreceptor dysfunction and death . changes in the retinal and choroidal blood flow may lead to an alteration of the retinal and choroidal vasculature in migraine . despite this , few studies have evaluated whether the retina and choroid are involved in migraine patients . the aim of this study was to compare the thickness of the retinal nerve fiber layer ( rnfl ) , macula , and choroid in the eyes of migraine patients with age - matched healthy subjects using enhanced depth imaging ( edi ) optical coherence tomography ( oct ) . this prospective , observational , cross - sectional study was conducted at the eye and neurology clinics at the kayseri training and research hospital on 76 patients who had previously been diagnosed as having migraine , with or without aura , according to the criteria of the headache international society . control subjects were required to have had fewer than three headaches in the past year and to have never experienced a migraine . the study protocol was approved by our institutional review board and performed according to the declaration of helsinki . written informed consent for participation in the study all selected eyes had a best - corrected visual acuity ( bcva ) of 0.20 or better ( logarithm of the minimum angle resolution ( logmar ) ) . a normal optic disc was defined as having a cup / disc area ratio less than or equal to 0.4 and a neuroretinal rim with no glaucomatous changes such as localized rim loss , thinning of the rim , or peripapillary hemorrhages seen ophthalmoscopically . the exclusion criteria were pregnancy , smoking , organic eye disease , previous ocular trauma , intraocular surgery , cataract , glaucoma , strabismus , eccentric fixation , laser treatment , neurological disorders , retinal or optic nerve disorders , and diabetic or hypertensive retinopathy . each participant underwent a comprehensive neurologic and ophthalmologic examination , which included the following tests : bcva ( logmar , early treatment diabetic retinopathy study ( etdrs ) chart ) , slit - lamp biomicroscopy , gonioscopy , dilated funduscopic examination with a 90-diopter lens , and intraocular pressure ( iop ) ( goldmann applanation tonometry ) , axial length ( al ) , and oct measurements . the al measurements were interpreted based on the signal - to - noise ratio ( snr ) above 2.0 and the appearance of the graphs . edi - oct software ( version 5.6.4.0 ; spectralis oct heidelberg engineering , dossenheim , germany ) was used to assess the thickness of the rnfl , macula , and choroid . the spectralis oct device was used to assess the peripapillary rnfl thickness and the macular thickness . the macular thickness ( m ) the fast macular thickness map included a 25-line raster volume scan , 20 20 degrees , and was centered on the fovea . in the raster scans utilized for the macular measurements , the scans were obtained in high - speed mode with the automated real - time ( art ) feature enabled and set at 9 frames . we selected the macular map analysis protocol on the spectralis to display the numeric averages of the measurements for each of the nine subfields as defined by the etdrs circle grid . the diameters of the concentric circles were 1 , 3 , and 6 mm for the macular scan . the software also calculated average thickness values ( m ) globally and for each of the 6 sectors centered on the optic disc ( temporal , temporal superior , temporal inferior , nasal , nasal inferior , and nasal superior ) . each section , consisting of 30 average scans , was obtained in a 15 30-degree rectangle centered at the macula . choroidal thickness was determined as the distance from the outer surface of the hyperreflective line , referred to as the retinal pigment epithelium layer , to the hyperreflective line of the inner scleral border . choroidal thickness was measured at the fovea , 1500 m nasal and 1500 m temporal to the fovea in a horizontal scan section . figures 1(a ) and 1(b ) show pictures of the scanning imaging obtained from two of our subjects . measurements were evaluated by two independent ophthalmologists ( sleyman demircan and mustafa ata ) , and the mean value was generated for analysis . this study included 76 eyes in the same number of patients in the migraine group , 36 in patients with aura and 40 in patients without aura , and 38 eyes in 38 participants in the control group . the mean ages were 38.8 11.6 , 37.6 15.7 , and 37.9 11.1 years in the mwa , mwoa , and control groups , respectively . the mean attack frequency was 3.1 1.6 attacks / month in the mwa group and 2.9 1.6 attacks / month in the mwoa group , respectively , and the mean duration of disease was 10.8 8.1 years and 11.1 7.9 years . there was no significant difference between the mean duration of disease ( p = 0.928 ) or the mean number of attacks ( p = 0.688 ) . there were also no statistically significant differences between the migraineurs and the healthy subjects in terms of sex , bcva , al , or iop ( table 1 ) . the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors were significantly less ( p 0.018 ) in the eyes of the migraine group than in those of the control group ( table 2 ) . the manova did not reveal any significant difference between the mwoa , mwa , and control groups ( p = 0.139 ) . the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . similarly , the mean choroid thickness ( m ) at the fovea , 1500 m nasal and 1500 m temporal to the fovea , was significantly less ( p < 0.0001 ) in the eyes of the migraine group than in those of the control group ( table 3 ) . however , there were no significant differences between the two groups in the mean macular thickness ( m ) in any sector ( table 4 ) . most current models of migraine pathogenesis claim that a condition of brain hyperresponsivity to several exogenous and endogenous stimuli may underlie the susceptibility to migraine attacks . however , the exact pathophysiological mechanisms leading to the onset of an attack remain under debate . cortical spreading depression ( csd ) is the underlying mechanism of the migraine aura characterized by brief neuronal excitation , which is followed by a prolonged inhibition of neuronal activity . it may also trigger the headache phase of migraine attacks by activating and sensitizing the trigeminovascular system ( tgvs ) , initiating a series of neural , vascular , and inflammatory events that result in pain [ 9 , 10 ] . the tgvs consists of the trigeminal nerve and nerve fibers that innervate the network of extra- and intracranial meningeal blood vessels and the brain stem . this system is thought to play an integral role in regulating vascular tone and in the transmission of pain signals . in detail , the peripheral terminations of the tgvs are located in extracranial soft tissues such as muscles , eyes , ears , skin , subcutaneous tissue , nasal cavities , arteries , and periosteum , as well as in intracranial structures or venous sinuses and the vagus and glossopharyngeal nerves . activation of the tgvs stimulates the release of neuropeptides from peripheral endings of the trigeminal nerve which cause the vascular and inflammatory changes associated with migraine pain . it is believed that these neuropeptides play a role in causing a sterile neurogenically driven inflammation of the meningeal blood vessels wall ( the dura mater ) , including mast cell degranulation and changes in postcapillary venules including extravasation of plasma proteins and platelet aggregation , and in maintaining the migraine pain . neurogenic plasma extravasation in both dura and the retina can be seen after electrical stimulation of the trigeminal ganglion in experimental animals [ 11 , 12 ] . this presumed neurogenic inflammation can also occur in the choroid , and dadaci et al . studies have detected an increased risk for ischemic stroke in patients who had migraine with aura [ 1518 ] . vasospasm emerging prior to or during the pain has been considered to occur concurrently in tissues located outside the brain , and , by extension , local infarctions have been considered to lead to histopathological and functional disorders at the tissue level . demonstrated a reduction in blood flow in the lower temporal artery in a patient who had a visual area defect in the left eye during a migraine attack . found a reduction in blood flow at the level of the central retinal and posterior ciliary arteries in migraine patients without aura relative to healthy individuals during intercritical migraine periods . reported that the mean rnfl average thickness parameter was significantly less in the migraine subjects than that in the control group , although there were no differences between the migraine subjects and controls in the mean rnfl thickness in the superior and inferior areas . found that the mean rnfl thickness in migraine patients was similar to that of healthy individuals , with only the thickness of the temporal quadrant . the rnfl thickness parameters correlate with the migraine disability assessment score ( midas ) , number of attacks , and length of migraine history . the average rnfl thicknesses for global and superior , inferior , and temporal quadrants were significantly thinner in the eyes of the length of migraine history at or above 15 years than in those of the length of migraine history below 15 years . attacks of migraine may be related to decreased blood flow in the retina and optic nerve . in addition , a statistically significant correlation was found between the migraine severity and rnfl thickness . found significant thinning in the rnfl thickness in the upper quadrant but found no differences in the foveal thickness and macular volume in female migraine patients relative to healthy women . we found that the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with the reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . found no correlation between reduction in rnfl thickness and duration and frequency of migraine . they proposed that the reduction in rnfl thickness strictly related to the presence of migraine . found no statistically significant differences between the control group and the migraine patients for the retinal thickness in any of the quadrants ( p > 0.05 ) . in this study , the average rnfl thickness was significantly thinner in the migraineurs than in the control subjects , and the anova did not reveal any significant difference between the mwoa , mwa , and control groups . we found that the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors were significantly less ( p 0.018 ) in the eyes of the migraine group than in those of the control group . however , the average global rnfl thickness was no statistically significant difference between the control group and the migraine patients . the manova did not reveal any significant difference between the mwoa , mwa , and control groups ( p = 0.139 ) . however , we did not find any differences between rnfl , macula , and choroid thicknesses in mwa and mwoa subgroups . this result may be explained by the fact that there is a common putative pathogenic mechanism for both migraine types . in a recent study palm - meinders et al . women in the migraine group had a higher prevalence and a greater increase of deep white matter hyperintensities than women in the control group . in addition , the number of migraines , frequency of migraines , migraine severity , type of migraine ( mwa or mwoa ) , and migraine therapy were not associated with lesion progression . similarly , we found no correlation between the mean duration of migraine , the mean number of attacks of the migraine , and type of migraine and reduction in rnfl thickness . a limitation of our study is that most of the treatments used for migraine attack are vasoactive ( i.e. at least theoretically , the migraine - ischemic insult link may be the consequence of the unfavourable effect of migraine - specific drugs ( i.e. therefore it may be speculated that their vasoactive properties are a confounding factor for our results of the study . showed that the average rnfl thickness was significantly correlated with midas . found that the visual analogue scale score of the migraine patients was not statistically significantly correlated with any of the parameters , while the length of migraine history was negatively correlated with the average rnfl thickness , supporting a possible association between these pathologies . found that the thinning of the rnfl and ganglion cell layer was detected only in the mwa group when groups were analysed . used sd - oct to investigate the rnfl thickness and macular changes in patients with chronic migraine ( cm ) without visual impairment in comparison to healthy controls . the average rnfl thickness was not significantly thinner in the patients with cm , but the mean superior quadrant rnfl thickness was significantly less in the cm patients than in healthy controls . we found that the mean duration of migraine or the mean number of attacks of the migraine patients was not statistically significantly correlated with reduction in the average rnfl thicknesses ( m ) for nasal and nasal - inferior sectors ( p > 0.05 ) . similar to our findings , migraine duration and the frequency of attacks were unaffected by the rnfl thickness , and the macular changes were not significantly different between the cm patients and healthy controls . found that rnfl thickness was significantly thinner only in the nasal quadrant in migraineurs relative to a control group , and there was no statistical difference between the two migraine subgroups . in addition , the reduction in the average rnfl thicknesses for nasal quadrant was not significantly correlated with the mean duration of migraine from diagnosis , the mean number , or the severity of attacks of the migraine patients . the discrepancy between the results of these studies may be explained by the difference in methods used and the migraine severity [ 2022 ] . al could influence the measured thickness of the retinal structures due to the reflectance directionality , which can degrade the accuracy of technologies assessing rnfl thickness . the choroid is responsible for most of the ocular blood flow , which is the highest of any tissue in the body , to supply the normal metabolic demands of the outer retina . migraine is known to reduce blood flow at the level of the central retinal and posterior ciliary arteries ; a thinning of the choroid layer is an expected clinical outcome in migraine patients . noted that an increased choroidal thickness during the migraine attack period could reflect an alteration of ocular circulation . however , the other studies reported a reduction in choroidal thickness during the migraine attack period in mwa and mwoa groups [ 33 , 34 ] . the discrepancy between the results of these studies may be explained by the fact that both groups of migraine evolve from hypoperfusion to hyperperfusion during their time course , although perhaps with a difference in intensity . like ekinci , we found that the choroidal thickness was significantly decreased in migraine patients , with or without aura , in comparison to healthy participants . this can be explained by the detrimental effect of migraine on the vascular system in chronic nature of the disease . a reduction in the rnfl thickness has been reported in migraine patients and is most likely a consequence of retinal ischemia . in contrast to the choroidal vessels , which have an intense autonomic innervation , the intraocular portion of the retinal vessels has no autonomic innervation . an alteration in the quality of the blood supply in the optic nerve or rnfl could be indirectly triggered to a greater or lesser extent by altered rheological characteristics of the blood , local vasospasm , or changes in the physiological and/or physical characteristics of the blood vessels implicated in the process . it could be hypothesized that an alteration in the quality of blood supply in the anterior optic nerve head leads to an oligemic - hypoxic insult , which then contributes to ganglion cell death . thus , alterations in these structures may be associated with diseases of the optic nerve . the chronic ischemic insult related to migraine pathogenic mechanisms may cause structural changes in the lamina cribrosa which have been implicated in the pathogenesis of glaucomatous optic neuropathy . the laminar region of the optic nerve head is the principal site of the retinal ganglion cell axonal insult in glaucoma . found that patients with normal tension glaucoma had reduction in superior and inferior quadrants , similar to our patients with mwa or mwoa . further studies are needed to determine the alterations in the lamina cribrosa in patients with migraine . in addition , alterations in the ocular and systemic circulation have been demonstrated in patients with glaucoma . the oct results in this study suggest that the migraine pathological process could have a disruptive impact on the posterior ocular structures in chronic nature or severity of the disease and support the theory that mwa and mwoa may be not distinct disorders because they lead to the same results [ 3941 ] . in conclusion , the peripapillary rnfl and choroid were significantly thinner in the migraine patients , with or without aura , than in the age- and sex - matched healthy control subjects . our study suggests that migraine leads to a reduction in the peripapillary rnfl thickness and to thinning in choroidal structures in presence of the migraine as a result of the chronic nature and severity of disease . these findings can be explained by a chronic ischemic insult related to migraine pathogenic mechanisms and these findings are considered supportive of the relationship between glaucoma and migraine .

black hole - neutron star ( bh - ns ) binaries are believed to be formed as a result of two supernovae in a massive binary system ( see for an alternative possibility ) . after their formation , the orbital separation decreases gradually due to the longterm gravitational radiation reaction ( i.e. , two objects are in an adiabatic inspiral motion ) , and eventually , two objects merge to be a black hole system . the lifetime of a binary in quasi - circular orbit is approximately written by ( see [ 156 , 155 ] or chapter 16 of ) 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { { \tau _ { { \text{gw } } } } = \frac{{5{c^5}}}{{256\;{g^3}}}\frac{{{r^4}}}{{({m_{{\text{bh } } } } + { m_{{\text{ns}}}}){m_{{\text{bh}}}}{m_{{\text{ns}}}}}}\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; } \\ { \approx 1.34 \times { { 10}^{10}}{\text{yrs}}{{\left ( { \frac{r}{{6 \times { { 10}^6}{\text{km } } } } } \right)}^4}{{\left ( { \frac{{{m_{{\text{bh}}}}}}{{6{m _ \odot } } } } \right)}^ { - 1}}{{\left ( { \frac{{{m_{{\text{ns}}}}}}{{1.4{m _ \odot } } } } \right)}^ { - 1}}{{\left ( { \frac{{{m_0}}}{{7.4{m _ \odot } } } } \right)}^ { - 1 } } , } \end{array}$$\end{document } where r , mbh , and mns are the orbital separation , masses of the bh and ns , respectively , and m0 = mbh + mns . the lifetime for a binary of elliptic orbits with the semi - major axis r is shorter than gw [ 156 , 155 ] . thus , if the initial semi - major axis is smaller than 10 km , the bh and ns merge within the hubble time scale after a substantial emission of gravitational waves . in most of the inspiral phase during which the binary separation gradually decreases due to the gravitational radiation reaction , two compact objects are well approximated by two point masses in an adiabatic orbit , because their radii are much smaller than the orbital separation ( finite - size effects , such as tidal deformation , are negligible ) and also the gravitational - radiation - reaction time scale is much longer than the orbital period ( cf . equation ( 2 ) with r gm0/c ) . the evolution through the inspiral phase is well understood within the post - newtonian ( pn ) approximation . on the other hand , throughout the late inspiral to the merger phases , the orbital evolution process depends significantly on their finite - size effects and the resulting modification on the interaction between the two objects . in addition , the adiabatic approximation for the orbital evolution becomes worse , because the gravitational - radiation - reaction time scale is as short as the orbital period ; the ratio of gw to the orbital period , porb , is approximately written as 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{\tau _ { { \rm{gw } } } } } \over { { p_{{\rm{orb } } } } } } \approx 1.8{\left({{r \over { 6g{m_0}/{c^2 } } } } \right)^{5/2}}{\left({{{{m_{bh } } } \over { 6{m _ \odot } } } } \right)^{- 1}}{\left({{{{m_{ns } } } \over { 1.4{m _ \odot } } } } \right)^{- 1}}{\left({{{{m_0 } } \over { 7.4{m _ \odot } } } } \right)^2},$$\end{document } and thus , for the orbit close to the last one with r 6 gm0/c , gw is comparable to porb . in particular , in the merger phase and subsequent remnant - formation phase , the dynamics of the system depends strongly on the structure of the ns ( the radius and density profile , or its equation of state ; hereafter eos ) and the bh spin , as well as on general relativistic gravity . this implies that a numerical study in the framework of general relativity is required for precisely understanding the final evolution phase of bh - ns binaries . bh - ns binaries have not been observed yet even in our galaxy in contrast to ns - ns binaries [ 205 , 131 ] . however , many of statistical studies based on the stellar evolution synthesis suggest that the coalescence will occur by 110% as frequently as that of ns - ns binaries in our galaxy and hence in the normal spiral galaxies [ 144 , 160 , 223 , 98 , 97 , 20 , 21 , 148 ] ( every 1010 years ) . in addition , coalescence in elliptic galaxies could contribute to the total coalescence rate of the universe by a significant fraction . this implies that coalescence is likely to occur frequently in the hubble volume , and therefore , the evolution process and the final fate of bh - ns binaries deserve a detailed theoretical study . in particular , the following two facts have recently enhanced the motivation for the study of bh - ns binaries : first , bh - ns binaries in close orbits are among the most promising sources for the large laser - interferometric gravitational - wave detectors , such as ligo [ 126 , 2 , 1 ] , virgo [ 222 , 4 , 3 ] , lcgt , and einstein telescope [ 91 , 92 ] : the frequency and amplitude of gravitational waves near the last orbit are estimated to give 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f \approx { \omega \over \pi } \approx 594\,{\rm{hz}}{\left({{{6g{m_0 } } \over { { c^2}r } } } \right)^{3/2}}{\left({{{{m_0 } } \over { 7.4{m _ \odot } } } } \right)^{- 1}},$$\end{document } 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \approx { { 4g{m_0}\mu } \over { { c^4}dr } } \approx 3.6 \times { 10^{- 22}}\left({{{{m_{bh } } } \over { 6{m _ \odot } } } } \right)\left({{{{m_{ns } } } \over { 1.4{m _ \odot } } } } \right)\left({{{6g{m_0 } } \over { { c^2}r } } } \right){\left({{d \over { 100{\rm{mpc } } } } } \right)^{- 1}},$$\end{document } where is the reduced mass of the binary defined by mbhmns / m0 , and d is the distance to the source . the frequency for the late inspiral orbits is just within the frequency - band sensitivity for the advanced gravitational - wave detectors , 103000 khz , and the amplitude of 10 is high enough that the signal of gravitational waves may be detected . the detection rate of bh - ns binaries will be 0.550 events per year for the advanced detectors such as advanced - ligo . to detect gravitational waves and to extract physical information from the gravitational - wave signal , theoretical templates this has motivated pn and numerical - relativity studies as well as two - body approximate general relativistic studies ( e.g. , [ 37 , 50 , 11 , 12 ] ) for the coalescing compact binaries . the second fact is that bh - ns binaries may be some of the progenitors of the central engine of gamma - ray bursts with short time duration 2s ( sgrb ) , for which the source is still unknown . to elucidate whether the merger of bh - ns binaries could be a promising source for the progenitor of the central engine , the final fate of bh - ns binaries is classified into two categories ; a ns is tidally disrupted by its companion bh before it is swallowed by the bh or a ns is simply swallowed by its companion bh in the final phase . there is a third possibility , in which stable mass transfer occurs after the onset of mass shedding of the ns by the bh tidal field . although this may be possible , numerical simulations performed so far have not shown this to be the case , as will be mentioned in section 1.6 . the final fate of a ns depends primarily on the mass of its companion bh and the compactness of the ns . when the bh mass is small enough or the ns radius is large enough , the ns will be tidally disrupted before it is swallowed by the bh . a necessary ( not sufficient ) condition for this is semi - quantitatively derived from the following analysis . mass shedding of a non - spinning ns occurs when the tidal force of its companion bh at the surface of the ns is stronger than the self - gravity of the ns . this condition is approximately ( assuming newtonian gravity ) written as 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2{{g{m_{{\rm{bh}}}}({c_r}{r_{ns } } ) } \over { { r^3}}}\gtrsim{{g{m_{{\rm{ns } } } } } \over { { { ( { c_r}{r_{ns}})}^2}}},$$\end{document } where rns is the circumferential radius of the ns at r . cr is a function of r and an eos - dependent parameter , which is larger than unity and denotes a degree of tidal deformation of the ns , i.e. , the semi - major axis is assumed to be elongated as crrns . the left - hand side denotes the tidal force by the bh at the surface of the ns and the right - hand side is the self - gravitational force of the ns at the inner edge of its surface . we emphasize here that equation ( 5 ) is the necessary condition for the onset of mass shedding , strictly speaking . tidal disruption occurs after substantial mass is stripped from the surface of the ns , during the decrease of the orbital separation due to gravitational - wave emission . thus , tidal disruption should occur for a smaller orbital separation ( larger orbital angular velocity ) than that derived from equation ( 5 ) . we also note that the ns radius is assumed to depend weakly on the ns mass . if the radius quickly increases with mass loss , tidal disruption may occur soon after the onset of mass shedding . assuming that the binary is in a circular orbit with the keplerian angular velocity , equation ( 5 ) may be written in terms of the angular velocity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega (= \sqrt { g{m_0}/{r^3}})$\end{document } as 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega ^2 } \geq 0.5c_{\rm{r}}^{- 3}{{g{m_{{\rm{ns } } } } } \over { r_{{\rm{ns}}}^3}}(1 + { q^{- 1}}),$$\end{document } where q denotes the mass ratio defined by mbh / mns . the latest general - relativistic numerical study for quasi - equilibrium states of bh - ns binaries derives a more quantitative result [ 209 , 210 ] ( see section 2 ) , 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega ^2 } \geq c_\omega ^2\left({{{g{m_{{\rm{ns } } } } } \over { r_{{\rm{ns}}}^3 } } } \right)(1 + { q^{- 1}}),$$\end{document } where the value of the constant , c , is 0.3 for a system composed of a ns of irrotational velocity field and a non - spinning bh . the small value of c ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c_\omega } ( < \sqrt { 0.5})$\end{document } ) indicates that mass shedding is enhanced by significant tidal deformation ( i.e. , cr > 1 ) and/or possibly by general relativistic gravity of the ns . equation ( 7 ) indicates that the frequency of gravitational waves at mass shedding is 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f = { \omega \over \pi } \geq 1.0{\rm{khz}}\left({{{{c_\omega } } \over { 0.3 } } } \right){\left({{{{m_{ns } } } \over { 1.4{m _ \odot } } } } \right)^{1/2}}{\left({{{{r_{{\rm{ns } } } } } \over { 12{\rm{km } } } } } \right)^{- 3/2}}\sqrt { 1 + { q^{- 1 } } } .$$\end{document } thus , tidal disruption is likely to occur for a high frequency f 1 ( 0.7 ) khz for a ns of mns = 1.4 m and rns = 12 ( 15 ) km , irrespective of the bh mass . up to now , we implicitly assume that orbits with arbitrary orbital separations may be possible for the binary system . however , bh - ns binaries always have the innermost stable circular orbit ( isco ) determined by the general relativistic effect , and hence , we should impose the condition that mass shedding ( and tidal disruption ) has to occur before the binary orbit reaches the isco in this analysis . according to pn analysis , a non - dimensional orbital compactness parameter , gm0/c , at the isco , is 0.10 for 1 q 5 for a system composed of a non - spinning bh and a ns . ( the tidal - deformation effect reduces this value by 1020% [ 209 , 210 ] ) . in the presence of the isco , the condition for the onset of mass shedding is written by 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal{c}_{{\rm{isco } } } } \equiv { { g{m_0}{\omega _ { { \rm{isco } } } } } \over { { c^3 } } } \geq { { g{m_0}\omega } \over { { c^3 } } } \geq { c_\omega}{\left({{{g{m_{{\rm{ns } } } } } \over { { r_{{\rm{ns}}}}{c^2 } } } } \right)^{3/2}}{(1 + q)^{3/2}}{q^{- 1/2}},$$\end{document } where isco is the angular velocity at the isco , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{{\rm{isco } } } } \sim 0.1$\end{document } for a system composed of non - spinning bh ( for a spinning bh , this could be much larger than 0.1 ; see section 1.2 ) . equation ( 9 ) is rewritten to give 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left({{{{{\mathcal c}_{{\rm{isco } } } } } \over { { c_\omega } } } } \right)^{2/3}}{{{q^{1/3 } } } \over { 1 + q } } \geq { { g{m_{{\rm{ns } } } } } \over { { r_{{\rm{ns}}}}{c^2 } } } \equiv \mathcal{c},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } denotes the compactness of the ns , which will be , according to nuclear physics theories for high - density matter , in the range between 0.12 and 0.25 [ 115 , 116 ] for the typical ns of mass 1.21.5 m. this estimate suggests that tidal disruption by a non - spinning bh could occur for a binary of a relatively low mass ratio of q 5 . equation ( 10 ) also shows that the conditions for the onset of mass shedding and for tidal disruption depend strongly on the compactness of the ns . in the above simple estimate , the tidal - deformation effect of the ns to the orbital motion is not taken into account . as a result of the tidal deformation , the gravitational force between two stars is modified , so are the orbital evolution and the criterion for tidal disruption . lai , rasio , and shapiro [ 111 , 113 , 112 ] thoroughly investigated the effects associated with tidal deformation in the newtonian framework with the ellipsoidal approximation . they found that the two - body attractive force is strengthen by the effect of the tidal deformation and , by this , the orbital separation of the isco is increased ( see [ 165 , 166 , 114 , 190 ] for related issues ) and that gravitational waveforms in the late inspiral phase are modified ( see [ 66 , 68 , 67 , 72 ] for related issues ) . ury and eriguchi confirmed the fact that the tidal force modifies the orbital motion of bh - ns binaries by numerically computing equilibrium states of a binary composed of a point mass and a fluid star . the essence is that in addition to the newtonian potential , which has the form r , the correction term of the form r appears when a ns is tidally deformed . the magnitude of this correction increases steeply with the decrease of the orbital separation , and modifies the location of the isco . also , the tidal effect accelerates the orbital evolution , because the orbital velocity ( and the centrifugal force ) has to be increased to maintain circular orbits in an enhanced attractive force , and then , the emissivity of gravitational waves is enhanced , leading to a shorter inspiral time . as shown by a simple estimate in the previous section 1.1 , the final fate of bh - ns binaries depends primarily on the mass ratio , q , and the compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document}. however , a detailed analysis has shown that the bh spin and the ns eos also play an important role in determining the final fate . a general relativistic study for the criterion of mass shedding ( note again that this is not the criterion of tidal disruption ) was first performed using an analysis of the tidal interaction between a fluid star and a kerr bh in circular orbit [ 71 , 137 , 135 , 191 , 225 , 94 , 69 , 70 , 154 ] . in this class of analysis , one handles a fluid star orbiting a kerr bh in a circular orbit ( the center of the fluid star has a geodesic motion around the kerr bh ) , and analyzes the structure of the star taking into account the tidal field of the kerr bh located at the center . specifically , one analyzes the euler equation in the following form 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{u_i } } \over { d\tau } } = - { 1 \over \rho}{{\partial p } \over { \partial { x^i } } } - { { \partial \phi } \over { \partial { x^i } } } - { c_{ij}}{x^j},$$\end{document } where is the affine parameter of a geodesic around the bh , x is a coordinate orthogonal to the geodesic , ui denotes the internal velocity of the fluid star , is the rest - mass density , p is the pressure , is the newtonian potential , which obeys = 4g , and cij determines the lowest - order tidal field . higher - order corrections of the tidal potential based on the manasse - misner formalism are also given in . the gravitational effect of the fluid star on the orbital motion , the gravitational radiation reaction , and the relativistic effect on the self - gravity of the star are in general neglected ( see for a more detailed phenomenological analysis ) . thus , this analysis is valid only for the case in which the bh mass is much larger than the ns mass and t the radius of the ns is relatively large : for the case of bh - ns binaries of mass ratio 10 , this analysis can provide only a qualitative or semi - quantitative nature of the tidal effect and orbital evolution . however , by this analysis , several important qualitative properties of the criterion of mass shedding have been found . one of the most important findings is that the criteria for the onset of mass shedding ( and/or for tidal disruption ) depend sensitively on the bh spin , a. the reason for this is that the angular velocity at the isco depends sensitively on the bh spin ; giscombh / c = 1/6 for the non - spinning bh whereas it is 1/2 for the bh of maximum possible spin , a = 1 [ 16 , 186 ] . here , the spin is non - dimensional and defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a \equiv c{j_{{\rm{bh}}}}/gm_{{\rm{bh}}}^2$\end{document } where jbh is the angular momentum of the bh . as equation ( 10 ) indicates , the increase of isco results in the increase of the critical value of q for mass shedding ( tidal disruption ) . indeed , analyses [ 191 , 225 ] indicate that the critical value for q ( or bh mass ) increases by a factor of 15 if the spin is changed from zero to the maximum value ( a = 1 ) ; e.g. , for an incompressible fluid star , the maximum possible mass of a bh , which can cause mass shedding , was derived as 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{bh } } } } = { c_m}{m _ \odot}{\left({{{{m_{{\rm{ns } } } } } \over { 1.4{m _ \odot } } } } \right)^{- 1/2}}{\left({{{{r_{{\rm{ns } } } } } \over { 10{\rm{km } } } } } \right)^{3/2}},$$\end{document } where cm 4.6 for a = 0 , 7.8 for a = 0.5 , 12 for a = 0.75 , 19 for a = 0.9 , and 68 for a = 1 . another important finding is that the criteria for mass shedding ( and tidal disruption ) depend on the ns eos even if the mass and radius of the ns are identical [ 225 , 94 ] : nss with stiffer eos , ( i.e. , with high adiabatic index ) have a relatively uniform density profile and are susceptible to tidal deformation , mass shedding , and tidal disruption by the bh tidal field . consequently , condition ( 12 ) is modified by the eos . in the calculation with compressible models [ 225 , 94 ] , the maximum possible mass of a bh , which can cause mass shedding , may be reduced by 1020% for soft eos ( with the relatively small adiabatic index ) . this implies that not only the compactness of the ns but also its eos , which is still unknown , is reflected in the tidal disruption event . general relativistic studies for quasi - equilibria of bh - ns binaries in addition show that the self - gravity of the ns significantly reduces the maximum mass of the bh for the onset of mass shedding ( see section 1.4 and figure 22 ) . the merger of bh - ns binaries ( more specifically , tidal disruption of a ns by a bh ) is physically and astrophysically an important phenomenon , and deserves a detailed study , because of at least three reasons as follows . gravitational waves emitted during tidal disruption of a ns will bring us invaluable information about the radius and the eos of the ns , because the orbital frequency at tidal disruption depends strongly on the compactness of the ns ( gmns / rnsc ) , e.g. , [ 107 , 109 ] . the masses of the ns and the bh will be determined by the data analysis for gravitational waves emitted in the inspiral phase ( see , e.g. , and also references cited in [ 96 , 180 ] ) . if the ns radius could be determined or constrained from the observation of gravitational waves emitted during tidal disruption , the resultant relation between the mass and the radius of the observed ns may be used for constraining the eos of the high - density nuclear matter [ 127 , 220 , 70 ] . therefore , the gravitational - wave observation for bh - ns binaries will provide a new tool for exploring high - density nuclear matter , which is totally independent of standard nuclear experiments . the issue here is to theoretically clarify the dependence of gravitational waveforms on the ns eos quantitatively ; theoretical templates for a variety of possible eos have to be prepared.a tidally - disrupted ns may form a disk or torus of mass larger than 0.01 m with the density 10 g / cm and the temperature 10 mev around the remnant bh , if tidal disruption occurs outside the isco . a system consisting of a spinning bh surrounded by a massive , hot , and dense torus has been proposed as one of the likely sources for the central engine of a grb [ 143 , 232 , 161 , 142 ] . for the merger of bh - ns binaries , the resulting disk is likely to be compact and its mass is relatively small , of order 0.1 m. since the lifetime of such a disk is likely to be short ( 1 s ) , bh - ns binaries are proposed to be the progenitor of sgrb . specifically , the merger of a low - mass bh and/or a rapidly - spinning bh , and its companion ns can be a candidate for the source of the central engine . according to the observational results by the swift and hete-2 satellites , the total energy of the sgrb is larger than 10 ergs , and typically 1010 ergs . this value may be explained if 110% of the thermal energy generated in a compact disk around a bh is converted to sgrb : 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l = { \eta _ { { \rm{eff}}}}{{g{m_{{\rm{bh}}}}\dot m } \over { { r_d } } } \approx 2 \times { 10^{49}}{\rm{ergs / s}}\left({{{{\eta _ { { \rm{eff } } } } } \over { { { 10}^{- 2 } } } } } \right)\left({{{10 \ ; g{m_{{\rm{bh } } } } } \over { r_{d}{c^2 } } } } \right)\left({{{\dot m } \over { { { 10}^{- 2}}{m _ \odot}/s } } } \right).$$\end{document } here , eff is the efficiency , rd is the typical inner radius of the disk , and is the mass accretion rate . the issue to be resolved is whether or not the mass and thermal energy of the torus formed after the merger are large enough for driving an sgrb of such huge total luminosity . the latest observations have also discovered that a longterm x - ray flare of duration 1010 s is often associated with an sgrb . another more challenging issue is whether or not such an activity may be explained in the bh - ns merger scenario.material ejected from a tidally - disrupted ns may be important for understanding the observed abundances of the heavy elements that are formed by rapid neutron capture in the r - process . one crucial question is whether it is possible for a fraction of material to escape from the system , because the situation is not well prepared for the mass ejection . tidal disruption of a ns occurs typically at an orbital separation of 10gmbh / c . for a test particle of mass m in a circular orbit around the bh with r = 10 gmbh / c , the total energy is approximately gmbhm/2r = 0.05 mc . for a free nucleon , 0.05 mc 50 mev . thus , the issues are , specifically , to answer whether it is possible to give about 50 mev energy to each nucleon and , if possible , to clarify what the relevant process is . gravitational waves emitted during tidal disruption of a ns will bring us invaluable information about the radius and the eos of the ns , because the orbital frequency at tidal disruption depends strongly on the compactness of the ns ( gmns / rnsc ) , e.g. , [ 107 , 109 ] . the masses of the ns and the bh will be determined by the data analysis for gravitational waves emitted in the inspiral phase ( see , e.g. , and also references cited in [ 96 , 180 ] ) . if the ns radius could be determined or constrained from the observation of gravitational waves emitted during tidal disruption , the resultant relation between the mass and the radius of the observed ns may be used for constraining the eos of the high - density nuclear matter [ 127 , 220 , 70 ] . therefore , the gravitational - wave observation for bh - ns binaries will provide a new tool for exploring high - density nuclear matter , which is totally independent of standard nuclear experiments . the issue here is to theoretically clarify the dependence of gravitational waveforms on the ns eos quantitatively ; theoretical templates for a variety of possible eos have to be prepared . a tidally - disrupted ns may form a disk or torus of mass larger than 0.01 m with the density 10 g / cm and the temperature 10 mev around the remnant bh , if tidal disruption occurs outside the isco . a system consisting of a spinning bh surrounded by a massive , hot , and dense torus has been proposed as one of the likely sources for the central engine of a grb [ 143 , 232 , 161 , 142 ] . for the merger of bh - ns binaries , the resulting disk is likely to be compact and its mass is relatively small , of order 0.1 m. since the lifetime of such a disk is likely to be short ( 1 s ) , bh - ns binaries are proposed to be the progenitor of sgrb . specifically , the merger of a low - mass bh and/or a rapidly - spinning bh , and its companion ns can be a candidate for the source of the central engine . according to the observational results by the swift and hete-2 satellites , the total energy of the sgrb is larger than 10 ergs , and typically 1010 ergs . this value may be explained if 110% of the thermal energy generated in a compact disk around a bh is converted to sgrb : 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l = { \eta _ { { \rm{eff}}}}{{g{m_{{\rm{bh}}}}\dot m } \over { { r_d } } } \approx 2 \times { 10^{49}}{\rm{ergs / s}}\left({{{{\eta _ { { \rm{eff } } } } } \over { { { 10}^{- 2 } } } } } \right)\left({{{10 \ ; g{m_{{\rm{bh } } } } } \over { r_{d}{c^2 } } } } \right)\left({{{\dot m } \over { { { 10}^{- 2}}{m _ \odot}/s } } } \right).$$\end{document } here , eff is the efficiency , rd is the typical inner radius of the disk , and is the mass accretion rate . the issue to be resolved is whether or not the mass and thermal energy of the torus formed after the merger are large enough for driving an sgrb of such huge total luminosity . the latest observations have also discovered that a longterm x - ray flare of duration 1010 s is often associated with an sgrb . another more challenging issue is whether or not such an activity may be explained in the bh - ns merger scenario . material ejected from a tidally - disrupted ns may be important for understanding the observed abundances of the heavy elements that are formed by rapid neutron capture in the r - process . one crucial question is whether it is possible for a fraction of material to escape from the system , because the situation is not well prepared for the mass ejection . tidal disruption of a ns occurs typically at an orbital separation of 10gmbh / c . for a test particle of mass m in a circular orbit around the bh with r = 10 gmbh / c , the total energy is approximately gmbhm/2r = 0.05 mc . for a free nucleon , 0.05 mc 50 mev . thus , the issues are , specifically , to answer whether it is possible to give about 50 mev energy to each nucleon and , if possible , to clarify what the relevant process is . there are two computational tasks necessary to deeply understand the nature of the final coalescence phase of bh - ns binaries . one of the important tasks is to quantitatively determine the tidal - deformation effect on the orbital evolution in the late inspiral phase and the criterion for the onset of mass shedding ( which is a necessary condition for tidal disruption ) . determining the degree of the tidal deformation in close orbits is a crucial task for deriving phenomenological two - body equations of motion including the tidal - deformation effect ( e.g. , ) . in the framework of newtonian gravity , a lot of effort was devoted to qualitatively determining the degree of the tidal deformation , its effects on the orbital motion , and the criterion of mass shedding [ 71 , 111 , 114 , 213 , 191 , 217 , 225 , 94 ] as reviewed in section 1.1 . although those approximate analyses are valuable for understanding the qualitative nature of coalescing close binaries , they can not be appropriate for a quantitatively strict understanding , because coalescing bh - ns binaries in close orbits are in a highly relativistic state . a fully general - relativistic study is obviously required . motivated by this fact , numerical computations of quasi - equilibrium states in general relativity have been performed in the past 5 years by several groups , after early attempts , which were done in an approximate formulation of extreme mass ratios [ 19 , 207 ] or in a preliminary formulation . the first results in general relativity were published in 2006 by three groups : taniguchi and collaborators , grandclment [ 82 , 83 ] , and shibata and ury [ 202 , 203 ] . all these works solved the hamiltonian and momentum constraint equations , and some components of einstein s equation ( see section 2 in detail ) . the first two groups employed the excision formulation ( in which the region inside the bh apparent horizon is excised from the computational domain ) and the last one employed the moving - puncture formulation . however , those early - stage results were unsatisfactory for accurately studying quasi - equilibrium sequences . taniguchi and collaborators treated only mildly - relativistic ns and the numerical computation was not very accurate . shibata and ury constructed bh - ns binaries only including the corotating motion for the internal velocity field of the ns . however , in subsequent work , this situation was soon improved . taniguchi and collaborators succeeded in accurately computing bh - ns binaries in quasi - equilibrium and investigated their nature in detail ; grandclment corrected his numerical code , and derived results as accurate as those of taniguchi et al . ; kyutoku et al . succeeded in constructing bh - ns binaries with the irrotational velocity field for the ns [ 197 , 106 ] developing a formulation originally proposed by shibata and ury [ 202 , 203 ] . all these groups computed sequences of quasi - equilibrium states and studied the nature of bh - ns binaries in close circular orbits ( see section 2 ) . except for the early work of shibata and ury [ 202 , 203 ] , all the computations have been done based on the spectral methods library , lorene , because this enables high - precision computation , e.g. , [ 27 , 28 , 84 , 85 ] . ( note also that grandclment and taniguchi are two of the main developers of the lorene library ) . the cornell - caltech group also developed a numerical code , based on a spectral method for the precise computation of bh - ns binaries in quasi - equilibrium states , extending their original code to bh - bh binaries [ 158 , 47 , 40 ] . up to now , this group has published quasi - equilibrium sequences only for the equal - mass case , which is not very realistic for bh - ns binaries , although their results for a variety of bh - ns binaries have been used as initial conditions for numerical - relativity simulations [ 58 , 57 , 74 ] . there are also two additional works on bh - bh / bh - ns binaries in quasi - equilibrium [ 216 , 8 ] . however , these works primarily discuss a numerical method for computing bh binaries in quasi - equilibrium , and do not shown any computational results for bh - ns binaries . the other important task is to clarify the evolution process in the last inspiral and merger phases . for this purpose , the numerical simulation is the unique approach because ( at least ) non - linear hydrodynamics including related physics has to be solved . the merger process of bh - ns binaries was first studied in the framework of newtonian or pseudo - newtonian gravity by several groups [ 95 , 124 , 123 , 121 , 122 , 171 , 169 , 174 ] . these works have qualitatively clarified the tidal disruption process , the subsequent formation process of a bh - disk system , properties of the remnant accretion disk , gravitational waveforms emitted during the merger , and a fraction of matter ejected from the system . earlier work [ 95 , 124 , 123 , 121 , 122 , 171 ] was performed modeling bh by a point mass of newtonian gravity . because of the absence of the isco around the point mass in newtonian gravity , the gravity in the vicinity of the bh was even qualitatively different from that in reality . they conclude that the ns might be tidally disrupted even in an orbit very close to the bh ( well inside a radius of 6 gmbh / c , which is unrealistic ) , and consequently , a massive remnant disk with mass larger than 0.1 m might be formed around the bh , irrespective of the mass ratio and the internal velocity field of the ns . in subsequent work [ 169 , 174 ] , the general - relativistic gravity of the bh was partly mimicked employing the paczyski - wiita potential for the point mass . in this work , it was found that massive disk formation with mass larger than 0.1 m was possible only for a binary system composed of a low - mass bh or of a spinning bh for the case that the bh mass is not low . these properties are qualitatively in agreement with those derived in recent fully general - relativistic simulations , and thus , for qualitatively or semi - qualitatively understanding the nature of the bh - ns merger , the pseudo - newtonian simulation is shown to be helpful . in recent years , approximately general relativistic simulations were also performed by two groups [ 64 , 65 , 164 ] . the derived qualitative features in the merger of bh - ns binaries agree with those from pseudo - newtonian studies as well as by the general relativistic studies described in section 3 . simulations in were carried out incorporating detailed microphysical processes such as finite - temperature eos and neutrino emission employing a neutrino leakage scheme . the neutrino luminosity from the bh - disk system formed in their simulations was found to be 1010 ergs / s for the first 1020 ms after the formation of the disks . this was the result for the case in which a hot and massive disk ( of temperature 10 mev and mass 0.20.7 m ) is formed . the high neutrino luminosity is encouraging for driving a sgrb via neutrino - antineutrino pair annihilation process . as remarked upon above , the massive disk formation in their model parameters would unlikely if general relativistic effects had been correctly taken into account . the high temperature also does not agree with a latest fully general relativistic result in which the simulation was performed with a similar eos . however , the results of suggested for the first time that if such a massive disk was indeed formed , the resulting bh - disk system was a promising candidate for the central engine of sgrb . also , their technique for handling the neutrino emission process becomes a useful guideline for detailed numerical studies in full general relativity ( e.g. , [ 183 , 185 ] ) . one interesting question for the final phase of bh - ns binaries is whether the stable mass transfer occurs in a close orbit ; specifically , the question is whether the mass accretion from a ns to its companion bh proceeds for a time scale longer than the orbital period while escaping tidal disruption or merger . this could occur if the orbital separation does not decrease due to the presence of mass accretion . as discussed in several works and clark and eardley originally guessed that stable mass transfer could occur for a binary of mass ratio larger than a critical value ( but smaller than a value determined by the presence of the isco ; cf . section 1.1 ) , based on their analytic pn analysis . in their analysis , the decrease of the orbital separation associated with gravitational - wave emission , which is one of the important processes in compact binaries of close orbits ( see equation ( 2 ) ) , was not taken into account . cameron and iben showed the importance of the gravitational radiation reaction for the onset of the stable mass transfer even in the close binary of white dwarfs [ 38 , 22 ] . in addition , benz et al . show that if the stripped mass of a mass - shedding star forms a disk around the companion primary star ( or contributes to the spin - up of the companion ) , the condition for the onset of the stable mass transfer is further restricted ( see below ) . the condition for the onset of stable mass transfer is roughly derived applying the analysis method of [ 38 , 22 ] to bh - ns binaries . here we will derive the approximate condition , briefly showing the essence of their analysis method . in the following , the analysis is performed in the newtonian - gravity framework ( except for the incorporation of the gravitational radiation reaction ) , and we assume that the mass element is not lost from the system nor forms a common envelope for simplicity ; the material stripped from a mass - shedding ns is assumed to fall into the companion bh or to form the disk surrounding the bh , because this is indeed the case as found in general relativistic simulations . we also assume that the ns radius does not change , because it indeed depends only weakly on the ns mass as long as the mass is larger than 0.4 m ( e.g. , [ 116 , 186 ] and figure 10 ) . for a very low - mass ns we do not consider such a low - mass ns here . in the newtonian framework , the total angular momentum of the system is written as 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_{{\rm{orb } } } } = \sqrt { { { gr } \over { { m_0 } } } } { m_{{\rm{bh}}}}{m_{{\rm{ns}}}},$$\end{document } where the mass of the disk around the bh is included in mbh . the time evolution of the angular momentum obeys the equation 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot j_{{\rm{orb } } } } = - { \dot j_{{\rm{gw } } } } - \dot s,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot j}_{{\rm{gw}}}}$\end{document } is the loss rate of the angular momentum by the gravitational - wave emission , written assuming that the system is composed of point masses , as 6.4m0(/m0)(gm0/cr ) , and ( 0 ) denotes the total increase rate of the angular momentum of the bh and a disk of the bh . is totally unknown ; to determine it , a numerical simulation is necessary . because it should be approximately proportional to ns ( the mass - shedding rate of the ns ) and mbh , we write = sgc bhmbh , where we use bh = ns . s is a constant of order unity if all the stripped mass elements contribute to spin - up of the bh or to forming a disk ; on the other hand , s = 0 if the stripped mass element does not contribute to the spin - up and the formation of a disk . the assumption s = 0 corresponds to the case in which we simply assume that the orbital momentum of the accreting material is simply added to the companion bh ( the orbital angular momentum of the bh is increased ) . using equation ( 14 ) with bh = ns > 0 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\dot j}_{{\rm{orb}}}}$\end{document } is written as 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\dot j_{{\rm{orb } } } } = { { \dot r } \over { 2r}}{j_{{\rm{orb } } } } + { { q - 1 } \over q}{{{{\dot m}_{{\rm{ns } } } } } \over { { { m}_{{\rm{ns}}}}}}{j_{{\rm{orb,}}}}$$\end{document } and then , we obtain 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\dot r } \over { 2r}}{j_{{\rm{orb } } } } = { { q - 1 } \over q}{{\left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert } \over { { m_{{\rm{ns}}}}}}{j_{{\rm{orb } } } } - { \dot j_{{\rm{gw } } } } - \dot s.$$\end{document } for stable mass transfer to occur , has to satisfy 0 . after a short calculation , the condition is written as 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert \geq } & { { { 16\pi } \over { 45\sqrt { 6}}}{{{q^2 } } \over { ( q - 1){{(q - 1)}^2}}}{{\left({{{6g{m_0 } } \over { { c^2}r } } } \right)}^{5/2}}\left({{{{m_{{\rm{ns } } } } } \over { { p_{{\rm{orb } } } } } } } \right ) } \\ { } & { + { { + { \alpha _ { \rm{s}}}q } \over { \sqrt 6 ( q - 1)}}{{\left({{{6g{m_0 } } \over { { c^2}r } } } \right)}^{1/2}}\left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert . } \\ \end{array}$$\end{document } here , the first and second terms on the right - hand side denote the effects of the gravitational radiation reaction , and of the spin - up or disk formation of the bh , respectively . mass shedding sets in when condition ( 7 ) thus , equation ( 18 ) may be rewritten in the form 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert \geq } & { { { 16\pi } \over { 45\sqrt 6}}{{{q^{7/6}}{{(q + 1)}^{1/2 } } } \over { ( q - 1)}}c_\omega ^{5/3}{{\left({{\mathcal{c } \over { 1/6 } } } \right)}^{5/2}}\left({{{{m_{{\rm{ns } } } } } \over { { p_{{\rm{orb } } } } } } } \right ) } \\ { } & { + { { + { \alpha _ { \rm{s}}}c_\omega ^{1/3 } } \over { \sqrt 6}}{{{q^{5/6}}{{(q + 1)}^{1/2 } } } \over { ( q - 1)}}{{\left({{\mathcal{c } \over { 1/6 } } } \right)}^{1/2}}\left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert . } \\ then the condition can be written in the form 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert { p_{{\rm{orb } } } } } \over { { m_{{\rm{ns } } } } } } \ge { { 16\pi } \over { 45\sqrt 6}}{{{q^{7/6}}{{(q + 1)}^{1/2 } } } \over { ( q - 1)}}c_\omega ^{5/3}{\left({{{\mathcal c } \over { 1/6 } } } \right)^{5/2}}.$$\end{document } the left - hand side denotes the fraction of mass shedding in one orbital period . for stable mass transfer to occur , it should be much smaller than unity ; at least smaller than unity . here , 3.6 q(q + 1)/(q 1 ) 5.4 for 2 thus , for a realistic value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.13 - 0.21$\end{document } , the right - hand side of equation ( 20 ) is 0.1 . this suggests that stable mass transfer may occur in principle with a short time scale ( in 10 orbits ) . however , we note that for a too large value of q , mass shedding is unlikely to occur outside the isco , as illustrated in section 1.1 ( but for a high - spin bh , this may be avoided ) . then , the condition for stable mass transfer becomes 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\left\vert { { { \dot m}_{{\rm{ns } } } } } \right\vert { p_{{\rm{orb } } } } } \over { { m_{{\rm{ns } } } } } } \geq { { 16\pi } \over { 45\sqrt 6}}{{{q^{7/6}}{{(q + 1)}^{1/2 } } } \over { ( q - 1)}}c_\omega ^{5/3}{\left({{{\mathcal c } \over { 1/6 } } } \right)^{5/2}}{\left [ { 1 - { { { \alpha _ { \rm{s}}}c_\omega ^{1/3}{q^{5/6}}{{(q + 1)}^{1/2 } } } \over { \sqrt 6 ( q - 1)}}{{\left({{c \over { 1/6 } } } \right)}^{1/2 } } } \right]^{- 1}}.$$\end{document } here , 2.3 q(q + 1)/(q 1 ) 3.1 for 2 q 10 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$c_\omega ^{1/3 } \lesssim 0.7$\end{document}. thus , the term [ ] is likely to be much larger than unity for s = o(1 ) , and because of this presence of the term associated with s = o(1 ) , stable mass transfer is more strongly restricted . this indicates that the possibility for the occurrence of stable mass transfer depends strongly on the accretion process of the mass - shedding fluid elements , e.g. , if the accreting material contributes mainly to the spin - up of the companion bh with s = 1 , the last term ( the term [ ] ) is 3 , and thus , the mass accretion rate has to be quite high for stable mass transfer to occur . however , with such a high mass - shedding rate , stable mass transfer would only for a short time scale , if at all . one interesting point is that to realize the onset of the stable mass transfer , the formation of the disk surrounding the bh or the spin - up of the bh has to be significantly suppressed . many newtonian simulations , even when including the gravitational radiation reaction by the quadrupole formula , have found that stable mass transfer occurs ( e.g. , the works by janka et al . and rosswog et al . [ 95 , 171 ] ) . show that the presence of the gravitational radiation reaction slightly prevents stable mass transfer , but this is not a very strong effect . however , their subsequent work [ 169 , 174 ] shows that the discovery of stable mass transfer seems to be due to the lack of correct general relativistic physics . they performed an improved simulation in which general relativistic corrections to the gravity of the bh were phenomenologically taken into account via a pseudo - newtonian prescription and showed that stable mass transfer was unlikely to occur , at least for the parameter space they considered . this shows that the gravitational radiation reaction alone does not prevent stable mass transfer , but this plus the strong two - body gravitational force in general relativity does . remember that in the presence of general relativistic two - body effects , the onset of mass shedding outside the isco is possible only for a small value of q , although for the stable mass transfer , a high value of q is required . this conclusion agrees with the results in fully general relativistic simulations ( see section 3 ) . general relativistic simulations , in which the mass accretion process to a bh is accurately followed , also show that the accretion of the stripped mass is used to spin up the bh . this is also an important property for preventing the onset of stable mass transfer ( see section 3 ) . it is worth noting that numerical simulations for bh - ns binaries with both a high bh spin with a > 0.9 and a high mass ratio with q 10 have not been performed yet . for such a case , a strong repulsive force associated with spin - orbit coupling is likely to decrease the orbital radius of the isco and also to increase the inspiral time scale in a close orbit . this effect may help the onset of stable mass transfer , and thus , further studies are still required on this topic . as illustrated above , general relativistic effects ( the general relativistic attractive force between two bodies and resulting presence of the isco , spin - orbit coupling effect , and the general relativistic self - gravity in the ns ) play a crucial role in the dynamics of a close binary system of a bh and a ns . up to merger , the dynamics are primarily determined by the strong general relativistic gravity , and thus , the orbital evolution in a close binary , the merger and tidal disruption processes , the criterion of tidal disruption , and the evolution of the tidally - disrupted ns material depend strongly on general relativistic effects . numerical computation in a fully general relativistic framework is obviously required for accurately and quantitatively understanding the nature of orbital evolution and the merger of bh - ns binaries ; although non - general - relativistic works have provided a qualitative insight for these systems . fortunately , the simulation in full general relativity is not a difficult task any longer , as reviewed in this paper . now there is no reason to employ approximate frameworks for the study of this system . since 2005 , which was the break - through year in the field of numerical relativity , the simulation of binaries composed of bhs has been feasible . soon after the first success for the simulation of bh - bh binaries , work on the merger of bh - ns binaries was published [ 202 , 203 , 197 , 62 , 58 , 63 , 194 , 57 , 107 , 108 , 41 , 74 , 154 , 109 ] . shibata and his collaborators ( hereafter the kyoto / tokyo ( kt ) group ) performed a fully general relativistic simulation for a bh - ns binary merger for the first time , extending their earlier works for ns - ns binaries [ 193 , 200 , 201 , 198 , 199 , 196 ] . soon after the success of the kt group , the university of illinois at urbana - champaign ( uiuc ) and caltech / cornell / cita / washington state university ( cccw ) groups also performed simulations for bh - ns binary merger . the uiuc group extended their earlier work on ns - ns binaries , and the cccw group extended their work for bh - bh binaries [ 30 , 31 , 181 ] incorporating the hydrodynamics equation solvers . in addition , in 2010 , the louisiana state university / brigham young university / perimeter institute / long island university / indiana university ( lbpli ) and albert einstein institute ( aei ) groups published their first results for the bh - ns binary merger [ 41 , 154 ] . all these groups will report with more sophisticated physics incorporating nuclear - theory - based eos , microphysical processes , and magnetic - field effects in the near future . thus , in the following sections , we focus only on reviewing the current status of the general relativistic studies ( see also for a review of the latest status of this field in 2010 ) . we review the current status of the study of quasi - equilibrium states of bh - ns binaries in general relativity . first , the formalisms for a solution of quasi - equilibrium states are described and then the resulting solution , its sequence , and implications are summarized . in section 3 , we review the current status of the numerical - relativity study of bh - ns binaries . in section 3.1 , we summarize the formulations , procedures , and numerical implementations employed in numerical simulations . in section 3.2 , the parameter space ( composed of masses of bh and ns , bh spin , and the eos of ns ) investigated so far is summarized . in sections 3.33.7 , the numerical results for the evolution process throughout the late inspiral to the merger , tidal disruption processes , properties of the remnant formed after the merger , and features of gravitational waveforms and gravitational - wave spectra are summarized . we recommend that readers who are interested only in these simulation results read section 3.33.7 . in this article , we adopt the following notations : latin and greek indices denote spatial and spacetime components , respectively . the geometrical units of c = g = 1 are used , whereas in other sections , c and g are recovered . g - the spacetime metric, g + nn- the three metric on a three dimensional hypersurface t , n - the timelike unit hypersurface normal , k ij - the extrinsic curvature on t , k - the trace of the extrinsic curvature , - the lapse function , - the shift vector , g - the determinant of g,- the determinant of ij ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { - g } = \alpha \sqrt y$\end{document},- the covariant derivative associated with g , d i - the covariant derivative associated with ij , t - the stress energy momentum tensor , - the baryon rest - mass density , - the specific internal energy of the fluid , p - the pressure of the fluid , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h \equiv 1 + \varepsilon + { p \over \rho}$\end{document } - the specific enthalpy of the fluid , k - the polytropic constant,- the adiabatic index , u - the four velocity of the fluid , mns and mbh- the gravitational masses of ns and bh in isolation , m b - the baryon rest mass of ns , m0 mns + mbh- the total mass at infinite separation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a \equiv { { c{j_{{\rm{bh } } } } } \over { gm_{{\rm{bh}}}^2}}$\end{document } - the non - dimensional spin of bh , j bh - the spin angular momentum of bh , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \equiv { { g{m_{{\rm{ns } } } } } \over { { c^2}{r_{{\rm{ns}}}}}}$\end{document } - the compactness of ns in isolation , r ns - the circumferential radius of spherical ns,- the orbital angular velocity , f - the frequency of gravitational waves,- the wavelength of gravitational waves . in this section , we first review formulations to construct bh - ns binaries in quasi - equilibrium , and then the representative numerical results derived so far . for a solution of bh - ns binaries in quasi - equilibrium , it is at least necessary to solve einstein s constraint equations and relativistic hydrostationary equations , supplemented by ns eos . to derive a better model of quasi - equilibrium states , a part of einstein s evolution equation , two approaches have been proposed to constructing bh - ns binaries in quasi - equilibrium . the main difference in those approaches comes from the method for handling the bh singularity . in one approach the bh singularity is eliminated from the computational domain by excising a coordinate sphere and by imposing equilibrium bh boundary conditions ( see also [ 10 , 81 ] for a related isolated - horizon formalism ) . in the other approach , , the metric quantities are decomposed into a singular part , which is written analytically and denotes contributions from a bh and a regular part , which is obtained by numerically solving the corresponding basic equations . we refer to [ 208 , 209 , 210 ] for the excision approach and to [ 202 , 203 , 106 ] for the puncture one . for a more detailed discussion of the decomposition of einstein s equation and the formalism , we refer to [ 29 , 231 , 44 ] , and for the hydrostatics [ 80 , 214 , 219 ] . in the excision approach , einstein s equations in the conformal thin - sandwich formalism are solved for constructing quasi - equilibrium configurations . the line element in the 3 + 1 form is written as 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { d{s^2 } = { g_{\mu \nu } } d{x^\mu } d{x^\nu } \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; } \\ { = - { \alpha ^2}d{t^2 } + { \gamma _ { ij}}(d{x^i } + \beta _ { { \text{com}}}^idt)(d{x^j } + \beta _ { { \text{com}}}^jdt ) , } \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{com}}}^i$\end{document } is the shift vector seen by a comoving observer . we note that the coordinates in the comoving observer frame are usually chosen for convenience in this field ( e.g. , [ 80 , 78 ] ) . the spatial metric ij is further decomposed into a conformal factor and a background spatial metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } as 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ij } } = { \psi ^4}{\tilde \gamma _ { ij}}.$$\end{document } here , the condition , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\det ( { { \tilde \gamma}_{ij } } ) = 1$\end{document } , is not always imposed . the shift vector seen by a comoving observer , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{com}}}^i$\end{document } , can be decomposed as 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { { \rm{com}}}^i = { \beta ^i } + \beta _ { { \rm{rot}}}^i,$$\end{document } where is the shift vector seen by an inertial observer and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{rot}}}^i$\end{document } is a rotating shift . the rotating shift is written as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{rot}}}^i = { ( \omega \times r)^i}$\end{document } , where is the orbital angular velocity vector of the binary system measured at infinity , and r a coordinate vector for which the origin is located at the center of mass of the binary system . the extrinsic curvature is defined by 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${k_{ij } } = - { 1 \over 2}{\mathcal{l}_n}{\gamma _ { ij}},$$\end{document } where n is the lie derivative along the unit normal to the hypersurface t . following [ 229 , 145 ] , it is split into the trace k and the traceless part aij as 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${k_{ij } } = { a_{ij } } + { 1 \over 3}{\gamma _ { ij}}k.$$\end{document } the traceless part is rewritten as 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_{ij } } = { \psi ^{- 2}}{\hat a_{ij}},\,{a^{ij } } = { \psi ^{- 10}}{\hat a^{ij}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat a}_{ij } } = { { \tilde \gamma}_{ik}}{{\tilde \gamma}_{jl}}{{\hat a}^{kl}}$\end{document}. the hamiltonian constraint then takes the form 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde \delta \psi = - 2\pi { \psi ^5}{\rho _ { \rm{h } } } + { 1 \over 8}\psi \tilde r + { 1 \over { 12}}{\psi ^5}{k^2 } - { 1 \over 8}{\psi ^{- 7}}{\hat a_{ij}}{\hat a^{ij}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \delta}$\end{document } denotes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}^{ij}}{{\tilde d}_i}{{\tilde d}_j}$\end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde d}_i}$\end{document } the covariant derivative with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document } the scalar curvature with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document}. equations ( 25 ) , ( 26 ) , and ( 27 ) yield 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a^{ij } } = { { { \psi ^6 } } \over { 2\alpha}}\left({{\partial _ t}{{\tilde \gamma}^{ij } } + { { \tilde d}^i}\beta _ { { \rm{com}}}^i + { { \tilde d}^j}\beta _ { { \rm{com}}}^i - { 2 \over 3}{{\tilde \gamma}^{ij}}{{\tilde d}_k}\beta _ { { \rm{com}}}^k } \right).$$\end{document } inserting equation ( 29 ) into the momentum constraint gives 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { \tilde \delta \beta _ { { \text{com}}}^i + \frac{1}{3}{{\tilde \gamma } ^{ik}}{{\tilde d}_k}({{\tilde d}_j}\beta _ { { \text{com}}}^j ) = - \frac{\alpha } { { { \psi ^6}}}{{\tilde d}_j}\left ( { \frac{{{\psi ^6}}}{\alpha } { \partial _ t}{{\tilde \gamma } ^{ij } } } \right ) + 16\pi \alpha { \psi ^4}{j^i } + \frac{4}{3}\alpha { { \tilde d}^i}k\;\;\ ; } \\ { \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\ ; - \left ( { { { \tilde d}^i}\beta _ { { \text{com}}}^j + { { \tilde d}^j}\beta _ { { \text{com}}}^i - \frac{2}{3}{{\tilde \gamma } ^{ij}}{{\tilde d}_k}\beta _ { { \text{com}}}^k } \right)\frac{\alpha } { { { \psi ^6}}}{{\tilde d}_j}\left ( { \frac{{{\psi ^6}}}{\alpha } } \right ) . } \end{array}$$\end{document } in addition to the hamiltonian and momentum constraints , the trace of the evolution equation of the extrinsic curvature is often employed as one of the field equations : 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}k - { { \mathcal l}_\beta}k = - { \psi ^{- 4}}({\tilde d^2}\alpha + 2{\tilde d_i}\ln \psi { \tilde d^i}\alpha ) + \alpha \left [ { 4\pi ( { \rho _ { \rm{h } } } + s ) + { \psi ^{- 12}}{{\hat a}_{ij}}{{\hat a}^{ij } } + { { { k^2 } } \over 3 } } \right].$$\end{document } for a given condition for k and tk , this equation reduces to an elliptic - type equation for , which possesses primary information of gravitational potential for an equilibrium or quasi - equilibrium configuration . the matter terms in the right - hand side of equations ( 28 ) , ( 30 ) , and ( 31 ) are derived from the projections of the stress - energy tensor t into the spatial hypersurface t , defined by 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho _ { \rm{h } } } = { n_\mu}{n_\nu}{t^{\mu \nu}},$$\end{document } 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j^i } = - \gamma _ \mu ^i{n_\nu}{t^{\mu \nu}},$$\end{document } 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{ij } } = { \gamma _ { i\mu}}{\gamma _ { j\nu}}{t^{\mu \nu}},$$\end{document } 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { \gamma ^{ij}}{s_{ij}}.$$\end{document } for an ideal fluid for which 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\mu \nu } } = ( \rho + \rho \varepsilon + p){u_\mu}{u_\nu } + p{g_{\mu \nu } } = \rho h{u_\mu}{u_\nu } + p{g_{\mu \nu}},$$\end{document } the following relation holds : 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rho _ { \rm{h } } } = \rho h{(\alpha { u^t})^2 } - p,$$\end{document } 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_i } = \rho h(\alpha { u^t}){u_i},$$\end{document } 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{ij } } = \rho h{u_i}{u_j } + p{\lambda _ { ij}}.$$\end{document } the set of equations ( 28)(31 ) has four functions that can be chosen freely ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\partial _ t}{{\tilde \gamma}^{ij}}$\end{document } , tk , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , and k. for computing quasi - equilibrium states , one usually assumes the presence of a helical killing vector , , and the absence of gravitational waves in the wave zone . under these assumptions , it is natural to choose the time direction so as to satisfy = ( /t ) ( in the comoving frame ) , and to set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\partial _ t}{{\tilde \gamma}^{ij}}$\end{document } and tk = 0 . for the choice of k and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , two ways have been proposed . the first one is to choose a maximal slicing k = 0 and to adopt a flat metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij } } = { f_{ij}}$\end{document } , for simplicity . the other choice is to use the kerr - schild metric for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } and k in the vicinity of the bh or in the whole computational space . one of the advantages of choosing maximal slicing and the flat metric background is that the source term becomes simple and falls off rapidly for r enough to obtain accurate results . the disadvantage is that it is not possible to construct the kerr bh even with a distant orbit , and moreover , the set of equations ( 28)(31 ) may have non - unique solutions [ 159 , 17 , 224 ] . in particular , the spin of a bh has two values for the same spin parameter r ( see equation ( 49 ) for the definition ) . the lower branch of the spin as a function of r , which should be physically reasonable because it approaches to the schwarzschild bh as r 0 , can reach only a 0.85 , much less than the maximum spin of a kerr bh . the advantage of using the kerr - schild metric for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } and k is that one can calculate a spinning bh with nearly maximum spin , a 1 . the disadvantage is that the source term becomes complicated and falls off slowly for r . because of this situation , it is not easy to derive the results as accurately as those with the conformal three metric , if one adopts this background metric in the whole computational space . to recover the accuracy , a modification of the kerr - schild metric seems to be necessary ; the metric is chosen to be nearly the kerr - schild one in the vicinity of the bh , whereas away from the bh , the metric should approach exponentially a conformally - flat metric and a maximal slicing [ 75 , 132 ] . in the following , we restrict ourselves to the case of maximal slicing and flat spatial background metric for simplicity . then , equations ( 28 ) , ( 30 ) , and ( 31 ) can be written as 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta \psi = - 2\pi { \psi ^5}{\rho _ { \rm{h } } } - { 1 \over 8}{\psi ^{- 7}}{\hat a_{ij}}{\hat a^{ij}},$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta { \beta ^i } + { 1 \over 3}{\partial ^i}({\partial _ j}{\beta ^j } ) = 16\pi \phi { \psi ^3}{j^i } + 2{\hat a^{ij}}{\partial _ j}(\phi { \psi ^{- 7}}),$$\end{document } 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta \phi = 2\pi \phi { \psi ^4}({\rho _ { \rm{h } } } + 2{\rm{s } } ) + { 7 \over 8}\phi { \psi ^{- 8}}{\hat a_{ij}}{\hat a^{ij}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\underline \delta$\end{document } and i denote the flat laplace operator and the partial derivative , and . note here that the shift vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{com}}}^i$\end{document } was replaced by in equation ( 41 ) , because the rotating shift \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{rot}}}^i$\end{document } does not contribute to the equation in the conformally - flat case . equation ( 29 ) becomes 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a^{ij } } = { { { \psi ^7 } } \over { 2\phi}}\left({{\partial ^i}{\beta ^j } + { \partial ^j}{\beta ^i } - { 2 \over 3}{f^{ij}}{\partial _ k}{\beta ^k } } \right),$$\end{document } where we have replaced \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{com}}}^i$\end{document } by for the same reason as above . to solve the gravitational field equations ( 40 ) , ( 41 ) , and ( 42 ) , it is necessary to impose appropriate boundary conditions on two different boundaries in the excision approach : outer boundaries at spatial infinity and inner boundaries on the bh horizons . assuming asymptotic flatness , the boundary conditions at spatial infinity are written as 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi \vert_{r \to \infty } = 1,$$\end{document } 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta ^i}\vert_{r \to \infty } = 0,$$\end{document } 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi \vert_{r \rightarrow \infty } = 1.$$\end{document } inner boundary conditions arise from the excision of the bh interior . the assumption that the bh is in equilibrium leads to a set of boundary conditions for the conformal factor and the shift vector ( see also [ 40 , 45 ] as well as the related isolated horizon formalism , e.g. , [ 10 , 81 ] ) . the boundary condition for the conformal factor is 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde s^k}{\tilde d_k}\ln \psi \vert _ { \mathcal{s } } = - { 1 \over 4}({{\tilde h}^{ij}}{{\tilde d}_i}{{\tilde s}_j } - { \psi ^2}l)\vert _ { \mathcal{s}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal s}^i } \equiv { \psi ^{- 2}}{{\tilde { \mathcal s}}^i}$\end{document } is the outward pointing unit vector normal to the excision surface and hij is the induced metric on the excision surface , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{ij } } \equiv { \psi ^4}{{\tilde h}_{ij } } = { \gamma _ { ij } } - { { \mathcal s}_i}{{\mathcal s}_j}$\end{document}. the quantity l is computed from the projection of the extrinsic curvature kij as l h kij . the boundary condition on the normal component of the shift vector is 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\beta _ { \top}}\vert _ { \mathcal{s } } = \alpha \vert _ { \mathcal{s}}.$$\end{document } note that the quantity is the the normal component of the shift vector in the comoving frame , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{com}}}^i$\end{document}. the tangential component must form a conformal killing vector of the conformal metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde h}_{ij}}$\end{document } on the excision surface . this can be achieved by choosing them to be killing vectors of a 2-sphere , 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { \vert \vert}^i\vert _ { \mathcal{s } } = { \omega _ r}{{\tilde \xi}^i},$$\end{document } where r is an arbitrary parameter related to the spin of the bh , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \xi}^i}$\end{document } is derived by solving the conformal killing equation on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde h}_{ij}}$\end{document}. because we choose a conformally - flat spatial background , equation ( 49 ) can be rewritten as 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\beta _ { \vert \vert}^i{\vert _ { \mathcal s } } = \epsilon_{jk}^i\omega _ r^j{x^k}.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ \tau ^j$\end{document } is a freely specifiable vector , related to the bh spin , and x is a cartesian coordinate centered on the 2-sphere . the quasi - local spin angular momentum associated with an approximate killing vector of hij is defined by 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{(\xi ) } } = { 1 \over { 8\pi}}\oint\nolimits_{\mathcal{s } } { ( { k_{ij } } - { \gamma _ { ij}}k){\xi ^j}{d^2}{s^i},}$$\end{document } where is the approximate killing vector that is found by solving the killing transport equations as described in [ 55 , 40 ] ( described the original formulation and reported a numerical result for bh - bh binaries based on the formulation of [ 55 , 47 ] ) , or by a new alternative method for finding approximate killing vectors of closed 2-spheres [ 48 , 132 ] . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega _ \tau ^j$\end{document } is iteratively determined until the quasi - local spin angular momentum of bh relaxes to a required value . for example , we can choose a neumann boundary condition 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde s^i}{\partial _ i}\phi { \vert _ \mathcal{s } } = 0$$\end{document } on the excision surface . a puncture method was proposed by brandt and brgmann to describe multiple bh with arbitrary linear momenta and spin angular momenta , extending the original work by brill and lindquist . moving - puncture approach [ 39 , 15 ] was revealed to be quite useful in dynamic simulations . here , we describe the puncture approach for quasi - equilibrium in the context of bh - ns binaries , which was originally developed by shibata and ury [ 202 , 203 ] and subsequently modified by kyutoku et al . . the puncture approach employs a mixture of the conformal thin - sandwich decomposition and the conformal transverse - traceless decomposition of einstein s equations . the trace part of the extrinsic curvature is set to zero ( maximal slicing ) and the three metric is assumed to be conformally flat in the work so far . ( 40 ) , ( 41 ) , and ( 42 ) as in the excision approach . in the puncture approach , the metric quantities , which appear in equations ( 40 ) and ( 42 ) , and , are decomposed into an analytic singular part and a regular part . the former part denotes the contribution from a bh and the latter one is obtained by numerically solving the basic equations . assuming that the puncture is located at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{\rm{p } } } = x_{\rm{p}}^k$\end{document } , and are given by 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi = 1 + { { { m_p } } \over { 2{r_{{\rm{bh } } } } } } + \phi , $ $ \end{document } 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi = 1 - { { { m_\phi } } \over { { r_{{\rm{bh } } } } } } + \eta , $ $ \end{document } where mp and m are positive constants of mass dimension , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r_{{\rm{bh } } } } = \vert { x^k } - x_{\rm{p}}^k\vert$\end{document } is a coordinate distance from the puncture . and denote the regular parts of and , respectively , and are determined by solving elliptic equations ( see equations ( 59 ) and ( 61 ) shown below ) . the quantity mp is an arbitrarily - chosen parameter called the puncture mass , while m is determined by the condition that the adm mass ( madm ) and the komar mass agree , 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\oint\nolimits_{r\rightarrow \infty } { { \partial _ i}\phi d{s^i } = - } \oint\nolimits_{r\rightarrow \infty } { { \partial _ i}\psi d{s^i } = } 2\pi { m_{adm}}.$$\end{document } also , ij is decomposed into singular and regular parts as 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat a_{ij } } = { \tilde d_i}{w_j } + { \tilde d_j}{w_i } - { 2 \over 3}{f_{ij}}{\tilde d_k}{w^k } + k_{ij}^{\rm{p}}.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k_{ij}^{\rm{p}}$\end{document } is the singular part , which denotes a weighted extrinsic curvature associated with the linear momentum , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_j^{{\rm{bh}}}$\end{document } , and spin , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$s_j^{{\rm{bh}}}$\end{document } , of the bh written by 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { k_{ij}^p = { 3 \over { 2r_{{\rm{bh}}}^2}}[{l_i}p_j^{{\rm{bh } } } + { l_j}p_i^{{\rm{bh } } } - ( { f_{ij } } - { l_i}{l_j}){l^k}p_k^{{\rm{bh } } } ] } \\ { + { 3 \over { r_{{\rm{bh}}}^3}}[{l_i}{\epsilon_{jkn } } + { l_j}{\epsilon_{ikn}}]{s^{k{\rm{bh}}}}{l^n}. } \\ \end{array}$$\end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${l^k } = x_{\rm{p}}^k/{r_{{\rm{bh}}}}$\end{document } and ijk is the completely anti - symmetric tensor on t . wi denotes an auxiliary three - dimensional function and w = fwj . the equation for wi is derived by substituting equation ( 56 ) into the momentum constraint equation . because the total linear momentum of the binary system should vanish , the linear momentum of the bh , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p_i^{{\rm{bh}}}$\end{document } , is related to that of the companion ns as 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_i^{{\rm{bh } } } = - \int { { j_i}{\psi ^6}dv,}$$\end{document } where the right - hand side denotes the ( minus ) linear momentum of the ns . the field equations to be solved are summarized as follows : 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta \phi = - 2\pi { \psi ^5}{\rho _ { \rm{h } } } - { 1 \over 8}{\psi ^{- 7}}{\hat a_{ij}}{\hat a^{ij}},$$\end{document } 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta { \beta ^i } + { 1 \over 3}{\partial ^i}({\partial _ j}{\beta ^j } ) = 16\pi \phi { \psi ^3}{j^i } + 2{\hat a^{ij}}{\partial _ j}(\phi { \psi ^{- 7}}),$$\end{document } 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta \eta = 2\pi \phi { \psi ^4}({\rho _ { \rm{h } } } + 2s ) + { 7 \over 8}\phi { \psi ^{- 8}}{\hat a_{ij}}{\hat a^{ij}},$$\end{document } 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underline \delta { w_i } + { 1 \over 3}{\partial _ i}({\partial _ j}{w^j } ) = 8\pi { \psi ^6}{j_{i}}.$$\end{document } we note that in the puncture approach ij is determined by solving equation ( 56 ) , not equation ( 43 ) , because ij is not straightforwardly defined for the location with = 0 when adopting equation ( 43 ) . in this approach , the elliptic equation for still has to be solved because is needed in solving hydrostatic equations . equations ( 59)(62 ) are elliptic in type , and hence , appropriate boundary conditions have to be imposed at spatial infinity . because of the asymptotic flatness , the boundary conditions at spatial infinity are written as 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi { \vert _ { r \rightarrow \infty } } = { \beta ^i}{\vert _ { r \rightarrow \infty } } = \eta { \vert _ { r \rightarrow \infty } } = { w^i}{\vert _ { r \rightarrow \infty } } = 0.$$\end{document } here it is assumed that the equations are solved in the inertial frame . in contrast to the case in which the excision approach is adopted , the inner boundary conditions do not have to be imposed in the puncture approach . this could be a drawback in this approach , because one can not impose physical boundary conditions ( e.g. , killing horizon boundary conditions ) for the bh . however , this could also be an advantage , because we do not have to impose a special condition for the geometric variables , and as a result , flexibility for adjusting a quasi - equilibrium state to a desired state is preserved . there are several versions for deriving the hydrostatic equations [ 26 , 9 , 192 , 215 , 80 , 204 ] . in this section , the equation of motion is written as 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \nu}t_\mu ^\nu = 0.$$\end{document } assuming a perfect fluid , equation ( 36 ) , the term in the left - hand side of the equation of motion is written as 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { \nabla _ \nu}t_\mu ^\nu = \rho { u^\nu}{\nabla _ \nu}(h{u_\mu } ) + h{u_\mu}{\nabla _ \nu}(\rho { u^\nu } ) + { \nabla _ \mu}p , } \\ { = \rho [ { u^\nu}{\nabla _ \nu}(h{u_\mu } ) + { \nabla _ \mu}h ] + h{u_\mu}{\nabla _ \nu}(\rho { u^\nu } ) + \rho t{\nabla _ \mu}s , } \\ \end{array}$$\end{document } where t is the temperature , and s is the entropy per baryon mass . to derive the second line of equation ( 65 ) , we use an equation of local thermodynamic equilibrium , 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$dh = tds + { 1 \over \rho}dp.$$\end{document } assuming that the entropy per baryon is constant everywhere inside the ns and using equation ( 65 ) , equation . ( 64 ) yields 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${u^\nu}{\nabla _ \nu}(h{u_\mu } ) + { \nabla _ \mu}h = 0,$$\end{document } where we use the equation of rest - mass conservation , 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \nu}(\rho { u^\nu } ) = 0.$$\end{document } equation ( 67 ) can be further modified . defining a spatial velocity in the comoving frame , the 4-velocity is written as 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${u^\mu } = { u^t}({\xi ^\mu } + { v^\mu}),$$\end{document } where is a helical killing vector , and we have a relation , n = 0 . u denotes the time component of u and is calculated through the normalization condition of u , uu = 1 . substituting equation ( 69 ) into equation ( 67 ) and using the relation , 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mathcal{l}_\xi}(h{u_\mu } ) = 0,$$\end{document } we obtain 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal l}_v}(h{u_\mu } ) + { \nabla _ \mu}\left({{h \over { { u^t } } } } \right ) = 0.$$\end{document } if the fluid motion in a ns is synchronized with the orbital motion , i.e. , the corotating case , we have = 0 . thus , equation ( 71 ) can be integrated once to yield 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h \over { { u^t } } } = \mathrm{constant}.$$\end{document } if the fluid motion in a ns is irrotational , we need to consider the vorticity of the fluid . the relativistic vorticity tensor is defined by 73\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { \mu \nu } } \equiv { \nabla _ \mu}(h{u_\nu } ) - { \nabla _ \nu}(h{u_\mu}).$$\end{document } for the irrotational flow , = 0 , there exists a scalar potential by which the term hu is expressed as 74\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h{u_\mu } = { \nabla _ \mu}\psi .$$\end{document } inserting equation ( 74 ) into equation ( 71 ) , we have 75\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \mu}\left({{{\mathcal l}_v}\psi + { h \over { { u^t } } } } \right ) = 0,$$\end{document } and thus , 76\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal l}_v}\psi + { h \over { { u^t } } } = { c_{\rm{e } } } = \mathrm{constant},$$\end{document } or , in another form , 77\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${u^\mu}{\nabla _ \mu}\psi + { h \over { { u^t } } } = { c_{\rm{e}}}.$$\end{document } it is interesting to note that if we use the cartan identity , 78\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^\mu}{\omega _ { \mu \nu } } = { { \mathcal l}_\xi}(h{u_\nu } ) - { \nabla _ \nu}(h{u_\mu}{\xi ^\mu}),$$\end{document } another form of the integrated euler equation , e.g. , equation ( 33 ) in , is obtained . because of the helical symmetry \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal l}_\xi}(h{u_\nu } ) = 0$\end{document } and the irrotational flow = 0 , the cartan identity yields 79\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \nu}(h{u_\mu}{\xi ^\mu } ) = 0,$$\end{document } and thus , 80\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h{u_\mu}{\xi ^\mu } = - { c_{\rm{e}}}.$$\end{document } this equation is equivalent to equation ( 77 ) . the next task is to derive an equation for the velocity potential . the term in the left - hand side of the equation of continuity ( 68 ) is rewritten as 81\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}{c } } { { \nabla _ \nu } ( \rho { u^\nu } ) = \frac{1}{{\sqrt { - g } } } { \mathcal{l}_u}(\rho \sqrt { - g } ) , } \\ = \frac{1}{{\alpha \sqrt \gamma } } { \mathcal{l}_v}(\rho { u^t}\alpha \sqrt \gamma ) , \\ = \frac{1}{\alpha } { d_i}(\rho \alpha { u^t}{v^i } ) . \end{array},$$\end{document } using equations ( 69 ) , ( 74 ) , and the expression for the helical killing vector , 82\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\xi ^\mu } = \alpha { n^\mu } + \beta _ { { \rm{com}}}^\mu , $ $ \end{document } the equation of continuity ( 68 ) is rewritten as 83\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d^i}{d_i}\psi + ( { d^i}\psi - h{u^t}\beta _ { { \rm{com}}}^i){d_i}\ln \left({{{\rho \alpha } \over h } } \right ) - { d_i}(h{u^t}\beta _ { { \rm{com}}}^i ) = 0,$$\end{document } where equation ( 81 ) is used . finally , we comment on the prescription for determining the constant in the right - hand side of equation ( 77 ) . for this task , the central value of the quantities in the left - hand side is usually used . here the center of the ns is defined as the location of the maximum baryon rest - mass density . equation ( 77 ) includes one more constant , which should be determined for each quasi - equilibrium figure ; the orbital angular velocity as found from equations ( 24 ) , ( 69 ) , ( 74 ) , and ( 82 ) . the first integral of the euler equation ( 77 ) includes information of the orbital angular velocity , , through equations . ( 24 ) , ( 69 ) , ( 74 ) , and ( 82 ) . should be determined from the condition that a binary system is in a quasi - equilibrium circular orbit . in the following , we describe two typical methods referring to the rotation axis of the binary system as the z - axis and to the axis connecting the bh s and ns s centers as the x - axis . in one of two typical methods , the force balance equation is derived from the condition that the central value of the enthalpy gradient for the ns is zero , 84\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left . { { { \partial \ln h } \over { \partial x } } } \right\vert _ { { \mathcal{o}_{{\rm{ns } } } } } } = 0,,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal o}_{{\rm{ns}}}}$\end{document } denotes the ns s center at which the pressure gradient and self - gravitational force of the ns are absent . thus , equation ( 84 ) may be regarded as the condition that the gravitational force from the bh at the ns s center is equal to the centrifugal force associated with the orbital motion , and hence , can be used to determine for a given set of gravitational field variables . in the other method , is determined by requiring the enthalpy at two points on the ns s surface along the x - axis to be equal to h = 1 on the surface . at these two points , the pressure is absent . namely , the sum of the gravitational force from the bh , self - gravitational force from the ns , and the centrifugal force associated with the orbital motion is balanced . these two conditions may be regarded as the conditions that determine ce and , and thus , can be determined for a given set of gravitational field variables . [ 208 , 209 , 210 , 214 ] confirmed that in both methods , an accurate numerical result can be computed with a reasonable number of iterations , and that the results by these two methods coincide within the convergence level of the enthalpy . equation ( 84 ) also depends on the location of the center of mass , because the rotating shift \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta _ { { \rm{rot}}}^i$\end{document } includes r , which is the radial coordinate measured from the center of mass of the binary system . to determine the location of the center of mass of a binary system , in the framework of the excision approach , taniguchi et al . [ 208 , 209 , 210 , 214 ] and grandclment [ 82 , 83 ] require that the linear momentum of the system vanishes 85\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^i } = { 1 \over { 8\pi}}\oint\nolimits_{r \rightarrow \infty}{k^{ij}}d{s_j } = 0,$$\end{document } where the maximal slicing condition , k = 0 , is assumed . the essence of this condition is as follows : for a hypothetical orbital angular velocity , , the total linear momentum of the system depends on the location of the center of mass , and hence , its location is determined by condition ( 85 ) . once the location of the center of mass is determined in an iteration step , the positions of the bh and ns are moved , keeping the separation , in order for the center of mass of the binary system to locate on the z - axis . in the puncture approach , the situation is totally different from the above , because condition ( 85 ) has already been used to calculate the linear momentum of the bh ; see equation ( 58 ) in section 2.1.2 . in this framework , there is no known natural , physical condition for determining the center of mass of the system . until now , three methods have been employed to determine the center of mass . in the first method , the dipole part of at spatial infinity is required to be zero [ 202 , 203 ] . however , it was found that in this condition , the angular momentum derived for a close orbit of m0 0.03 is 2% smaller than that derived by the 3pn approximation for q = 3 . because the 3pn approximation should be an excellent approximation of general relativity for a fairly distant orbit , as such m0 0.03 , the obtained initial data deviates from the true quasi - circular state , and hence , the initial orbit would be eccentric . in the second method , the azimuthal component of the shift vector at the location of the puncture is required to be equal to ; a corotating gauge condition at the location of the puncture is imposed . however , the angular momentum derived for a close orbit of m0 0.03 is also 2% smaller than that derived by the 3pn relation for a larger mass ratio q 2 . the disagreement is larger for the larger mass ratio . such initial conditions are likely to deviate from the true quasi - circular state and hence the orbital eccentricity is large as well . in the last method , the center of mass is determined in a phenomenological manner : one imposes a condition that the total angular momentum of the binary system for a given value of m0 agrees with that derived by the 3pn approximation . this condition can be achieved by appropriately choosing the position of the center of mass . with this method , the drawback in the previous two methods , i.e. , the angular momentum becoming smaller than the expected value , is overcome . recent numerical simulations by the kt group have been performed employing initial conditions obtained by this method , and showed that the binary orbit is not very eccentric with these initial conditions ( cf . a wide variety of eos has been adopted for the study of quasi - equilibrium states of bh - ns binaries , which are employed as initial conditions of numerical simulations ( see section 3 ) . however , the only eos used for the study of quasi - equilibrium sequences has been the polytrope , 86\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p = \kappa { \rho ^\gamma}.$$\end{document } where is a polytropic constant and is the adiabatic index . hereafter , we review only the numerical results in this eos . for the polytropic eos , we have the following natural units , i.e. , polytropic units , to normalize the length , mass , and time scales : 87\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{{\rm{poly } } } } = { \kappa ^{1/(2\gamma - 2)}}.$$\end{document } because the geometric units with g = c = 1 are adopted , the polytropic units rpoly normalize all of the length , mass , and time scales . even though the eos used for constructing sequences is only the polytrope , a lot of quasi - equilibria with several eos have been derived as initial data for the merger simulations . the several key quantities that are necessary in the quantitative analysis of quasi - equilibrium sequences are summarized in this section ; the irreducible mass and spin of bh , the baryon rest mass of ns , the adm mass , the komar mass , and the total angular momentum of the system . it is reasonable to consider that the irreducible mass and spin of the bh and the baryon rest mass of the ns are conserved during the inspiral of bh - ns binaries . in addition , temperature and entropy of the ns may be assumed to be approximately zero because the thermal effects of not - young ns are negligible to their structure , i.e. , it is reasonable to use a fixed cold eos throughout the sequence . for such a sequence , the adm mass , the komar mass , and the total angular momentum of the system vary with the decrease of the orbital separation . we classify the study by seven parameters and summarize in table 1 : table 1summary for the study of quasi - equilibrium sequences.(1)(2)(3)(4)(5)(6)(7)taniguchi et al . ( 2007 ) cf , mirex00.088 , 0.1451 , 2 , 3 , 5 , 10taniguchi et al . ( 2008 ) cf , mirex00.109 , 0.120 , 0.132,1 90.145 , 0.160 , 0.178grandclmentcf , mirex00.075 , 0.100 , 0.125,5(2006 ) , ( 2007 ) 0.150shibata & ury ( 2006 ) cf , mcopu00.1392.47shibata & ury ( 2007 ) cf , mcopu00.139 , 0.1482.47 , 3.08kyutoku et al . ( 2009 ) cf , mirpu00.132 , 0.145 , 0.1601 , 3 , 5cf , mirex00.1441foucart et al . this is defined by 88\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{irr } } } } \equiv \sqrt { { { { a_{{\rm{eh } } } } } \over { 16\pi}},}$$\end{document } where aeh is the proper area of the bh event horizon . because it is not possible to numerically calculate aeh for a study of quasi - equilibrium configuration , people in the numerical relativity community approximate this area with that of the apparent horizon , aeh , which is computed from an integral on the apparent horizon s , 89\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_{{\rm{ah } } } } = \int\nolimits_{\mathcal s}{\psi ^4}{d^2}x.$$\end{document } in the presence of a helical killing vector , it is reasonable to consider that aeh agrees with aeh ( at least approximately ) . in the framework of the excision approach , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } corresponds to the excision surface . on the other hand , it is necessary to determine the apparent horizon in the framework of the puncture approach ( although it is not a difficult task ) . the spin is determined by the quasi - local spin angular momentum of the bh , which is given in equation ( 51 ) , i.e. , 90\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{(\xi ) } } = { 1 \over { 8\pi}}\oint\nolimits_{\mathcal{s}}({k_{ij } } - { \gamma _ { ij}}k){\xi ^j}{d^2}{s^i}.$$\end{document } this is defined by 91\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{\rm{b } } } = \int { \rho { u^t}\sqrt { - g } { d^3}x,}$$\end{document } where g is the determinant of g. assuming the absence of mass loss , this should be conserved . the adm mass in isotropic cartesian coordinates may be computed from 92\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{adm } } } } = - { 1 \over { 2\pi}}\oint\nolimits_{r \rightarrow \infty}{\partial ^i}\psi d{s_i}.$$\end{document } in the conformal thin - sandwich formalism employed so far , the komar mass is written as 93\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{komar } } } } = - { 1 \over { 4\pi}}\oint\nolimits_{r \rightarrow \infty } { { \partial ^i } } \alpha d{s_i},$$\end{document } where the shift vector is assumed to fall off sufficiently rapidly . york defined this quantity by 94\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_i } = { 1 \over { 16\pi}}{\epsilon_{ijk}}\oint\nolimits_{r \rightarrow \infty } { ( { x^j}{k^{kl } } - { x^j}{k^{jl}})d{s_l } } , $ $ \end{document } where x is a spatial cartesian coordinate relative to the center of mass of the binary system . in addition , the definition of the linear momentum ( which is usually set to be zero ) is the same as equation ( 85 ) , 95\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^i } = { 1 \over { 8\pi}}\oint\nolimits_{r \rightarrow \infty } { { k^{ij}}d{s_j}},$$\end{document } where the maximal slicing condition , k = 0 , is assumed . then , the binding energy of the binary system is often defined by 96\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e_{\rm{b } } } = { m_{{\rm{adm } } } } - { m_0},$$\end{document } where m0 is the adm mass of the binary system at infinite orbital separation , m0 mbh + mns ( see section 1.8 for the definition ) . for a non - spinning bh , mbh coincides with the irreducible mass mirr . for a spinning bh , the relation among mbh , mirr , and a is given by 97\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{{\rm{bh } } } } = { m_{{\rm{irr}}}}{\left [ { { 2 \over { 1 + { { ( 1 + { a^2})}^{1/2 } } } } } \right]^{1/2}}.$$\end{document } in order to measure a global error in the numerical results , the virial error is often defined as the fractional difference between the adm and komar masses , 98\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta m = \left\vert { { { { m_{{\rm{adm } } } } - { m_{{\rm{komar } } } } } \over { { m_{{\rm{adm } } } } } } } \right\vert .$$\end{document } here , we note the presence of a theorem , which states that for the helical symmetric spacetime , the adm mass and komar mass are equal ( e.g. , [ 76 , 204 ] ) . to determine the orbit at the onset of mass shedding of a ns , gourgoulhon et al . [ 80 , 211 , 212 ] defined a sensitive mass - shedding indicator ( in the context of ns - ns binaries ) as 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi \equiv { { { { ( \partial ( \ln \,h)/\partial r)}_{{\rm{eq } } } } } \over { { { ( \partial ( \ln \,h)/\partial r)}_{{\rm{pole}}}}}}.$$\end{document } here , the numerator of equation ( 99 ) , ( (ln h)/r)eq is the radial derivative of the enthalpy in the equatorial plane at the surface along the direction toward the companion , and the denominator , ( (lnh)/r)pole , is that at the surface of the pole . the radial coordinate r is measured from the center of the ns . for a spherical ns at infinite separation , = 1 , while = 0 indicates the formation of a cusp , and hence , the onset of mass shedding . taniguchi et al . [ 208 , 209 , 210 ] analyze this parameter for identifying the mass - shedding limit of bh - ns binaries . only a small parameter space has been surveyed for the study of quasi - equilibrium sequences . we classify the study by seven parameters in table 1 : the eos of the nsthe spatial background metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } and the extrinsic curvature kthe notation of cf means the conformally - flat condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij } } = { f_{ij}}$\end{document } , and that of m denotes the maximal slicing k = 0.the notation of ks means the kerr - schild metric . in this case , the extrinsic curvature k is also set to that derived from the kerr - schild metric.the state of the fluid flow in the nsthe notation of co means a corotating ns , and that of ir denotes an irrotational ns.the method to handle the bh , i.e. , the excision or the puncturethe notation of pu means the puncture approach , and that of ex denotes the excision approach.the bh spin athe compactness of the ns \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document}the mass ratio q because all the papers published so far employed the = 2 polytrope for the eos , we do not include the eos in table 1 . the spatial background metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } and the extrinsic curvature k the notation of cf means the conformally - flat condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij } } = { f_{ij}}$\end{document } , and that of the notation of ks means the kerr - schild metric . in this case , the extrinsic curvature k is also set to that derived from the kerr - schild metric . the state of the fluid flow in the ns the notation of co means a corotating ns , and that of ir denotes an irrotational ns . the method to handle the bh , i.e. , the excision or the puncture the notation of pu means the puncture approach , and that of ex denotes the excision approach . the compactness of the ns \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } as found in table 1 , the surveyed parameter space is quite narrow , in particular for the eos of the ns and the bh spin ; complete sequences with a 0 have not yet been derived , and moreover , bh - ns binaries in quasi - equilibrium in which the bh spin takes a high value > 0.9 or the bh spin and orbital angular momentum vector misalign have not been studied yet . these are issues left for the future . for the initial data of the merger simulation several works , summarized in table 1 , have been done to calculate quasi - equilibrium sequences . in this section , we focus on the results reported in because a systematic survey for a wider parameter space was done only there . figure 1 displays contours of the conformal factor for a bh - ns binary with mass ratio q = 3 and ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(j / m_0 ^ 2)$\end{document}. this contour plot shows the configuration at the smallest orbital separation , for which taniguchi s code can achieve a converged solution . the thick solid circle on the left - hand side denotes the position of the excised surface ( the apparent horizon ) , while that on the right - hand side denotes the position of the ns surface . a saddle point presents between the bh and ns , and for this close orbit , it is located in the vicinity of the ns surface , suggesting that the orbit of the binary is close to the mass - shedding limit . the value of on the excised surface is not constant because a neumann boundary condition ( 47 ) is imposed . figure 1contours of the conformal factor in the equatorial plane for the innermost configuration with q = 3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } shown in . contours of the conformal factor in the equatorial plane for the innermost configuration with q = 3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } shown in . the cross figure 2 shows the binding energy ( eb / m0 ) and the total angular momentum ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar m}_b } = 0.15({\mathcal c } = 0.145)$\end{document } ) as functions of the orbital angular velocity ( m0 ) for a ns with baryon rest mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } and for mass ratio q = 3 . the solid curves with the filled circles denote the numerical results , and the dashed curves , the results in the 3pn approximation . the numerical sequences terminate at an orbit of cusp formation ( i.e. , at an orbit of the mass - shedding limit ) before the isco is encountered , i.e. , before a turning point ( minimum ) of the binding energy appears . figure 2binding energy eb / m0 ( left panel ) and total angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j / m_0 ^ 2$\end{document } ( right panel ) as functions of m0 for binaries of mass ratio q = 3 and ns mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar m}_{\rm{b } } } = { ( } 0.15{)}({\mathcal c } = { ( } 0.145{)})$\end{document } . the solid curve with filled circles show numerical results , and the dashed curve denotes the results in the 3pn approximation . binding energy eb / m0 ( left panel ) and total angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$j / m_0 ^ 2$\end{document } ( right panel ) as functions of m0 for binaries of mass ratio q = 3 and ns mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar m}_{\rm{b } } } = { ( } 0.15{)}({\mathcal c } = { ( } 0.145{)})$\end{document } . the solid curve with filled circles show numerical results , and the dashed curve denotes the results in the 3pn approximation . from the qualitative argument described in section 1.1 , the binary separation dms at the onset of mass shedding of a ns can be approximately derived as ( see equation ( 5 ) ) 100\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{{d_{{\rm{ms } } } } } \over { { m_{{\rm{bh } } } } } } \simeq { 1 \over { { q^{2/3}}\mathcal{c}}}.$$\end{document } if dms is greater by a sufficient amount than the isco separation disco , we may expect the ns to start shedding mass and to be tidally disrupted before being swallowed by the bh . relation ( 100 ) suggests that the mass - shedding separation decreases with increasing mass ratio , q , and compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(j / m_0 ^ 2)$\end{document}. it is natural to expect to encounter minima in the binding energy and total angular momentum for binaries with sufficiently large mass ratio and ns compactness . figure 3 shows the binding energy ( eb / m0 ) and the total angular momentum \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bar m}_{\rm{b } } } = { ( } 0.15{)}({\mathcal c } = { ( } 0.145{)})$\end{document } as functions of the orbital angular velocity ( m0 ) for a ns with baryon rest mass \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.120$\end{document } and for q = 5 . the ns compactness is the same as , but the mass ratio is larger than , that shown in figure 2 . in this model , the binary encounters an isco before the onset of mass shedding , i.e. , we see minima in the binding energy and angular momentum just before the end of the sequence . figure 3the same as figure 2 but for the sequence of mass ratio q = 5 . the same as figure 2 but for the sequence of mass ratio q = 5 . figure 3 shows that the turning points in the binding energy and the total angular momentum appear simultaneously to within numerical accuracy . this fact is more clearly seen in figure 4 in which the binding energy versus total angular momentum for sequences of mass ratio q = 5 but with different ns compactness is plotted . a simultaneous turning point in as suggested by equation ( 100 ) , turning points are not found for small compactness ( e.g. , the case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } ) , since the sequences terminate at mass shedding before encountering an isco . note that 3pn sequences can not identify mass shedding and therefore always exhibit turning points . figure 4the binding energy as a function of total angular momentum for binaries of mass ratio q = 5 , and different ns compactness . the binding energy as a function of total angular momentum for binaries of mass ratio q = 5 , and different ns compactness . one of the most important questions in the context of bh - ns binaries is whether the coalescence leads to mass shedding of the ns before reaching an isco , or whether the ns is swallowed by the bh before the onset of mass shedding . this question arises from the perspective of gravitational - wave observations and from the relation with sgrb . gravitational waveforms in the final inspiral phase depend strongly on this issue , and hence , a precise understanding of this is necessary to predict gravitational waveforms theoretically . for launching a sgrb , the formation of an accretion disk surrounding the bh is one of the most promising models , and can occur only if the ns is disrupted prior to reaching an isco . exploring this issue quantitatively requires dynamic simulations , and the results of such simulations are reviewed in section 3 . however , the study of quasi - equilibrium sequences can also provide a guide to where separation mass shedding or dynamic merger occurs . in the following , specifically , we will review qualitative expressions that may be used to predict whether a bh - ns binary of arbitrary mass ratio and ns compactness encounters an isco before shedding mass or not . first , we summarize the results for the binary separation and the orbital angular velocity at which mass shedding from the ns surface occurs . in newtonian gravity and semi - relativistic approaches , simple equations may be introduced to fit the effective radius of a roche lobe [ 225 , 94 , 151 , 60 ] . in [ 202 , 203 ] a fitting equation is introduced for binaries composed of a non - spinning bh and a corotating ns in general relativity . in this section , we review how to derive a fitting equation from data in for a non - spinning bh and an irrotational ns . to derive a fitting formula , we need to determine the orbital angular velocity at the mass - shedding limit . however , it is not possible to construct cusp - like configurations by the numerical code used in , which is based on a spectral method and accompanied by the gibbs phenomena . this is also the case for a configuration with smaller values of 0.5 , even though a cusp - like configuration does not appear ( here is a mass - shedding indicator defined by equation ( 99 ) ) . thus , the data points for such close orbits have to be determined by extrapolation . for this purpose , taniguchi et al . tabulated as a function of the orbital angular velocity and extrapolated the sequence to = 0 by using fitting polynomial functions to find the orbits at the onset of mass shedding . figure 5 shows an example of such extrapolations for sequences of ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } with mass ratios q = 1 , 2 , 3 , and 5 . from the extrapolated results toward = 0 , the orbital angular velocity at the mass - shedding limit is approximately determined for each set of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } and q. figure 5extrapolation of sequences for ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } to the mass - shedding limit ( = 0 ) . the thick curves are sequences constructed using numerical data , and the thin curves are extrapolated sequences . note that the horizontal axis is the orbital angular velocity in polytropic units , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \omega = \omega { r_{{\rm{poly}}}}$\end{document}. extrapolation of sequences for ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } to the mass - shedding limit ( = 0 ) . the thick curves are sequences constructed using numerical data , and the thin curves are extrapolated sequences . note that the horizontal axis is the orbital angular velocity in polytropic units , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \omega = \omega { r_{{\rm{poly}}}}$\end{document}. to derive a fitting formula for the orbital angular velocities at the mass - shedding limit for all the values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } and q , the qualitative newtonian expression of equation ( 7 ) is useful . by fitting sequence data to this expression , taniguchi et al . determined the value of c for = 2 polytropic eos as 0.270 , i.e. , 101\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\overline \omega _ { { \rm{ms } } } } = 0.270{{{c^{3/2 } } } \over { { { \bar m}_{{\rm{ns}}}}}}{(1 + { q^{- 1}})^{1/2}},$$\end{document } or equivalently 102\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{ms}}}}{m_0 } = 0.270\mathcal{c}^{3/2}(1+q){(1 + { q^{- 1}})^{1/2}},$$\end{document } figure 6 shows the results of the fitting for the mass - shedding limit . the solid curve denotes equation ( 101 ) and the points are the numerical results . figure 6fits of the mass - shedding limit by the analytic expression ( 101 ) . the mass - shedding limit for each ns compactness and mass ratio is computed by the extrapolation of the numerical data . the mass - shedding limit for each ns compactness and mass ratio is computed by the extrapolation of the numerical data . it may be interesting to note that the value of c = 0.270 is the same as that found for quasi - equilibrium sequences of ns - ns binaries in general relativity in and of bh - ns binaries in general relativity where the ns is corotating in [ 202 , 203 ] . the value of c = 0.270 could be widely used for an estimation of the orbital angular velocity at the mass - shedding limit of ns in a relativistic binary system with = 2 . one of the most important pieces of information for relativistic close binaries is the binary separation ( or the orbital angular velocity ) at which the minimum of the binding energy appears , corresponding to the isco . the minimum point is located by fitting three nearby points of the sequence to a second - order polynomial , because the numerical data is discrete and does not necessarily give the exact minimum . a simple empirical fitting that predicts the angular velocity isco at the isco for an arbitrary companion orbiting a bh one searches for an expression with three free parameters that express isco function of the mass ratio q and the compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.5$\end{document } of the companion . then the three parameters are determined by matching to three known values of isco , namely , ( 1 ) that of a test particle orbiting a schwarzschild bh , iscom0 = 6 ( for q = ) , ( 2 ) that of an equal - mass bh - bh system as computed in , iscom0 = 0.1227 ( for q = 1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.1452$\end{document } ) , and finally ( 3 ) that of a bh - ns configuration as computed in , iscom0 = 0.0854 ( for q = 5 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } ) . a further requirement arises from the fact that for a test particle ( with q = ) , the expression should be independent of the companion s compactness . a good fit to the numerical data is found in with the expression 103\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{isco}}}}{m_0 } = 0.0680\left [ { 1 - { { 0.444 } \over { { q^{0.25}}}}\left({1 - 3.54{\mathcal{c}^{1/3 } } } \right ) } \right],$$\end{document } as demonstrated in figure 7 . the exponents of q and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } in equation ( 103 ) are empirically determined by requiring that the fitted curves lie near the data points for all the models . the agreement is not perfect , but sufficient for finding the orbital angular velocity at the isco within 10% error . figure 7fits of the minimum point of the binding energy curve by expression ( 103 ) . fits of the minimum point of the binding energy curve by expression ( 103 ) . combining equations ( 102 ) and ( 103 ) the binary encounters an isco before reaching mass shedding or the ns reaches the mass - shedding limit . figure 8 illustrates the final fate of bh - ns binaries for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \omega}$\end{document}. the solid curve denotes the orbital angular velocity at the mass - shedding limit , and the long - dashed one denotes it at the isco . as seen from equations ( 102 ) and ( 103 ) , both of these curves depend on the mass ratio q , but in different ways , which leads to the intersection of the two curves . an inspiraling binary evolves along horizontal lines towards increasing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , starting at the left and moving toward the right , until reaching either the isco or the mass - shedding limit . after the binary reaches the isco for sufficiently large mass ratio , we can not predict the fate of the ns , because it is in a dynamic plunge phase ( see section 3 ) , but nevertheless the mass - shedding limit for unstable quasi - equilibrium sequences is included as the dotted curve in figure 8 . as shown in figure 8 , the model with mass ratio q = 6 ( dotted - dashed line ) encounters the isco , while that of q = 3 ( dot - dot - dashed line ) ends up at the mass - shedding limit . the intersection between the mass - shedding and isco curves marks a critical point that separates the two distinct fates of the binary inspiral . figure 8an example of the boundary between the mass - shedding limit and the isco . the selected model is the case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document}. the solid curve denotes the mass - shedding limit , and the long - dashed one the isco for each mass ratio as a function of the orbital angular velocity in polytropic units . the dotted curve denotes the mass - shedding limit for unstable quasi - equilibrium sequences . the selected model is the case of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document}. the solid curve denotes the mass - shedding limit , and the long - dashed one the isco for each mass ratio as a function of the orbital angular velocity in polytropic units . the dotted curve denotes the mass - shedding limit for unstable quasi - equilibrium sequences . when we eliminate m0 from equations ( 102 ) and ( 103 ) , we can draw a curve of the critical mass ratio , which separates bh - ns binaries that encounter an isco before reaching mass shedding , and vice versa , as a function of the compactness of the ns . the equation , which gives the critical curve is expressed as 104\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.270{\mathcal{c}^{3/2}}(1 + q){(1 + { q^{- 1}})^{1/2 } } = 0.0680\left [ { 1 - { { 0.444 } \over { { q^{0.25}}}}\left({1 - 3.54{\mathcal{c}^{1/3 } } } \right ) } \right],$$\end{document } and the curve that separates those two regions is shown in figure 9 . if the mass ratio of a bh - ns binary is larger than the critical one , the quasi - equilibrium sequence terminates by encountering the isco , while if smaller , it ends at the mass - shedding limit of the ns . figure 9critical mass ratio , which separates bh - ns binaries that encounter an isco before reaching mass shedding and undergoing tidal disruption , as a function of the compactness of ns . critical mass ratio , which separates bh - ns binaries that encounter an isco before reaching mass shedding and undergoing tidal disruption , as a function of the compactness of ns . this figure is drawn for the model of = 2 polytropic eos . in this section , we have given an overview of the current status of the study of quasi - equilibrium bh - ns binary sequences in general relativity . broadly speaking , two formulations have been proposed to construct quasi - equilibrium bh - ns binaries . the difference in these formulations comes primarily from the difference in the method for handling the bh singularity , the excision approach or the puncture approach . in these formulations , only five of ten components of einstein s equation are solved : constraint equations and the slicing condition . deriving an improved formulation , in which full components of einstein s equation are solved , is left for the future ( but see [ 204 , 46 ] for proposed formulations and [ 218 , 219 ] for a study of ns - ns binaries ) . for the quasi - equilibrium sequences numerically derived so far , we mainly review those in , because it is the only systematic study . in particular , we highlight a curve of the critical mass ratio , which separates bh - ns binaries that encounter an isco before reaching mass shedding , and vice versa , as a function of the compactness of the ns . such a critical curve clearly classifies the possible final fate of bh - ns binaries , which depends on the ratio and the compactness of the ns for a given eos of ns and bh spin . as seen in table 1 , the parameter space surveyed so far is quite narrow . a systematic study of quasi - equilibrium sequences has been done only for binaries composed of a non - spinning bh and an irrotational ns with = 2 polytropic eos . systematic numerical results for such a study will be quite helpful for predicting the final fate of bh - ns binaries and for checking the results derived in numerical simulations . specifically , it is necessary to study in detail quasi - equilibrium sequences of binaries composed of a spinning bh and a ns with an eos other than single polytrope . by the year 2010 , five groups aei , cccw , kt , lbpli , and uiuc groups have succeeded in fully general - relativistic simulations for bh - ns binaries and provided a variety of the numerical results . the results of these groups agree qualitatively with each other and have clarified the basic picture for the merger and tidal disruption processes , although quantitatively , minor differences ( e.g. , on the remnant disk mass ) have been reported . they have provided the criteria for tidal disruption , the final mass and spin of the bh left after the merger , the remnant disk mass , gravitational waveforms , and gravitational - wave spectrum . in the following sections , we review the methods of numerical relativity employed in these groups , the basic picture of the merger and tidal disruption processes clarified to date , and the resulting gravitational waveforms and spectra , separately . as shown in table 1 , the studies of quasi - equilibrium and its sequences for bh - ns binaries have been performed by several groups . in the numerical simulations , quasi - equilibrium is employed as the initial condition ( besides a minor modification of the quasi - equilibrium state , reviewed below ) . the five simulation groups , aei , cccw , kt , lbpli , and uiuc , employ the quasi - equilibrium states derived by different groups as summarized in table 2 . depending on the implementations on which each group relies , the type of the initial condition is different . table 3 classifies the type of the initial data by the following six parameters : the eos of the nsthe notation of = 2 or = 2.75 means the single polytropic eos with an adiabatic index of = 2 or = 2.75.the notations of pwpoly and sh mean the piecewise polytropic eos and the shen s eos , respectively.the spatial background metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij } } = { f_{ij}}$\end{document } and the extrinsic curvature kthe notation of cf means the conformally - flat condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , and that of m means the maximal slicing k = 0.the notation of ks indicates the kerr - schild metric . in this case , the extrinsic curvature k is also set to that derived from the kerr - schild metric.the state of the fluid flow in the nsthe notation of co means a corotating ns , and that of ir an irrotational ns.the method to treat the bh , i.e. , the moving puncture or the excisionthe notations of pu and ex mean that the moving - puncture and the excision approaches are used in the numerical simulation , respectively . expu means that the initial condition is prepared in the excision approach , but the simulation is done in the moving - puncture formulation ( see below).presence or absence of the bh spin a and misalignment angle between the spin axis and orbital angular momentum vector.the mass ratio q table 2quasi - equilibrium study groups by which the initial data of each simulation group is supplied.simulation groupquasi - equilibrium studyaeigrandclment [ 82 , 83]cccwfoucart et al . [ 209 , 210 ] table 3summary of the initial data used in simulations performed so far . note that lbpli s data includes magnetic fields.group & ref.(1)(2)(3)(4)(5)(6)kt = 2cf , mcopua = 0q 2.5kt = 2cf , mcopua = 0q 2.5 , 3.1kt = 2cf , mirpua = 0q 2.5 , 3.1kt = 2cf , mirpua = 0q 3.1kt = 2cf , mirpua = 0q = 1.5 , 2 , 3 , 4 , 5kt pwpolycf , mirpua = 0q = 2 , 3kt pwpolycf , mirpua = 0,0.5 , 0.25 , 0.5 , 0.75q = 2 , 3 , 4 , 5uiuc = 2cf , mirexpua = 0q = 2 , 3uiuc = 2cf , mirexpua = 0 0.5 , 0.75q = 1 , 3 , 5cccw = 2ksirexa = 0q = 1cccw = 2 , 2.75 , shksirexa = 0.5q = 3cccw = 2ksirexa = 0 , 0.5 , 0.9q = 3 = 0 , 20 , 40 , 60 , 80lbpli = 2cf , mirexa = 0.5q = 5aei = 2cf , mirexpua = 0q = 5 the notation of = 2 or = 2.75 means the single polytropic eos with an adiabatic index of = 2 or = 2.75 . the notations of pwpoly and sh mean the piecewise polytropic eos and the shen s eos , respectively . the spatial background metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij } } = { f_{ij}}$\end{document } and the extrinsic curvature k the notation of cf means the conformally - flat condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } , and that of m means the maximal slicing k = 0 . the notation of ks indicates the kerr - schild metric . in this case , the extrinsic curvature k is also set to that derived from the kerr - schild metric . the state of the fluid flow in the ns the notation of co means a corotating ns , and that of ir an irrotational ns . the method to treat the bh , i.e. , the moving puncture or the excision the notations of pu and ex mean that the moving - puncture and the excision approaches are used in the numerical simulation , respectively . expu means that the initial condition is prepared in the excision approach , but the simulation is done in the moving - puncture formulation ( see below ) . presence or absence of the bh spin a and misalignment angle between the spin axis and orbital angular momentum vector . quasi - equilibrium study groups by which the initial data of each simulation group is supplied . when a quasi - equilibrium configuration constructed by the excision approach is used as initial data for a simulation code based on the moving - puncture approach , one more step is needed before starting the simulations because the data inside the bh excision surface does not exist , and thus , one needs to fill the bh interior by extrapolating all field values radially from the excision surface toward the bh center . in the extrapolation procedure , one has to employ a method in which any constraint violation introduced inside the bh is not allowed to affect the exterior spacetime . two groups proposed such a procedure [ 34 , 61 ] , and the uiuc group used the method they developed in their simulations . strictly speaking , the quasi - equilibrium derived in the framework of section 2 is not realistic because an approaching radial velocity driven by gravitational radiation reaction is not taken into account . if we adopt such quasi - equilibrium data as the initial condition , the trajectories obtained in the numerical simulation usually result in a slightly eccentric orbit . one prescription to suppress this error is to choose an initial condition in which the binary separation is large enough that the effect of the radial velocity is not serious . another method to reduce the eccentricity was proposed by the cccw group [ 157 , 75 ] as described in the following . ( 1 ) at the beginning of their procedure , they prepare a quasi - equilibrium data , which has zero approaching velocity r = 0r = 0 and orbital angular velocity 0 . ( 2 ) the second step is to evolve the quasi - equilibrium initial data for at least one and a half orbits . then one records the time derivative of the measured coordinate separation between the center of the compact objects , (t ) and fits (t ) to a function of the form 105\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\dot d = { a_0 } + { a_1}t + b\sin ( \omega t + \phi),$$\end{document } where the parameters a0 , a1 , b , , and are all determined by the fitting . the a0 + a1 t part denotes the smooth inspiral , while the b sin(t + ) part denotes the unwanted oscillations due to the eccentricity of the orbit . for a nearly circular newtonian orbit , the eccentricity e of the orbit can be written as 106\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e - { b \over { \omega { d_0}}},$$\end{document } where d0 = d(t = 0 ) . this implies that reducing the orbital eccentricity is equivalent to reducing b. ( 3 ) the final step is to add the corrections to the approaching velocity 0 and the orbital angular velocity using the parameters , which appear in equation ( 105 ) . such corrections should be chosen so that the eccentricity - induced initial radial velocity and radial acceleration can be removed . namely , the initial radial velocity is changed as 107\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \dot a_0 } = - { { b\sin \phi } \over { { d_0}}},$$\end{document } and the initial orbital angular velocity as 108\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \omega _ 0 } = - { { b\omega \cos \phi } \over { 2{d_0}{\omega _ 0}}}.$$\end{document } using the corrected approaching radial velocity and the orbital angular velocity for the initial step ( 1 ) , they iterated the procedure and obtained a better initial data . they showed that going twice through the iterative method described above , the orbital eccentricity is reduced by about an order of magnitude . one is the baumgarte - shapiro - shibata - nakamura ( bssn ) formalism [ 140 , 195 , 18 ] together with the moving - puncture gauge condition [ 39 , 15 , 35 , 136 ] , and the other is the generalized harmonic ( gh ) formalism ( see also [ 77 , 86 , 162 , 163 ] ) . the aei , kt , and uiuc groups employ the bssn formalism , while the cccw and lbpli groups use the gh formalism . these two formalisms are different from the standard 3 + 1 formalism , which was found to be inappropriate in numerical relativity because stable and longterm numerical simulations are not feasible due to the presence of an unphysical growth mode excited even by a tiny numerical truncation error . the bssn formalism , the original version of which was first proposed by nakamura in 1987 , is in a sense a modified version of the 3 + 1 formalism . the essence in this formalism is to adopt not only the three metric ( ij ) and the extrinsic curvature ( kij ) but also a conformal factor of the three metric ( or = log( ) or w = or = ) , the trace part of the extrinsic curvature ( k ) , and the first spatial derivative of the three metric ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_i } = { { \tilde \gamma}_{ij , j}}$\end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}^i } = - { { \tilde \gamma}^{ij}}_{,j}$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}_{ij}}$\end{document } is the conformal three metric ) as new independent variables , and then , to appropriately rewrite the basic equations using these new variables . with this prescription , the evolution equations for the conformal three metric , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}^i}$\end{document } , and the conformal traceless extrinsic curvature , ij (kij kij/3 ) , are written in the form of simple wave equations . this prescription leads to avoiding the spurious growth of unphysical modes by numerical truncation errors and enables one to perform a stable and longterm evolution for a variety of systems . typical basic equations are described in appendix a. the basic equations of the gh formalism are similar to those employed in the pn approximation . using the harmonic or generalized harmonic gauge condition , 109\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \mu}{\nabla ^\mu}{x^\nu } = { h^\nu},$$\end{document } where h is the arbitrary function , einstein s equation is written in a hyperbolic form of the spacetime metric g. the main modification , analogous to the introduction of fi or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_i } - { { \tilde \gamma}_{ij , j } } = 0$\end{document } in the bssn formalism , is treating h as independent functions . the other modification is to introduce the first spatial derivative of the metric components as new independent variables di = ig to make the gh formulation first - order . in both formalisms , the number of constraint equations is increased as a result of defining the new variables , in addition to the hamiltonian and momentum constraints . for the bssn formalism , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\rm det}({{\tilde \gamma}_{ij } } ) = 1,{{\tilde \gamma}^{ij}}{{\tilde a}_{ij } } = 0$\end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde \gamma}^i } + { { \tilde \gamma}^{ij}}_{,j } = 0$\end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\kappa _ i}\rho _ i^{{\gamma _ i } } = { \kappa _ i}_{+ 1}\rho _ i^{{\gamma _ { i + 1}}}$\end{document } are new constraints . for the gh formalism , re - defining a spatial con - formal factor , adopting more than fourth - order finite - differencing schemes , and employing an appropriate moving - puncture gauge condition [ 39 , 15 , 221 , 35 ] are required for evolving bhs accurately and stably . in such a bssn - puncture formulation , although a coordinate singularity may appear in the center of the bh , this can be effectively ( and , in a sense , fortunately ) excised in the moving - puncture gauge condition . consequently , one can evolve the whole computational region without artificially excising inside the bh horizon ( i.e. , without special numerical treatments for the inside of the bh horizon ) . on the other hand , in the code of the cccw and lbpli groups using gh formalism , the gauge , similar to the moving - puncture gauge , has not yet been developed . these groups employ the excision technique , i.e. , a region inside the apparent horizon is cut out of the computation domain and replaced with an inner boundary . pretorius has successfully simulated orbiting bh - bh binaries using the gh formalism with excision for the first time [ 162 , 163 ] . subsequently , the cccw group has successfully simulated a longterm inspiral and merger of bh - bh binaries using a high - accuracy spectral method [ 181 , 206 ] , verifying that this technique is also robust for handling bh spacetimes . for the evolution of orbiting ns and a disk surrounding bhs formed after the merger , hydrodynamics ( or magnetohydrodynamics ) equations have to be solved . for a hydrodynamic simulation specifically , one has to solve the continuity equation for the rest - mass density , the relativistic euler and energy equations for the four velocity and internal energy ( or h ) , i.e. , 110\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \mu}(\rho { u^\mu } ) = 0,$$\end{document } 111\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { i\nu}}{\nabla _ \mu}{t^{\mu \nu } } = 0,$$\end{document } 112\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_\nu}{\nabla _ \mu}{t^{\mu \nu } } = 0,$$\end{document } where t is the stress - energy tensor . for the perfect fluid , it is written as 113\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t^{\mu \nu } } = \rho h{u^\mu}{u^\nu } + p{g^{\mu \nu}}.$$\end{document } for a magnetohydrodynamics simulation , we have to solve maxwell s equation as well . in ideal magnetohydrodynamics , only the induction equation for magnetic - field components if one needs to follow the evolution of the lepton number densities , the following additional continuity equations for the leptons ( the electron number density and/or the total lepton number density ) have to be solved 114\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ \mu}(\rho { y_a}{u^\mu } ) = \rho { s_a},$$\end{document } where ya denotes a lepton number fraction per baryon and sa is the corresponding production / annihilation rates of the lepton in the baryon rest frame , which is associated with neutrino emission and absorption ( e.g. , [ 175 , 176 , 172 , 173 , 170 , 146 , 183 , 184 , 185 ] ) . the hydrodynamic equation is schematically written in the form 115\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial u } \over { \partial t } } + { { \partial { f^i } } \over { \partial { x^i } } } + { s_{\rm{u } } } = 0,$$\end{document } where u represents the set of the evolved variables and f are the associated transport fluxes . the third term of equation ( 115 ) denotes the source term , which is usually the coupling term between the hydrodynamic variables and geometric quantities or the first derivative of the metric . the third term is evaluated in a straightforward manner , which usually does not cause numerical instabilities in a straightforward evaluation ( as long as an appropriate time step is chosen ) . the cccw , lbpli , and uiuc groups employ the scheme of harten , lax , and van leer ( hll ) , the aei group employs several schemes prepared in their whisky library [ 13 , 14 , 11 , 12 ] , and the kt group employs the scheme of kurganov and tadmor . in handling the transport term , one further needs to determine the method of the interpolation by which the values of hydrodynamic quantities at the cell surfaces are specified . all these groups currently employ the third - order piecewise parabolic interpolation . employing realistic eos for modeling ns is a key ingredient in accurately determining the final fate after the onset of the merger and in deriving realistic gravitational waveforms in the merger phase . however , the eos of the nuclear matter beyond the normal nuclear density is still uncertain due to the lack of strong constraints obtained from experiments and observations ( e.g. , [ 115 , 116 , 117 ] and see for a recent devlopment ) . hence , we do not know exactly the detailed physical state of ns such as density and composition profiles as functions of radius for a given mass and the relation between the mass and the radius . gravitational waveforms emitted in the tidal - disruption phase as well as properties of the remnant bh - disk system such as the mass and the typical density of the disk depend strongly on the eos , as shown in the following . the primary reason for this is that these depend strongly on the relation between the mass and the radius of the ns , the sensitivity of a ns to the tidal force by its companion bh depends primarily on its radius , e.g. , a ns of larger radii ( with a stiffer eos ) will be disrupted at a larger orbital separation ( or a lower orbital frequency ; see equations in section 1 ) ; if the tidal disruption of a ns occurs at a larger distance , more material will be widely spread around its companion bh , and consequently , a high - mass remnant disk is likely to be formed ; also , the gravitational - wave frequency at tidal disruption , which will be one of the characteristic frequencies , is lower for a ns of larger radius . in the study of binaries composed of ns , rather than require one to prepare a realistic eos , we should consider exploring the possibility of determining the ns eos by gravitational - wave observation , as discussed in [ 127 , 220 , 168 , 70 ] . for this purpose , we need to prepare theoretical templates of gravitational waves employing a wide variety of possible eos for the ns matter , and systematically perform a wide variety of simulations employing a large number of plausible eos . then , the next task is to determine what kinds of eos should be employed in their theoretical work . thermal energy per baryon inside ns , except for newly - born ones , is believed to be much lower than the fermi energy of the constituent particles , because a young ns is quickly cooled down by neutrino emission ( e.g. , [ 116 , 99 ] ) . this implies that we can safely neglect thermal effects on the ns in the inspiral and early merger phases , and can employ a cold eos , for which the pressure , p , the specific internal energy , , and other thermodynamic quantities are written as functions of the rest - mass density . in particular , employing the cold eos is appropriate for the case in which the merger does not result in the formation of a disk surrounding the remnant bh , i.e. , for the case in which the ns is simply swallowed by the companion bh , because a heating process , such as shock heating , never plays a role . by contrast , to follow a longterm evolution of tidally disrupted material , which forms a disk around the remnant bh , the finite - temperature effects of the eos and the neutrino cooling have to be taken into account . this is because the temperature of the disk material is likely to be increased significantly by shock or a longterm viscous heating , while the density of the disk is lower than that of the ns , and as a result , the fermi energy is lowered , resulting in a situation where the roles of the pressures by degenerate electrons and thermal gas become less important and increasingly important , respectively . because the simulation for bh - ns binaries in general relativity is still in an early phase ( by 2010 ) , much work has been done with a rather simple ( not very realistic ) eos . the often - used eos in the early phase of this study within the numerical - relativity community is the -law eos , 116\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p = ( \gamma - 1)\rho \varepsilon , $ $ \end{document } with = 2 . in this eos , the initial condition for the ns is determined by the polytropic eos , p = ( see section 2 ) . however , this eos is known to disagree with typical nuclear - theory - based eos for a small value of 2 ( e.g. , and figure 10 ) . figure 10the relation between the mass and the circumferential radius of a spherical ns with piecewise polytropic eos for which parameters are described in table 4 . for comparison , the curve for the = 2 polytropic eos with /c = 2 10 in cgs units is also plotted ( dotted curve ) . note that if the observational result of ( if the presence of an 1.97 m ns ) is confirmed by 100% , some of the soft eos displayed here will be ruled out . the relation between the mass and the circumferential radius of a spherical ns with piecewise polytropic eos for which parameters are described in table 4 . for comparison , the curve for the = 2 polytropic eos with /c = 2 10 in cgs units is also plotted ( dotted curve ) . note that if the observational result of ( if the presence of an 1.97 m ns ) is confirmed by 100% , some of the soft eos displayed here will be ruled out . this is a phenomenologic ally parameterized eos , which approximately reproduces cold nuclear - theory - based eos at a high density ( above the nuclear density ) only with a small number of polytropic constants and indices [ 167 , 168 , 150 ] ( see also [ 87 , 128 ] ) , i.e. , 117\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(\rho ) = { \kappa _ i}{\rho ^{{\gamma _ i}}}{\rm{for}}{\rho _ { i - 1 } } \leq \rho < { \rho _ i}(1 \leq i \leq n),$$\end{document } where n is the number of the pieces used to parameterize an eos and i denote boundary densities for which appropriate characteristic values are assigned . here , 0 = 0 and n . i are the polytropic constants and i the adiabatic indices for each piece . at each boundary density , = i ( i = 1 , , n 1 ) , the pressure is required to be continuous , i.e. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = { m_{{\rm{ns}}}}/{r_{{\rm{ns}}}}$\end{document}. thus , if one gives k1 , i , and i ( i = 1 , , n ) , the eos is totally determined . for the zero - temperature eos , the first law of thermodynamics ( d = pd(1/ ) or dh = dp/ ) holds , and thus , and h are also determined except for the choice of the integration constants , which are fixed by the continuity condition of ( hence equivalently h ) at each i . recently , it has been shown that the piecewise polytropic eos composed of one piece in the crust region and three pieces in the core region approximately reproduces most of nuclear - theory - based eos at a high density . here , three pieces in the core region are required to reproduce a high - mass ns for which inner and outer cores could have different stiffness due to the variation of the properties of the high - density nuclear matter ( 2 10 g / cm ) . thus , if one focuses on a ns of relatively low mass , a smaller number of pieces is acceptable ; see [ 168 , 107 ] for the two - pieces case . table 4 lists the parameters employed in [ 168 , 107 ] , and figure 10 shows the relation between the mass and the radius of a spherical ns for these piecewise polytropic eos . we note that using a single polytrope for the low - density eos is justified to the extent that the radius and deformability of the ns as well as resulting gravitational waveforms in the merger phase are insensitive to the low - density eos . 2 is the adiabatic index in the core region and p is the pressure at the fiducial density fidu = 10 g / cm , which determines the polytropic constant 2 of the core region and 1 : the critical rest - mass density separating the crust and core regions . r135 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{135}}$\end{document } are the circumferential radius and the compactness of the ns with mns = 1.35 m.model2log10p [ g / cm]1 [ 10 g / cm]mmax [ m]r135 [ km ] \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{135}}$\end{document } 2h3.013.950.70332.83515.230.1309h3.013.551.2322.24912.270.1624hb3.013.451.4172.12211.610.1718hbs2.713.451.0691.92611.570.1723hbss2.413.450.68541.70111.450.1741b3.013.351.6302.00310.960.1819bs2.713.351.2691.79910.740.1856bss2.413.350.85471.56610.270.1940 the parameters and key ingredients for a piecewise polytropic eos employed in . 2 is the adiabatic index in the core region and p is the pressure at the fiducial density fidu = 10 g / cm , which determines the polytropic constant 2 of the core region and 1 : the critical rest - mass density separating the crust and core regions . r135 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}_{135}}$\end{document } are the circumferential radius and the compactness of the ns with mns = 1.35 m. in the presence of shocks during the merger phase , the assumption of zero - temperature breaks down . thus , in addition to the piecewise polytropic part , a correction term has to be added . an often - used minimum prescription is to simply add the following term to the pressure [ 199 , 168 , 107 ] 118\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_{{\rm{th } } } } = ( { \gamma _ { { \rm{th } } } } - 1)\rho { \varepsilon _ { { \rm{th}}}},$$\end{document } where th is an adiabatic index for the thermal part , and th = cold with cold being determined by the piecewise polytropic eos . this type of the piecewise polytropic eos is employed by the kt group for a wide variety of parameters [ 107 , 109 ] . to self - consistently take into account thermal effects , which play an important role in the evolution of a disk formed after tidal disruption occurs , the best way is probably to employ a finite - temperature eos , which is derived by a detailed model based on nuclear physics . this type of eos is usually described in table form , e.g. , as 119\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p - p(\rho , t,{y_e}){\rm{and}}\varepsilon = \varepsilon ( \rho , t , y_{e}),$$\end{document } where t and ye are the temperature and the electron fraction [ 119 , 188 , 189 ] . here , approximately speaking , is determined by the continuity equation ( 110 ) , and t by the internal energy , which is essentially determined by the energy equation ( 112 ) in general relativity . ye is determined by the continuity equation for the electron fraction ( 114 ) . to solve this equation correctly , we have to take into account neutrino emission / absorption , by which the electron fraction is varied . multidimensional numerical - relativity simulations have reached a level of incorporating the effects of neutrino emission / absorption only quite recently [ 183 , 185 ] . rather , most multidimensional numerical - relativity simulations have been performed without solving neutrino transfer equations and the equation for ye [ 146 , 53 , 54 , 74 , 149 ] , but an a priori assumption for ye as a function of other variables such as and t is imposed . along this line ( under the assumption that ye is unchanged in the fluid rest frame or ye is determined by the -equilibrium ) , the cccw group performed the first simulation taking into account the finite - temperature effect . recently , sekiguchi has developed a leakage scheme of neutrinos in general relativistic simulation [ 183 , 184 , 185 ] and solves the equation for ye taking into account neutrino emission for the first time . this technique will be applied to the simulation of bh - ns binaries in the near future . one is the scale associated with the size of compact objects , which is gmbh / c for bh and rns 58gmns / c for ns . the other is the wavelength of gravitational waves , . for a binary in a circular orbit of angular velocity , the wavelength for the dominant quadrupole mode ( l = |m| = 2 mode ) is 120\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda = { { \pi c } \over \omega } \approx 105{{g{m_0 } } \over { { c^2}}}{\left({{{g{c^{- 3}}\omega { m_0 } } \over { 0.03 } } } \right)^{- 1}},$$\end{document } where m0 = mbh + mns . a longterm numerical - relativity simulation is typically performed with initial angular velocity gcm0 0.02 for ns - ns binaries , and 0.03 for bh - ns binaries . thus , is larger than gm0/c by a factor of 100 . to accurately compute the evolution of an orbiting bh and ns resolving the structure of each object , the grid spacing should be smaller than 0 gmbh/20c and rns/40 , respectively . in addition , the outer boundary along each axis of the computation domain should be larger than the wavelength of gravitational waves to accurately take into account gravitational radiation reaction and to accurately extract gravitational waves in the wave zone . then , if one adopts a uniform grid with grid spacing 0 , the total grid number in one direction should be larger than 121\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\begin{array}{*{20}c } { { \lambda \over { { \delta _ 0 } } } \approx \max \left [ { 2100(1 + { q^{- 1}})\left({{{g{c^{- 3}}\omega { m_0 } } \over { 0.03 } } } \right)^{- 1 } } \right . } , \\ { \left . { 700(1 + q){{\left({{{{c^2}{r_{{\rm{ns}}}}/g{m_{{\rm{ns } } } } } \over 6 } } \right)}^{- 1}}{{\left({{{g{c^{- 3}}\omega { m_0 } } \over { 0.03 } } } \right)}^{- 1 } } } \right ] . } \\ \end{array}}$$\end{document } in three spatial dimensions , the required grid number is more than ( 2/0 ) . for such a simulation , a quite high computational cost is required even in the present computational resources . it should also be pointed out that in such studies , a survey for a large parameter space composed of the mass and the spin of the bh , and the mass and eos of the ns is necessary ( see section 3.2 ) . for this purpose , it is very important to make every effort to save computational costs for each run . therefore , to efficiently perform a large number of numerical simulations with the finite - differencing method , adaptive mesh refinement ( amr ) , such as that proposed by berger and oliger , is an indispensable technique . in this technique , one prepares several refinement levels of cartesian boxes ( or other geometrical domains ) with different grid resolutions ; usually in the smaller boxes , the grid resolution is higher . at such a fine refinement level , the bh and the ns are evolved with sufficiently high resolution with grid spacing 0 . on the other hand , the propagation of gravitational waves , which have a wavelength much longer than the size of the compact objects , is followed in large - size boxes ( in the coarser levels ) with a large grid spacing , which is still much smaller than the gravitational wavelength ( say , /10 ) . mesh refinement techniques in numerical relativity have been developed by several groups ( e.g. , [ 93 , 182 , 35 , 228 ] ) . the aei and uiuc groups use the < monospace > carpet</monospace > module of the < monospace > cactus</monospace > code developed primarily by schnetter , the kt group the < monospace > sacra</monospace > code developed by yamamoto , and the < monospace > lbpli</monospace > group the < monospace > had</monospace > code . recently , two independent codes ( < monospace > whisky</monospace > , which employs < monospace > carpet</monospace > , and < monospace > sacra</monospace > ) were compared , performing simulations of ns - ns binaries , and it was illustrated that the amr scheme in < monospace > carpet</monospace > and < monospace > sacra</monospace > work well at the same level . the cccw group employs a two - grid technique , which was originally proposed by the meudon - valencia - mpa team . in this approach , geometric - field quantities are evolved with the multi - patch pseudo - spectral method and the hydrodynamic equations are solved using a standard finite - volume scheme on a second grid . in solving the equations for the geometric field , the computational domain encompassing the local wave zone is prepared , while the hydrodynamic equations are solved in the region where the matter presents . there are many free parameters that specify a bh - ns binary ; the mass and spin of the bh , and the mass of the ns . furthermore , the eos of the ns is still unknown , and thus , it should also be regarded as one of the free parameters . here , the internal motion of the ns is believed to be close to an irrotational velocity field [ 24 , 104 ] because the viscosity of the ns matter is too small to realize a corotational velocity field , and , in addition , the typical spin angular momentum of the ns ( prot = 0.11 s ) is slow enough that we can safely assume that the ns spin is zero ( because an orbital period of 10 ms for m0 10 m is much shorter than prot ) . however , we have to accept a wide possible range for other parameters and eos of ns . in particular , the mass and spin of a bh in a realistic binary system are totally unknown , and hence , a survey for a wide parameter space is required to fully understand the nature of bh - ns coalescence and to derive a complete catalog of possible gravitational waveforms . in the first phase , all groups employed the -law eos in the form p = ( 1) with the special value of = 2 , for which the initial condition is prepared by using the polytropic eos p = . in this eos , physical parameters are non - dimensional quantities such as mass ratio , q = mbh / mns , and compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } , because can be freely chosen . since 2009 , the early work of the kt group was done with the -law eos for a = 0 and for a wide range of q and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.145 \leq { \mathcal c } \leq 0.178$\end{document } ; 1.5 q 5 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(q , a,{\mathcal c})$\end{document}. since 2009 , the kt group has employed a piecewise polytropic eos [ 167 , 150 ] with a wide variety of eos parameters ( see table 4 ) . simulations have been systematically performed employing this eos for a wide range of ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.12 \lesssim { \mathcal c } \lesssim 0.19$\end{document } ) [ 107 , 109 ] ; 2 q 5 , 0.5 a 0.75 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(q , a,{\mathcal c})$\end{document}. ( here , the negative value of the spin implies that the bh spin and orbital angular momentum vector are anti - parallel . ) the simulations of the uiuc group were performed employing the -law eos with = 2 . the uiuc group has chosen in total nine parameter sets for ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.088$\end{document } ) as follows ; q = 1 , 2 , 3 , and 5 , a = 0 , 0.5 , and 0.75 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(q , a,{\mathcal c})$\end{document } and 0.145 . the cccw group performed simulations employing two types of eos ; -law eos with = 2 and 2.75 , and shen s eos , which is a tabulated eos derived in a relativistic mean field theory [ 188 , 189 ] . they focused on special parameter sets of ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \approx 0.145$\end{document } ) as q = 1 and 3 , a = 0 and 0.5 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(q , a,{\mathcal c } ) = ( 5,0.5,0.1)$\end{document } and 0.174 . in their latest work , they focused on the case q = 3 and a = 0.5 , paying particular attention to the dependence of the merger process on the eos and on the misalignment angle of the bh spin and orbital angular momentum axes . the lbpli group has performed one simulation to date , using the -law eos with = 2 , and with ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.1 - 0.17$\end{document } ) = ( 5 , 0.5 , 0.1 ) . in their first work , the compactness was chosen to be small and not very realistic . in this simulation , magnetic fields in the ideal magnetohydrodynamics mhd approximation were incorporated , but they did not play an important role . the aei group has performed simulations using -law eos with = 2 , and with q = 5 , a = 0 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } . to summarize , the total number of simulations is still small , although a systematic survey is required to fully understand the complete picture of the coalescence of bh - ns binaries . in particular , many simulations were performed in a not very realistic setting using a simple -law eos and small values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document}. as mentioned in section 1 , tidal disruption is more subject to the less compact ns , and hence , it should be in particular cautioned that a simulation with unphysically - small values of compactness may lead to an incorrect conclusion that tidal disruption and subsequent disk formation are easily achieved . nevertheless , the simulations performed so far have clarified a basic picture for the merger process of bh - ns binaries , the properties for the remnant , gravitational waveforms , and gravitational - wave spectrum . in the following , as mentioned in section 1 , broadly speaking , the final fates of bh - ns binaries are divided into two classes . one in which the ns is tidally disrupted before it is swallowed by the companion bh and the other is that the ns is simply swallowed by the bh . figures 11 and 12 display snapshots of the rest - mass density profiles and the location of the apparent horizon on the equatorial plane at selected time slices for two typical cases . for these results , figure 11 illustrates the process in which the ns is tidally disrupted before the binary reaches the isco and then a disk is formed surrounding the companion bh . for this model , mbh = 2.7 m , a = 0 , mns = 1.35 m , and rns = 15.2 km ( eos 2h ) ; the mass ratio ( q = 2 ) is small and the ns radius is large . for this setting , the ns is significantly tidally deformed in close orbits , and eventually , mass shedding from an inner cusp of the ns sets in far outside the isco . after a substantial fluid element is removed from the inner cusp , the ns is tidally disrupted outside the isco . it should be emphasized that tidal disruption does not occur immediately after the onset of mass shedding in this case . tidal disruption occurs for an orbital separation smaller than that for the onset of mass shedding . figure 11evolution of the rest - mass density profile in units of g / cm and the location of the apparent horizon on the equatorial plane for a model with mbh = 2.7 m , a = 0 , mns = 1.35 m , and rns = 15.2 km ( eos 2h ) . the colored panel on the right - hand side of each figure shows log10( ) . figure 12the same as figure 11 but for a model with mbh = 4.05 m , a = 0 , mns = 1.35 m , and rns = 11.0 km ( eos b ) . evolution of the rest - mass density profile in units of g / cm and the location of the apparent horizon on the equatorial plane for a model with mbh = 2.7 m , a = 0 , mns = 1.35 m , and rns = 15.2 km ( eos 2h ) . the colored panel on the right - hand side of each figure shows log10( ) . this figure is taken from . the same as figure 11 but for a model with mbh = 4.05 m , a = 0 , mns = 1.35 m , and rns = 11.0 km ( eos b ) . after tidal disruption occurs , the material of the ns forms a one - armed spiral . as a result of angular momentum transport in the arm , a large amount of material spreads outward , and after the spiral arm is wound from the differential rotation , a disk of approximately axisymmetric configuration is formed around the bh located approximately at the center . however , because of the presence of a non - axisymmetric structure at its formation , however , the disk does not completely relax to an axisymmetric state in the rotational period 10 ms . rather , a one - armed spiral of small amplitude is present for a long time , and helps gradually transporting angular momentum outward , resulting in a gradual mass infall into the bh . however , the mass accretion time scale is much longer than the rotational period , and hence , the disk remains quasi - stationary for; 10 ms . this evolution process agrees qualitatively with that found in the study of the longterm evolution of bh - disk systems [ 90 , 103 ] . the tidal disruption process as illustrated in figure 11 is qualitatively common for the model with a low - mass bh or a large - radius ns or a high - spin bh . however , quantitative details depend on the parameters of the binary . for a small mass ratio ( q 2 ) with a = 0 , the typical size of the disk ( the region with > 10 g / cm ) is 50100 km with maximum density 10 g / cm for a disk of mass 0.1 m as shown in figure 11 . the disk relaxes to a nearly axisymmetric configuration in a short time duration , approximately equal to the rotational period around the bh . for example , for a large mass ratio with a high bh spin ( e.g. , q = 5 and a = 0.75 ) , the typical size is also 100 km , but the maximum density is smaller than those for the smaller value of q ; the time scale until the disk relaxes to an axisymmetric configuration is relatively long . a remarkable point is that the tidal debris of relatively low density 10 g / cm could be ejected to a distance of 100 km [ 63 , 57 , 41 , 109 ] , i.e. , a wider but less dense disk is formed ( see also section 3.3.3 ) . figure 12 illustrates the case in which the ns is not tidally disrupted before it is swallowed by the bh . for this model , mbh = 4.05 m , a = 0 , mns = 1.35 m , and rns = 11.0 km ( eos b in table 4 ) . in this case , the ns is tidally deformed only in a close orbit . then , mass shedding sets in for a too close orbit to induce subsequent tidal disruption outside the isco . as a result , these processes help exciting a non - axisymmetric fundamental quasi - normal mode ( qnm ) of the remnant bh . the mass of the disk formed after the onset of the merger is negligible ( much smaller than 0.01 m ) , because the bh simply engulfs the ns . generally speaking , the final fate depends on the location where mass shedding of the ns sets in . if the location is in the vicinity of or inside the isco , most of the ns material falls into the companion bh , and a bh with negligible surrounding material is the outcome . with the increase of the orbital separation at the onset of mass shedding , the mass of the material surrounding the bh increases . note again that the mass shedding has to set in sufficiently outside the isco to induce tidal disruption , because tidal disruption occurs only after substantial mass is removed from the ns . the effect of the bh spin significantly modifies the orbital evolution process in the late inspiral phase and merger dynamics , as first demonstrated by the uiuc group . figure 13 shows the trajectories of the bh and ns for models with q = 3 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } , and a = 0 ( left ) and a = 0.75 ( right ) . the initial orbital angular velocity is the same for two models . for the binary composed of a non - spinning ( a = 0 ) bh and ns , the merger occurs after about 4 orbits , whereas for the case with a spinning bh ( a = 0.75 ) , it occurs after about 6 orbits . for the case with a spinning bh qualitatively , these differences may be explained primarily by the presence of a spin - orbit coupling effect , which is accompanied by an additional repulsive force for a > 0 ( and attractive force for a < 0 ) , and thus , reduces the attractive force between two objects ( see the equations of motion for two - body systems in the context of the pn approximation [ 102 , 226 , 100 ] ) . in the presence of this additional repulsive force , centrifugal forces should be reduced for a given orbital separation . this slows down the orbital velocity , and therefore , the luminosity of gravitational waves is decreased and orbital evolution due to gravitational radiation reaction is delayed ( the lifetime of the binary becomes longer ) . in addition , the orbital radius at the isco around the bh is decreased ( and the absolute value of the binding energy at the isco around the bh is increased ) due to the spin - orbit coupling effect ( e.g. , ) . this further helps to increase the lifetime of the binary because it evolves as a result of gravitational radiation reaction and hence has to emit more gravitational waves to reach the isco . figure 13trajectories of the bh and ns coordinate centroids for models with q = 3 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } , and a = 0 ( left ) and a = 0.75 ( right ) . the bh coordinate centroid corresponds to the centroid of the bh , and the ns coordinate centroid denotes a mass center . trajectories of the bh and ns coordinate centroids for models with q = 3 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } , and a = 0 ( left ) and a = 0.75 ( right ) . the bh coordinate centroid corresponds to the centroid of the bh , and the ns coordinate centroid denotes a mass center . this longer lifetime for a binary with a spinning bh enhances the possibility of tidal disruption , and the final outcome is modified . figure 14 displays the contour curves and the location of the remnant bh for the same models as in figure 13 . for both models , the ns is tidally disrupted outside the isco and a disk is formed . for the spinning bh case ( a = 0.75 ) , a more extended , more massive , and denser disk is the outcome . for the non - spinning case ( a = 0 ) , the disk mass is only 4% of the total rest mass whereas for a = 0.75 , it is 13% ( see section 3.4 for details of the remnant disk ) . this is probably due to the effect that the physical radius of the isco ( or specific angular momentum of a particle orbiting the isco ) around the spinning bh is smaller than that for the non - spinning bh and also that the radial approaching velocity at tidal disruption is smaller for a spinning bh because of the repulsive nature of the spin - orbit coupling effect . the contour curves , velocity fields ( arrows ) , and bh ( solid circles ) are plotted . this figure is taken from . a remnant bh - disk system for models shown in figure 13 . the contour curves , velocity fields ( arrows ) , and bh ( solid circles ) are plotted . the cccw group subsequently studied the effects of bh spin on the final remnant with several eos . they reached a similar conclusion about the orbital evolution and final outcome to that of the uiuc group even for the case in which bh spin is slightly smaller , a = 0.5 . this conclusion was reconfirmed also by the kt group for a wide variety of piecewise polytropic eos and mass ratios . therefore , a moderately large bh spin , a = 0.5 , is substantial enough for modifying the merger process and enhancing the disk formation . the cccw group also performed a simulation with a = 0.9 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.18$\end{document } and found that tidal disruption occurs far outside the isco and the resulting disk mass is very high , 36% of the total rest mass ( see section 3.4 ) . the kt group also found that for binaries composed of a high - spin bh ( a = 0.75 ) and ns , tidal disruption may occur for a large value of mass ratio , q 5 , for a wide variety of ns eos as far as it gives \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = { ( } 0.144 { ) } - 0.173$\end{document}. this implies that tidal disruption of a ns may be possible for a large bh mass over a wide area . in such case , the material of the tidally - elongated ns is swallowed from a relatively narrow region of the bh surface . as will be discussed in section 3.6 , this helps excite a non - axisymmetric fundamental qnm of the remnant bh . on the other hand , for the non - spinning bh case for which tidal disruption occurs only for a small bh , the material of a tidally - elongated ns is always swallowed by a wide region of the bh surface . this difference is reflected in gravitational waveforms and spectra , as predicted in [ 177 , 178 ] ( in which a bh perturbation study was performed ) . to clarify the fact described above , kyutoku et al . generated figures 1517 , which show the evolution of the rest - mass density profile for q = 3 , mns = 1.35 m , and eos hb ( cf . the evolution processes shown in figures 15 and 17 are similar to those in figures 11 and 12 , respectively . figure 15 shows the case in which mass shedding of the ns occurs at an orbit sufficiently far from the bh . subsequently , the ns is extensively elongated , a one - armed spiral is formed , and then the spiral arm composed of dense material is wound around the bh . the material located in the outer region of the spiral arm forms a disk , while that in the inner region falls into the bh . the infall of the dense material proceeds from a wide region of the bh surface as seen in the fourth panel of figure 11 . by contrast , for a = 0.5 ( see figure 17 ) , tidal disruption does not occur and more than 99.99% of the ns matter falls into the bh from a narrow region of the bh horizon and in a short time scale . figure 15the same as figure 11 but for a model with mbh = 4.05 m , a = 0.75 , mns = 1.35 m , and rns = 11.6 km ( eos hb ) . this figure is taken from . the same as figure 11 but for a model with mbh = 4.05 m , a = 0.75 , mns = 1.35 m , and rns = 11.6 km ( eos hb ) . the figure is taken from . the same as figure 15 but for a model with a = 0.5 . this figure is taken from . the same as figure 16 but for a model with a = 0.5 . the type of merger process for a = 0.5 shown in figure 16 is qualitatively new . tidal disruption occurs in a relatively close orbit ( in the vicinity of the bh isco ) . the subsequent evolution process is similar to that for a = 0.75 , but the infall of dense ns material to the bh occurs from a relatively narrow region ( see the second to fourth panels of figure 16 ) . eventually , the infall proceeds from a wide region of the bh surface , but the density of the infalling material seems to be too low to enhance a qnm oscillation of the bh significantly ( see the fifth panel of figure 16 ) . this feature is often found for a binary of high - mass ratio and high bh spin . to date , three types of merger process have been found . type - i : the bh mass is low or the bh spin is high , and the ns is tidally disrupted for an orbit far from the bh isco ; type - ii : the bh mass is not low , the bh spin is small ( or a < 0 ) , and the ns is not tidally disrupted ; type - iii : the bh mass is not low , the bh spin ( a > 0 ) is high , and the ns is tidally disrupted for an orbit close to the bh isco . these differences in the infall process are well reflected in gravitational waveforms and spectra . figure 18schematic pictures for the three types of merger process that have been found to date . left ( type - i ) : the ns is tidally - disrupted and the extent of the tidally disrupted material is as large as or larger than the bh surface area . middle ( type - ii ) : the ns is not tidally disrupted and simply swallowed by the bh . right ( type - iii ) : the ns is tidally disrupted and the extent of the tidally disrupted material in the vicinity of the bh horizon is smaller than the bh surface area . the solid black sphere is the bh , the blue distorted ellipsoid is the ns , the solid red circle is the location of the bh isco , and the dashed circle is the location of the tidal - disruption limit . schematic pictures for the three types of merger process that have been found to date . left ( type - i ) : the ns is tidally - disrupted and the extent of the tidally disrupted material is as large as or larger than the bh surface area . middle ( type - ii ) : the ns is not tidally disrupted and simply swallowed by the bh . right ( type - iii ) : the ns is tidally disrupted and the extent of the tidally disrupted material in the vicinity of the bh horizon is smaller than the bh surface area . the solid black sphere is the bh , the blue distorted ellipsoid is the ns , the solid red circle is the location of the bh isco , and the dashed circle is the location of the tidal - disruption limit . the effect of spin orientation , which is misaligned with that of the orbit rotation axis , was studied for the first time . they performed simulations for a = 0 , 0.5 , and 0.9 , and q = 3 with -law eos ( = 2 ) , and found that the remnant disk mass decreases sensitively with increase of the inclination ( misalignment ) angle for given values of a and q ( see figure 20 ) . this is quite natural because the spin - orbit coupling force is proportional to sl , where s and l denote bh spin and orbital angular momentum vectors , respectively , and the radius of the isco around the bh approaches that for a = 0 with increasing of the inclination angle . thus , the bh spin effect becomes less important with the increase of the inclination angle . they also found that the inclination angle is significantly decreased after a substantial mass of the ns falls into the companion bh , implying that the angular momentum vector of the remnant disk misaligns only modestly with the bh spin vector ( by 20 ) . this is also quite natural because the orbital angular momentum is as large as or larger than the spin angular momentum of the bh for small mass ratios like q = 3 . however , for a high value of q for which the fraction of the bh spin angular momentum in the total angular momentum is large , this conclusion will be modified . the initial inclination angle will not be significantly modified and an inclined disk , which subsequently precesses around the spinning bh , may be the outcome . a tail of a one - armed spiral formed at tidal disruption often extends far away from the bh , in particular for the case in which the bh spin is high . the cccw group reported that for q = 3 , a = 0.5 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { l^3}/g{m_0}}$\end{document } ( i.e. , for realistic values of the compactness ) , a tidal tail of mass 0.010.1 m goes to a distance l = 2002000 km away from the bh before falling back to the central region . they also reported that the fall - back time scale was 200 ms for l = 2000 km assuming that the material obeys geodesic motion . here , 200 ms agrees roughly with the dynamic infall time scale \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { { l^3}/g{m_0}}$\end{document}. this indicates that the time scale of mass accretion from the disk onto the bh is much longer than the rotational period of the disk in the vicinity of the bh 10 ms . such a time scale may be explained by the time scale of the fall - back motion . the lbpli group also estimated the fall - back time for their simulation with q = 5 , a = 0.5 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.16 - 0.18$\end{document } . in their simulation , the compactness of the ns was assumed to be smaller than that for a realistic ns , and thus , the formation of the tidal tail can be significantly enhanced . they reported that for a large fraction of the material 0.05 m , the fall - back time may be longer than 1 s. however , they followed the motion of the tidal tail only for a short time duration ( 16.3 ms ; i.e. , they did not show that the material really went far away , l 10 km ) , and in addition , did not describe the detail of the method for estimating the fall - back time . ( note that a fall - back time longer than 1 s is realized only for an element , which reaches a distance from the bh of l 10 km . ) hence , their conclusion should be confirmed by a longer - term simulation in the future . their finding in the framework of numerical relativity that the disk can extend to a large distance 10 gm0/c for a high bh spin was qualitatively new . the kt group performed simulations for mns = 1.35 m , q = 25 , a = 0.75 with several piecewise polytropic eos . they found that even for a binary with a ns of realistic compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \approx 0.145$\end{document } , the disk can extend to 500 km ( which is approximately the location of the outer boundary of the computational domain in their simulations ) . thus , for the merger of a rapidly - spinning bh and a ns , it may be concluded that a widely - spread disk is formed , and the lifetime of the accretion disk will be fairly long 0.1 s. the kt groups also found that the density of the disk decreases with increase of q ( or the bh mass ) ; for a high - mass bh , a widely spread but less dense disk is formed . the dependence of the merger process on the ns eos comes primarily from the fact that the ns radius depends sensitively on the eos . for a ns with a large radius , tidal disruption ( and subsequent disk formation ) this fact was clearly shown in the works by the kt group [ 107 , 109 ] , performed employing a variety of the piecewise polytropic eos . the eos also determines the density profile of a ns . even if the radius is the same for a given mass , the density profiles for two ns may be different if the hypothetical eos is different . the kt group showed that a ns with a small adiabatic index for the core region , with which the density is concentrated in the central region , is less subject to tidal disruption than that with a larger adiabatic index ( with relatively uniform density profile ) , even if the radius and mass are identical ; e.g. , for the piecewise polytropic eos listed in table 4 , a ns with a smaller value of 2 is less subject to tidal disruption . the reason for this is that the star with a high degree of central density concentration is less subject to tidal deformation , as reviewed in section 1.2 . the cccw group performed a simulation incorporating a tabulated finite - temperature eos for the first time . the advantage of this approach is that one can determine the temperature and composition , such as electron fraction in the disk formed after tidal disruption occurs . this will be useful for discussing the possibility that the remnant bh - disk system could be a central engine of an sgrb . to avoid taking into account the effects of neutrino emission , they assumed that the system is in -equilibrium or that the electron fraction is unchanged in the fluid - moving frame . in the former and latter , they assumed that the system is in either of the following two limiting cases ; the weak interaction time scale is either much shorter or much longer than the dynamic time scale , respectively . irrespective of the eos , they found that the remnant disk is neutron rich with ye 0.10.2 and the temperature is only moderately high ( maximum is 10 mev with the average 3 mev ) with the maximum density 10 g / cm and disk mass 0.1 m. perhaps , because of the relatively low mass and density of the disk , the temperature is not as high as that found in a newtonian simulation with detailed microphysics . during merger , ns material falls into its companion bh , and then , the mass and spin of the bh vary . because a large fraction of the ns material falls into the bh for most of the numerical experiments performed so far and the total energy of gravitational waves emitted ( egw ) is much smaller than the total mass energy of the system , the final bh mass becomes roughly mbh + mns mdisk egw mbh + mns = m0 . ( the disk mass mdisk and egw are less than 10% of the initial total mass m0 . ) the total orbital angular momentum for two point masses in a circular orbit with an angular velocity is 122\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${j_{{\rm{orb } } } } = { { { g^{2/3}}{m_{{\rm{bh}}}}{m_{{\rm{ns } } } } } \over { { { ( \omega { m_o})}^{1/3}}}}.$$\end{document } thus , the non - dimensional spin parameter of the system is approximately written as 123\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_f } = { { c{g^{- 1}}{j_{{\rm{orb } } } } + m_{{\rm{bh}}}^2a } \over { m_0 ^ 2 } } = { { { { ( g{c^{- 3}}\omega { m_0})}^{- 1/3}}q + a{q^2 } } \over { { { ( 1 + q)}^2}}},$$\end{document } where we assume that the bh spin aligns with the orbital angular momentum vector . if the mass and angular momentum of the remnant disk , and loss by gravitational waves may be neglected , af will be equal to the spin of the remnant bh . at the onset of the merger or at tidal disruption , the angular velocity becomes gcm0 0.050.1 , and thus , ( gcm0 ) is in a narrow range of 2.12.7 . this implies that af is primarily determined by q and a. equation ( 123 ) gives rather qualitative estimate for the spin of the remnant bh . nevertheless , it still gives a good approximate value of the final spin with the choice of ( gcm0 ) = 3 as large as the remnant disk mass is small . with this choice , af = 0.67 , 0.56 , 0.48 , and 0.42 for q = 2 , 3 , 4 , and 5 and for a = 0 . these values agree with the results derived by the cccw , kt [ 194 , 107 , 108 ] , and uiuc groups within the error of a = 0.010.02 . for a large bh spin with a 0.5 , a disk of a large mass ( 0.1 m ) is often formed even for q 3 5 ( see below ) . in such cases , equation ( 123 ) however , this equation still captures the qualitative tendency of the final spin ; e.g. , for small bh spin , the final spin is determined by the value of q and the larger values of q results in smaller final bh spin ; for larger values of q with a large bh spin a 0.5 , the final bh spin is primarily determined by the initial bh spin . the mass and characteristic density of the remnant disk surrounding the bh depend sensitively on the mass ratio ( q ) , the bh spin ( a ) , and the eos ( or the compactness ) of the ns . figure 19 displays a result of the disk mass as a function of the ns compactness for q = 2 ( left ) and q = 3 ( right ) for various piecewise polytropic eos and for various values of a , reported by the kt group . this shows that the disk mass decreases steeply and systematically with the increase of the compactness irrespective of a and q. figure 19left : disk mass at 10 ms after the onset of merger as a function of ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } with various piecewise polytropic eos and with various values of a for q = 2 . the simulations were performed by the kt group and the figure is taken from . figure 20left : summary of the remnant disk mass as a function of the bh spin for a fixed eos ( = 2 eos ) , ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } , and mass ratio ( q = 3 ) , computed by the uiuc , cccw , and kt groups . the vertical axis shows the fraction of the disk mass mr > rah / mb where mb is the baryon rest mass of the ns . cccwin shows the results by the cccw group with inclination angle of the bh spin , 40 , 60 , and 80 degrees ( from upper to lower points ) . right : the same as the left panel but for the disk mass in the solar mass unit for more compact ns \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c } \approx 0.172)$\end{document } with a piecewise polytropic eos ( hb ) . the disk mass is measured at t 10 ms after the onset of the merger ( for the -law eos , mns is assumed to be 1.4 m ) . figure 21left : disk mass as a function of ns compactness for various values of q with a = 0.75 . these simulations were performed by the kt group and this figure is taken from . left : disk mass at 10 ms after the onset of merger as a function of ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } with various piecewise polytropic eos and with various values of a for q = 2 . the simulations were performed by the kt group and the figure is taken from . left : summary of the remnant disk mass as a function of the bh spin for a fixed eos ( = 2 eos ) , ns compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } , and mass ratio ( q = 3 ) , computed by the uiuc , cccw , and kt groups . the vertical axis shows the fraction of the disk mass mr > rah / mb where mb is the baryon rest mass of the ns . cccwin shows the results by the cccw group with inclination angle of the bh spin , 40 , 60 , and 80 degrees ( from upper to lower points ) . right : the same as the left panel but for the disk mass in the solar mass unit for more compact ns \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c } \approx 0.172)$\end{document } with a piecewise polytropic eos ( hb ) . the disk mass is measured at t 10 ms after the onset of the merger ( for the -law eos , mns is assumed to be 1.4 m ) . left : disk mass as a function of ns compactness for various values of q with a = 0.75 . the left panel of figure 20 plots together the results obtained by the uiuc , cccw , and kt groups for = 2 eos with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } and q = 3 ( the revised result by the kt group is plotted here ; results in early work by the kt group do not agree with the result shown here [ 228 , 194 ] ; see below for the reason ) . this shows that the disk mass increases steeply with bh spin ( a ) for given values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \sim 0.17$\end{document } and q. the results by these three groups agree approximately with each other for a = 0 . the cccw group showed for the first time that the disk mass decreases with the increase of the inclination angle of the bh spin , and toward the limit to 90 degree , the disk mass approaches to that of a = 0 . the right panel of figure 20 plots the results by the kt group for different compactness ( using eos hb with mns = 1.35 m ; see table 4 ) . this shows again that the disk mass increases with the increase of the bh spin , and also that for high bh spin ( e.g. , a = 0.75 ) , the disk mass is larger than 0.1 m even for q = 5 with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c } \approx 0.145)$\end{document}. figure 21 shows disk mass as a function of ns compactness for a = 0.75 and a = 0.5 as performed by the kt group . a steep decrease in disk mass with increasing compactness is found irrespective of the values of a and q. simulations for bh - ns binaries with a spinning bh for particular values of the compactness or mass ratio were also performed by the cccw and uiuc groups for a = 0.5 and 0.75 , respectively . the results of the cccw group for a = 0.5 and q = 3 with various eos agree with those of figure 21 within 1020% . we note that the disk mass may be different in the different eos even with the same compactness of the ns , because the density profile of the ns , and the resulting susceptibility to the bh tidal force is different . thus , this disagreement is reasonable . also , the results of the uiuc group for a = 0.75 , q = 3 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } = 0.145 with the -law eos ( the disk mass 0.15 mb ) agree with the relation expected from figure 21 within 20% error . to clarify the dependence of the disk mass on relevant parameters , we consider three types of comparisons . first , we consider the case in which a , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } , and the eos are fixed , but q is varied . in this case for example , for a = 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } with = 2 eos , the disk mass is larger ( smaller ) than 0.01 mb for q 4 ( q 4 ) [ 63 , 74 , 154 ] . however , we should point out the exception to this rule , because for the case in which a large - mass disk is formed , this rule may not hold . for example , the comparison between the left and right panels of figure 19 shows that for relatively small compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.16$\end{document } , a large - mass disk is formed for high bh spins and the disk mass depends only weakly on the value of q for given values of a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document}. second , we consider the case in which q and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } are fixed , but a is varied . the uiuc group compared the results for a = 0.5 , 0 , and 0.75 for q = 3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } with the -law eos ( = 2 ) , and the resulting disk mass at 10 ms after the onset of the merger is 0.008 , 0.039 , and 0.15 mb for a = 0.5 , 0 , and 0.75 , respectively . the cccw group performed a similar study for a = 0 , 0.5 , and 0.9 for q = 3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.144$\end{document } with the -law eos ( = 2 ) , and found that the disk mass at 10 ms after the onset of the merger is 0.034 , 0.126 , and 0.360 mb for a = 0 , 0.5 , and 0.9 , respectively . both groups found the systematic steep increase of the disk mass with the increase of the bh spin ( cf . this was also reconfirmed by the kt group ( see the right panel of figure 19 and figure 21 ) . third , we consider the case in which a and q are fixed , but the compactness , c , is varied . systematic work was recently performed by the kt group [ 107 , 108 , 109 ] , employing a piecewise polytropic eos . figures 19 and 21 show that the disk mass decreases monotonically with the increase of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } for given values of a and q. for producing a disk of mass larger than 0.01 m for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a = 0,\,{\mathcal c}$\end{document } should be smaller than 0.18 for q = 2 and 0.16 for q = 3 according to their results . for a = 0.75 , the condition is significantly relaxed . finally , the kt group [ 107 , 108 ] found that the disk mass depends not only on the compactness but weakly on the density profile . for the ns with a more centrally concentrated density profile , the disk mass is smaller . the reason for this is that if the degree of the central mass concentration is smaller , tidal deformation is enhanced and it encourages earlier tidal disruption after the onset of mass shedding . it is interesting to note that for a = 0.75 and mns 1.4 m , the mass of a disk surrounding a bh can be larger than 0.1 m for q 4 with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.18$\end{document } and for q 5 with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.17$\end{document}. for q = 2 , the disk mass will be 0.1 m for any realistic ns with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.20$\end{document}. for a bh of higher spin , the disk mass will be even larger ( cf . in addition , the disk mass does not decrease steeply with the increase of q for such a high spin . in particular , for a small value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } , the disk mass depends very weakly on q. all these results indicate that the disk mass is likely to be large even for a higher value of q( 5 ) with a high bh spin ( a 0.75 ) . the maximum density of the disk increases monotonically with the disk mass . for a disk mass larger than 0.1 m , the maximum density is larger than 10 g / cm for q 4 and 10 g / cm for q = 5 . hence , a high - mass disk with a relatively small value of q 4 is likely to be universally opaque against thermal neutrinos for the typical geometrical thickness of the disk ; a neutrino - dominated accretion disk is the outcome and this is favorable for copious neutrino emission . this leads to the conclusion that the coalescence of bh - ns binaries with a high - spin bh with a 0.75 and q 4 is a promising progenitor for forming a bh plus a massive disk system ; that is the candidate for a central - engine of sgrb . before closing this subsection , we should note that different groups have reported different quantitative results for the disk mass in their earlier work , which has been improved upon . for example , the earlier work of the kt group presented a small disk mass [ 228 , 194 ] . this is mainly due to an unsuitable choice of computational domain and partly due to a spurious numerical effect associated with insufficient resolution and an unsuitable choice of amr grid . the first work of the uiuc group also underestimated the disk mass . first , the fluid elements in the disk have to acquire a sufficiently large specific angular momentum which is larger than that at the isco of the remnant bh . the material that forms a disk obtains angular momentum by a hydrodynamic angular momentum transport process from the inner part of the material . this implies that such a transport process has to be accurately computed in a numerical simulation . however , it is well known that this is one of the challenging tasks in computational astrophysics . second , to avoid spurious loss and transport of the angular momentum , a high - resolution computation is required . however , the disk material is located in a relatively distant orbit around the central bh . in the amr scheme , which is employed in all the groups , the resolution in this region is usually poorer than that in the central region . this might induce a spurious loss of angular momentum and resulting decrease of disk mass , even by a factor of 2 . however , these issues are being resolved with the improvement of computational resources , the efficiency of the numerical code , and the skill for computation with the amr algorithm . as mentioned in section 1 , tidal disruption occurs after the onset of mass shedding and after a substantial fraction of the ns material is removed from the inner cusp by the bh tidal field . it is important to emphasize again that the condition of tidal disruption is in general different from that of mass shedding for bh - ns binaries and that tidal disruption could occur in a more restricted condition than that for mass shedding , as mentioned in sections 1 and 2 . to determine the condition of tidal disruption , a dynamic simulation ( not a study of quasi - equilibrium or equilibrium ) is necessary . it is not easy to strictly determine the condition of tidal disruption , because its concept is not as clear as that of mass shedding . one way to determine the condition is to use the property of gravitational waveforms in the merger phase ; if the cutoff frequency is smaller than the frequency of a quasi - normal mode ( qnm ) of the remnant bh , we may conclude that tidal disruption occurs ( see section 3.6 for details of the cutoff frequency ) . another way is to use the property of the merger remnant ; we may recognize that tidal disruption occurs if the mass of a remnant disk surrounding a bh is substantial , say larger than 1% of the total rest mass at t 10 ms after the onset of mass shedding . we employ both criteria here to determine whether tidal disruption occurs or not . first , we summarize the criterion for the case in which the bh spin is zero ( a = 0 ) . in this case , the condition of tidal disruption depends strongly on the mass ratio and compactness of the ns . according to the criterion associated with a gravitational - wave spectrum ( see figure 28 , as an example ) , the condition of tidal disruption is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.19$\end{document } for q = 2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.16$\end{document } for q = 3 . figure 19 , which displays the result of the disk mass for a = 0 with several piecewise polytropic eos , also illustrates that the condition of tidal disruption depends strongly on the values of q and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } : the approximate condition for a = 0 is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.18 - 0.19$\end{document } for q = 2 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.16$\end{document } for q = 3 . we note that for q = 3 , this conclusion is consistent with those of the cccw and uiuc groups . the aei group studied the case of q = 5 with -law eos ( = 2 ) and showed that the condition is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.13 - 0.14$\end{document } for q = 5 . for a spinning bh , in particular for the case in which the spin vector aligns with the orbital angular momentum vector , the condition for tidal disruption is highly relaxed . here , we focus only on the case in which the bh spin vector is aligned with the orbital angular momentum vector . from the disk mass as a function of compactness shown in figure 21 derived by the kt group for q = 2 , tidal disruption occurs irrespective of the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}(\lesssim 0.2)$\end{document } for a 0.5 . however , for a counter - rotating spin ( a = 0.5 ) , the criterion for tidal disruption is significantly restricted ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c } \lesssim { ( } 0.16{)})$\end{document } ) . for a = 0.75 , tidal disruption occurs irrespective of the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}(\lesssim 0.2)$\end{document } as far as q 4 . finally , we compare the conditions for tidal disruption and mass shedding for a = 0 . figure 22 plots threshold curves of tidal disruption , mass shedding in general relativity , and mass shedding in a tidal approximation in the plane of ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } , q ) . if the value of q or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is smaller than that of the threshold curves , tidal disruption or mass shedding occurs . the points with error bar approximately denote the numerical results for the criterion of tidal disruption , based on the results by the kt group for q = 2 , by the cccw , kt , and uiuc groups for q = 3 , and by the aei group for q = 5 . the solid and dashed curves denote the critical curves for the onset of mass shedding for = 2 eos in general relativity [ 209 , 210 ] , and for the incompressible fluid in a tidal approximation ( see equation ( 12 ) ) , respectively . this shows that , for a realistically compact ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.13 \lesssim { \mathcal c } \lesssim 0.21$\end{document } , the condition for tidal disruption is more restricted than that for mass shedding . the primary reason for this is that for such large compactness with a = 0 , tidal disruption occurs only for a small value of q. for such a system , the time scale for gravitational radiation reaction is as short as the orbital period in close orbits . this indicates that at the onset of mass shedding , the radial approaching velocity induced by gravitational radiation reaction is high enough to significantly decrease ( increase ) the orbital radius ( angular frequency ) during the subsequent mass - shedding phase up to final tidal disruption . if the fraction of this decrease in the orbital radius is large enough to enforce the orbit inside the isco , tidal disruption is prohibited . this mechanism makes the condition of tidal disruption for bh - ns binaries more restricted than that of mass shedding . figure 22a summary for the conditions of tidal disruption and mass shedding for a = 0 in the plane of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c},q)$\end{document}. if the value of q or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is smaller than that of the threshold curves shown here , they occur . the points with error bars ( and the dotted curve ) approximately denote the numerical results for tidal disruption , based on results by the kt group for q = 2 , by the cccw , kt , and uiuc groups for q = 3 , and by the aei group for q = 5 . the solid and dashed curves denote the critical curves for the onset of mass shedding for = 2 eos in general relativity [ 209 , 210 ] , and for the incompressible fluid in a tidal approximation ( see equation ( 12 ) ) , respectively . a summary for the conditions of tidal disruption and mass shedding for a = 0 in the plane of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c},q)$\end{document}. if the value of q or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is smaller than that of the threshold curves shown here , they occur . the points with error bars ( and the dotted curve ) approximately denote the numerical results for tidal disruption , based on results by the kt group for q = 2 , by the cccw , kt , and uiuc groups for q = 3 , and by the aei group for q = 5 . the solid and dashed curves denote the critical curves for the onset of mass shedding for = 2 eos in general relativity [ 209 , 210 ] , and for the incompressible fluid in a tidal approximation ( see equation ( 12 ) ) , respectively . on the other hand , if the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is small , tidal disruption may occur even for a large value of q. with increase of q , the ratio of the time scale of gravitational radiation reaction to the orbital period at the isco increases ( e.g. , equation ( 2 ) ) . for such a high - q case , the effect of orbital decrease by gravitational radiation reaction after the onset of mass shedding becomes relatively minor , and therefore , the critical curves of tidal disruption and mass shedding approach each other . for a bh - ns binary with high bh spin , mass shedding may occur even for high mass ratio ( say q = 10 for a 0.9 , e.g. , equation ( 12 ) ) . for such a case , the conditions for tidal disruption and mass shedding may approximately agree with each other . if this is the case , the quasi - equilibrium study plays an important role in determining the condition of mass shedding , because this also gives the ( approximate ) condition of tidal disruption . figure 23 displays the typical gravitational waveforms for a = 0 , which clearly reflect the features of the orbital evolution and subsequent merger processes ( tidal disruption or not ) as described in the following . in the early inspiral phase in which r rns and r gmbh / c , two objects behave like point masses . in addition , general relativistic effects to the orbital motion are not extremely strong . for such a phase , the signal of gravitational waves is the chirp signal that can be well reproduced by the pn approximation for the two - body problem . figure 23gravitational waveforms observed along the axis perpendicular to the orbital plane for q = 3 and a = 0 with very stiff ( 2h ) , stiff ( h ) , moderate ( hb ) , and soft ( b ) eos . the solid and dashed curves denote the numerical results and results derived by the taylor - t4 formula . the left and right axes show the normalized amplitude ( cdh / gm0 ) and physical amplitude for d = 100 mpc , respectively . gravitational waveforms observed along the axis perpendicular to the orbital plane for q = 3 and a = 0 with very stiff ( 2h ) , stiff ( h ) , moderate ( hb ) , and soft ( b ) eos . the solid and dashed curves denote the numerical results and results derived by the taylor - t4 formula . the left and right axes show the normalized amplitude ( cdh / gm0 ) and physical amplitude for d = 100 mpc , respectively . in which the finite - size effect is still negligible but general relativistic gravity between two objects plays a role , it is known that the simple pn analysis fails to provide a precise waveform . comparisons of the waveforms derived through pn analysis and through numerical computation for bh - bh binaries [ 36 , 30 , 31 , 5 , 179 ] propose that a better waveform is phenomenologically derived using the taylor - t4 formula . this method requires a special summation method of pn high - order terms in the equations of motion , which include gravitational radiation reaction effects in an adiabatic approximation . first , one needs to calculate the evolution of the orbital angular velocity (t ) through x(t ) = [ gc(t)m0 ] up to 3.5pn order by solving the following ordinary differential equations 124\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{dx } \over { dt } } = { { 64\nu { x^3 } } \over { 5g{c^{- 3}}{m_0}}}f(x),$$\end{document } where f(x ) is a polynomial of x as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 + \sigma _ { i = 2}^7{a_i}{x^{i/2}}$\end{document } and ai denote coefficients ( functions of mass and spin of compact objects ) . is the ratio of the reduced mass to the total mass m0 , = q/(1 + q ) . for a solution of x(t ) , then , the orbital phase (t ) is derived by integrating the following equation 125\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d\theta } \over { dt } } = { { { x^{3/2 } } } \over { g{c^{- 3}}{m_0}}}.$$\end{document } after x(t ) and (t ) are obtained , the complex gravitational - wave amplitude h of ( l , m ) = ( 2 , 2 ) mode is calculated up to 3pn order using the formula of . figure 23 shows that gravitational waveforms in the late inspiral phase before the onset of the merger ( or tidal disruption ) indeed agree with the result derived by the taylor - t4 formula for the bh - ns binaries , as in the case of bh - bh binaries . ( note that in the first few wave cycles , the agreement is not very good . this is because the initial condition given for their simulation was not in an exact quasi - circular orbit . ) the waveforms may deviate from the prediction by the taylor - t4 formula before the onset of the merger for a small value of q or for a large ns radius . the reason for this is that the ns is tidally deformed by the bh , and , as a result , the pure taylor - t4 formula , in which the tidal - deformation effect is not taken into account , is not a good formula for such a phase . the features of gravitational waveforms in the final inspiral phase are summarized later . by contrast , for a sufficiently large value of q or for a sufficiently compact ns , the tidal - deformation effect is negligible , and hence , the waveforms are quite similar to those for a bh - bh binary as mentioned above . for a small degree of tidal deformation and mass shedding , most of the ns material falls into the bh simultaneously ( this case corresponds to type - ii according to the definition of figure 18 ) . in such a case , a fundamental qnm of a bh is excited ( see the waveform for the soft eos in figure 23 ) , and the highest frequency of gravitational waves is determined by the qnm . the degree of the qnm excitation depends strongly on the degree of tidal deformation and mass shedding . the primary reason for this is that a phase cancellation is concerned in the excitation ; here , the phase cancellation is the amount that the gravitational waves emitted in a non - coherent manner ( with different phases ) interfere with each other to suppress the amplitude of gravitational waves [ 141 , 187 , 139 ] . with increasing degree of mass shedding , the phase cancellation effect plays an increasingly important role and the amplitude of the qnm - ringdown gravitational waves decreases . for the case in which a ns is tidally disrupted far outside the isco , this effect is significantly enhanced because the ns material does not simultaneously fall into the bh . rather , a widely spread material , for which the density is much smaller than the typical ns s density , falls into the bh from a wide region of the bh surface spending a relatively long time duration ( this case corresponds to type - i according to the definition of figure 18 ) . here , it is appropriate to point out why infall occurs from a wide region of the bh surface ; the bh mass is small for the case in which mass shedding occurs for a = 0 , and thus , the areal radius of the bh is smaller than or as small as the ns radius . all these effects are discouraging for efficiently exciting a qnm , and therefore , the amplitude of the qnm - ringdown gravitational waves is strongly suppressed for the case in which tidal disruption occurs ( see the waveform for eos 2h in figure 23 ) . for the case in which tidal disruption occurs , the highest frequency of gravitational waves is approximately determined by the orbital frequency at tidal disruption , not by the frequency of a qnm . one important remark here is that this highest , characteristic frequency is not in general determined by the frequency at the onset of mass shedding . even after the onset of mass shedding , the ns continues to be a self - gravitating star for a while and gravitational waves associated with an approximately - inspiral motion are emitted . after a substantial fraction of gravitational waves is emitted and thus , the orbital separation becomes sufficiently small , tidal disruption occurs . at such a moment , the amplitude of the gravitational waves damps steeply , and hence , the highest frequency of gravitational waves should be determined by the tidal - disruption event . the qualitative features summarized above depend on the bh spin ; for binaries composed of a bh of high spin , tidal disruption may occur for a high mass ratio , and hence , the infall process of the tidally - disrupted material into the bh may be qualitatively modified . this is well reflected in gravitational waveforms , as described in the next section 3.6.2 . figure 24 plots gravitational waveforms for q = 3 with the same stiff eos ( hb eos ) and with the same initial angular velocity ( gcm0 = 0.030 ) but with different values of the bh spin . this obviously shows that with increasing the bh spin , the lifetime of the binary system increases and hence the number of wave cycles increases . this is explained primarily by the spin - orbit coupling effect ( see also section 3.3 ) , which brings a repulsive force into the bh - ns binary for the prograde spin of the bh . due to the presence of this repulsive force , the orbital separation of the isco ( the absolute value of the binding energy there ) can be smaller ( larger ) than that for the non - spinning bh . this effect increases the lifetime of the binary , and furthermore , enhances the chance for tidal disruption of the ns because a circular orbit with a closer orbital separation is allowed . second , the repulsive force reduces the orbital velocity for a given separation , because the centrifugal force may be weaker for a given separation to maintain a quasi - circular orbit . the decrease of the orbital velocity results in the decrease of the gravitational - wave luminosity , and this decelerates the orbital evolution as a result of gravitational radiation reaction , making the lifetime of the binary longer and increasing the number of cycles of gravitational waves . we note that all these effects are also clearly reflected in the gravitational - wave spectrum , as is shown in section 3.7 . figure 24the same as figure 23 but for q = 3 and a = 0.75 ( top left ) , 0.5 ( top right ) , 0 ( bottom left ) , and 0.5 ( bottom right ) with hb eos . this figure is taken from . the same as figure 23 but for q = 3 and a = 0.75 ( top left ) , 0.5 ( top right ) , 0 ( bottom left ) , and 0.5 ( bottom right ) with hb eos . figure 24 shows that for a 0 , a ringdown waveform associated with a qnm of the bh is clearly seen , whereas for a = 0.75 , such a feature is absent . this reflects the fact that tidal disruption of the ns occurs for a = 0.75 far outside the isco , whereas it does not for a 0 . for a = 0.5 tidal disruption occurs near the isco and a large fraction of the ns material falls into the bh . the infall occurs approximately simultaneously and proceeds from a narrow region of the bh surface . this new type appears for the case in which the bh mass ( or mass ratio q ) is large enough that the surface of the event horizon is wider than the extent of the infalling material , as explained in section 3.3.2 ( see figure 18 ) . the inspiral waveform matches well to that of the taylor - t4 formula for binaries composed of a spinning bh as well as for a non - spinning bh . figure 24 also shows that matching is achieved as well as for a = 0 , irrespective of the spin , except for the final phase just before the onset of the merger . as in the case of a = 0 , the deviation of gravitational waveforms from the prediction by the taylor - t4 formula is enhanced with increasing degree of tidal deformation , and with subsequent mass shedding and tidal disruption . the final fate of the ns in bh - ns binaries is clearly reflected in the spectrum of gravitational waves . general qualitative features of the gravitational - wave spectrum for bh - ns binaries composed of non - spinning bh are summarized as follows . for the early stage of the inspiral phase , during which the orbital frequency is 1 khz ( r/12 km ) and the pn point - particle approximation works well , the gravitational - wave spectrum is approximately reproduced by the taylor - t4 formula . for this phase , the spectrum amplitude of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{{\rm{eff } } } } \equiv f\tilde h(f)$\end{document } decreases as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f^{- { n_i}}}$\end{document } where ni 1/6 for f 1 khz and the value of ni increases with f for f 1 khz ( r/12 km ) . as the orbital separation decreases , both the non - linear effect of general relativity and the finite - sized effect of the ns come into play , and thus , the pn point - particle approximation breaks down . when tidal disruption ( not mass shedding ) occurs for a relatively large separation ( e.g. , for a ns of stiff eos or for a small value of q ) , the amplitude of the gravitational - wave spectra damps above a cutoff the cutoff frequency is equal to a frequency in the middle of the inspiral phase with fcut 12 khz for this case ( it is lower than the frequency at the isco ) . the cutoff frequency depends on the binary parameters as well as on the eos of the ns . a more strict definition of fcut was given by the kt group and will be reviewed below . by contrast , if tidal disruption does not occur or occurs at a close orbit near the isco , the spectrum amplitude for a high frequency region ( f 1 khz ) is larger than that predicted by the taylor - t4 formula ( i.e. , the value of ni decreases and can even become negative ) . in this case , an inspiral - like motion may continue even inside the isco for a dynamic time scale and gravitational waves with a high amplitude are emitted . this is reflected in the fact that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\tilde h(f)$\end{document } becomes a slowly varying function of f for 1 khz f fcut , where fcut 23 khz . a steep damping of the spectra for f fcut is universally observed , and for softer eos with smaller ns radius , the frequency of fcut is higher for a given mass of bh and ns . this cutoff frequency is determined by the frequency of gravitational waves emitted when the ns is tidally disrupted for the stiff eos or by the frequency of a qnm of the remnant bh for the soft eos . therefore , the cutoff frequency provides potential information for a eos through the tidal - disruption event of the ns , in particular for the stiff eos . figure 25 , plotted by the uiuc group , clearly illustrates the facts described above . the top panel ( case e ) plots the spectrum for q = 1 , in which the ns is tidally disrupted far outside the isco . in this case , the spectrum damps at f 1 khz at which the tidal disruption occurs . the bottom panel ( case d ) plots the spectrum for q = 5 , in which the ns is not tidally disrupted . in this case , the steep damping of the spectrum at f 2 khz is determined by the swallowing of the ns by the companion bh , and thus , the cutoff frequency is characterized by ringdown gravitational waves associated with the qnm of the remnant bh . because the finite - sized effect of the ns is not very important in this case , the gravitational - wave spectrum is similar to that of the bh - bh binary merger with the same mass ratio ( q = 5 ( case a ) , the cutoff frequency , at which the steep damping of heff sets in , is different from that for the bh - bh binary with the same mass ratio . this implies that tidal deformation and disruption play an important role in the merger process and in determining the gravitational waveform . figure 25the gravitational - wave spectrum for q = 1 ( case e ) , 3 ( case a ) , and 5 ( case d ) with a = 0 and -law eos with = 2 . the solid curve shows the spectrum of a 2.5pn and numerical waveforms , while the dotted curve shows the contribution from the numerical waveform only . the dashed curve is the analytic fit derived in from analysis of bh - bh binaries composed of a non - spinning bh with the same values of q as the bh - ns . the heavy solid curve is the effective strain of advanced ligo . to set physical units , a rest mass is assumed to be mb = 1.4 m ( r 13 km ) for the ns and a source distance of d = 100 mpc . this figure is taken from . the gravitational - wave spectrum for q = 1 ( case e ) , 3 ( case a ) , and 5 ( case d ) with a = 0 and -law eos with = 2 . the solid curve shows the spectrum of a 2.5pn and numerical waveforms , while the dotted curve shows the contribution from the numerical waveform only . the dashed curve is the analytic fit derived in from analysis of bh - bh binaries composed of a non - spinning bh with the same values of q as the bh - ns . the heavy solid curve is the effective strain of advanced ligo . to set physical units , a rest mass is assumed to be mb = 1.4 m ( r 13 km ) for the ns and a source distance of d = 100 mpc . as described above , the cutoff frequency at which the steep damping of the spectrum occurs will bring us the information for the degree of tidal deformation and where tidal disruption occurs in a close orbit just before the merger . the degree of tidal deformation and the frequency at which tidal disruption occurs depend on the eos of the ns . motivated by this idea , the kt group performed a wide variety of simulations , changing the mass ratio , eos , and bh spin , and systematically analyzed the resulting gravitational waveforms . figure 26 plots the spectrum as a function of the frequency for q = 2 , mns = 1.35 m , and with a variety of eos for a = 0 . irrespective of the eos , the spectrum has the universal feature mentioned above . however , the cutoff frequency , at which the steep damping sets in , depends strongly on the eos . figure 26spectra of gravitational waves from bh - ns binaries for q = 2 , a = 0 , and mns = 1.35 m with various eos . the bottom axis denotes the normalized dimensionless frequency fm0(= gfm0/c ) and the left axis the normalized amplitude c \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c^2}f\tilde h(f)d / g{m_0}$\end{document}. the top axis denotes the physical frequency f in hz and the right axis the effective amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\tilde h(f)$\end{document } observed at a distance of 100 mpc from the binaries . the short - dashed sloped line plotted in the upper left region denotes a planned noise curve of advanced - ligo optimized for 1.4 m ns - ns inspiral detection ( standard ) , the long - dashed slope line denotes a noise curve optimized for burst detection ( broadband ) and the dot - dashed slope line plotted in the lower right region denotes a planned noise curve of the einstein telescope ( et ) [ 91 , 92 ] . the upper transverse dashed line is the spectrum derived by the quadrupole formula and the lower one is the spectrum derived by the taylor - t4 formula , respectively . spectra of gravitational waves from bh - ns binaries for q = 2 , a = 0 , and mns = 1.35 m with various eos . the bottom axis denotes the normalized dimensionless frequency fm0(= gfm0/c ) and the left axis the normalized amplitude c \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c^2}f\tilde h(f)d / g{m_0}$\end{document}. the top axis denotes the physical frequency f in hz and the right axis the effective amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\tilde h(f)$\end{document } observed at a distance of 100 mpc from the binaries . the short - dashed sloped line plotted in the upper left region denotes a planned noise curve of advanced - ligo optimized for 1.4 m ns - ns inspiral detection ( standard ) , the long - dashed slope line denotes a noise curve optimized for burst detection ( broadband ) and the dot - dashed slope line plotted in the lower right region denotes a planned noise curve of the einstein telescope ( et ) [ 91 , 92 ] . the upper transverse dashed line is the spectrum derived by the quadrupole formula and the lower one is the spectrum derived by the taylor - t4 formula , respectively . the features of gravitational - wave spectra for a = 0 are schematically summarized in figure 27 . here , three curves are plotted assuming that the masses of the bh and the ns are all the same with a = 0 and with a relatively small value of q , but the ns eos is different . the curves ( i)-a , ( i)-b , and ( ii ) schematically denote the gravitational - wave spectra for the stiff , moderately stiff , and soft eos . for ( i)-a and ( i)-b , the damping of the spectrum is determined by tidal disruption . in this case , the spectrum is characterized simply by exponential damping for f fcut . we refer to a spectrum of this type as type - i . for the case ( ii ) , on the other hand , tidal disruption does not occur , and the cutoff frequency is determined by the qnm of the remnant bh . in this case , for a frequency slightly smaller than fcut , the amplitude of the spectrum slightly increases with the frequency , that is a characteristic feature seen for the spectrum of bh - bh binaries ( e.g. , ) . figure 27schematic figure for the gravitational - wave spectrum for the same masses of bh and ns with different eos of ns for a = 0 . for ( i)-a , tidal disruption does not occur and the ns is simply swallowed by the bh . we refer to the spectra ( i)-a and ( i)-b as type - i and the spectrum ( ii ) as type - ii . for a = 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.13 \lesssim { \mathcal c } \lesssim 0.2$\end{document } , the type - i spectrum is seen only for small values of the mass ratio with q 3 . the filled and open circles denote the cutoff frequencies associated with tidal disruption and qnm , respectively . schematic figure for the gravitational - wave spectrum for the same masses of bh and ns with different eos of ns for a = 0 . for ( i)-a , tidal disruption does not occur and the ns is simply swallowed by the bh . we refer to the spectra ( i)-a and ( i)-b as type - i and the spectrum ( ii ) as type - ii . for a = 0 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0.13 \lesssim { \mathcal c } \lesssim 0.2$\end{document } , the type - i spectrum is seen only for small values of the mass ratio with q 3 . the filled and open circles denote the cutoff frequencies associated with tidal disruption and qnm , respectively . to quantitatively analyze the cutoff frequency and to strictly study its dependence on the eos , the kt group [ 194 , 107 , 109 ] fits all the spectra by a function with seven free parameters 126\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \tilde h}_{{\rm{fit}}}}(f ) = { { \tilde h}_{3{\rm{pn}}}}(f){e^{- { { ( f/{f_{{\rm{ins}}}})}^{{\sigma _ { { \rm{ins } } } } } } } } } \\ { + { { a{m_0 } } \over { df}}{e^{- { { ( f/{f_{cut}})}^{{\sigma _ { { \rm{cut}}}}}}}}[1 - { e^{- { { ( f/{f_{ins2}})}^{{\sigma _ { { \rm{ins}}2 } } } } } } ] , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde h}_{3{\rm{pn}}}}(f)$\end{document } is the fourier spectrum calculated by the taylor - t4 formula and fins , fins2 , fcut , ins , ins2 , cut , and a are free parameters . the first and second terms on the right - hand side of equation ( 126 ) denote spectrum models for the inspiral and merger phases , respectively . these free parameters are determined by searching the minimum for a weighted norm defined by 127\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sum\limits_i { { { \left\{{[{f_i}\tilde h({f_i } ) - { f_i}{{\tilde h}_{{\rm{fit}}}}({f_i})]f_i^{1/3 } } \right\}}^2},}$$\end{document } where i denotes the data point for the spectrum . among these seven free parameters , they focus on fcut because it depends most strongly on the compactness \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } and the ns eos . figure 28 plots fcutm0 , obtained in this fitting procedure , as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } for a = 0 . also the typical qnm frequencies , fqnm , of the remnant bh for q = 2 and 3 are plotted by the two horizontal lines , which show that the values of fcutm0 for compact models ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } ) with q = 3 agree approximately with fqnm and indicates that fcut for these models are irrelevant to tidal disruption . for q = 3 , fcutm0 depends on the eos only for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathcal c } \lesssim 0.16)$\end{document}. by contrast , fcutm0 for q = 2 depends strongly on ns compactness , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.16$\end{document } , irrespective of mns for a wide range of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document}. figure 28gcfcut m0 as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.19$\end{document } in logarithmic scales . the solid line is obtained by a linear fitting of the data for q = 2 and 2 = 3 . the short - dashed and long - dashed lines show approximate frequencies of the qnm of the remnant bh for q = 2 and q = 3 , respectively . gcfcut m0 as a function of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } \lesssim 0.19$\end{document } in logarithmic scales . the solid line is obtained by a linear fitting of the data for q = 2 and 2 = 3 . the short - dashed and long - dashed lines show approximate frequencies of the qnm of the remnant bh for q = 2 and q = 3 , respectively . an interesting finding in is that the following relation approximately holds for the identical value of q , 128\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\ln ( g{c^{- 3}}{f_{{\rm{cut}}}}{m_0 } ) = ( 3.87 \pm 0.12)\ln c + ( 4.03 \pm 0.22).$$\end{document } namely , fcutm0 is approximately proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal c}^{3.9}}$\end{document}. this is a note - worthy point because the power of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } is much larger than a well - known factor 1.5 , which is expected from the relation for the mass - shedding limit presented in section 1 ( cf . ( for binaries composed of spinning bh , this power is smaller than 3.9 , but it is still much larger than 1.5 . ) this difference indicates that the cutoff frequency is not determined simply by mass shedding . qualitatively , this increase in the power is natural because the duration of a ns for the survival against tidal disruption after the onset of mass shedding is in general longer for a more compact ns due to a stronger central condensation of the density profile . figure 26 illustrates that fcut is rather high , 2 khz , for a variety of eos , and thus , the dependence of fcut on the eos for a = 0 appears only for a high frequency . the reason for this is that for a = 0 , tidal disruption can occur only for a small mass ratio ( and thus for a small total mass ) with a typical ns mass of 1.31.4 m ; see equation ( 8) . the effective amplitude of gravitational waves at f = 2 khz is 2 10 for a hypothetical distance to the source of 100 mpc . the amplitude is smaller than the noise level of advanced gravitational - wave detectors , but such a signal will be detectable to next - generation detectors such as einstein telescope [ 91 , 92 ] . the gravitational - wave spectrum is qualitatively and quantitatively modified by the bh spin . in the following , we focus only on the case in which the bh spin and orbital angular momentum vectors align , because gravitational waves for the misaligned case have not yet been studied in detail . figure 29 shows the same relation as in figure 26 but for q = 3 , mns = 1.35 m and a = 0.75 , 0.5 , 0 , and 0.5 with hb eos ( cf . table 4 ; left ) and for q = 4 , a = 0.75 , and mns = 1.35 m with various eos ( right ) . the left panel of figure 29 shows that the spectrum shapes for a 0 , a = 0.5 , and a = 0.75 are qualitatively different ; for a 0 , the exponential damping above a cutoff frequency , which is determined by the fundamental qnm of the remnant bh , is seen . in this case , this is the type - ii spectrum according to the classification in figure 27 . on the other hand , for a = 0.75 , the cutoff frequency ( fcut 1.5 khz ) the spectrum for a = 0.5 is neither type - i nor type - ii . in this case , there are two typical frequencies . one is at f 2 khz , above which the spectrum amplitude sinks , and the other is at f 3 khz , above which the spectrum amplitude steeply damps . the first frequency is determined primarily by the frequency at which tidal disruption occurs , and the second one is the qnm frequency of the remnant bh . we call this new type of spectrum type - iii ( according to the definition of figure 18 ) . in the right panel of figure 30 , we summarize three types of gravitational - wave spectrum . figure 29left : the same as figure 26 but for q = 3 , mns = 1.35 m , and a = 0.75 , 0.5 , 0 , and 0.5 with a moderately stiff eos ( eos hb ) . right : the same as the left panel but for q = 4 , a = 0.75 , and mns = 1.35 m with various eos . the thin dashed curves show the noise curves for lcgt , advanced ligo , broadband - designed advanced ligo , and einstein telescope ( from upper to lower ) . figure 30left : schematic figure of the type - i gravitational - wave spectrum for a high - frequency side with different values of the bh spin but for the same masses of bh and ns , and ns eos . with increase of the bh spin , the cutoff frequency decreases and the amplitude below the cutoff frequency increases . type - i ( i ) and type - ii ( ii ) are the same as those shown in figure 27 . type - iii ( iii ) is shown for the case in which the bh spin is high and the mass ratio ( and thus the area of the bh horizon ) is large . the filled and open circles denote the cutoff frequencies associated with tidal disruption and with a qnm , respectively . left : the same as figure 26 but for q = 3 , mns = 1.35 m , and a = 0.75 , 0.5 , 0 , and 0.5 with a moderately stiff eos ( eos hb ) . right : the same as the left panel but for q = 4 , a = 0.75 , and mns = 1.35 m with various eos . the thin dashed curves show the noise curves for lcgt , advanced ligo , broadband - designed advanced ligo , and einstein telescope ( from upper to lower ) . left : schematic figure of the type - i gravitational - wave spectrum for a high - frequency side with different values of the bh spin but for the same masses of bh and ns , and ns eos . with increase of the bh spin , the cutoff frequency decreases and the amplitude below the cutoff frequency increases . type - i ( i ) and type - ii ( ii ) are the same as those shown in figure 27 . type - iii ( iii ) is shown for the case in which the bh spin is high and the mass ratio ( and thus the area of the bh horizon ) is large . the filled and open circles denote the cutoff frequencies associated with tidal disruption and with a qnm , respectively . for the type - iii spectrum , we refer to the first ( lower ) typical frequency as the cutoff frequency in the following . in this definition , with increasing bh spin , the cutoff frequency decreases and the amplitude of the gravitational - wave spectrum for f fcut increases . these two effects are preferable for gravitational - wave detection by planned advanced laser - interferometric detectors , because their sensitivity is better for smaller frequencies around f = fcut . these quantitative changes come again from the spin - orbit coupling effect ( see also sections 3.3 and 3.6 ) , as explained in the following . due to the spin - orbit coupling effect , which brings a repulsive force into bh - ns binaries ( for the prograde spin ) , the orbital velocity for a given separation is reduced , because the centrifugal force may be weaker for a given separation to maintain a quasi - circular orbit . due to the decrease of the orbital velocity ( a ) the orbital angular velocity at a given separation is decreased and ( b ) the luminosity of gravitational waves is decreased . effect ( a ) results in the decrease of the cutoff frequency at which tidal disruption occurs . effect ( b ) decelerates the orbital evolution as a result of gravitational radiation reaction , resulting in a longer lifetime of the binary system and in increase in the number of the gravitational - wave cycle . this effect increases the amplitude of the gravitational - wave spectrum of f < fcut for a > 0 . a more quantitative explanation follows . from the relation of the luminosity of gravitational waves , the power spectrum of gravitational waves is written as 129\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{de } \over { df}}\propto{[f\tilde h(f)]^2}.$$\end{document } in the 1.5 pn approximation , de / df in the inspiral phase is written as 130\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{de } \over { df } } = { { { c^5 } } \over { 3g{{(\pi f)}^2}}}{q \over { { { ( 1 + q)}^2}}}{(g{c^{- 3}}\pi { m_0}f)^{5/3}}\left [ { 1 + ( g{c^{- 3}}\pi { m_0}f){\bf{\hat s } } \cdot { \bf{\hat l}}{{20{q^2 } + 15q } \over { 3{{(1 + q)}^2 } } } } \right],$$\end{document } where we write only the terms associated with the lowest - order spin - orbit coupling term assuming that the ns spin is zero , and omit other terms . and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\bf{\hat l}}}$\end{document } are unit vectors of the bh spin and orbital angular momentum , respectively . thus , for a given frequency f , |de / df| is larger in the presence of the prograde spin ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\bf{\hat s } } \cdot { \bf{\hat l } } > 0)$\end{document } ) than for a = 0 . the binary evolves due to gravitational - wave emission , and hence , the binary with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{\hat s } } \cdot { \bf{\hat l } } > 0$\end{document } has to emit more gravitational waves than for a = 0 to increase the frequency ( to decrease the orbital separation ) . in addition , equation ( 129 ) shows that de / df is proportional to the square of the effective amplitude \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(f\tilde h(f))$\end{document}. therefore , the effective amplitude for a given frequency with > 0 should be larger than that for a = 0 . this agrees completely with the numerical results . for binaries composed of a high - spin bh , tidal disruption can occur outside the isco even for a high - mass bh for a variety of eos . thus , the dependence of fcut on the eos is clearly seen even for a high value of q. the right panel of figure 29 shows the spectrum for q = 4 , mns = 1.35 m , and a = 0.75 with four different eos . for all the eos , tidal disruption occurs , and the value of fcut depends strongly on the eos . for eos 2h and h , the spectra are type - i , but for eos hb and b ( stiff eos ) , they are type - iii . thus , for a high bh spin , a type - i or type - iii spectrum is often seen even for a high value of q. with the increase of q ( for a canonical mass of a ns 1.4 m ) , the value of fcut decreases ( cf . equation ( 7 ) ) , and the effective amplitude at f = fcut increases as the total mass increases . the right panel of figure 29 indeed illustrates that fcut is smaller than 2 khz irrespective of the eos , and also , that the effective amplitude at f 2 khz is as large as the noise curve of the advanced detector for a hypothetical distance of d = 100 mpc . for an even higher spin , say a 0.9 , tidal disruption is likely to occur for a higher bh mass with q 10 . for such a case , the amplitude of gravitational waves at tidal disruption is likely to be high enough to be observable irrespective of eos for d = 100 mpc . this indicates that a bh - ns binary with a high bh spin will be a promising experimental field for constraining the eos of high - density nuclear matter , when advanced gravitational - wave detectors are in operation . the inspiral and merger of bh - ns binaries are among promising sources for kilometer size laser - interferometric gravitational - waves detectors . the merger remnant is also a possible candidate for the progenitor of the central engine of sgrb . bh - ns binaries are also invaluable experimental fields for studying high - density nuclear matter through astronomical observations . to derive accurate gravitational waveforms in the late inspiral and merger phases , and to explore the compact - binary - merger hypothesis for the central engine of sgrb , the numerical simulation in full general relativity , taking into account realistic physics , is the unique approach . we review the progress and current status of numerical simulations for bh - ns binaries , and summarize the current understanding obtained from numerical results . the following is a summary as of june 2011 : in the final phase of bh - ns binaries , the ns is either tidally disrupted or swallowed by the companion bh . for a typical compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.14 - 0.20$\end{document } , with zero bh spin , tidal disruption occurs outside the isco only for a small mass ratio of q 3 . for the case in which tidal disruption occurs , the final remnant is a bh surrounded by a disk of relatively small mass ( say 0.1 m).the effects associated with bh spin enhance the possibilities for tidal disruption and for disk formation . even for a moderately large spin a = 0.5 , the criterion of tidal disruption for the mass ratio is relaxed to q 5 , for , e.g. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.17$\end{document}. in addition the effects of bh spin increase the mass of a remnant disk surrounding a bh to 0.10.3 m for a typical ns of mass mns = 1.35 m with a = 0.75 and q 5 . for a favorable condition , such as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } and a = 0.9 , the disk mass may be 0.5 m .for a binary composed of a high - spin bh with high mass ratio , the disk , if it is formed , has a spread structure . however , the simulations so far ( in which detailed microphysical effects such as neutrino wind are not taken into account ) have not shown any evidence that a fraction of ns material escapes from the system.the final merger process is well reflected in gravitational waveforms . up to now , it has been found that there are at least three types of gravitational waveform . ( i ) for the case in which tidal disruption occurs far outside the isco , the amplitude of gravitational waves steeply damps in the middle of the late inspiral phase , and ringdown gravitational waves associated with a qnm of the remnant bh are not seen clearly . in which tidal disruption does not occur outside the isco , ringdown gravitational waves associated with a qnm of the remnant bh are clearly seen in the final phase of the gravitational - wave signal . one is that the amplitude of the gravitational waves steeply damps and the ringdown signal of a qnm is not seen clearly . this is the case that the mass ratio q ( and thus bh mass ) is small , and the resulting type of the gravitational waveform is type - i . for a large value of q ( for a high bh mass ) , the disrupted material falls into the bh from a narrow region of the bh surface . in such cases , both the feature of steep damping associated with tidal disruption and ringdown gravitational waves associated with a qnm are seen . this type is referred to as type-iii.reflecting that there are three types of gravitational waveforms , the gravitational - wave spectrum is also classified into three types . for type - i and type - iii , the gravitational - wave spectrum is characterized by the cutoff frequency associated with tidal disruption . for a given set of masses of bh and ns , and bh spin , thus , if the cutoff frequencies are determined for a detected signal of gravitational waves , the eos of the ns will be constrained . the cutoff frequency is higher than 1 khz ( e.g. , equation ( 8) ) . the frequency is lower for a ns of larger radii or for a rapidly spinning bh with a large bh mass . in particular , for a binary composed of a high - spin bh , the effective amplitude at f = fcut is enhanced by the spin - orbit coupling effect . this effect is favorable for detecting a gravitational - wave signal at the cutoff frequency by advanced detectors . in the final phase of bh - ns binaries , the ns is either tidally disrupted or swallowed by the companion bh . for a typical compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.14 - 0.20$\end{document } , with zero bh spin , tidal disruption occurs outside the isco only for a small mass ratio of q 3 . for the case in which tidal disruption occurs , the final remnant is a bh surrounded by a disk of relatively small mass ( say 0.1 m ) . the effects associated with bh spin enhance the possibilities for tidal disruption and for disk formation . even for a moderately large spin a = 0.5 , the criterion of tidal disruption for the mass ratio is relaxed to q 5 , for , e.g. , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.17$\end{document}. in addition the effects of bh spin increase the mass of a remnant disk surrounding a bh to 0.10.3 m for a typical ns of mass mns = 1.35 m with a = 0.75 and q 5 . for a favorable condition , such as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c } = 0.145$\end{document } and a = 0.9 , the disk mass may be 0.5 m . for a binary composed of a high - spin bh with high mass ratio , the disk , however , the simulations so far ( in which detailed microphysical effects such as neutrino wind are not taken into account ) have not shown any evidence that a fraction of ns material escapes from the system . the final merger process is well reflected in gravitational waveforms . up to now , it has been found that there are at least three types of gravitational waveform . ( i ) for the case in which tidal disruption occurs far outside the isco , the amplitude of gravitational waves steeply damps in the middle of the late inspiral phase , and ringdown gravitational waves associated with a qnm of the remnant bh are not seen clearly . in which tidal disruption does not occur outside the isco , ringdown gravitational waves associated with a qnm of the remnant bh are clearly seen in the final phase of the gravitational - wave signal . one is that the amplitude of the gravitational waves steeply damps and the ringdown signal of a qnm is not seen clearly . this is the case that the mass ratio q ( and thus bh mass ) is small , and the resulting type of the gravitational waveform is type - i . for a large value of q ( for a high bh mass ) , the disrupted material falls into the bh from a narrow region of the bh surface . in such cases , both the feature of steep damping associated with tidal disruption and ringdown gravitational waves associated with a qnm are seen . this type is referred to as type - iii . reflecting that there are three types of gravitational waveforms , the gravitational - wave spectrum is also classified into three types . for type - i and type - iii , the gravitational - wave spectrum is characterized by the cutoff frequency associated with tidal disruption . for a given set of masses of bh and ns , and bh spin , thus , if the cutoff frequencies are determined for a detected signal of gravitational waves , the eos of the ns will be constrained . the cutoff frequency is higher than 1 khz ( e.g. , equation ( 8) ) . the frequency is lower for a ns of larger radii or for a rapidly spinning bh with a large bh mass . in particular , for a binary composed of a high - spin bh , the effective amplitude at f = fcut is enhanced by the spin - orbit coupling effect . this effect is favorable for detecting a gravitational - wave signal at the cutoff frequency by advanced detectors . first , more realistic modeling of ns is required because numerical studies have been performed with quite simple eos and microphysics up till now . for more realistic modeling of bh - ns binaries ( in particular for modeling formation and evolution processes of a disk surrounding a bh ) , more physical eos should be taken into account ; we have to take into account finite - temperature eos , neutrino process , and magnetic fields ( accurately evolving magnetic field configurations ) . second , there is still a wide range of the parameter space that has not been studied . in particular , binaries of high bh spin ( a > 0.9 ) is close to unity , the ns may be tidally disrupted even for a high mass ratio q 20 ; cf . for such a high - mass binary , tidal disruption occurs at a relatively - low gravitational - wave frequency 1 khz . this is favorable for observing the tidal - disruption event by gravitational - wave detectors , and thus , this deserves intense study . . indicated it feasible to perform a simulation with a high spin a 0.99 using a simple prescription ( see also ) . third , only one study has been done for the merger process of the binaries in which the bh spin and orbital angular momentum vectors misalign . in particular , any study of gravitational waveforms has not been done for this case . finally , it is necessary to optimize simulation codes to efficiently and accurately perform a large number of longterm simulations for a wide range of parameter space . this is required for preparing template sets of gravitational waves that are used for gravitational - wave data analysis . work along this line has recently begun in 2010 [ 153 , 110 ] , and in the next several years , it will be encouraged because the preparation of theoretical templates is an urgent task for advanced gravitational - wave detectors .

we review the current status of general relativistic studies for the coalescence of black hole - neutron star ( bh - ns ) binaries . first , procedures for a solution of bh - ns binaries in quasi - equilibrium circular orbits and the numerical results , such as quasi - equilibrium sequence and mass - shedding limit , of the high - precision computation , are summarized . then , the current status of numerical - relativity simulations for the merger of bh - ns binaries is described . we summarize our understanding for the merger and/or tidal disruption processes , the criterion for tidal disruption , the properties of the remnant formed after the tidal disruption , gravitational waveform , and gravitational - wave spectrum .

Introduction Quasi-Equilibrium States Numerical Simulations

black hole - neutron star ( bh - ns ) binaries are believed to be formed as a result of two supernovae in a massive binary system ( see for an alternative possibility ) . in particular , the following two facts have recently enhanced the motivation for the study of bh - ns binaries : first , bh - ns binaries in close orbits are among the most promising sources for the large laser - interferometric gravitational - wave detectors , such as ligo [ 126 , 2 , 1 ] , virgo [ 222 , 4 , 3 ] , lcgt , and einstein telescope [ 91 , 92 ] : the frequency and amplitude of gravitational waves near the last orbit are estimated to give 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f \approx { \omega \over \pi } \approx 594\,{\rm{hz}}{\left({{{6g{m_0 } } \over { { c^2}r } } } \right)^{3/2}}{\left({{{{m_0 } } \over { 7.4{m _ \odot } } } } \right)^{- 1}},$$\end{document } 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h \approx { { 4g{m_0}\mu } \over { { c^4}dr } } \approx 3.6 \times { 10^{- 22}}\left({{{{m_{bh } } } \over { 6{m _ \odot } } } } \right)\left({{{{m_{ns } } } \over { 1.4{m _ \odot } } } } \right)\left({{{6g{m_0 } } \over { { c^2}r } } } \right){\left({{d \over { 100{\rm{mpc } } } } } \right)^{- 1}},$$\end{document } where is the reduced mass of the binary defined by mbhmns / m0 , and d is the distance to the source . to elucidate whether the merger of bh - ns binaries could be a promising source for the progenitor of the central engine , the final fate of bh - ns binaries is classified into two categories ; a ns is tidally disrupted by its companion bh before it is swallowed by the bh or a ns is simply swallowed by its companion bh in the final phase . the latest general - relativistic numerical study for quasi - equilibrium states of bh - ns binaries derives a more quantitative result [ 209 , 210 ] ( see section 2 ) , 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega ^2 } \geq c_\omega ^2\left({{{g{m_{{\rm{ns } } } } } \over { r_{{\rm{ns}}}^3 } } } \right)(1 + { q^{- 1}}),$$\end{document } where the value of the constant , c , is 0.3 for a system composed of a ns of irrotational velocity field and a non - spinning bh . as a result of the tidal deformation , the gravitational force between two stars is modified , so are the orbital evolution and the criterion for tidal disruption . as shown by a simple estimate in the previous section 1.1 , the final fate of bh - ns binaries depends primarily on the mass ratio , q , and the compactness of the ns , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document}. thus , this analysis is valid only for the case in which the bh mass is much larger than the ns mass and t the radius of the ns is relatively large : for the case of bh - ns binaries of mass ratio 10 , this analysis can provide only a qualitative or semi - quantitative nature of the tidal effect and orbital evolution . general relativistic studies for quasi - equilibria of bh - ns binaries in addition show that the self - gravity of the ns significantly reduces the maximum mass of the bh for the onset of mass shedding ( see section 1.4 and figure 22 ) . the merger of bh - ns binaries ( more specifically , tidal disruption of a ns by a bh ) is physically and astrophysically an important phenomenon , and deserves a detailed study , because of at least three reasons as follows . for the merger of bh - ns binaries , the resulting disk is likely to be compact and its mass is relatively small , of order 0.1 m. since the lifetime of such a disk is likely to be short ( 1 s ) , bh - ns binaries are proposed to be the progenitor of sgrb . if the ns radius could be determined or constrained from the observation of gravitational waves emitted during tidal disruption , the resultant relation between the mass and the radius of the observed ns may be used for constraining the eos of the high - density nuclear matter [ 127 , 220 , 70 ] . for the merger of bh - ns binaries , the resulting disk is likely to be compact and its mass is relatively small , of order 0.1 m. since the lifetime of such a disk is likely to be short ( 1 s ) , bh - ns binaries are proposed to be the progenitor of sgrb . one of the important tasks is to quantitatively determine the tidal - deformation effect on the orbital evolution in the late inspiral phase and the criterion for the onset of mass shedding ( which is a necessary condition for tidal disruption ) . taniguchi and collaborators succeeded in accurately computing bh - ns binaries in quasi - equilibrium and investigated their nature in detail ; grandclment corrected his numerical code , and derived results as accurate as those of taniguchi et al . all these groups computed sequences of quasi - equilibrium states and studied the nature of bh - ns binaries in close circular orbits ( see section 2 ) . the cornell - caltech group also developed a numerical code , based on a spectral method for the precise computation of bh - ns binaries in quasi - equilibrium states , extending their original code to bh - bh binaries [ 158 , 47 , 40 ] . up to now , this group has published quasi - equilibrium sequences only for the equal - mass case , which is not very realistic for bh - ns binaries , although their results for a variety of bh - ns binaries have been used as initial conditions for numerical - relativity simulations [ 58 , 57 , 74 ] . however , these works primarily discuss a numerical method for computing bh binaries in quasi - equilibrium , and do not shown any computational results for bh - ns binaries . these works have qualitatively clarified the tidal disruption process , the subsequent formation process of a bh - disk system , properties of the remnant accretion disk , gravitational waveforms emitted during the merger , and a fraction of matter ejected from the system . the derived qualitative features in the merger of bh - ns binaries agree with those from pseudo - newtonian studies as well as by the general relativistic studies described in section 3 . one interesting question for the final phase of bh - ns binaries is whether the stable mass transfer occurs in a close orbit ; specifically , the question is whether the mass accretion from a ns to its companion bh proceeds for a time scale longer than the orbital period while escaping tidal disruption or merger . up to merger , the dynamics are primarily determined by the strong general relativistic gravity , and thus , the orbital evolution in a close binary , the merger and tidal disruption processes , the criterion of tidal disruption , and the evolution of the tidally - disrupted ns material depend strongly on general relativistic effects . numerical computation in a fully general relativistic framework is obviously required for accurately and quantitatively understanding the nature of orbital evolution and the merger of bh - ns binaries ; although non - general - relativistic works have provided a qualitative insight for these systems . soon after the first success for the simulation of bh - bh binaries , work on the merger of bh - ns binaries was published [ 202 , 203 , 197 , 62 , 58 , 63 , 194 , 57 , 107 , 108 , 41 , 74 , 154 , 109 ] . we review the current status of the study of quasi - equilibrium states of bh - ns binaries in general relativity . first , the formalisms for a solution of quasi - equilibrium states are described and then the resulting solution , its sequence , and implications are summarized . in section 3 , we review the current status of the numerical - relativity study of bh - ns binaries . in sections 3.33.7 , the numerical results for the evolution process throughout the late inspiral to the merger , tidal disruption processes , properties of the remnant formed after the merger , and features of gravitational waveforms and gravitational - wave spectra are summarized . in this section , we first review formulations to construct bh - ns binaries in quasi - equilibrium , and then the representative numerical results derived so far . for a solution of bh - ns binaries in quasi - equilibrium , it is at least necessary to solve einstein s constraint equations and relativistic hydrostationary equations , supplemented by ns eos . a wide variety of eos has been adopted for the study of quasi - equilibrium states of bh - ns binaries , which are employed as initial conditions of numerical simulations ( see section 3 ) . the several key quantities that are necessary in the quantitative analysis of quasi - equilibrium sequences are summarized in this section ; the irreducible mass and spin of bh , the baryon rest mass of ns , the adm mass , the komar mass , and the total angular momentum of the system . [ 80 , 211 , 212 ] defined a sensitive mass - shedding indicator ( in the context of ns - ns binaries ) as 99\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\chi \equiv { { { { ( \partial ( \ln \,h)/\partial r)}_{{\rm{eq } } } } } \over { { { ( \partial ( \ln \,h)/\partial r)}_{{\rm{pole}}}}}}.$$\end{document } here , the numerator of equation ( 99 ) , ( (ln h)/r)eq is the radial derivative of the enthalpy in the equatorial plane at the surface along the direction toward the companion , and the denominator , ( (lnh)/r)pole , is that at the surface of the pole . the compactness of the ns \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } as found in table 1 , the surveyed parameter space is quite narrow , in particular for the eos of the ns and the bh spin ; complete sequences with a 0 have not yet been derived , and moreover , bh - ns binaries in quasi - equilibrium in which the bh spin takes a high value > 0.9 or the bh spin and orbital angular momentum vector misalign have not been studied yet . the solid curve denotes the mass - shedding limit , and the long - dashed one the isco for each mass ratio as a function of the orbital angular velocity in polytropic units . if the mass ratio of a bh - ns binary is larger than the critical one , the quasi - equilibrium sequence terminates by encountering the isco , while if smaller , it ends at the mass - shedding limit of the ns . in this section , we have given an overview of the current status of the study of quasi - equilibrium bh - ns binary sequences in general relativity . such a critical curve clearly classifies the possible final fate of bh - ns binaries , which depends on the ratio and the compactness of the ns for a given eos of ns and bh spin . by the year 2010 , five groups aei , cccw , kt , lbpli , and uiuc groups have succeeded in fully general - relativistic simulations for bh - ns binaries and provided a variety of the numerical results . they have provided the criteria for tidal disruption , the final mass and spin of the bh left after the merger , the remnant disk mass , gravitational waveforms , and gravitational - wave spectrum . in the following sections , we review the methods of numerical relativity employed in these groups , the basic picture of the merger and tidal disruption processes clarified to date , and the resulting gravitational waveforms and spectra , separately . , a ns of larger radii ( with a stiffer eos ) will be disrupted at a larger orbital separation ( or a lower orbital frequency ; see equations in section 1 ) ; if the tidal disruption of a ns occurs at a larger distance , more material will be widely spread around its companion bh , and consequently , a high - mass remnant disk is likely to be formed ; also , the gravitational - wave frequency at tidal disruption , which will be one of the characteristic frequencies , is lower for a ns of larger radius . to summarize , the total number of simulations is still small , although a systematic survey is required to fully understand the complete picture of the coalescence of bh - ns binaries . nevertheless , the simulations performed so far have clarified a basic picture for the merger process of bh - ns binaries , the properties for the remnant , gravitational waveforms , and gravitational - wave spectrum . thus , for the merger of a rapidly - spinning bh and a ns , it may be concluded that a widely - spread disk is formed , and the lifetime of the accretion disk will be fairly long 0.1 s. the kt groups also found that the density of the disk decreases with increase of q ( or the bh mass ) ; for a high - mass bh , a widely spread but less dense disk is formed . this leads to the conclusion that the coalescence of bh - ns binaries with a high - spin bh with a 0.75 and q 4 is a promising progenitor for forming a bh plus a massive disk system ; that is the candidate for a central - engine of sgrb . the points with error bars ( and the dotted curve ) approximately denote the numerical results for tidal disruption , based on results by the kt group for q = 2 , by the cccw , kt , and uiuc groups for q = 3 , and by the aei group for q = 5 . figure 23 shows that gravitational waveforms in the late inspiral phase before the onset of the merger ( or tidal disruption ) indeed agree with the result derived by the taylor - t4 formula for the bh - ns binaries , as in the case of bh - bh binaries . general qualitative features of the gravitational - wave spectrum for bh - ns binaries composed of non - spinning bh are summarized as follows . because the finite - sized effect of the ns is not very important in this case , the gravitational - wave spectrum is similar to that of the bh - bh binary merger with the same mass ratio ( q = 5 ( case a ) , the cutoff frequency , at which the steep damping of heff sets in , is different from that for the bh - bh binary with the same mass ratio . figure 30left : schematic figure of the type - i gravitational - wave spectrum for a high - frequency side with different values of the bh spin but for the same masses of bh and ns , and ns eos . left : schematic figure of the type - i gravitational - wave spectrum for a high - frequency side with different values of the bh spin but for the same masses of bh and ns , and ns eos . due to the spin - orbit coupling effect , which brings a repulsive force into bh - ns binaries ( for the prograde spin ) , the orbital velocity for a given separation is reduced , because the centrifugal force may be weaker for a given separation to maintain a quasi - circular orbit . we review the progress and current status of numerical simulations for bh - ns binaries , and summarize the current understanding obtained from numerical results .

currently , one of the biggest worries of our society is the future of the climate . common belief supported by an impressive 97% consensus is that our planet is heating up at an accelerated rate , caused by the rapid increase in carbon dioxide ( co2 ) concentration in the atmosphere , henceforth called [ co2 ] . this increased carbon dioxide finds its origin in human activity ; humans burn fossil fuels , thereby injecting large quantities of carbon into the troposphere by converting it into co2 . the co2 contributes to the greenhouse effect of our atmosphere and it is believed that the anthropogenic co2 will heat up the planet by up to six degrees during this century ( page 45 of ipcc 2007 report ) . here for that , while the subject is the atmosphere , we do not have to go into much detail of atmospheric science . there are observations one can make about climate systems , even without going into technical details . the model of anthropogenic global warming ( agw ) stands or falls with this idea that temperature t is strongly correlated to [ co2 ] by the so - called greenhouse effect . even though it is highly questionable to summarize the entire climate system in these two scalar properties both [ co2 ] and t show fractal - like variations in space and time this is often done . the contribution of co2 to the greenhouse effect can easily be estimated to be about 3.6% . the total greenhouse effect is also well known ; without our atmosphere our planet would be 32 degrees colder , as can be calculated on basis of a radiation balance and albedo of our planet . we arrive at a similar value if we use statistics and do a linear regression on contemporary [ co2 ] and temperature data , the maximum of the effect we can thus expect in a linear model when doubling the concentration artificially by burning up fossil fuels . this is below the global warming models even if we were to use a linear model . that is because the greenhouse effect is governed by absorption of light which is expected to follow the beer - lambert law : the absorption is highly sublinear ; twice as much co2 will not cause twice as much absorption . the classical arrhenius ' greenhouse law actually states that the forcing is logarithmic . yet , later models incorporating nonlinear positive - feedback effects as proposed by many climate scientists do predict a super - linear behavior and come up with an estimate of between 1.1 and 6.4 degrees heating for the next century as caused by our carbon dioxide injection into the atmosphere . the positive feedback can come from secondary effects such as an increase in water in the atmosphere , a strong greenhouse agent , or a co2-degassing of ground in the permafrost regions when these thaw . climate scientists are basing these conclusions mainly on research of the so - called finite - elements type , dividing the system in cells that interact , the same way the weather is studied . such systems are complicated , but by tuning the processes and parameters that are part of the simulations they manage to explain the actual climate data to an impressive accuracy , as evidenced by the quality of pictures presented in the official climate reports ; see , for example , the ipcc 2007 report where simulation and reality are as good as indistinguishable , and , moreover , alarmingly , they conclude that the recent rise in temperature can only be attributed to co2 . yet , the problem with such simulations is that even if they do simulate the past , no insight is gained into the system unless the simulations themselves are analyzed ; that is , we have just deferred the problem . this kind of simulations , if not properly analyzed and interpreted , could be in fact a devastating tool for the progress of understanding . from a philosophical point of view , the fact that the past was explained very accurately does not guarantee the same quality for the prediction of the future . small deviations in parameters and initial conditions or assumptions made in the simulations can cause huge changes in the outcome . if we have a chaotic circuit with , for instance , critical feedback , we can go to our spice or cadence simulator and find the parameters of our components that exactly explain the behavior of our circuit , so far so good . the problem is that if we now go back and switch on the same circuit , we will get a different result . ( just take an operational amplifier with 100% positive feedback , in the saturated state , the output voltage may reach the positive supply voltage as easily as the negative supply voltage , either case can be simulated . ) an additional problem is that even the parameters themselves are not constant and seem to change without any apparent reason , for instance , the el nio phenomena in the climate . this is one of the reasons electronic engineers talk about phase margins , the zone in nyquist plots , real versus imaginary parts of gain , that should be avoided because the circuit will become unpredictable even if it may be simulated perfectly . in fact , recent temperature data fall way out of the prediction margins of earlier models . in view of the discussion above , this does not come as a surprise . where extrapolation from the 2007 ipcc report predicted 2011 to be a year with an anomaly of close to one degree ( 0.95c is our personal estimate based on figure 2.5 of the ipcc 2007 report ) , in reality the anomaly is closer to zero . since 1998 , the hottest year in recent history , the planet has actually been cooling , something that was not foreseen by the predictions of 2007 where a continuing exponential increase in temperature was forecasted by the then generally accepted model . the scientific community is now going back to its drawing boards and fine - tunes its models to new perfection and manages to simulate the new data as well . the correctness of this statement is evidenced by the fact that there now apparently exist many models that explain the data up to a certain point in time ; every correction of the model that is still consistent with earlier data proves this . apparently , there is a manifold of models that can explain certain data quite satisfactorily ( but that diverge for future predictions ) . in view of this , one should be reluctant in making strong claims about the correctness of any model . just like in the weather , where the same simulation - evaluation techniques are used , we can only hope to get the predictions reasonably under control after thousands of iterations between predictions and reality . before we get it right , it will take at least some hundreds of centuries if we uniquely use the approach of finite - elements calculations on supercomputers . in the meantime , we should not see any climate models as proven indisputable facts . a skeptic approach to any scientific model is not an illness ; it is an essential ingredient in science . theories are correct until proven wrong . ideas that stand up to scrutiny are more likely to be correct than ideas one is supposed not to question . still , undeniably , a strong correlation is found between the co2 concentrations and the temperatures as measured by gas - analysis in drillings in ice shelves ; see , for example , the data of the pangaea project suggesting that one is the function of the other for the past hundreds of thousands of years . however , proving only statistical correlation , it is not clear from these data which one comes first . generally , correlation does not mean causation : in particular , in our case , are temperature variations the result of [ co2 ] variations , or vice versa ? while the data are consistent with the model of agw , they can not serve as proof of these models . in fact , upon closer scrutiny , the temperature always seems to be ahead of co2 variations . see figure 1 , where a detail of the temperature and [ co2 ] history as measured by ice - trapped gases is plotted , picturing the most blatant example of this effect . a simulation ( dashed line ) is also shown with an exponential - decay convolution of 15 kyr , quite adequately reproducing the results . indermhle and coworkers made a full statistical analysis and find a value of 900 yr for the delay and moreover note that this value is roughly in agreement with findings by fischer et al . who reported a time lag of co2 to the vostok temperature of ( 600 400 ) yr during early deglacial changes in the last 3 transitions glacial - interglacial . this is inexplicable in the framework of global warming models and we honestly start having some legitimate doubts . the apparent time lag may possibly be due to a calibration problem of the measurements , and indeed corrections have been made to the data since then , to make [ co2 ] variations and temperature variations coincide . while these corrections are the result of circular reasoning where the magnitude is found by modeling the behavior of ice based on climate models and the climate models are based on the ice behavior these corrections are not even sufficient to remove our doubts . if the correlations are true and we continue to claim that temperature variations are the result of [ co2 ] variations , something is still not correct . the vostok data of figure 1 show a sensitivity of 10 degrees for 50 ppm [ co2 ] . note that contemporary [ co2 ] variations are of the order of 80 ppm rise from the preindustrial value . we are thus in for a 16-degree temperature - rise . the fact that we did not reach that level means that either co2 is not climate forcing , or that there is a delay between [ co2 ] variations ( cause ) and temperature variations ( effect ) . to then get a rough idea of the magnitude of this delay , in 25 years , only 2.5% ( 0.4 of 16 degrees ) of this rise occurred . the relaxation time is thus ( 25 years)/ln(0.975 ) , which is about 1000 years . in other words , either the vostok plots should show a delay between [ co2 ] and t of the order of 1000 years , or the carbon dioxide is not climate forcing . the data , however , show a delay of 900 years or zero , the latter value resulting from questionable corrections . as far as we know , no correction was proposed to result in the + 1000 yr delay necessary to explain contemporary behavior . what is more , modern correlation figures such as those given in figure 1 also include methane ch4 ( available at noaa paleoclimatology ) and , remarkably , this methane shows the same correlation with [ co2 ] and t. this leaves us flabbergasted . we know that methane is also ( assumed to be ) a strong climate - forcing greenhouse agent . the enigma is , then , how did the information from the [ co2 ] variations percolate to [ ch4 ] variations ? was this information from [ co2 ] transmitted to the methane through the temperature variations ? in other words , [ ch4 ] variations are the result of t variations , rather than their cause ? then we may equally assume that [ co2 ] variations are the effect of t variations rather than their cause . there are several mechanisms that may explain such an inverse phase relation , such as outgassing of co2 ( and ch4 ) from the warming oceans and thawing permafrost ; the correlation between [ co2 ] and [ ch4 ] then stems from a common underlying cause . if that is the case , artificially changing the co2 in the atmosphere will not change the temperature of our planet , just like heating up a can of soda will liberate the gases contained therein into the atmosphere , while increasing the concentrations of gases above the can of soda will not raise its temperature . this unidirectional relation between temperature and gas concentrations is what is called henry 's law ; the ratio of concentrations of gas dissolved in the liquid and mixed in the air above it in equilibrium is a parameter that depends on temperature . al - anezi and coworkers have studied this effect in more detail in a laboratory setup under various conditions of salinity and pressure , and so forth . for co2 in and above water an increase in temperature will cause outgassing with a proportionality that is consistent with the correlation found by the historic correlations of global temperature and co2 in the atmosphere . also , fischer and coworkers find the delay of [ co2 ] relative to t , as discussed above , likely caused by this ocean outgassing effects and find that at colder times , the delay is longer , which is itself consistent with arrhenius - like behavior of thermally - activated processes , characteristic of the vast majority of chemical reactions occurring in nature . in the presence of an alternative explanation , there is room for doubt in the agw ideas that increased [ co2 ] will cause an increased temperature . inspired by this uncertainty in the ( anthropogenic ) global warming model , we tried to see if we can find more evidence for this failure of the cause - and - effect idea . we looked at the recent historic climate data ( from just before the agw model prevalence ) and meticulously measured [ co2 ] data and came to the same conclusion , as we will present here . we started with the data of a climate report from before the global warming claims . we deem these data more reliable since they were for sure not produced under the tutelage of a political committee ( ipcc ) . at least we are more convinced about the neutrality of the scientists reporting these data . moreover , the work contains all the useful data and these are even available online . the ideas presented here do not need recent data and thus we refrained from looking at them altogether . the authors of the work , balling and coworkers , analyzed the global warming ( without capitals because it is not the name of a model ) and concluded the following : our analysis reveal a statistically significant warming of approximately 0.5c over the period 1751 to 1995 . the period of most rapid warming in europe occurred between 1890 and 1950 , no warming was observed in the most recent half century . note that at the onset of the global warming ideas , no warming was observed that can be correlated to the ( accelerated ) increase of [ co2 ] . note also that since 1998 it has not warmed up at all , as confirmed by satellite data ( 1998 was the warmest year ) , in spite of the continuing exponential increase in atmospheric co2 . coworkers then went on to analyze the increase in temperature as a function of the time of the year for the data between 1851 and 1991 . the first thing we note is that , while there has been an average of warming , this is not spread equally over the year . in fact , summer months have become cooler . without knowing the underlying reason , this is remarkable , since [ co2 ] there are seasonal fluctuations of the co2 concentrations ; see the black dots which represent the monthly [ co2 ] fluctuations relative to the yearly average at the mauna loa site ( source : noaa , visited 2008 ) . these rapid fluctuations are mainly attributed to biological activity ( the northern hemisphere has more land and in colder times in winter more plants are converted into co2 and in warmer times in summer more photosynthesis takes place converting co2 into biomass , i.e. , [ co2 ] is a natural function of temperature ) . part of the fluctuations , however , is attributed to human activity ( in winter the northern hemisphere where more people live is cold and humans thus burn more fuel to warm their houses , i.e. , [ co2 ] is a function of temperature ) . as a side note , these two things show us that it is very straightforward to understand how [ co2 ] can be a function of temperature , in these cases through biological activity , including that of humans , in this case resulting in a rapid inverse proportionality ( warmer less co2 ) . other long - term processes such as degassing of oceans can have opposite effects , that is , warmer more co2 . while we bear this in mind , we will continue the reasoning of anthropogenic global warming and assume an opposite correlation , that is , temperature is a function of [ co2 ] , and analyze the oscillations . while the natural oscillations have always existed and thus do not result in seasonal oscillations of global warming , the human - caused fluctuations should be represented in the temperature fluctuations . what we would expect in the framework of agw is that all months have warmed up ( because of general injection of anthropogenic co2 into the atmosphere ) , but winter months a little bit more ( because of seasonal fluctuations of these injections ) . as a response to the sinusoidal [ co2 ] oscillations , a sinusoidal oscillation in temperature is to be expected that is ( i ) offset vertically by an amount to make it fully above the zero line ; ( ii ) offset ( delayed in time ) by a time that can be up to 3 months maximum , as will be discussed here . neither is the case . comparing the monthly fluctuations in temperature increase with monthly fluctuations in [ co2 ] we see again that the latter lags behind , this time by about 3 months ( to be precise , fitting sine curves to the data gives a difference of 2.9 months ) . after all , months are periodic but upon second thought , this is not possible . electronic engineers model things with electronic circuits and this case of temperature and co2 is also very adequately studied by such circuits . using an equivalent electronic circuit does not mean that the processes are electronic , but that they can be modeled by such circuits , as in an analog computer . ( the appendix gives the mathematical link between a relaxation model and the equivalent electronic circuit . ) in this case we have a model between driving force ( either [ co2 ] , as we are wo nt to believe , or temperature t ) and the response ( resp . , t or [ co2 ] ) . for instance , an increase in [ co2 ] will cause an increase in t by the greenhouse effect . this is necessarily a simple relaxation system , where the changes of the force cause the system to be off - equilibrium until a new equilibrium is reached . for instance , co2 has to diffuse to places where it can do its temperature effect . there can even be more than a single relaxation process , with a complex kinetics similar to a multistage nuclear decay . the fact is that one of the relaxation times is dominant , and we can describe the relaxation by a single relaxation time ( i.e. , the sum of all relaxation times ) . as long as there is no resonance in the system ( something that can only be achieved with positive feedback ) it will behave as described here . we will model our climate system with a simple electronic relaxation system consisting of a resistance and a capacitance , r and c , respectively ; see figure 3 . the product of the two yields the relaxation time , = rc . at the entrance of this system we connect our oscillating driving voltage vi(t ) ( representing , e.g. , [ co2 ] oscillations ) , in which t is time . the response is measured as the charge q(t ) in the capacitor which represents , for instance , the temperature variations . this charge is also measured by the output voltage by the standard capacitor relation v = q / c . thus our output voltage vo(t ) represents the response ( e.g. , temperature ) . applying a sinusoidal input signal , vi(t ) sin(2ft) [ co2 ] ( t ) ( with f the frequency of oscillation ) , we get a sinusoidal wave at the output , with the same frequency , but with a phase at the output that is not equal to the phase at the input signal , vo(t ) sin(2ft + ) . the phase difference is directly and uniquely determined by the relaxation time of the system and the oscillation frequency f ; see figure 3 . for very low oscillating frequencies , the system can easily relax and the phase of the output signal is equal to that of the input signal . for increased frequencies or for the maximum phase difference for infinite frequencies or infinite relaxation time is exactly one - quarter period . in our case one - quarter period is thus 3 months and that is the maximum delay we can expect between driving force and response . for relaxation times much longer than the oscillating period of one year , that is actually the delay one expects . , the comparable system of solar radiation and temperature comparable in that the oscillating period is one year and both deal with the weather and climate has a delay of one month ; the solar radiation and temperature oscillate with one year period , but the warmest day is nearly everywhere one month after the day with the most daylight and the on average coldest day is one month after the day with least daylight . in figure 3(c ) we see that the relaxation time of the { radiation temperature } system therefore must be about 0.1 year ( 1.2 months ) . in the plot we can get a similar estimation value of the relaxation time of the atmosphere temperature through daily oscillations . as a rough figure , the temperature drops by about 4 degrees at night in about 8 hours after the sun has set . assume that the relaxation upon this step - like solar radiation is a simple exponential ( situation b shown in the appendix ) and would finish eventually at close to absolute zero ( say 10 kelvin ) and starts at 290 k ( thus a total amplitude of 280 k ) : 4 degrees in 8 hours , we solve the equation ( 1)(280 k)exp(8 hours)=(2804)k , which yields 23 days , close to the value found above from yearly oscillations . going back to the data of [ co2 ] and temperature ( figure 2 ) we can now understand the behavior , that is , the phase difference , but only if we assume the temperature to be the driving force . for instance , for some reason the temperature has increased more in winter months , and , as a result , to the natural [ co2 ] oscillations has been added a component with a maximum in spring months . the alternative , [ co2 ] being the driving force and a delay of 9 months ( 3 quarter periods ) , is mathematically not possible . another explanation , which we do not consider as a valid alternative , is that the temperature might be lagging behind [ co2 ] if it has a negative gain , that is , [ co2 ] increments lower the temperature . this negative sign of the gain would add another 180 phase shift and a total apparent phase shift of 270 would be possible . this goes even more against agw models , although we do not see an easy physical explanation for how co2 might lower the temperature . this simple analysis opposes the hypothesis that [ co2 ] is causing serious temperature rises . as said , the model assumes that no resonance occurs that can possibly cause longer delay times . this , in our opinion , is a valid assumption since resonance is not likely . first of all , for this strong positive feedback , resonance effects should be observable , which experimentally is not the case . although many climate scientists have proposed positive feedbacks as discussed in the introduction and they make heavy use of them in order to explain and model the needed nonlinear behavior of the greenhouse effect , this goes against intuition . in a chaotic system scientists of any plumage , when making such simulations , know this : if they change their parameters just slightly ( sometimes even in the scale of the numerical resolution of their floating point numbers ) , the outcome can be hugely different . there is also an experimental argument against positive feedback factors , namely , the conscientious satellite measurements ; see , for instance , the work of lindzen and choi , spencer , or wielicki et al . . these , in fact , prove a negative feedback in the climate system . without feedback , in standard theory , if earth warms up ( by global warming in a radiation imbalance ) , the temperature rises and the outward earth radiation increases by a certain amount , until establishing a new equilibrium . in the agw model , a positive feedback of the the following form is used : if the temperature increases , the outward earth radiation is less than that predicted by standard theory or the incoming solar radiation increases because of reasons like cloud ( non)forming , thus increasing the temperature even further . the contrary can also happen : in negative feedback , if the planet heats up by a radiation imbalance for whatever reason , new channels of earth radiation can be opened or incoming solar radiation blocked ( for instance , by increased cloud cover ) , thus reducing the temperature with respect to standard theory . as demonstrated by the scientists mentioned above , the earth climate is a negative - feedback autostabilizing system , while they do not identify any specific feedback mechanisms . this is also in agreement with the fact that , whereas the conditions on our planet have significantly changed over the geological history ( the sun for instance has been 25% less bright than today ) , the climate has been rather stable , always restoring from climate perturbations to median values instead of saturating in extreme values ; the latter would be expected in a thermal - runaway positive - feedback climate system . note that , if large positive feedback exists , the temperature is unstable and will change until it saturates , that is , until negative feedback becomes important . in other words , it is technically not even possible that we are in a positive - feedback situation , considering the stable temperatures . ( compare this to the positive - feedback of a shopaholic buying always makes him buy even more his funds are acceleratingly depleted or his credit increasingly rising , until the banks put a lid on his spending , i.e. , negative feedback . ) we must be in a negative - feedback situation and lindzen and choi , spencer , and wielicki et al . have proven this by measurements . negative feedback was already argued to be significant when the consensus of the scientists was for a global cooling ; see the work of idso . additional arguments against positive feedback come from the fact that every day , and every year , the temperature is brought off equilibrium . at night these temperature disturbances are much larger and much faster than those that may have been produced by greenhouse gases ( 20 degrees / day or 30 degrees / year versus 0.7 degrees/100 years ) . the same accounts for co2 disturbances . the human - caused co2 seems insignificant compared to the large and noisy emissions naturally occurring on this planet ( only the accumulated effect of the tiny human - originated co2 is supposed to have an effect ) . to give an idea , segalstad established that of the current rise in [ co2 ] levels relative to the preindustrial level only 12 ppm is attributable to human activity while 68 ppm is attributed to natural phenomena . these fluctuations are also visible in the extensive summary of beck and show that even in recent history the [ co2 ] levels were sometimes higher than the modern values , while as everyone knows , the human emissions have monotonously increased , showing that these huge fluctuations can only have a natural origin . relevant for the discussion here , the fluctuations would rapidly push the climate off equilibrium if it were unstable . yet , in spite of these huge disturbances , both in temperature and co2 , the equilibrium is restored every day and every year and every century . had the earth climate been a positive - feedback system , in summer or in winter the temperature would have been in a runaway situation , unrecoverable in the following compensating half - period . the reason is that the climate is a negative - feedback system that stabilizes itself . one might think that the seasonal fluctuations are too fast to be causing a runaway scenario and that before the system runs away it already recovers . if starting oscillations are much faster than the response time of the system , the effective amplitude is reduced , but in a runaway system they will be amplified up to the point of saturation . the system can only be stable if the feedback factor at that specific frequency is not positive . look at it like this : in the first half of the year , it is hot and the system tries to runaway . in the second half of the year it is colder and it will restore , but it has a minute memory that the temperature has already run off a little in the first half and the second half therefore does not compensate completely . in the first year chance will determine which one , but if the system is unstable ( positive feedback ) , the system will run away . like the metastable system of a ball placed on top of a hill . it can only stay there in the absence of noise or any fluctuation in general . in conclusion relevant to the current work , such negative feedback will make any delay longer than 1/4 period impossible . thus , the fact that we find a delay close to a quarter period means that ( i ) the temperature signal is the origin for [ co2 ] signal ( or the two are uncorrelated ) and ( ii ) the relaxation time linking the two is ( much ) longer than the period ( 12 months ) of oscillation . moreover , even if positive feedback were present , for the resonance itself to be significant , the oscillating frequency needs to be close to the resonance frequency , that is , 12 months . it is highly unlikely that the natural frequency of the climate-[co2 ] system is close to the 12-month - periodic driving force , even more so since also the long - term ice - drilling data need to be explained somehow , where delays of several thousands of years are observed . in our analysis , relaxation times of several thousands of years will explain both the ice - drilling data and the yearly temperature and [ co2 ] oscillations . finally , the set of data we used is rather limited . future research should tell if the ideas presented here can stand up to scrutiny when more recent data and pan - global data are used . as a note in proof , humlum et al . have recently investigated correlation between temperature and [ co2 ] variations on the time scale of decades , similarly concluding that [ co2 ] changes are delayed in relation to temperature and can therefore not be the reason for temperature changes . in conclusion , the idea tested here that [ co2 ] is the cause of temperature changes does not pass our signal analysis . it goes a little too far to say that what we present here is proof for the opposite , namely , that [ co2 ] is the effect of temperature , but our analysis does not contradict this .

the primary ingredient of the anthropogenic global warming hypothesis , namely , the assumption that additional atmospheric carbon dioxide substantially raises the global temperature , is studied . this is done by looking at the data of temperature and co2 , both in the time domain and in the phase domain of periodic data . bicentenary measurements are analyzed and a relaxation model is introduced in the form of an electronic equivalent circuit . the effects of this relaxation manifest themselves in delays in the time domain and correlated phase shifts in the phase domain . for extremely long relaxation time constants , the delay is maximally one - quarter period , which for the yearly - periodic signal means 3 months . this is not in line with the analyzed data , the latter showing delays of 9 ( 3 ) months . these results indicate a reverse function of cause and effect , with temperature being the cause for atmospheric co2 changes , rather than their effect . these two hypotheses are discussed on basis of literature , where it was also reported that co2 variations are lagging behind temperature variations .

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

common belief supported by an impressive 97% consensus is that our planet is heating up at an accelerated rate , caused by the rapid increase in carbon dioxide ( co2 ) concentration in the atmosphere , henceforth called [ co2 ] . the co2 contributes to the greenhouse effect of our atmosphere and it is believed that the anthropogenic co2 will heat up the planet by up to six degrees during this century ( page 45 of ipcc 2007 report ) . the model of anthropogenic global warming ( agw ) stands or falls with this idea that temperature t is strongly correlated to [ co2 ] by the so - called greenhouse effect . even though it is highly questionable to summarize the entire climate system in these two scalar properties both [ co2 ] and t show fractal - like variations in space and time this is often done . the total greenhouse effect is also well known ; without our atmosphere our planet would be 32 degrees colder , as can be calculated on basis of a radiation balance and albedo of our planet . we arrive at a similar value if we use statistics and do a linear regression on contemporary [ co2 ] and temperature data , the maximum of the effect we can thus expect in a linear model when doubling the concentration artificially by burning up fossil fuels . this is below the global warming models even if we were to use a linear model . yet , later models incorporating nonlinear positive - feedback effects as proposed by many climate scientists do predict a super - linear behavior and come up with an estimate of between 1.1 and 6.4 degrees heating for the next century as caused by our carbon dioxide injection into the atmosphere . climate scientists are basing these conclusions mainly on research of the so - called finite - elements type , dividing the system in cells that interact , the same way the weather is studied . such systems are complicated , but by tuning the processes and parameters that are part of the simulations they manage to explain the actual climate data to an impressive accuracy , as evidenced by the quality of pictures presented in the official climate reports ; see , for example , the ipcc 2007 report where simulation and reality are as good as indistinguishable , and , moreover , alarmingly , they conclude that the recent rise in temperature can only be attributed to co2 . yet , the problem with such simulations is that even if they do simulate the past , no insight is gained into the system unless the simulations themselves are analyzed ; that is , we have just deferred the problem . this kind of simulations , if not properly analyzed and interpreted , could be in fact a devastating tool for the progress of understanding . from a philosophical point of view , the fact that the past was explained very accurately does not guarantee the same quality for the prediction of the future . small deviations in parameters and initial conditions or assumptions made in the simulations can cause huge changes in the outcome . ( just take an operational amplifier with 100% positive feedback , in the saturated state , the output voltage may reach the positive supply voltage as easily as the negative supply voltage , either case can be simulated . ) an additional problem is that even the parameters themselves are not constant and seem to change without any apparent reason , for instance , the el nio phenomena in the climate . this is one of the reasons electronic engineers talk about phase margins , the zone in nyquist plots , real versus imaginary parts of gain , that should be avoided because the circuit will become unpredictable even if it may be simulated perfectly . in fact , recent temperature data fall way out of the prediction margins of earlier models . the correctness of this statement is evidenced by the fact that there now apparently exist many models that explain the data up to a certain point in time ; every correction of the model that is still consistent with earlier data proves this . just like in the weather , where the same simulation - evaluation techniques are used , we can only hope to get the predictions reasonably under control after thousands of iterations between predictions and reality . a skeptic approach to any scientific model is not an illness ; it is an essential ingredient in science . still , undeniably , a strong correlation is found between the co2 concentrations and the temperatures as measured by gas - analysis in drillings in ice shelves ; see , for example , the data of the pangaea project suggesting that one is the function of the other for the past hundreds of thousands of years . generally , correlation does not mean causation : in particular , in our case , are temperature variations the result of [ co2 ] variations , or vice versa ? while the data are consistent with the model of agw , they can not serve as proof of these models . in fact , upon closer scrutiny , the temperature always seems to be ahead of co2 variations . see figure 1 , where a detail of the temperature and [ co2 ] history as measured by ice - trapped gases is plotted , picturing the most blatant example of this effect . indermhle and coworkers made a full statistical analysis and find a value of 900 yr for the delay and moreover note that this value is roughly in agreement with findings by fischer et al . this is inexplicable in the framework of global warming models and we honestly start having some legitimate doubts . the apparent time lag may possibly be due to a calibration problem of the measurements , and indeed corrections have been made to the data since then , to make [ co2 ] variations and temperature variations coincide . if the correlations are true and we continue to claim that temperature variations are the result of [ co2 ] variations , something is still not correct . the vostok data of figure 1 show a sensitivity of 10 degrees for 50 ppm [ co2 ] . note that contemporary [ co2 ] variations are of the order of 80 ppm rise from the preindustrial value . the fact that we did not reach that level means that either co2 is not climate forcing , or that there is a delay between [ co2 ] variations ( cause ) and temperature variations ( effect ) . to then get a rough idea of the magnitude of this delay , in 25 years , only 2.5% ( 0.4 of 16 degrees ) of this rise occurred . the relaxation time is thus ( 25 years)/ln(0.975 ) , which is about 1000 years . in other words , either the vostok plots should show a delay between [ co2 ] and t of the order of 1000 years , or the carbon dioxide is not climate forcing . the data , however , show a delay of 900 years or zero , the latter value resulting from questionable corrections . in other words , [ ch4 ] variations are the result of t variations , rather than their cause ? then we may equally assume that [ co2 ] variations are the effect of t variations rather than their cause . if that is the case , artificially changing the co2 in the atmosphere will not change the temperature of our planet , just like heating up a can of soda will liberate the gases contained therein into the atmosphere , while increasing the concentrations of gases above the can of soda will not raise its temperature . this unidirectional relation between temperature and gas concentrations is what is called henry 's law ; the ratio of concentrations of gas dissolved in the liquid and mixed in the air above it in equilibrium is a parameter that depends on temperature . al - anezi and coworkers have studied this effect in more detail in a laboratory setup under various conditions of salinity and pressure , and so forth . for co2 in and above water an increase in temperature will cause outgassing with a proportionality that is consistent with the correlation found by the historic correlations of global temperature and co2 in the atmosphere . also , fischer and coworkers find the delay of [ co2 ] relative to t , as discussed above , likely caused by this ocean outgassing effects and find that at colder times , the delay is longer , which is itself consistent with arrhenius - like behavior of thermally - activated processes , characteristic of the vast majority of chemical reactions occurring in nature . in the presence of an alternative explanation , there is room for doubt in the agw ideas that increased [ co2 ] will cause an increased temperature . inspired by this uncertainty in the ( anthropogenic ) global warming model , we tried to see if we can find more evidence for this failure of the cause - and - effect idea . we looked at the recent historic climate data ( from just before the agw model prevalence ) and meticulously measured [ co2 ] data and came to the same conclusion , as we will present here . we started with the data of a climate report from before the global warming claims . the ideas presented here do not need recent data and thus we refrained from looking at them altogether . the authors of the work , balling and coworkers , analyzed the global warming ( without capitals because it is not the name of a model ) and concluded the following : our analysis reveal a statistically significant warming of approximately 0.5c over the period 1751 to 1995 . the period of most rapid warming in europe occurred between 1890 and 1950 , no warming was observed in the most recent half century . note that at the onset of the global warming ideas , no warming was observed that can be correlated to the ( accelerated ) increase of [ co2 ] . note also that since 1998 it has not warmed up at all , as confirmed by satellite data ( 1998 was the warmest year ) , in spite of the continuing exponential increase in atmospheric co2 . coworkers then went on to analyze the increase in temperature as a function of the time of the year for the data between 1851 and 1991 . the first thing we note is that , while there has been an average of warming , this is not spread equally over the year . without knowing the underlying reason , this is remarkable , since [ co2 ] there are seasonal fluctuations of the co2 concentrations ; see the black dots which represent the monthly [ co2 ] fluctuations relative to the yearly average at the mauna loa site ( source : noaa , visited 2008 ) . , [ co2 ] is a natural function of temperature ) . part of the fluctuations , however , is attributed to human activity ( in winter the northern hemisphere where more people live is cold and humans thus burn more fuel to warm their houses , i.e. , [ co2 ] is a function of temperature ) . as a side note , these two things show us that it is very straightforward to understand how [ co2 ] can be a function of temperature , in these cases through biological activity , including that of humans , in this case resulting in a rapid inverse proportionality ( warmer less co2 ) . while we bear this in mind , we will continue the reasoning of anthropogenic global warming and assume an opposite correlation , that is , temperature is a function of [ co2 ] , and analyze the oscillations . while the natural oscillations have always existed and thus do not result in seasonal oscillations of global warming , the human - caused fluctuations should be represented in the temperature fluctuations . as a response to the sinusoidal [ co2 ] oscillations , a sinusoidal oscillation in temperature is to be expected that is ( i ) offset vertically by an amount to make it fully above the zero line ; ( ii ) offset ( delayed in time ) by a time that can be up to 3 months maximum , as will be discussed here . comparing the monthly fluctuations in temperature increase with monthly fluctuations in [ co2 ] we see again that the latter lags behind , this time by about 3 months ( to be precise , fitting sine curves to the data gives a difference of 2.9 months ) . after all , months are periodic but upon second thought , this is not possible . electronic engineers model things with electronic circuits and this case of temperature and co2 is also very adequately studied by such circuits . ( the appendix gives the mathematical link between a relaxation model and the equivalent electronic circuit . ) this is necessarily a simple relaxation system , where the changes of the force cause the system to be off - equilibrium until a new equilibrium is reached . there can even be more than a single relaxation process , with a complex kinetics similar to a multistage nuclear decay . the fact is that one of the relaxation times is dominant , and we can describe the relaxation by a single relaxation time ( i.e. the product of the two yields the relaxation time , = rc . at the entrance of this system we connect our oscillating driving voltage vi(t ) ( representing , e.g. the response is measured as the charge q(t ) in the capacitor which represents , for instance , the temperature variations . applying a sinusoidal input signal , vi(t ) sin(2ft) [ co2 ] ( t ) ( with f the frequency of oscillation ) , we get a sinusoidal wave at the output , with the same frequency , but with a phase at the output that is not equal to the phase at the input signal , vo(t ) sin(2ft + ) . the phase difference is directly and uniquely determined by the relaxation time of the system and the oscillation frequency f ; see figure 3 . for very low oscillating frequencies , the system can easily relax and the phase of the output signal is equal to that of the input signal . for increased frequencies or for the maximum phase difference for infinite frequencies or infinite relaxation time is exactly one - quarter period . in our case one - quarter period is thus 3 months and that is the maximum delay we can expect between driving force and response . , the comparable system of solar radiation and temperature comparable in that the oscillating period is one year and both deal with the weather and climate has a delay of one month ; the solar radiation and temperature oscillate with one year period , but the warmest day is nearly everywhere one month after the day with the most daylight and the on average coldest day is one month after the day with least daylight . in figure 3(c ) we see that the relaxation time of the { radiation temperature } system therefore must be about 0.1 year ( 1.2 months ) . in the plot we can get a similar estimation value of the relaxation time of the atmosphere temperature through daily oscillations . assume that the relaxation upon this step - like solar radiation is a simple exponential ( situation b shown in the appendix ) and would finish eventually at close to absolute zero ( say 10 kelvin ) and starts at 290 k ( thus a total amplitude of 280 k ) : 4 degrees in 8 hours , we solve the equation ( 1)(280 k)exp(8 hours)=(2804)k , which yields 23 days , close to the value found above from yearly oscillations . going back to the data of [ co2 ] and temperature ( figure 2 ) we can now understand the behavior , that is , the phase difference , but only if we assume the temperature to be the driving force . the alternative , [ co2 ] being the driving force and a delay of 9 months ( 3 quarter periods ) , is mathematically not possible . another explanation , which we do not consider as a valid alternative , is that the temperature might be lagging behind [ co2 ] if it has a negative gain , that is , [ co2 ] increments lower the temperature . this negative sign of the gain would add another 180 phase shift and a total apparent phase shift of 270 would be possible . this , in our opinion , is a valid assumption since resonance is not likely . first of all , for this strong positive feedback , resonance effects should be observable , which experimentally is not the case . although many climate scientists have proposed positive feedbacks as discussed in the introduction and they make heavy use of them in order to explain and model the needed nonlinear behavior of the greenhouse effect , this goes against intuition . in a chaotic system scientists of any plumage , when making such simulations , know this : if they change their parameters just slightly ( sometimes even in the scale of the numerical resolution of their floating point numbers ) , the outcome can be hugely different . there is also an experimental argument against positive feedback factors , namely , the conscientious satellite measurements ; see , for instance , the work of lindzen and choi , spencer , or wielicki et al . without feedback , in standard theory , if earth warms up ( by global warming in a radiation imbalance ) , the temperature rises and the outward earth radiation increases by a certain amount , until establishing a new equilibrium . in the agw model , a positive feedback of the the following form is used : if the temperature increases , the outward earth radiation is less than that predicted by standard theory or the incoming solar radiation increases because of reasons like cloud ( non)forming , thus increasing the temperature even further . this is also in agreement with the fact that , whereas the conditions on our planet have significantly changed over the geological history ( the sun for instance has been 25% less bright than today ) , the climate has been rather stable , always restoring from climate perturbations to median values instead of saturating in extreme values ; the latter would be expected in a thermal - runaway positive - feedback climate system . additional arguments against positive feedback come from the fact that every day , and every year , the temperature is brought off equilibrium . to give an idea , segalstad established that of the current rise in [ co2 ] levels relative to the preindustrial level only 12 ppm is attributable to human activity while 68 ppm is attributed to natural phenomena . these fluctuations are also visible in the extensive summary of beck and show that even in recent history the [ co2 ] levels were sometimes higher than the modern values , while as everyone knows , the human emissions have monotonously increased , showing that these huge fluctuations can only have a natural origin . relevant for the discussion here , the fluctuations would rapidly push the climate off equilibrium if it were unstable . yet , in spite of these huge disturbances , both in temperature and co2 , the equilibrium is restored every day and every year and every century . if starting oscillations are much faster than the response time of the system , the effective amplitude is reduced , but in a runaway system they will be amplified up to the point of saturation . the system can only be stable if the feedback factor at that specific frequency is not positive . look at it like this : in the first half of the year , it is hot and the system tries to runaway . in the second half of the year it is colder and it will restore , but it has a minute memory that the temperature has already run off a little in the first half and the second half therefore does not compensate completely . in the first year chance will determine which one , but if the system is unstable ( positive feedback ) , the system will run away . thus , the fact that we find a delay close to a quarter period means that ( i ) the temperature signal is the origin for [ co2 ] signal ( or the two are uncorrelated ) and ( ii ) the relaxation time linking the two is ( much ) longer than the period ( 12 months ) of oscillation . moreover , even if positive feedback were present , for the resonance itself to be significant , the oscillating frequency needs to be close to the resonance frequency , that is , 12 months . it is highly unlikely that the natural frequency of the climate-[co2 ] system is close to the 12-month - periodic driving force , even more so since also the long - term ice - drilling data need to be explained somehow , where delays of several thousands of years are observed . in our analysis , relaxation times of several thousands of years will explain both the ice - drilling data and the yearly temperature and [ co2 ] oscillations . have recently investigated correlation between temperature and [ co2 ] variations on the time scale of decades , similarly concluding that [ co2 ] changes are delayed in relation to temperature and can therefore not be the reason for temperature changes . in conclusion , the idea tested here that [ co2 ] is the cause of temperature changes does not pass our signal analysis . it goes a little too far to say that what we present here is proof for the opposite , namely , that [ co2 ] is the effect of temperature , but our analysis does not contradict this .

according to a national health interview survey in 20032005 , different forms of arthritis affected approximately 46 million adults in the united states . the total cost estimate attributable to arthritis and related conditions is close to $ 128 billion . osteoarthritis ( oa ) , the most common form of arthritis , is characterized by the destruction of articular cartilage . the main constituents of articular or joint cartilage are type ii collagen and various proteoglycans , such as aggrecan , chondroitin sulfate , and hyaluronan . the tensile strength of articular cartilage is due to the highly constrained supersecondary triple - helical structure of type ii collagen . this triple - helical structure also makes collagen resistant to hydrolysis by the majority of human proteinases , with the exception of several matrix metalloproteinases ( mmp-1 , -8 , and -13 , known as collagenases , and membrane type 1 mmp ) . in native joint cartilage , type ii collagen fibrils are protected from cleavage by tight association with molecules of aggrecan . in arthritic cartilage , aggrecan is hydrolyzed by members of another family of metalloproteases , adamts-1 , -4 , and -5 , known as aggrecanases . aggrecanolysis removes aggrecan molecules from type ii collagen fibrils , which makes collagenolysis possible . mmp-13 has been shown to be the main collagenase responsible for degradation of articular cartilage during oa and therefore represents a target for drug development . multiple attempts to develop mmp-13 inhibitor - based drugs have failed mostly due to the dose - limiting side effects collectively known as musculoskeletal syndrome ( mss ) . while the exact cause of mss is not known , it is believed to be due to the lack of selectivity of drug candidates toward other members of the mmp family as well as related metalloenzymes . high structural similarity of the catalytic domains of mmps and the fact that the majority of chemistry efforts focused on active site zn - binding groups as a basis for the mmp inhibitors resulted in clinical trial failures mainly due to the off - target inhibition by drug candidates . despite the prevalence of zinc chelators among mmp inhibitors , there are examples of inhibitors that do not act by binding the active site zinc , but rather bind via so - called exosites or allosteric sites . aventis discovered a pyrimidinedicarboxamide that had low micromolar potency for mmp-13 and no activity against other mmps when tested at 100 m . the potency of this compound was further improved to low nanomolar without loss of selectivity . pfizer reported discovery of highly selective nanomolar range mmp-13 inhibitors based on pyrimidinedione and quinazolinone scaffolds acting via binding to the same s1 exosite . furthermore , pyrimidinedione derivatives were efficacious and safe in rabbit and dog models of oa . similarly , alantos pharmaceuticals identified a new class of highly selective non - zinc - binding mmp-13 inhibitors . although selective mmp-13 inhibitors have been described by alantos , aventis , boehringer , pfizer , and wyeth , important pharmacokinetic ( pk ) and/or other data have not been reported for many of these compounds , and no clinical studies have appeared . for example , no pk or mss data have been reported for the aventis and wyeth compounds . the first series of pfizer compounds , while exhibiting good pk and mss data , were tested against a limited number of mmps . in similar fashion , the boehringer compounds exhibited good pk data but were tested against a limited number of mmps , and not at all in an mss model . the alantos compounds exhibited excellent mmp selectivity and good pk data , but were not tested in an mss model . only the second series of pfizer compounds were reported to exhibit excellent mmp selectivity and good pk and mss data . however , as mentioned above , no clinical studies have been reported for the pfizer compounds . in our hands , we found the primary pfizer compound ( table 1 ) to have low solubility ( it could only be tested at a maximal concentration of 2.5 m ) , and it inhibited cytochrome p450 1a2 . most recently , the takeda pharmaceutical co. reported yet another non - zinc - binding inhibitor of mmp-13 that acts via binding to the s1 site . the lead of the series , compound 26c , exhibited subnanomolar activity against mmp-13 and good oral availability ; however , the type of inhibition was not published . most of the above inhibitors possess large scaffolds that bind in the mmp-13 s1 subsite ( chart 1 ) . the structure of a non - zinc - binding mixed inhibitor from alantos has not been published ( compound als 1 - 0635 ) . neither the type of inhibition nor the binding site has been published for boehringer compound 3 . recently , we reported the results of an hts campaign that resulted in the discovery of a selective mmp-13 inhibitor ( 1 ; table 1 ) , which was further optimized as a result of medicinal chemistry efforts ( 2 and 3 ; table 1 ) . in the studies reported herein , we mechanistically characterized compounds 1 , 2 , and 3 , assessed their selectivity against an expanded enzymatic panel , and evaluated their ability to prevent type ii collagen degradation . the binding of compound 2 to mmp-13 was determined directly by using single - crystal x - ray diffraction . previously , we reported the outcome of an hts campaign that resulted in a discovery of selective mmp-13 inhibitor 1 ( table 1 ) . interestingly , this was the only compound that was found to be a more effective inhibitor of hydrolysis of a triple - helical , collagen - model substrate compared to a short , linear substrate . this finding suggested that compound 1 was mechanistically different from the rest of the hts leads . additionally , as a result of a structure activity relationship ( sar ) study of the 1 chemotype , we identified 2 and 3 , which exhibited improved potency against mmp-13 ( table 1 ) and selectivity against antitargets mmp-1 and mmp-8 ( ic50 > 40 m ) . we have now utilized known selective inhibitors of mmp-13 to benchmark our lead compound . more specifically , we examined ( e)-4-((1-methyl-2,4-dioxo-6-(3-phenylprop-1-enyl)-1,2-dihydroquinazolin-3(4h)-yl)methyl)benzoic acid , reported by pfizer as compound 2 , and n , n - bis(4-fluoro-3-methylbenzyl)pyrimidine-4,6-dicarboxamide , reported by aventis as compound 4 , which are referred to herein as 5 and 4 , respectively . compounds 13 exhibited low to submicromolar ki values ( see table 1 ) , whereas 4 and 5 had ki values of 0.06 0.02 and 0.01 0.0 m , respectively , when tested with the triple - helical fthp-15 substrate . interestingly , all tested compounds , with the exception of 6 ( n - hydroxyacetamide ( aha ) ) , inhibited mmp-13 hydrolysis of fthp-15 via a noncompetitive mechanism ( figure 1 ) . this finding suggests that , similarly to 4 and 5 , compounds 13 do not bind to the active site zn and possibly bind outside of an active site ( i.e. , to an exosite ) . burke plot of inhibition of mmp-13 fthp-15 hydrolysis by ( a ) 1 , ( b ) 2 , ( c ) 3 , ( d ) 4 , ( e ) 5 , and ( f ) 6 . note the lines of best fit crossing at the y - axis indicative of noncompetitive inhibition for all compounds except aha . all ki values were determined by nonlinear regression ( hyperbolic equation ) analysis using the mixed inhibition model , which allows for simultaneous determination of the mechanism of inhibition . burke analysis . to test whether 1 binds to the active site zn , a known zn binder , 6 ( aha ) , a competitive millimolar range mmp-13 inhibitor ( see table 1 ) , was used in combination with 1 following a previously described methodology.6 ( aha ) was used in the range of 01.25 mm in combination with 1 in the range of 02 m . when initial velocities from this experiment were organized in a yonetani theorell plot , they formed a series of intersecting lines of best fit ( figure 2a ) . in yonetani theorell plots , the intersecting lines indicate simultaneous ( i.e. , mutually nonexclusive ) binding of both inhibitors to the enzyme . for any mutually nonexclusive pair of inhibitors , x = bki when [ i ] is plotted on the x - axis . rearrangement of this equation allows us to calculate the value of b using ki values obtained from independent experiments with fthp-15 as the substrate ( table 1 ) . the resulting b value was < 1 , which indicated that 6 and 1 bind synergistically ( i.e. , binding of one inhibitor increases the affinity of binding of the second inhibitor ) . yonetani theorell plot of mmp-13 fthp-15 hydrolysis in the presence of ( a ) 6 and 1 , ( b ) 4 and 1 , and ( c ) 5 and 1 . note the nonparallel lines of best fit indicating mutually nonexclusive binding by two inhibitors . . 1 was subsequently tested in combination with 4 , which is a noncompetitive , non - zn - binding inhibitor of mmp-13 with a nanomolar ki value ( table 1 ) . compound 4 binds within the s1 specificity loop of the mmp-13 cat domain , which is a known exosite . surprisingly , 1 exhibited mutually nonexclusive binding when cotested with 4 ( figure 2b ) . similarly , 1 binding to mmp-13 was synergistic with that of 5 ( b < 1 , figure 2c ) . the structure of the mmp-13 cat compound 2 inhibitor complex containing two protomers ( a and b ) in the asymmetric unit was refined to a resolution of 1.66 with r and rfree values of 0.134 and 0.200 , respectively ( supplemental table 3 , supporting information ) . as observed in other mmp-13 cat inhibitor complexes in this crystal system , the first eight residues of the two protomers engaged in extensive interprotomer interactions around a 153 axis of rotation , an asymmetry that caused these residues to differ somewhat in conformation . four residues at the c - terminus of protomer b were not observed in protomer a. the two protomers are otherwise remarkably similar , aligning a root - mean - square deviation ( rmsd ) of 0.18 for 391 backbone atom target pairs . annealed omit maps confirmed that 2 was bound in two distinct binding sites ( figure 3a ) . compound 2 is observed in both protomers in the s1 specificity pocket ( figure 3b ) , while in protomer a a second 2 binds near the s1 and s2 subsites ( designated s1/s2 * ) within the substrate binding cleft ( figure 3c ) . the oxygen atoms of formate from the reservoir solution chelated the catalytic zinc ion in a mode similar to that of 6 to prevent protein self - proteolysis . structure of the mmp-13 cat compound 2 complex revealing the distinct binding sites in protomer a ( see the text ) . ( a ) annealed omit map with coefficients fo fc contoured at 2.5 superimposed on the refined model of the mmp-13 cat2 complex . 2 was left out of the phase calculation . the surface of the protein is blue , and the s1 loop is green . the catalytic zinc ion is a violet sphere , and formate from the reservoir solution is shown as cyan sticks . formate from the reservoir solution chelates the zinc ion and forms hydrogen bonds with a nearby water molecule and the side chain of the catalytic glutamic acid ( e233 ) hidden beneath the surface . the color scheme is the same as that in panel a , and the view is rotated only slightly around the vertical compared to that of panel a. gray dashed lines represent selected van der waals contacts ( < 4.6 ) , and the blue dashed lines represent hydrogen - bonding interactions . ( c ) a weighted electron density with coefficients 2mfo dfc superimposed on the s1/s2 * 2 binding site in the refined model of the complex . the color scheme is the same as that in panel a except y176 , f189 , and p190 , which make the majority of contacts with 2 , are highlighted as dark green sticks . hydrogen bonds are shown as orange dashes , and metal ligand interactions are shown as yellow dashes . ( d ) superposition of compounds 2 ( yellow ) , 4 ( white ) , and 5 ( pink ) in the s1 binding site . a second molecule of 2 can be seen on the other side of catalytic zinc in the s1/s2 * site . ( e ) superposition of compound 2 ( yellow ) with hydroxamic acid - based inhibitors ( pdb code 456c , red ; pdb code 830c , cyan ) that do not intrude deeply into the s1 specificity loop . the difference in positions of residues 248251 ( disordered in structure 830c ) in the two classes of inhibitors suggests the s1 specificity loop is conformationally dynamic in the uninhibited enzyme . compound 2 intruded deeply into the hydrophobic s1 pocket such that its p - methylphenyl moiety pointed toward the substrate binding cleft and contacted the hydrophobic region composed of four residues ( leu185 , val219 , pro242 , and tyr244 ) . there was also a stacking interaction between the imidazole ring of his222 and the compound 2 phenyl ring . the cyclopentene ring moiety pointed toward the s1 specificity loop and contacted the hydrophobic portions of residues leu218 , leu239 , thr245 , tyr246 , thr247 , phe252 , and pro255 . the central s16 atom of 2 accepted a hydrogen bond from the amide nitrogen of thr245 ( 3.4 ) , while the n19 atom of 2 accepted a hydrogen bond from the carbonyl oxygen of thr245 ( 2.7 ) . there were two water - mediated hydrogen bond networks that played roles in 2 binding . a cluster of three water molecules ( left side of 2 in figure 3b ) connected the carbonyl oxygen atoms of mmp-13 cat residues pro236 , ala238 , and ile243 and the side chain hydroxyl moiety of thr245 to inhibitor 2 atoms s16 , n18 , and n19 . the 2 o17 carbonyl oxygen atom accepted a hydrogen bond from the amide nitrogen of thr247 ( 3.3 ) . a second , four - water cluster ( right side of 2 in figure 3b ) connected the 2 o17 atom to the mmp-13 s1 specificity loop ( thr247 side chain hydroxyl oxygen , met253 amide nitrogen , and lys249 , his251 , and met253 carbonyl oxygens ) . this hydrogen bond network was essential to stabilize the otherwise flexible s1 specificity loop to define the s1 specific pocket size for inhibitor 2 . the second molecule of 2 resided in the substrate binding cleft on the opposite side of the catalytic zinc ion relative to the s1 specificity pocket . the p - methylphenyl moiety of 2 faced away from the catalytic center and interacted with the hydrophobic region composed of tyr176 , phe189 , and pro190 . an amide nitrogen proton pointed into the aromatic hole . to assess the potency of lead compounds with a cognate substrate , compounds 13 were tested in an assay utilizing type ii collagen conjugated to fitc . all compounds exhibited dose - dependent responses in both fthp-15 ( figure 4a ) and type ii collagen assays ( figure 4b ) with hill slopes close to 1.0 . compounds 1 and 2 appeared to be more potent in the type ii collagen assay than in the fthp-15 assay ( type ii collagen ic50 = 0.7 0.2 and 0.4 0.1 m versus fthp-15 ic50 = 3.4 0.2 and 2.4 0.1 m , respectively ) . compounds 3 and 4 were equipotent in these two assays ( type ii collagen ic50 = 2.3 0.2 and 0.07 0.01 m versus fthp-15 ic50 = 2.8 0.1 and 0.11 0.01 m , respectively ) . interestingly , while 4 clearly appeared more potent in the fthp-15 assay than compounds of the 1 chemotype , the difference in apparent potency was much less pronounced in the type ii collagen assay . response curves characteristic of partial inhibitors in the collagen assay ( figure 4b ) . in the fthp-15 assay compounds 13 appeared to be partial inhibitors , whereas 4 inhibited hydrolysis completely ( figure 4a ) . results of ( a ) fthp-15 , ( b ) type ii collagen , and ( c ) cartilage explant assays . in the fthp-15 assay ( a ) compounds of the q series appear to be partial inhibitors , whereas 4 inhibits hydrolysis completely . in the type ii collagen assay ( b ) all four compounds exhibit concentration response curves characteristic of partial inhibitors . 4 clearly appeared more potent in the fthp-15 assay than compounds of the 1 chemotype , while the difference in apparent potency was much less pronounced in the type ii collagen assay . to assess the ability of compounds 13 to prevent cartilage degradation , in vitro bovine cartilage digestion the three compounds inhibited cartilage degradation to different degrees : 69 17% , 59 50% , and 38 20% inhibition at 10 m 13 , respectively , and 70 8% inhibition for edta at 50 m ( figure 4c ) . one of the primary reasons for failures of mmp inhibitors in clinical trials was the lack of selectivity . to assess the selectivity of compounds 13 , they were tested against a panel of 30 proteases in 12-point 3-fold dilution dose response curve format in a single dose . compounds 13 were tested in the 20 m to 0.1 nm range , while 4 and 5 were tested in the 5 m to 0.028 nm range . when tested against 15 metalloproteases ( figure 5a ) , the only enzyme inhibited in excess of 20% by all five tested compounds was mmp-13 . compounds 13 appear to have activity in excess of 10% against mmp-2 , mmp-3 , mmp-9 , mmp-12 , and ace ; however , examination of the full range of tested concentrations ( 20 m to 0.1 nm ) revealed the absence of dose - dependent responses ( supplemental figure 1a , supporting information ) and therefore must represent an artifact . in addition to the metalloprotease panel , compounds were tested against representatives of the serine , cysteine , and aspartic classes of proteases ( figure 5b ) . all tested compounds exhibited activity below 10% inhibition for all tested enzymes , except 2 with caspase 5 and cathepsin k and 5 with cathepsin k. upon further inspection , the activity of 2 against caspase 5 and 5 against cathepsin k proved to be artifactual ( supplemental figure 1b ) . in contrast , compound 2 exhibited a semblance of dose - dependent response against cathepsin k , with the highest inhibitory activity reaching 20% at 20 m . test compounds were screened in 12-point 3-fold dilution dose response curve format . the activity of each enzyme was determined as the conversion ( % ) of the substrate to product after 3 h of incubation . ( b ) serine , cysteine , and aspartic protease panel results at the highest compound concentration tested . success or failure of drug candidate molecules in the clinic greatly depends on whether they induce toxicity . to assess possible toxicity , compounds 13 were screened against a panel of cytochrome p450 ( cyp ) enzymes in seven - point dose response format in duplicate . compounds 1 and 2 exhibited low micromolar potency against cyp 1a2 , 2c9 , and 2c19 ( table 3 ) , while 3 , 4 , and 5 did not inhibit any of the tested cyps in excess of 50% at inhibitor concentrations of 20 , 20 , and 2.5 m , respectively . examination of the activity of tested compounds at maximal tested concentrations revealed that 3 inhibited cyp 1a2 and 2c9 at 29% and 17% , respectively ( figure 6 ) . compound 4 inhibited all cyps in excess of 10% , except 3a4 , when tested with midazolam substrate ( figure 6 ) . compound 5 was tested at a maximal concentration of 2.5 m due to solubility concerns . at this concentration results of cytochrome p450 inhibition profiling . to determine the metabolic stability of 13 to predict potential problems , the compounds sunitinib is an orally available inhibitor of receptor tyrosine kinases used for the treatment of renal cell carcinoma ( rcc ) and imatinib - resistant gastrointestinal stromal tumor ( gist ) and , therefore , provides a good comparison for metabolic stability studies . inhibitors 13 exhibited low metabolic stability in the microsomes of all three species tested , with the best half - life of 10.4 min exhibited by 1 in human liver microsomes ( table 4 ) . compounds 13 are thioethers and , therefore , could potentially be metabolized in vivo by several members of cyp or flavin - containing monooxygenase ( fmo ) families to sulfoxides or sulfones , which could lead to the limitation of oral bioavailability due to a loss of activity . to test this hypothesis , we attempted to synthesize the sulfone and sulfoxide derivatives of 2 ; however , they proved to be unstable and decomposed during attempts at chromatographic purification . as a result of an hts campaign and subsequent sar studies , we identified a series of mmp-13 inhibitors that exhibited a good selectivity profile toward other members of the mmp family . a lack of obvious zn - binding moieties , preferential inhibition of triple - helical peptide hydrolysis , and a partial inhibitor profile of a lead of a series , 1 , suggested inhibition by a mechanism other than competitive . indeed , mechanistic characterization of these compounds revealed that they inhibit mmp-13 via a noncompetitive mechanism . the noncompetitive mechanism of inhibition of mmp-13 by our lead compounds in combination with results of the dual inhibition study with a zn binder ( aha ) strongly suggested the possibility of binding outside of an active site at a so - called exosite . exploration of these exosites was suggested as a possible way to find selective mmp inhibitors . indeed , pfizer and aventis identified small - molecule inhibitors of mmp-13 that bind within one of these exosites to the so - called specificity loop and inhibit catalysis by inducing rigidity . yonetani theorell analysis of dual inhibition kinetic data showed that binding of 1 does not preclude binding of either 4 or 5 to mmp-13 , but in fact made it stronger as evidenced by parameter b < 1 . this finding suggested either formation of a quaternary complex between two inhibitors , mmp-13 , and the fthp-15 substrate or binding of 1 to an exosite other than the specificity loop . crystallographic analyses revealed a second binding region for 2 ( the s1 and s2 subsites ) that is novel compared to those of all known mmp-13 inhibitors . in addition , alignment of all known mmp-13inhibitor structures to our mmp-132 structure showed that the molecular scaffold of 2 is the smallest in the s1 pocket . superposition of 2 , 4 , and 5 in their respective binding poses in the s1 subsite suggested that 2 and 4 or 5 can not simultaneously reside in the s1 subsite ( figure 3d ) . since both 4 and 5 have much greater affinity than 2 , this suggested that when 4 or 5 is present in the s1 subsite , 2 can only bind to the second binding site , explaining the lack of mutually exclusive binding ( figure 2b , c ) between 2 and 4 or 5 . to understand the effects of zinc - binding and non - zinc - binding inhibitors on mmp-13 , we overlaid mmp-13 complexed with 2 with mmp-13 complexed with 4 and 5 ( figure 3d ) and two hydroxamate - based inhibitors ( figure 3e ) . the analysis of the superimposed structures revealed that the specificity loop , where the s1 binding site is located , is more disordered in mmp-13 complexed with hydroxamate - based inhibitors , which can be explained by the fact that hydroxamate - based inhibitors do not significantly protrude into the specificity loop . knowledge of the novel binding site of compounds 13 will facilitate sar studies to achieve greater potencies for new analogues . indeed , despite demonstrating a good selectivity profile comparable to that of compounds developed by aventis ( 4 ) and pfizer ( 5 ) , compounds 13 have 10100 times lower affinity than 4 and 5 . however , compounds 13 are significantly smaller than 4 and 5 , suggesting that greater affinity can be achieved by increasing the size of future analogues of 13 . ligand efficiency indices have been utilized as the rational approach to the optimization of compound properties . one of the most frequently used methods is based on the combination of the binding efficiency index ( bei , pki / mw ) and surface - binding efficiency index ( sei , pki / psa ) , which allows monitoring of the dependence of affinity on the size and polar surface of the molecule . sei and bei values of an idealized compound ( ki = 1 nm , psa = 50 ) are used as a way to guide the optimization process . as evidenced by table 5 , the bei values of 13 are similar to those of 4 and 5 , whereas the sei values of 13 are greater than those of 4 and 5 . compound 2 exhibits sei and bei values that best approximate the values of idealized compound 7 . comparison of sei versus bei values ( figure 7 ) suggested that , to achieve greater potency , both the size and polar surface area of future analogues of 2 should be increased . mapping of surface - binding and binding efficiency indices for compounds of the 1 series and study controls . key : red circle , 1 ; purple circle , 5 ; blue circle , 4 ; green circle , 3 . the red diagonal line represents an optimization plane as described in ref ( 49 ) . thus , prevention of collagen degradation in vitro provides an important estimate of the ability of mmp-13 inhibitors to modify the course of the disease . compounds 13 modulated mmp-13 activity with apparent potencies in the submicromolar and low micromolar ranges . to determine whether mmp-13 inhibition translated into prevention of articular cartilage degradation , compounds 13 were studied in a cartilage explant assay . the cartilage explant assay is a well - established in vitro system for testing of prevention of cartilage degradation . compounds 13 inhibited cartilage degradation at a level comparable to that of edta , a broad - spectrum metalloprotease inhibitor . these data suggested that compounds 13 can be potentially protective against cartilage degradation in vivo . development of roughly 30 antiarthritic drugs has been discontinued due to the occurrence of mss as a result of mmp inhibitor dosing in the clinical trials . mss does not appear to be caused by the off - target inhibition of a single enzyme , but rather a combination of several mmps and possibly other related enzymes . among enzymes proposed to play role in mss development are mmp-1 , mmp-2 , mmp-9 , mt1-mmp , and adamalysins . with that in mind , 13 were screened against an expanded panel of proteases , which included proposed arthritis antitargets ( mmp-1 , mmp-2 , mmp-9 , and mt1-mmp ) and representative adamalysins ( adam10 and adam17 ( tace ) ) . compound 5 was previously tested in the rat model of mss and was found to be safe at plasma concentrations well above those needed to protect articular cartilage and therefore represents an excellent control for a selectivity study . importantly , 13 exhibited a selectivity profile similar to that of 5 ; in addition to sparing all arthritis antitargets , they did not inhibit mmp-3 and mmp-8 ( important cancer antitargets ) . while the usefulness of this protease panel in predicting mss occurrence has not been established , the similarity of the protease inhibition profile of compounds 13 to that of 5 suggested a similar in vivo safety profile . this hypothesis , of course , will have to be tested in one of the existing animal mss models . adverse effects during animal and human testing account for more than 20% of cases where drug development had to be terminated early . drug drug or drug food interactions due to the inhibition or induction of members of the cytochrome p450 enzyme family are believed to be one of the main causes of these effects . approximately 95% of all drug metabolism is attributable to just five representatives of the cyp family , with the 3a4 enzyme being responsible for almost half of these reactions . it is therefore encouraging that compounds 13 did not inhibit 3a4 when tested with the midazolam substrate . 1 and 2 exhibited some inhibition of 3a4 when testosterone was used as the substrate ( 14% and 20% , respectively ) , whereas 3 showed very little activity ( 3% inhibition ) . overall , 3 exhibited significantly lower cyp activity as compared to 1 and 2 . interestingly , the inhibitory activity of compound 3 was either the same as or lower than that of 4 and 5 . while the overall good inhibition profile of compounds 13 is encouraging , these data suggested the possibility of in vivo drug drug interactions due to the inhibition of 3a4 , 1a2 , 2c9 , and 2c19 . compounds 13 exhibited low metabolic stability when exposed to microsomes , and their sulfone and sulfoxide metabolites were too unstable to be tested for mmp-13 inhibition ; therefore , their activity against mmp-13 could not be ascertained . this suggests that future optimization studies should be focused not just on the improvement of potency , but also on the metabolic stability of future analogues to preempt the lack of in vivo efficacy of future mmp-13 inhibitors deriving from 13 due to their metabolic instability . future studies will ascertain the need for a thioether in the inhibitors and its effect on metabolic stability . there are examples of thioether - containing drugs and preclinical candidates with good in vivo efficacy , suggesting that metabolic stability of the molecule is a function of the structure and properties of the molecule as a whole , and not just one particular atom in the structure . the lead compound of this novel class of mmp-13 inhibitors , 1 , was discovered as a result of an hts campaign under the auspices of the nih roadmap initiative . additional compounds belonging to the same chemotype were identified as a result of a medicinal chemistry optimization effort as a part of the same program . as a result of the characterization studies presented here , it was determined that compounds 13 inhibit mmp-13 via a noncompetitive mechanism with the possibility of binding to two exosites . type ii collagen and cartilage protective effects exhibited by these mmp-13 inhibitors in vitro indicated the possibility of these compounds being efficacious in future in vivo studies . these compounds were also highly selective when tested against a panel of 30 proteases , which , in combination with a good cyp inhibition profile , suggested low off - target toxicity and drug drug interactions in humans . mmp-13 inhibitors were synthesized and characterized as described elsewhere . [ ( pyrimidine-4,6-dicarboxylic acid bis(4-fluoro-3-methylbenzamide ) ] was obtained from emd biosciences / calbiochem ( product no . 444283 ; san diego , ca ) . the synthesis , purification , and characterization of the fluorogenic triple - helical peptide substrate ( fthp-15 ) have been described . full - length recombinant human pro - mmp-13 ( rhmmp-13 ) was purchased from r&d systems ( catalog no . the zymogen form of mmp-13 was converted to the active form by incubating pro - mmp-13 with 1 mm ( p - aminophenyl)mercuric acid ( apma ) for 2 h at 37 c . the stock of active mmp-13 was diluted to 1 m and stored at 80 c . proteases used in the protease profiling panel were from r&d systems ( ace , ace2 , adam10 , adam17 , furin , ide , neprilysin , thrombin , upa ) , invitrogen ( bace ) , biomol ( mmp-13 , -79 , and -1214 , caspases 13 and 57 ) , and calbiochem ( factor xa , cathepsin d , l , s , and k ) . cyp450 substrates bupropion , ( s)-mephenytoin , and midazolam were obtained from bd biosciences discovery labware ( woburn , ma ) , while tacrine , amodiaquine , diclofenac , dextromethorphan , and testosterone were purchased from sigma - aldrich ( st . cyp450 control inhibitors ketoconazole , sulfaphenazole , and ( s)-benzylnirvanol were obtained from bd biosciences discovery labware , while 7,8-benzoflavone and quinidine were purchased from sigma - aldrich . probe substrate metabolites were from bd biosciences discovery labware with the exception of 1-hydroxytacrine , which was purchased from sigma - aldrich . stable labeled isotope internal standards were from bd biosciences discovery labware , and bucetin was from sigma - aldrich . all other reagents were purchased from sigma - aldrich . pooled human liver microsomes ( hlm , catalog no . 452161 ; 20 mg / ml protein concentration ) were from bd biosciences discovery labware . fthp-15 and mmp-13 working solutions were prepared in enzyme assay buffer ( eab ; 50 mm tris hcl , ph 7.5 , 100 mm nacl , 10 mm cacl2 , 0.05% brij-35 ( sigma - aldrich ) ) . all reactions were conducted in 384-well white polystyrene plates ( greiner , north carolina , catalog no . 784076 ) . determinations of inhibition constants and modalities were conducted by incubating the range of fthp-15 substrate concentrations ( 225 m ) with 4 nm mmp-13 at room temperature in the presence of varying concentrations of inhibitors . fluorescence was measured on a tecan safire monochromator microplate reader using excitation = 324 nm and emission = 393 nm . rates of hydrolysis were obtained from plots of fluorescence versus time using data points from only the linear portion of the hydrolysis curve . all kinetic parameters were calculated using graphpad prism , version 5.01 ( graphpad software , inc . , la jolla , ca ) . km values were determined by nonlinear regression analysis using the one - site hyperbolic binding model and additionally evaluated by linear analysis . all ki values were determined by nonlinear regression ( hyperbolic equation ) analysis using the mixed inhibition model , which allows for simultaneous determination of the mechanism of inhibition . the mechanism of inhibition was determined using the parameter derived from a mixed - model inhibition by graphpad prism . a matrix of two different inhibitor combinations was created in 384-well white polystyrene plates by serially diluting them in eab . mmp-13 and fthp-15 were then added , resulting in 4 nm and 8 m final assay concentrations , respectively . rates of hydrolysis were obtained from plots of fluorescence versus time using data points from only the linear portion of the hydrolysis curve . data from kinetic experiments utilizing a matrix of two different inhibitor concentrations were fitted to the yonetani theorell equation : where vij is the initial velocity of fthp-15 hydrolysis in the presence of both inhibitors , v0 is the initial velocity of fthp-15 hydrolysis in the uninhibited reaction , ki and kj are the dissociation constants for inhibitors i and j , respectively , and b is the parameter that describes the effect of binding of inhibitor i on the affinity of inhibitor j. when two inhibitors bind in a mutually exclusive manner , b = . when two inhibitors bind completely independently of each other , b = 1 . synergistic or antagonistic binding of two inhibitors yields b < 1 or b > 1 , respectively . the initial rates of fthp-15 hydrolysis in the presence of two inhibitors were arranged as dixon plots with 1/vij plotted as a function of [ i ] in the presence of varying concentrations of inhibitor j. the recombinant mmp-13 catalytic domain ( mmp-13 cat , residues 104274 ) was expressed , purified , and refolded as described . the reconstituted enzyme , which was fully functional in the modified knight substrate assay , was concentrated to 0.5 mg / ml ( mmp-13-cat 280 = 28 590 mol cm ) and made 25% ( v / v ) in glycerol for storage at 80 c . immediately prior to crystallization trials , the glycerol was removed from the protein by dialysis against buffer containing 20 mm tris ( ph 8.0 ) , 5 mm cacl2 , and 50 mm nacl . a 100 mm stock solution of 2 in dmso was added to obtain a 5:1 inhibitor : protein stoichiometric ratio . after incubation on ice for 1 h , the sample was concentrated to 12 mg / ml and screened in sitting drops using 1344 unique conditions coming from commercially available crystallization screening kits ( qiagen inc . , valencia , ca ) . a phoenix crystallization robot ( art robbins instruments , sunnyvale , ca ) was used to dispense the protein sample and crystallization reagents . the initial conditions were systematically optimized , and rod - shaped single crystals suitable for x - ray diffraction work were grown at 4 c from drops containing protein solution in a 3:1 ratio ( v / v ) with reservoir solution consisting of 0.1 m tris ( ph 8.5 ) , 1.5 m ammonium formate , and 1014% peg4000 . suitable specimens were extracted with nylon loops , swept through reservoir solution made 20% ( v / v ) in glycerol as a cryoprotectant , and flash - cooled in liquid nitrogen . diffraction data were measured at beamline 24-id - c at the advanced photon source , argonne , il , equipped with a pilatus 6 m detector and processed with the program xds . initial phases were obtained via the molecular replacement method as implemented in phaser using the mmp-13 cat coordinates of protein data bank entry 3zxh(63 ) as the search model . simulated annealing and individual anisotropic temperature factor refinement with the phenix suite of programs were followed by manual adjustment of the model using the program coot . the positions of 2 molecules bound to mmp-13 cat were illuminated in difference fourier electron density maps and were subsequently cross - validated in annealed omit maps . the model was refined using stereochemical restraints generated with the electronic ligand builder and optimization workbench ( elbow ) . structure factors and refined coordinates are deposited in the protein data bank under accession code 4l19 . ligand efficiency indices were calculated according to type ii collagen conjugated to fitc was purchased from sigma - aldrich ( catalog no . type ii collagen in eab using an frd ib workstation ( aurora discovery , carlsbad , ca ) . a 100 nl volume of 75% dmso/25% water containing the test compounds was dispensed using a 384-head pintool system ( gnf systems , san diego , ca ) . reactions were initiated by addition of 10 l of 40 nm mmp-13 in eab . after 22 h of incubation at 37 c , the emission fluorescence was read on a perkinelmer viewlux ( perkinelmer , turku , finland ) microplate reader ( excitation = 480 nm , emission = 530 nm ) . test compounds were assayed in triplicate using 10-point 3-fold serial dilutions . for each compound , fluorescence data were fitted with a four - parameter equation describing a sigmoidal dose response curve with adjustable baseline using the graphpad prism , version 5.01 , suite of programs . the ic50 values were generated from fitted curves by solving for the x - intercept at the 50% inhibition level of the y - intercept . bovine articular cartilage was procured by articular engineering , llc ( skokie , il ) . cartilage pieces ( 3 mm ) were frozen and thawed three times in ln2 to render them nonviable followed by trypsin digestion to remove aggrecan as described elsewhere . after 4 h of digestion , the cartilage was rinsed with 10% fbs and pbs . the cartilage was incubated with 100 nm mmp-13 in a 96-well polystyrene sterile nontreated plate ( falcon no . 1172 ) in the presence or absence of the compounds for 18 h at 37 c and 5% co2 . test compounds were assayed in triplicate at a single concentration , 50 m for edta and 10 m for compounds 13 . collagen degradation products in the buffer were measured using the c1,2c enzyme - linked immunosorbent assay ( ibex , montreal , quebec , canada , catalog no . absorbance data were normalized to wells containing undigested cartilage ( 100% inhibition ) and mmp-13-digested cartilage ( 0% inhibition ) to obtain inhibition values . assays were performed at nanosyn , http://www.nanosyn.com . the highest concentrations of compounds to be used in the assays were determined . the compounds were solubilized at 50 m in protease assay buffers , and the absorbance at = 540 nm was measured and compared to that of buffer alone . the assay began by dispensing 5 l of test compounds in assay buffer followed by 10 l of the respective protease . enzymes were allowed to incubate with the test compounds for 30 min at 25 c . the assays were initiated by addition of 10 l of various concentrations of the respective peptide substrates . the assays were incubated for 3 h at 25 c and terminated by addition of 45 l of quenching solution containing sds , edta , and iodoacetate . quenched assay solution was used for analysis on the microfluidic - based labchip 3000 ( caliper life sciences , inc . test compounds were screened in 12-point 3-fold dilution dose response curve format in singlicate . the activity of each enzyme was determined as the conversion ( % ) of the substrate to product after 3 h of incubation . the activity in the presence of a compound was plotted as a function of the compound concentration , and the curves were fitted using the built - in dose response model algorithm of xlfit software ( idbs ) . the ic50 values of the compounds were determined as the concentrations that resulted in 50% enzyme activity when compared to the activity of the control samples ( without a compound ) . where applicable , the hill slopes were calculated using the xlfit software . experiments were conducted in duplicate at 37 c using 96-well polypropylene reaction plates incubated on a heating block ( mecour , groveland , ma ) . test compound concentrations were 0 , 0.03 , 0.1 , 0.3 , 1 , 3 , 10 , and 20 m for compounds 14 and 0 , 0.003 , 0.01 , 0.03 , 0.1 , 0.3 , 1.0 , and 2.5 m for 5 . incubation mixtures ( 400 l ) contained probe substrate , inhibitor , hlm , and an nadph - regenerating system ( 1.3 mm nadp , 3.3 mm glucose 6-phosphate , 0.4 u / ml glucose 6-phosphate dehydrogenase , 3.3 mm magnesium chloride ) in 0.1 m potassium phosphate buffer ( ph 7.4 ) . reactions were initiated by the addition of diluted hlm and terminated by combining 4 parts sample with 1 part stop solution ( stable labeled isotope internal standard containing 0.1% formic acid in acetonitrile ) and placing them on ice . protein concentrations , incubation times , and substrate concentrations are listed in table 2 . metabolites were quantified by using the biocius rapidfire ultra - high - throughput mass spectrometer at biocius life sciences inc . ( woburn , ma ) using a proprietary sample purification and injection system coupled to a sciex api-4000 triple - quadrupole mass spectrometer at the transitions shown in table 2 . samples were separated on an spe column ( reversed - phase c4 chemistry ) with mobile phases of 0.09% formic acid/0.01% trifluoroacetic acid in water and 0.09% formic acid/0.01% detection metabolites and internal standard mass - to - charge ratios are listed in table 2 . test compound activity against cyp450 enzymes was calculated as follows : where r + i is the area ratio of probe substrate metabolite formed in the presence of the inhibitor and r i is the area ratio of probe substrate metabolite formed in the absence of the inhibitor . ic50 values were calculated by linear interpolation as follows : where lconcn is the highest concentration of inhibitor that results in < 50% inhibition , hconcn is the lowest concentration of inhibitor that results in > 50% inhibition , l is the inhibition ( % ) associated with lconc , and h is the inhibition ( % ) associated with hconc . metabolic stability was evaluated by incubating 1 m compound with 1 mg / ml hepatic microsomes ( human , rat , and mouse ) in 100 mm potassium phosphate buffer , ph 7.4 . the final incubation volume was 300 l , and 40 l aliquots were removed at 0 , 5 , 10 , 20 , 40 , and 60 min . the removed aliquot was added to 160 l of acetonitrile to stop the reaction and precipitate the protein . nadph dependence of the reaction is evaluated in parallel incubations without nadph . at the end of the assay , the samples were centrifuged through a 0.45 m filter plate ( millipore solventer low binding hydrophilic plates , catalog no . ms / ms . the data were log transformed , and the results are reported as half - lives .

matrix metalloproteinase 13 ( mmp-13 ) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development . as a result of high - throughput screening and structure activity relationship studies , we identified a novel , highly selective class of mmp-13 inhibitors ( compounds 1 ( q ) , 2 ( q1 ) , and 3 ( q2 ) ) . mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range . crystallographic analyses revealed two binding modes for compound 2 in the mmp-13 s1 subsite and in an s1/s2 * subsite . type ii collagen- and cartilage - protective effects exhibited by compounds 1 , 2 , and 3 suggested that these compounds might be efficacious in future in vivo studies . finally , these compounds were also highly selective when tested against a panel of 30 proteases , which , in combination with a good cyp inhibition profile , suggested low off - target toxicity and drug drug interactions in humans .

Introduction Results Discussion Experimental Procedures

osteoarthritis ( oa ) , the most common form of arthritis , is characterized by the destruction of articular cartilage . the main constituents of articular or joint cartilage are type ii collagen and various proteoglycans , such as aggrecan , chondroitin sulfate , and hyaluronan . the tensile strength of articular cartilage is due to the highly constrained supersecondary triple - helical structure of type ii collagen . mmp-13 has been shown to be the main collagenase responsible for degradation of articular cartilage during oa and therefore represents a target for drug development . high structural similarity of the catalytic domains of mmps and the fact that the majority of chemistry efforts focused on active site zn - binding groups as a basis for the mmp inhibitors resulted in clinical trial failures mainly due to the off - target inhibition by drug candidates . pfizer reported discovery of highly selective nanomolar range mmp-13 inhibitors based on pyrimidinedione and quinazolinone scaffolds acting via binding to the same s1 exosite . similarly , alantos pharmaceuticals identified a new class of highly selective non - zinc - binding mmp-13 inhibitors . although selective mmp-13 inhibitors have been described by alantos , aventis , boehringer , pfizer , and wyeth , important pharmacokinetic ( pk ) and/or other data have not been reported for many of these compounds , and no clinical studies have appeared . the first series of pfizer compounds , while exhibiting good pk and mss data , were tested against a limited number of mmps . in similar fashion , the boehringer compounds exhibited good pk data but were tested against a limited number of mmps , and not at all in an mss model . in our hands , we found the primary pfizer compound ( table 1 ) to have low solubility ( it could only be tested at a maximal concentration of 2.5 m ) , and it inhibited cytochrome p450 1a2 . most of the above inhibitors possess large scaffolds that bind in the mmp-13 s1 subsite ( chart 1 ) . recently , we reported the results of an hts campaign that resulted in the discovery of a selective mmp-13 inhibitor ( 1 ; table 1 ) , which was further optimized as a result of medicinal chemistry efforts ( 2 and 3 ; table 1 ) . in the studies reported herein , we mechanistically characterized compounds 1 , 2 , and 3 , assessed their selectivity against an expanded enzymatic panel , and evaluated their ability to prevent type ii collagen degradation . previously , we reported the outcome of an hts campaign that resulted in a discovery of selective mmp-13 inhibitor 1 ( table 1 ) . additionally , as a result of a structure activity relationship ( sar ) study of the 1 chemotype , we identified 2 and 3 , which exhibited improved potency against mmp-13 ( table 1 ) and selectivity against antitargets mmp-1 and mmp-8 ( ic50 > 40 m ) . more specifically , we examined ( e)-4-((1-methyl-2,4-dioxo-6-(3-phenylprop-1-enyl)-1,2-dihydroquinazolin-3(4h)-yl)methyl)benzoic acid , reported by pfizer as compound 2 , and n , n - bis(4-fluoro-3-methylbenzyl)pyrimidine-4,6-dicarboxamide , reported by aventis as compound 4 , which are referred to herein as 5 and 4 , respectively . compounds 13 exhibited low to submicromolar ki values ( see table 1 ) , whereas 4 and 5 had ki values of 0.06 0.02 and 0.01 0.0 m , respectively , when tested with the triple - helical fthp-15 substrate . interestingly , all tested compounds , with the exception of 6 ( n - hydroxyacetamide ( aha ) ) , inhibited mmp-13 hydrolysis of fthp-15 via a noncompetitive mechanism ( figure 1 ) . burke plot of inhibition of mmp-13 fthp-15 hydrolysis by ( a ) 1 , ( b ) 2 , ( c ) 3 , ( d ) 4 , ( e ) 5 , and ( f ) 6 . to test whether 1 binds to the active site zn , a known zn binder , 6 ( aha ) , a competitive millimolar range mmp-13 inhibitor ( see table 1 ) , was used in combination with 1 following a previously described methodology.6 ( aha ) was used in the range of 01.25 mm in combination with 1 in the range of 02 m . yonetani theorell plot of mmp-13 fthp-15 hydrolysis in the presence of ( a ) 6 and 1 , ( b ) 4 and 1 , and ( c ) 5 and 1 . 1 was subsequently tested in combination with 4 , which is a noncompetitive , non - zn - binding inhibitor of mmp-13 with a nanomolar ki value ( table 1 ) . the structure of the mmp-13 cat compound 2 inhibitor complex containing two protomers ( a and b ) in the asymmetric unit was refined to a resolution of 1.66 with r and rfree values of 0.134 and 0.200 , respectively ( supplemental table 3 , supporting information ) . compound 2 is observed in both protomers in the s1 specificity pocket ( figure 3b ) , while in protomer a a second 2 binds near the s1 and s2 subsites ( designated s1/s2 * ) within the substrate binding cleft ( figure 3c ) . the color scheme is the same as that in panel a except y176 , f189 , and p190 , which make the majority of contacts with 2 , are highlighted as dark green sticks . ( d ) superposition of compounds 2 ( yellow ) , 4 ( white ) , and 5 ( pink ) in the s1 binding site . a second molecule of 2 can be seen on the other side of catalytic zinc in the s1/s2 * site . ( e ) superposition of compound 2 ( yellow ) with hydroxamic acid - based inhibitors ( pdb code 456c , red ; pdb code 830c , cyan ) that do not intrude deeply into the s1 specificity loop . compound 2 intruded deeply into the hydrophobic s1 pocket such that its p - methylphenyl moiety pointed toward the substrate binding cleft and contacted the hydrophobic region composed of four residues ( leu185 , val219 , pro242 , and tyr244 ) . a cluster of three water molecules ( left side of 2 in figure 3b ) connected the carbonyl oxygen atoms of mmp-13 cat residues pro236 , ala238 , and ile243 and the side chain hydroxyl moiety of thr245 to inhibitor 2 atoms s16 , n18 , and n19 . a second , four - water cluster ( right side of 2 in figure 3b ) connected the 2 o17 atom to the mmp-13 s1 specificity loop ( thr247 side chain hydroxyl oxygen , met253 amide nitrogen , and lys249 , his251 , and met253 carbonyl oxygens ) . to assess the potency of lead compounds with a cognate substrate , compounds 13 were tested in an assay utilizing type ii collagen conjugated to fitc . compounds 1 and 2 appeared to be more potent in the type ii collagen assay than in the fthp-15 assay ( type ii collagen ic50 = 0.7 0.2 and 0.4 0.1 m versus fthp-15 ic50 = 3.4 0.2 and 2.4 0.1 m , respectively ) . interestingly , while 4 clearly appeared more potent in the fthp-15 assay than compounds of the 1 chemotype , the difference in apparent potency was much less pronounced in the type ii collagen assay . in the fthp-15 assay compounds 13 appeared to be partial inhibitors , whereas 4 inhibited hydrolysis completely ( figure 4a ) . results of ( a ) fthp-15 , ( b ) type ii collagen , and ( c ) cartilage explant assays . in the type ii collagen assay ( b ) all four compounds exhibit concentration response curves characteristic of partial inhibitors . to assess the selectivity of compounds 13 , they were tested against a panel of 30 proteases in 12-point 3-fold dilution dose response curve format in a single dose . when tested against 15 metalloproteases ( figure 5a ) , the only enzyme inhibited in excess of 20% by all five tested compounds was mmp-13 . compounds 13 appear to have activity in excess of 10% against mmp-2 , mmp-3 , mmp-9 , mmp-12 , and ace ; however , examination of the full range of tested concentrations ( 20 m to 0.1 nm ) revealed the absence of dose - dependent responses ( supplemental figure 1a , supporting information ) and therefore must represent an artifact . in addition to the metalloprotease panel , compounds were tested against representatives of the serine , cysteine , and aspartic classes of proteases ( figure 5b ) . to assess possible toxicity , compounds 13 were screened against a panel of cytochrome p450 ( cyp ) enzymes in seven - point dose response format in duplicate . compounds 1 and 2 exhibited low micromolar potency against cyp 1a2 , 2c9 , and 2c19 ( table 3 ) , while 3 , 4 , and 5 did not inhibit any of the tested cyps in excess of 50% at inhibitor concentrations of 20 , 20 , and 2.5 m , respectively . inhibitors 13 exhibited low metabolic stability in the microsomes of all three species tested , with the best half - life of 10.4 min exhibited by 1 in human liver microsomes ( table 4 ) . compounds 13 are thioethers and , therefore , could potentially be metabolized in vivo by several members of cyp or flavin - containing monooxygenase ( fmo ) families to sulfoxides or sulfones , which could lead to the limitation of oral bioavailability due to a loss of activity . to test this hypothesis , we attempted to synthesize the sulfone and sulfoxide derivatives of 2 ; however , they proved to be unstable and decomposed during attempts at chromatographic purification . as a result of an hts campaign and subsequent sar studies , we identified a series of mmp-13 inhibitors that exhibited a good selectivity profile toward other members of the mmp family . a lack of obvious zn - binding moieties , preferential inhibition of triple - helical peptide hydrolysis , and a partial inhibitor profile of a lead of a series , 1 , suggested inhibition by a mechanism other than competitive . indeed , mechanistic characterization of these compounds revealed that they inhibit mmp-13 via a noncompetitive mechanism . the noncompetitive mechanism of inhibition of mmp-13 by our lead compounds in combination with results of the dual inhibition study with a zn binder ( aha ) strongly suggested the possibility of binding outside of an active site at a so - called exosite . indeed , pfizer and aventis identified small - molecule inhibitors of mmp-13 that bind within one of these exosites to the so - called specificity loop and inhibit catalysis by inducing rigidity . crystallographic analyses revealed a second binding region for 2 ( the s1 and s2 subsites ) that is novel compared to those of all known mmp-13 inhibitors . superposition of 2 , 4 , and 5 in their respective binding poses in the s1 subsite suggested that 2 and 4 or 5 can not simultaneously reside in the s1 subsite ( figure 3d ) . since both 4 and 5 have much greater affinity than 2 , this suggested that when 4 or 5 is present in the s1 subsite , 2 can only bind to the second binding site , explaining the lack of mutually exclusive binding ( figure 2b , c ) between 2 and 4 or 5 . indeed , despite demonstrating a good selectivity profile comparable to that of compounds developed by aventis ( 4 ) and pfizer ( 5 ) , compounds 13 have 10100 times lower affinity than 4 and 5 . thus , prevention of collagen degradation in vitro provides an important estimate of the ability of mmp-13 inhibitors to modify the course of the disease . development of roughly 30 antiarthritic drugs has been discontinued due to the occurrence of mss as a result of mmp inhibitor dosing in the clinical trials . mss does not appear to be caused by the off - target inhibition of a single enzyme , but rather a combination of several mmps and possibly other related enzymes . with that in mind , 13 were screened against an expanded panel of proteases , which included proposed arthritis antitargets ( mmp-1 , mmp-2 , mmp-9 , and mt1-mmp ) and representative adamalysins ( adam10 and adam17 ( tace ) ) . compound 5 was previously tested in the rat model of mss and was found to be safe at plasma concentrations well above those needed to protect articular cartilage and therefore represents an excellent control for a selectivity study . while the usefulness of this protease panel in predicting mss occurrence has not been established , the similarity of the protease inhibition profile of compounds 13 to that of 5 suggested a similar in vivo safety profile . drug drug or drug food interactions due to the inhibition or induction of members of the cytochrome p450 enzyme family are believed to be one of the main causes of these effects . approximately 95% of all drug metabolism is attributable to just five representatives of the cyp family , with the 3a4 enzyme being responsible for almost half of these reactions . while the overall good inhibition profile of compounds 13 is encouraging , these data suggested the possibility of in vivo drug drug interactions due to the inhibition of 3a4 , 1a2 , 2c9 , and 2c19 . there are examples of thioether - containing drugs and preclinical candidates with good in vivo efficacy , suggesting that metabolic stability of the molecule is a function of the structure and properties of the molecule as a whole , and not just one particular atom in the structure . the lead compound of this novel class of mmp-13 inhibitors , 1 , was discovered as a result of an hts campaign under the auspices of the nih roadmap initiative . additional compounds belonging to the same chemotype were identified as a result of a medicinal chemistry optimization effort as a part of the same program . as a result of the characterization studies presented here , it was determined that compounds 13 inhibit mmp-13 via a noncompetitive mechanism with the possibility of binding to two exosites . type ii collagen and cartilage protective effects exhibited by these mmp-13 inhibitors in vitro indicated the possibility of these compounds being efficacious in future in vivo studies . these compounds were also highly selective when tested against a panel of 30 proteases , which , in combination with a good cyp inhibition profile , suggested low off - target toxicity and drug drug interactions in humans . proteases used in the protease profiling panel were from r&d systems ( ace , ace2 , adam10 , adam17 , furin , ide , neprilysin , thrombin , upa ) , invitrogen ( bace ) , biomol ( mmp-13 , -79 , and -1214 , caspases 13 and 57 ) , and calbiochem ( factor xa , cathepsin d , l , s , and k ) . data from kinetic experiments utilizing a matrix of two different inhibitor concentrations were fitted to the yonetani theorell equation : where vij is the initial velocity of fthp-15 hydrolysis in the presence of both inhibitors , v0 is the initial velocity of fthp-15 hydrolysis in the uninhibited reaction , ki and kj are the dissociation constants for inhibitors i and j , respectively , and b is the parameter that describes the effect of binding of inhibitor i on the affinity of inhibitor j. when two inhibitors bind in a mutually exclusive manner , b = . the initial rates of fthp-15 hydrolysis in the presence of two inhibitors were arranged as dixon plots with 1/vij plotted as a function of [ i ] in the presence of varying concentrations of inhibitor j. the recombinant mmp-13 catalytic domain ( mmp-13 cat , residues 104274 ) was expressed , purified , and refolded as described . the reconstituted enzyme , which was fully functional in the modified knight substrate assay , was concentrated to 0.5 mg / ml ( mmp-13-cat 280 = 28 590 mol cm ) and made 25% ( v / v ) in glycerol for storage at 80 c . the highest concentrations of compounds to be used in the assays were determined . the compounds were solubilized at 50 m in protease assay buffers , and the absorbance at = 540 nm was measured and compared to that of buffer alone . the activity in the presence of a compound was plotted as a function of the compound concentration , and the curves were fitted using the built - in dose response model algorithm of xlfit software ( idbs ) . metabolites were quantified by using the biocius rapidfire ultra - high - throughput mass spectrometer at biocius life sciences inc .

obesity increases the risk of heart failure ( hf)1 and cardiovascular death in the general population.2 however , a significant survival benefit of obesity in patients with established hf has been extensively described in different settings : short and longterm followup , acute and chronic hf , and in reduced and preserved ejection fraction.3 , 4 , 5 , 6 , 7 the prevalence of overweight or obesity in patients with hf can reach about 35% to 45%8 , 9 when defined using body mass index ( bmi ) . among weight indices , bmi has been shown to properly correlate with the thickness of the subcutaneous fat layer10 and has been rapidly adopted as the primary obesity index in hf . other parameters used for measuring body composition ( waist circumference , skinfold estimates of percentage of body fat , dexa , and bioelectrical impedance analysis ) have also demonstrated an independent relationship with mortality in hf patients,11 , 12 , 13 although none has proved to be clearly superior to bmi ; thus , the use of bmi in hf remains routine clinical practice . despite the potential benefits of weight loss ( wl ) in obese and overweight patients , this recommendation may not apply to patients with hf . indeed , both the american college of cardiology / american heart association and the european society of cardiology guidelines in hf do not provide conclusive recommendations about wl.14 , 15 moreover , the prognostic impact of wl in obese patients with hf is not completely known , despite possible beneficial effects of wl on hemodynamics , cardiac structure , diastolic function , and even systolic function.16 robust evidence regarding the relation of wl and longterm prognosis in patients with chronic hf is missing.17 , 18 , 19 accordingly , our aim was to analyze the prevalence of wl over a 1year followup period and its impact on mortality in a large , reallife , outpatient population with hf , with special attention to obese patients . all consecutive , ambulatory patients referred between august 1 , 2001 and september 30 , 2011 to a structured hf clinic at a university hospital , irrespective of etiology , with available weight data at the first visit and at a 1year followup visit were included in an outpatient setting . the criteria for clinical practice referral to the hf clinic have been reported elsewhere.20 , 21 briefly , the criteria were hf with at least one hospitalization and/or reduced left ventricular ejection fraction ( lvef ) of < 40% . most patients were referred from cardiology and internal medicine departments , and fewer were from the emergency room / shortstay unit or other hospital departments . less than 10% of patients were admitted to the hf unit due to asymptomatic , reduced lvef after acute myocardial infarction . bmi was analyzed according to weight and height using the formula weight ( kg)/(height [ m ] ) . patients were classified according to bmi into 4 strata following the criteria defined by the world health organization ( who technical report series , no 854 , geneva , 1999 ) as follows : low weight ( bmi < 20.5 kg / m ) , normal weight ( bmi 20.5 to < 25.5 kg / m ) , overweight ( bmi 25.5 to < 30 kg / m ) , and obese ( bmi 30 kg / m ) . significant wl was defined as a loss of 5% from the initial weight ( ie , baseline ) to end of first year of followup . all patients were seen regularly during followup visits at the hf clinic according to their clinical needs . the followup visit schedule ( nurses and physicians ) has been reported elsewhere.21 , 22 losing weight with dietary restrictions was not routinely advised for overweight , nor for the majority of obese patients , although a healthy diet and physical exercise is always recommended . the nutritionist at the hf unit was primarily involved in undernourished patients with lack of appetite . patients in nyha class ii to iv were evaluated by a rehabilitation physician and an exercise program was offered . nevertheless , less than one third of the patients in the present study actually participated in such an established program . all patients provided written consent for analytical samples and the use of their clinical data for research purposes . the study was performed in compliance with laws protecting personal data in accordance with the international guidelines on clinical investigation of the world medical association 's declaration of helsinki , it was approved by the local ethics committee , and all patients signed informed consent . at the first visit , we recorded the patient 's demographic characteristics , baseline clinical status , physical examination data , and treatment following a standardized protocol the number and causes of death during followup were obtained from clinical records at the hf clinic , other hospital departments , other hospital records , or by contacting the patient 's relatives . continuous variables were described by the meansd , or median and 25th to 75th percentiles ( q1q3 ) for cases with skewed distribution . statistical differences between groups were assessed using the test for categorical variables , student t test for continuous variables with normal distribution , or the mann cox proportional hazards regression analyses were performed using allcause mortality and also cardiovascular mortality as the dependent variable and significant wl , as defined , as the independent variable . afterwards , multivariable analyses were also performed , including as covariates age , sex , new york heart association ( nyha ) functional class , hf duration , lvef , etiology of hf , diabetes , baseline bmi , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists . these analyses were repeated after categorizing bmi in 2 groups : obese or nonobese , which included underweight , normal weight , and overweight . also , adjusted survival curves for allcause and cardiovascular death were plotted according to the presence or absence of significant wl for both obese and nonobese patients . finally , the cox regression multivariable analyses were repeated using standardized wl as continuous variable ( with 1 sd decrease ) . statistical analyses were performed using spss 15 ( spss inc , chicago , il ) . all consecutive , ambulatory patients referred between august 1 , 2001 and september 30 , 2011 to a structured hf clinic at a university hospital , irrespective of etiology , with available weight data at the first visit and at a 1year followup visit were included in an outpatient setting . the criteria for clinical practice referral to the hf clinic have been reported elsewhere.20 , 21 briefly , the criteria were hf with at least one hospitalization and/or reduced left ventricular ejection fraction ( lvef ) of < 40% . most patients were referred from cardiology and internal medicine departments , and fewer were from the emergency room / shortstay unit or other hospital departments . less than 10% of patients were admitted to the hf unit due to asymptomatic , reduced lvef after acute myocardial infarction . bmi was analyzed according to weight and height using the formula weight ( kg)/(height [ m ] ) . patients were classified according to bmi into 4 strata following the criteria defined by the world health organization ( who technical report series , no 854 , geneva , 1999 ) as follows : low weight ( bmi < 20.5 kg / m ) , normal weight ( bmi 20.5 to < 25.5 kg / m ) , overweight ( bmi 25.5 to < 30 kg / m ) , and obese ( bmi 30 kg / m ) . significant wl was defined as a loss of 5% from the initial weight ( ie , baseline ) to end of first year of followup . all patients were seen regularly during followup visits at the hf clinic according to their clinical needs . the followup visit schedule ( nurses and physicians ) has been reported elsewhere.21 , 22 losing weight with dietary restrictions was not routinely advised for overweight , nor for the majority of obese patients , although a healthy diet and physical exercise is always recommended . the nutritionist at the hf unit was primarily involved in undernourished patients with lack of appetite . patients in nyha class ii to iv were evaluated by a rehabilitation physician and an exercise program was offered . nevertheless , less than one third of the patients in the present study actually participated in such an established program . all patients provided written consent for analytical samples and the use of their clinical data for research purposes . the study was performed in compliance with laws protecting personal data in accordance with the international guidelines on clinical investigation of the world medical association 's declaration of helsinki , it was approved by the local ethics committee , and all patients signed informed consent . at the first visit , we recorded the patient 's demographic characteristics , baseline clinical status , physical examination data , and treatment following a standardized protocol . the number and causes of death during followup were obtained from clinical records at the hf clinic , other hospital departments , other hospital records , or by contacting the patient 's relatives . continuous variables were described by the meansd , or median and 25th to 75th percentiles ( q1q3 ) for cases with skewed distribution . statistical differences between groups were assessed using the test for categorical variables , student t test for continuous variables with normal distribution , or the mann cox proportional hazards regression analyses were performed using allcause mortality and also cardiovascular mortality as the dependent variable and significant wl , as defined , as the independent variable . afterwards , multivariable analyses were also performed , including as covariates age , sex , new york heart association ( nyha ) functional class , hf duration , lvef , etiology of hf , diabetes , baseline bmi , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists . these analyses were repeated after categorizing bmi in 2 groups : obese or nonobese , which included underweight , normal weight , and overweight . also , adjusted survival curves for allcause and cardiovascular death were plotted according to the presence or absence of significant wl for both obese and nonobese patients . finally , the cox regression multivariable analyses were repeated using standardized wl as continuous variable ( with 1 sd decrease ) . statistical analyses were performed using spss 15 ( spss inc , chicago , il ) . of 1322 patients admitted to the hf unit , a total of 1000 patients ( 72.7% men ; mean age 65.812.1 years ) were included in the study . causes for noninclusion were as follows : 139 patients died during the first year of followup , 152 did not attend the 1year visit , and 31 had no weight available ( wheelchair or impossibility to stand up ) . the patients were predominantly male , with a median duration of hf of 10.5 months ( q1q3 248 months ) , and a mean lvef of 32.412.6% . one hundred seventy patients ( 17% ) experienced significant wl during the first year of followup . figure 1 illustrates the percentage of patients with wl 5% , showing a significantly higher rate in overweight and obese patients . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; crt , cardiac resynchronization therapy ; dcm , dilated cardiomyopathy ; hf , heart failure ; hscrp , highsensitivity creactive protein ; hstnt , high sensitivity cardiac troponin t ; hyp , hypertension ; icd , implantable cardiac defibrillator ; ihd , ischemic heart disease ; lvef , left ventricular ejection fraction ; meds , medications ; mra , mineralocorticoid receptor antagonist ; ntprobnp , nterminal probrain natriuretic peptide ; nyha , new york heart association ; st2 , highsensitivity soluble st2 ; stfr , soluble transferrin receptor ; wl , weight loss . ntprobnp available in 422 patients ; st2 available in 340 patients ; hscrp , hstnt , and stfr available in 332 patients . significant wl was considered the loss of 5% of the initial weight during the first year of followup . in addition to sex , most were related to hf severity and treatment . in a limited sample of patients , we have data on biomarkers , without differences between both groups ; only highsensitivity troponin t tended to be higher in patients with significant wl ( p=0.05 , table 1 ) . after 3 years of followup from the first visit ( ie , 2 years after the second weight assessment ) , 174 patients ( 17.4% ) died121 from cardiovascular causes ( 54 worsening hf , 30 sudden death , 11 acute myocardial infarction , 5 stroke , and 21 other ) , 40 from noncardiovascular causes , and 13 from unknown causes . mortality in significant wl patients was significantly higher than in patients without significant wl ( 27.6% versus 15.3% , p<0.001 ) . this was basically due to cardiovascular mortality ( 20.0% versus 10.9% , respectively , p=0.01 ) , whereas differences in noncardiovascular mortality were nonsignificant ( 6.0% versus 3.7% , respectively , p=0.16 ) . death due to worsening hf was 11.2% and 4.2% , respectively , p<0.001 . in univariable cox regression analysis , patients with significant wl had 2fold allcause and cardiovascular higher mortality ( hazard ratio [ hr ] 1.95 [ 95% ci 1.392.72 ] , p<0.001 and hr 2.06 [ 95% ci 1.393.06 ] , p<0.001 , respectively ) . in the multivariable model adjusted by and age , sex , bmi , nyha functional class , lvef , hf duration , ischemic etiology , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists , significant wl remained highly and independently associated with higher allcause mortality ( hr 1.89 [ 95% ci 1.322.68 ] , p<0.001 ) ( table 2 ) . among obese hf patients , significant wl was associated with an even higher risk of allcause death ( adjusted hr 2.38 [ 95% ci 1.314.32 ] , p=0.004 ) than that observed in nonobese patients ( adjusted hr 1.83 [ 95% ci 1.162.89 ] , p=0.01 ) ( table 2 ) . figure 2 shows adjusted survival curves for allcause death relative to the presence or absence of significant wl for nonobese and obese patients . multivariable cox regression analysis for allcause death acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . adjusted survival curves for allcause death according to the presence of significant weight loss . a , nonobese patients . survival curves plotted from the multivariate analysis that included age , sex , new york heart association functional class , left ventricular ejection fraction , etiology of heart failure , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists as covariates . focus in cardiovascular death provided similar results : significant wl remained highly and independently associated with higher cardiovascular mortality ( hr 1.89 [ 95% ci 1.292.90 ] , p=0.003 ) ( table 3 ) , and again among obese hf patients , significant wl was associated with higher risk of cardiovascular death ( adjusted hr 2.51 [ 95% ci 1.235.14 ] , p=0.01 ) than that observed in nonobese patients ( adjusted hr 1.75 [ 95% ci 1.003.06 ] , p<0.05 ) ( table 3 ) . figure 3 shows adjusted survival curves for cardiovascular death relative to the presence or absence of significant wl for nonobese and obese patients . tables 4 and 5 show cox regression multivariable analyses for allcause and cardiovascular death , respectively , using standardized wl as continuous variable ( with 1 sd decrease ) . multivariable cox regression analysis for cardiovascular death acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; hr , hazard ratio ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . survival curves plotted from the multivariate analysis that included age , sex , new york heart association functional class , left ventricular ejection fraction , etiology of heart failure , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitorsangiotensin ii receptor blockers , and mineralocorticoid receptor antagonists as covariates . multivariable cox regression analysis for allcause death , using weight loss as continuous variable acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; hr , hazard ratio ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . multivariable cox regression analysis for cardiovascular death , using weight loss as continuous variable acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . thirteen patients excluded because of unknown cause of death . per 1 sd decrease in weight . the main conclusion of this study may seem perplexing , which is that significant wl might be not beneficial even in obese patients with hf . this finding may , in part , provide a rationale for the obesity paradox in hf , which is extensively described,3 , 4 , 5 , 6 , 7 not universally observed,22 , 23 , 24 , 25 , 26 and incompletely understood . obese patients with hf are thought to have metabolic reserves to better tolerate the catabolic stress of hf , thus explaining the better prognosis . however , the occurrence of unintentional wl may be a surrogate for the loss of metabolic reserves in obese patients and may be the trigger for adverse clinical outcomes . on the other hand , being obese while having hf seems incongruous with undernourishment , which is associated with a very poor prognosis in patients with chronic hf.27 indeed , it is possible that wl secondary to malnutrition might exacerbate underlying undernourishment . purposeful wl is usually recommended for patients with hf and morbid obesity . as suggested by lavie et al , this seems particularly sound for those with a bmi 40 kg / m and reasonable for most hf patients with bmi of 35 kg / m.16 a few reports with small sample populations exist on the effect of intentional weight reduction on quality of life and cardiac function in patients with hf . mariotti et al evaluated the impact of a planned body weight reduction plan on quality of life and cardiac function in 34 obese and overweight chronic hf patients through a 6month nutritional and physical activity program . those patients who achieved a loss of at least 3 kg ( about 3.2% of wl ) showed a significant improvement in lvef and mean nyha functional class and quality of life . the study did not evaluate mortality.28 another small randomized clinical trial evaluated the benefit of losing weight via a lipase inhibitor assisted diet in 21 severely obese patients . significant wl ( 5% absolute reduction in body weight ) and improvement in the 6minute walking test and functional class were observed in the treated group at 12 weeks.29 again , no analysis of mortality could be performed due to the small sample size and short followup . data on unintentional wl in obese patients are even scarcer . in a posthoc analysis of the solvd trial , anker et al30 were the first to suggest that any wl independent of the patients ' weight at baseline is related to poor survival , although no specific comment on obese patients was reported . more recently , rossignol et al31 observed significant wl in 16.4% and 15.7% of patients from the gissihf and valheft studies , respectively , with results quite similar to ours . as in valheft , we found that significant wl ( using similar criteria ) was associated with female sex , worse nyha functional class , higher bmi , and less use of blockers . it has been suggested that blockers may exert anticachexic effects by inhibiting catecholamineinduced lipolysis.32 we also found an association between significant wl and treatment with mineralocorticoid receptor antagonists and loop diuretics the latter of which was also observed in gissihf and with digoxin , treatments all associated with the severity of hf . whether biomarkers are useful to mirror or as surrogate markers of wl is unknown . song et al33 showed an independent association between highsensitivity creactive protein and unintentional wl in 243 patients with hf enrolled during an index hospitalization for hf exacerbation ( or , 1.49 ; 95% ci , 1.151.92 ) . in the valheft biomarker substudy , both nterminal probrain natriuretic peptide and highsensitivity creactive protein were associated with significant wl.31 in our study , we found no association between significant wl and any of these 2 biomarkers or with st2 and soluble transferrin receptor . only highsensitivity troponin t tended to be higher in patients with significant wl ( p=0.05 ) , which may eventually indicate myocyte damage caused by wl itself . from a prognostic point of view , significant wl was independently associated with mortality , with an increased risk of death of 20% ( gissihf ) to 150% ( valheft).31 in our study , the risk of death for significant wl after adjustment in the multivariable model was 89% higher . very remarkably , among obese patients ( bmi 30 kg / m ) , significant wl was associated with an even higher risk of death ( 138% increase ) than in nonobese patients ( 83% increase ) . this finding was very unexpected , even more so considering that lowweight patients were not excluded from the analysis . in an analysis of the charm study,34 those patients with 5% or greater wl in 6 months had a > 50% increase in hazard both for cardiovascular and for other causes of death compared to those with stable weight . the impact of wl on mortality was not significantly related to bmi , although wl carried a particularly high risk in patients who were already lean at study entry . in our study , in contrast , cardiovascular mortality was almost doubled and the patients with highest adjusted hazard of death were the obese patients . remarkably , in our study the higher mortality rate observed in patients with significant wl was mainly caused by worsening hf . on the other hand , although noncardiovascular death was nonnegligible in our series ( 23% ) , it was nonsignificantly higher in patients with significant wl . the rica registry35 analyzed weight changes in 731 patients admitted with acute hf during a 1year followup . these results , observed in the acute setting in decompensated patients , are in disagreement with our data and those of others obtained in ambulatory , stable patients without overt congestion . finally , it has been suggested that incorporating physical activity and exercise training into a purposeful wl program might be of benefit in patients with hf.36 in the hfaction study , nonsignificant reductions in allcause mortality and hospitalization were observed across bmi categories , although no association with wl was identified.9 further studies are needed to ascertain whether this strategy might improve outcomes in obese patients with hf . the present data are based on bmibased wl without further anthropometric characterization ( muscle or fat mass wasting assessments ) , and we can not fully ascertain whether wl was in part intentional or nonintentional . however , the result of intentional wl is difficult to address , and most of the analyses performed in hf trials included both intentional and nonintentional wl as we did . we defined significant wl as a loss of 5% of the initial weight during the first year of followup , as previously described.31 , 34 , 35 this cutoff can be considered arbitrary as were all the definitions used in previous studies ; however , no definite cutoff exists . we only found statistically significant differences between patients with and without significant wl in nyha functional class , sex , and baseline bmi ; nevertheless , we can not completely discard the possibility that they were sicker from the start , since significant wl is just a marker of such a situation without playing a causative role . further studies are needed to fully clarify the putative causative role of significant wl in outcomes . we have no data on what happened to weight during followup between year 1 and year 3 or the time of death , and we only analyzed weight loss during the first year of followup . in the present study , we focused on stable outpatients , but whether subclinical congestion may have influenced our results remains a caveat . although our population is a general population attending a hf unit , the unit is located at a tertiary university hospital and the cohort patients are mainly male and of ischemic etiology , with the great majority having had a hospital admission in the previous year or a depressed lvef . therefore , we can not disregard the possibility of bias due to selection of patients who may not necessarily represent the general hf population . the present data are based on bmibased wl without further anthropometric characterization ( muscle or fat mass wasting assessments ) , and we can not fully ascertain whether wl was in part intentional or nonintentional . however , the result of intentional wl is difficult to address , and most of the analyses performed in hf trials included both intentional and nonintentional wl as we did . we defined significant wl as a loss of 5% of the initial weight during the first year of followup , as previously described.31 , 34 , 35 this cutoff can be considered arbitrary as were all the definitions used in previous studies ; however , no definite cutoff exists . we only found statistically significant differences between patients with and without significant wl in nyha functional class , sex , and baseline bmi ; nevertheless , we can not completely discard the possibility that they were sicker from the start , since significant wl is just a marker of such a situation without playing a causative role . further studies are needed to fully clarify the putative causative role of significant wl in outcomes . we have no data on what happened to weight during followup between year 1 and year 3 or the time of death , and we only analyzed weight loss during the first year of followup . in the present study , we focused on stable outpatients , but whether subclinical congestion may have influenced our results remains a caveat . although our population is a general population attending a hf unit , the unit is located at a tertiary university hospital and the cohort patients are mainly male and of ischemic etiology , with the great majority having had a hospital admission in the previous year or a depressed lvef . therefore , we can not disregard the possibility of bias due to selection of patients who may not necessarily represent the general hf population . weight loss of 5% during 1 year in ambulatory hf patients was associated with an ominous prognosis in the subsequent 2 years . significant wl might not be beneficial in obese hf patients , and indiscriminate advice to lose weight in this population might not be indicated . further studies are needed to ascertain whether wl and the mode of wl achievement might ultimately be beneficial in obese or even very obese patients with hf . this work was supported by redes temticas de investigacin cooperativa en salud ( retics ) , and red cardiovascular ( rd12/0042/0047 ) .

backgroundin heart failure ( hf ) , weight loss ( wl ) has been associated with an adverse prognosis whereas obesity has been linked to lower mortality ( the obesity paradox ) . the impact of wl in obese patients with hf is incompletely understood . our objective was to explore the prevalence of wl and its impact on longterm mortality , with an emphasis on obese patients , in a cohort of patients with chronic hf.methods and resultsweight at first visit and the 1year followup and vital status after 3 years were assessed in 1000 consecutive ambulatory , chronic hf patients ( 72.7% men ; mean age 65.812.1 years ) . significant wl was defined as a loss of 5% weight between baseline and 1 year . obesity was defined as body mass index 30 kg / m2 ( n=272 ) . of the 1000 patients included , 170 experienced significant wl during the first year of followup . mortality was significantly higher in patients with significant wl ( 27.6% versus 15.3% , p<0.001 ) . in univariable cox regression analysis , patients with significant wl had 2fold higher mortality ( hazard ratio 1.95 [ 95% ci 1.392.72 ] , p<0.001 ) . in multivariable analysis , adjusting for age , sex , body mass index , new york heart association functional class , left ventricular ejection fraction , hf duration , ischemic etiology , diabetes , and treatment , significant wl remained independently associated with higher mortality ( hazard ratio 1.89 [ 95% ci 1.322.68 ] , p<0.001 ) . among obese patients with hf , significant wl was associated with an even more ominous prognosis ( adjusted hazard ratio for death of 2.38 [ 95% ci 1.314.32 ] , p=0.004 ) than that observed in nonobese patients ( adjusted hazard ratio 1.83 [ 95% ci 1.162.89 ] , p=0.01).conclusionsweight loss 5% in patients with chronic hf was associated with high longterm mortality , particularly among obese patients with hf .

Introduction Subjects and Methods Design and Study Population Mortality Assessment Statistical Analysis Results Discussion Limitations Conclusions Sources of Funding Disclosures

obesity increases the risk of heart failure ( hf)1 and cardiovascular death in the general population.2 however , a significant survival benefit of obesity in patients with established hf has been extensively described in different settings : short and longterm followup , acute and chronic hf , and in reduced and preserved ejection fraction.3 , 4 , 5 , 6 , 7 the prevalence of overweight or obesity in patients with hf can reach about 35% to 45%8 , 9 when defined using body mass index ( bmi ) . despite the potential benefits of weight loss ( wl ) in obese and overweight patients , this recommendation may not apply to patients with hf . indeed , both the american college of cardiology / american heart association and the european society of cardiology guidelines in hf do not provide conclusive recommendations about wl.14 , 15 moreover , the prognostic impact of wl in obese patients with hf is not completely known , despite possible beneficial effects of wl on hemodynamics , cardiac structure , diastolic function , and even systolic function.16 robust evidence regarding the relation of wl and longterm prognosis in patients with chronic hf is missing.17 , 18 , 19 accordingly , our aim was to analyze the prevalence of wl over a 1year followup period and its impact on mortality in a large , reallife , outpatient population with hf , with special attention to obese patients . all consecutive , ambulatory patients referred between august 1 , 2001 and september 30 , 2011 to a structured hf clinic at a university hospital , irrespective of etiology , with available weight data at the first visit and at a 1year followup visit were included in an outpatient setting . the criteria for clinical practice referral to the hf clinic have been reported elsewhere.20 , 21 briefly , the criteria were hf with at least one hospitalization and/or reduced left ventricular ejection fraction ( lvef ) of < 40% . patients were classified according to bmi into 4 strata following the criteria defined by the world health organization ( who technical report series , no 854 , geneva , 1999 ) as follows : low weight ( bmi < 20.5 kg / m ) , normal weight ( bmi 20.5 to < 25.5 kg / m ) , overweight ( bmi 25.5 to < 30 kg / m ) , and obese ( bmi 30 kg / m ) . significant wl was defined as a loss of 5% from the initial weight ( ie , baseline ) to end of first year of followup . the followup visit schedule ( nurses and physicians ) has been reported elsewhere.21 , 22 losing weight with dietary restrictions was not routinely advised for overweight , nor for the majority of obese patients , although a healthy diet and physical exercise is always recommended . at the first visit , we recorded the patient 's demographic characteristics , baseline clinical status , physical examination data , and treatment following a standardized protocol the number and causes of death during followup were obtained from clinical records at the hf clinic , other hospital departments , other hospital records , or by contacting the patient 's relatives . afterwards , multivariable analyses were also performed , including as covariates age , sex , new york heart association ( nyha ) functional class , hf duration , lvef , etiology of hf , diabetes , baseline bmi , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists . all consecutive , ambulatory patients referred between august 1 , 2001 and september 30 , 2011 to a structured hf clinic at a university hospital , irrespective of etiology , with available weight data at the first visit and at a 1year followup visit were included in an outpatient setting . the criteria for clinical practice referral to the hf clinic have been reported elsewhere.20 , 21 briefly , the criteria were hf with at least one hospitalization and/or reduced left ventricular ejection fraction ( lvef ) of < 40% . patients were classified according to bmi into 4 strata following the criteria defined by the world health organization ( who technical report series , no 854 , geneva , 1999 ) as follows : low weight ( bmi < 20.5 kg / m ) , normal weight ( bmi 20.5 to < 25.5 kg / m ) , overweight ( bmi 25.5 to < 30 kg / m ) , and obese ( bmi 30 kg / m ) . significant wl was defined as a loss of 5% from the initial weight ( ie , baseline ) to end of first year of followup . the followup visit schedule ( nurses and physicians ) has been reported elsewhere.21 , 22 losing weight with dietary restrictions was not routinely advised for overweight , nor for the majority of obese patients , although a healthy diet and physical exercise is always recommended . at the first visit , we recorded the patient 's demographic characteristics , baseline clinical status , physical examination data , and treatment following a standardized protocol . afterwards , multivariable analyses were also performed , including as covariates age , sex , new york heart association ( nyha ) functional class , hf duration , lvef , etiology of hf , diabetes , baseline bmi , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists . of 1322 patients admitted to the hf unit , a total of 1000 patients ( 72.7% men ; mean age 65.812.1 years ) were included in the study . causes for noninclusion were as follows : 139 patients died during the first year of followup , 152 did not attend the 1year visit , and 31 had no weight available ( wheelchair or impossibility to stand up ) . one hundred seventy patients ( 17% ) experienced significant wl during the first year of followup . figure 1 illustrates the percentage of patients with wl 5% , showing a significantly higher rate in overweight and obese patients . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; crt , cardiac resynchronization therapy ; dcm , dilated cardiomyopathy ; hf , heart failure ; hscrp , highsensitivity creactive protein ; hstnt , high sensitivity cardiac troponin t ; hyp , hypertension ; icd , implantable cardiac defibrillator ; ihd , ischemic heart disease ; lvef , left ventricular ejection fraction ; meds , medications ; mra , mineralocorticoid receptor antagonist ; ntprobnp , nterminal probrain natriuretic peptide ; nyha , new york heart association ; st2 , highsensitivity soluble st2 ; stfr , soluble transferrin receptor ; wl , weight loss . significant wl was considered the loss of 5% of the initial weight during the first year of followup . in a limited sample of patients , we have data on biomarkers , without differences between both groups ; only highsensitivity troponin t tended to be higher in patients with significant wl ( p=0.05 , table 1 ) . after 3 years of followup from the first visit ( ie , 2 years after the second weight assessment ) , 174 patients ( 17.4% ) died121 from cardiovascular causes ( 54 worsening hf , 30 sudden death , 11 acute myocardial infarction , 5 stroke , and 21 other ) , 40 from noncardiovascular causes , and 13 from unknown causes . mortality in significant wl patients was significantly higher than in patients without significant wl ( 27.6% versus 15.3% , p<0.001 ) . in univariable cox regression analysis , patients with significant wl had 2fold allcause and cardiovascular higher mortality ( hazard ratio [ hr ] 1.95 [ 95% ci 1.392.72 ] , p<0.001 and hr 2.06 [ 95% ci 1.393.06 ] , p<0.001 , respectively ) . in the multivariable model adjusted by and age , sex , bmi , nyha functional class , lvef , hf duration , ischemic etiology , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists , significant wl remained highly and independently associated with higher allcause mortality ( hr 1.89 [ 95% ci 1.322.68 ] , p<0.001 ) ( table 2 ) . among obese hf patients , significant wl was associated with an even higher risk of allcause death ( adjusted hr 2.38 [ 95% ci 1.314.32 ] , p=0.004 ) than that observed in nonobese patients ( adjusted hr 1.83 [ 95% ci 1.162.89 ] , p=0.01 ) ( table 2 ) . multivariable cox regression analysis for allcause death acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . survival curves plotted from the multivariate analysis that included age , sex , new york heart association functional class , left ventricular ejection fraction , etiology of heart failure , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitors angiotensin ii receptor blockers , and mineralocorticoid receptor antagonists as covariates . focus in cardiovascular death provided similar results : significant wl remained highly and independently associated with higher cardiovascular mortality ( hr 1.89 [ 95% ci 1.292.90 ] , p=0.003 ) ( table 3 ) , and again among obese hf patients , significant wl was associated with higher risk of cardiovascular death ( adjusted hr 2.51 [ 95% ci 1.235.14 ] , p=0.01 ) than that observed in nonobese patients ( adjusted hr 1.75 [ 95% ci 1.003.06 ] , p<0.05 ) ( table 3 ) . multivariable cox regression analysis for cardiovascular death acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; hr , hazard ratio ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . survival curves plotted from the multivariate analysis that included age , sex , new york heart association functional class , left ventricular ejection fraction , etiology of heart failure , diabetes , and treatment with blockers , angiotensinconverting enzyme inhibitorsangiotensin ii receptor blockers , and mineralocorticoid receptor antagonists as covariates . multivariable cox regression analysis for allcause death , using weight loss as continuous variable acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; hr , hazard ratio ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . multivariable cox regression analysis for cardiovascular death , using weight loss as continuous variable acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin ii receptor blocker ; bmi , body mass index ; hf , heart failure ; lvef , left ventricular ejection fraction ; mra , mineralocorticoid receptor antagonist ; nyha , new york heart association . the main conclusion of this study may seem perplexing , which is that significant wl might be not beneficial even in obese patients with hf . this finding may , in part , provide a rationale for the obesity paradox in hf , which is extensively described,3 , 4 , 5 , 6 , 7 not universally observed,22 , 23 , 24 , 25 , 26 and incompletely understood . obese patients with hf are thought to have metabolic reserves to better tolerate the catabolic stress of hf , thus explaining the better prognosis . on the other hand , being obese while having hf seems incongruous with undernourishment , which is associated with a very poor prognosis in patients with chronic hf.27 indeed , it is possible that wl secondary to malnutrition might exacerbate underlying undernourishment . as suggested by lavie et al , this seems particularly sound for those with a bmi 40 kg / m and reasonable for most hf patients with bmi of 35 kg / m.16 a few reports with small sample populations exist on the effect of intentional weight reduction on quality of life and cardiac function in patients with hf . mariotti et al evaluated the impact of a planned body weight reduction plan on quality of life and cardiac function in 34 obese and overweight chronic hf patients through a 6month nutritional and physical activity program . those patients who achieved a loss of at least 3 kg ( about 3.2% of wl ) showed a significant improvement in lvef and mean nyha functional class and quality of life . significant wl ( 5% absolute reduction in body weight ) and improvement in the 6minute walking test and functional class were observed in the treated group at 12 weeks.29 again , no analysis of mortality could be performed due to the small sample size and short followup . in a posthoc analysis of the solvd trial , anker et al30 were the first to suggest that any wl independent of the patients ' weight at baseline is related to poor survival , although no specific comment on obese patients was reported . more recently , rossignol et al31 observed significant wl in 16.4% and 15.7% of patients from the gissihf and valheft studies , respectively , with results quite similar to ours . as in valheft , we found that significant wl ( using similar criteria ) was associated with female sex , worse nyha functional class , higher bmi , and less use of blockers . it has been suggested that blockers may exert anticachexic effects by inhibiting catecholamineinduced lipolysis.32 we also found an association between significant wl and treatment with mineralocorticoid receptor antagonists and loop diuretics the latter of which was also observed in gissihf and with digoxin , treatments all associated with the severity of hf . song et al33 showed an independent association between highsensitivity creactive protein and unintentional wl in 243 patients with hf enrolled during an index hospitalization for hf exacerbation ( or , 1.49 ; 95% ci , 1.151.92 ) . in the valheft biomarker substudy , both nterminal probrain natriuretic peptide and highsensitivity creactive protein were associated with significant wl.31 in our study , we found no association between significant wl and any of these 2 biomarkers or with st2 and soluble transferrin receptor . only highsensitivity troponin t tended to be higher in patients with significant wl ( p=0.05 ) , which may eventually indicate myocyte damage caused by wl itself . from a prognostic point of view , significant wl was independently associated with mortality , with an increased risk of death of 20% ( gissihf ) to 150% ( valheft).31 in our study , the risk of death for significant wl after adjustment in the multivariable model was 89% higher . very remarkably , among obese patients ( bmi 30 kg / m ) , significant wl was associated with an even higher risk of death ( 138% increase ) than in nonobese patients ( 83% increase ) . the impact of wl on mortality was not significantly related to bmi , although wl carried a particularly high risk in patients who were already lean at study entry . in our study , in contrast , cardiovascular mortality was almost doubled and the patients with highest adjusted hazard of death were the obese patients . remarkably , in our study the higher mortality rate observed in patients with significant wl was mainly caused by worsening hf . on the other hand , although noncardiovascular death was nonnegligible in our series ( 23% ) , it was nonsignificantly higher in patients with significant wl . finally , it has been suggested that incorporating physical activity and exercise training into a purposeful wl program might be of benefit in patients with hf.36 in the hfaction study , nonsignificant reductions in allcause mortality and hospitalization were observed across bmi categories , although no association with wl was identified.9 further studies are needed to ascertain whether this strategy might improve outcomes in obese patients with hf . we defined significant wl as a loss of 5% of the initial weight during the first year of followup , as previously described.31 , 34 , 35 this cutoff can be considered arbitrary as were all the definitions used in previous studies ; however , no definite cutoff exists . we only found statistically significant differences between patients with and without significant wl in nyha functional class , sex , and baseline bmi ; nevertheless , we can not completely discard the possibility that they were sicker from the start , since significant wl is just a marker of such a situation without playing a causative role . we have no data on what happened to weight during followup between year 1 and year 3 or the time of death , and we only analyzed weight loss during the first year of followup . although our population is a general population attending a hf unit , the unit is located at a tertiary university hospital and the cohort patients are mainly male and of ischemic etiology , with the great majority having had a hospital admission in the previous year or a depressed lvef . we defined significant wl as a loss of 5% of the initial weight during the first year of followup , as previously described.31 , 34 , 35 this cutoff can be considered arbitrary as were all the definitions used in previous studies ; however , no definite cutoff exists . we only found statistically significant differences between patients with and without significant wl in nyha functional class , sex , and baseline bmi ; nevertheless , we can not completely discard the possibility that they were sicker from the start , since significant wl is just a marker of such a situation without playing a causative role . we have no data on what happened to weight during followup between year 1 and year 3 or the time of death , and we only analyzed weight loss during the first year of followup . although our population is a general population attending a hf unit , the unit is located at a tertiary university hospital and the cohort patients are mainly male and of ischemic etiology , with the great majority having had a hospital admission in the previous year or a depressed lvef . weight loss of 5% during 1 year in ambulatory hf patients was associated with an ominous prognosis in the subsequent 2 years . significant wl might not be beneficial in obese hf patients , and indiscriminate advice to lose weight in this population might not be indicated . further studies are needed to ascertain whether wl and the mode of wl achievement might ultimately be beneficial in obese or even very obese patients with hf .

cell lines and culture conditions : four cell lines ( provided by the university of wisconsin - madison ) , ctac ( canine thyroid carcinoma , the ohio state university ) , d-17 ( canine osteosarcoma , american type culture collection ) , cml-1 ( canine melanoma , auburn university ) and cmt-12 ( canine mammary gland carcinoma , auburn university ) were used . all cell lines were grown under standard culture conditions with dulbecco s modified eagle medium ( dmem ; mediatech masassas , herndon , va , u.s.a . ) supplemented with 10% fetal bovine serum ( fbs ) , penicillin - streptomycin and 5% carbon dioxide at 37c . confluent cells were subcultured every 37 days after detaching the cells with 0.1% trypsin and 0.02% ethylenediaminetetraacetic acid in phosphate buffered saline ( pbs ; mediatech ) . loperamide hydrochloride : loperamide hydrochloride was purchased from sigma ( st . louis , mo , u.s.a . ) . stock solutions of 50 mm were prepared in dimethyl sulfoxide ( dmso ) and stored at 20c . fresh dilutions of loperamide hydrochloride were made from the stock solutions in media supplemented with 10% fbs for each experiment such that the dmso concentration did not exceed 0.2% as per standard experimental procedures , which has previously been shown to have no effect on growth of these cell lines [ 9 , 11 ] . inhibition of cellular proliferation assay : the effect of loperamide on cellular proliferation was evaluated using a bioreductive fluorometric assay ( alamar blue , molecular probes , invitrogen , carlsbad , ca , u.s.a . ) . between 1,500 and 6,000 cells per well were plated and grown in 10% fbs media in triplicate or quadruplicate in 96-well tissue culture treated microtiter plates and incubated for 1224 hr at 37c . number of cells plated per well was dependent on the rate of growth of cell lines in the flask . media were removed , and various concentrations of loperamide diluted in 10% fbs supplemented media were added . cell viability was determined at 24 , 48 and 72 hr following addition of loperamide . at these time points , 20 l of alamar blue was added to each well , and the plates were incubated at 37c for 8 hr . after incubation , plates were read using a spectrophotometric microplate reader ( biotek synergy 4 ; biotek instruments , winooski , vt , u.s.a . ) . the ic50 was determined by nonlinear regression analysis fitting to a dose - response curve using a computer software program ( prism 4 , graphpad software , la jolla , ca , u.s.a . ) . to complement cell viability assessment , a second set of cells for each cell line these cells were plated at various concentrations between 75,000200,000 cells per well depending on growth rate in the flask . after 24 hr , these cells were treated with two different drug concentrations ( 10 m and 100 m ) , while a control group was treated with media alone . the cells were observed with light microscopy and photographed at 6 , 24 and 48 hr after addition of loperamide . apoptosis analysis : the proportion of cells undergoing apoptosis was evaluated using 7-aad ( 10 g / ml ; molecular probes , invitrogen ) . cells were plated in 6-well plates at concentrations ranging from 25,000 to 150,000 cells / well depending on inherent growth rate to ensure that cells did not overgrow by the time of the assay . the cells were incubated for 1224 hr at 37c , at which time media were removed , and various concentrations of loperamide ( 0 , 10 , and 30 m ) diluted in 10% media were added . cells were then evaluated with flow cytometry at 6 , 24 and 48 hr post addition of drug . immediately prior to analysis , media were removed , and the attached cells were rinsed with cold pbs , trypsinized and resuspended in media . the cell suspension was centrifuged at 186 g at 7c for 7 min , after which the cell recovery was enumerated with an automated cell counter ( cellometer , nexcelom , lawrence , ma , u.s.a . ) . supernatant was removed , and the cells were resuspended in 100 l of cold pbs . one hundred l of 7-aad , prepared in a supplemented buffer ( 0.1% bovine serum albumen ( bsa ) , 0.1% nan3 and 1.0% fbs ) , was added . the cells were incubated on ice for 1530 min and evaluated using a lsr - ii flow cytometer with facsdiva 6.0 software ( bd biosciences , san jose , ca , u.s.a . ) . a minimum of 10,000 events were collected per sample , and reported values were a percentage of total cells counted . cells were discriminated into three populations , live cells and early apoptotic or late apoptotic / necrotic cells based on size and 7-aad uptake as described previously [ 15 , 25 ] . briefly , viable cells were of moderate size with minimal 7-aad uptake , while early apoptotic cells had moderate dye uptake and smaller size , and late apoptotic / necrotic cells were smallest in size with most intense uptake . the excitation wavelength was 488 nm with emission detected with a photomultiplier equipped with a 695/40 band pass filter . cell cycle analysis : cells were grown and treated with loperamide for cell cycle analysis in the same manner as for apoptosis analysis ( 0 , 10 and 30 m ) and allowed to incubate for 6 , 24 and 48 hr . cells were harvested as above for apoptosis analysis and were evaluated with a commercial cell cycle kit ( bd cycletest plus dna kit , bd biosciences ) . briefly , cells were suspended in a sodium citrate , sucrose and dimethyl sulfoxide buffer . the cell suspension was centrifuged for 5 min at 300 g at room temperature , the supernatant was removed , and the cells were resuspended in the same buffer solution . this wash step was repeated a second time , and the resulting suspension was then centrifuged for 5 min at 300 g at 23c 5c , and the supernatant was removed . fifty l of trypsin in a spermine tetrahydrochloride buffer was added for the digestion of cell membranes and cytoskeletal elements and allowed to react for 10 min . after incubation , 40 l of a trypsin inhibitor and rnase buffer was added and incubated for 10 min , after which 40l of cold ( 28c ) propidium iodide was added and allowed to incubate for another 10 min . the samples were placed on ice and immediately analyzed using flow cytometry to detect propidium iodide fluorescence , using an excitation of 488 nm and emission detected through a 575/26 band pass filterand analyzed with flowjo software ( tree star , ashland , or , u.s.a . ) to determine cell cycle parameters . chemosensitization assay : cells were grown as harvested as for the cellular proliferations assays . after plating , cells were allowed to adhere for 1224 hr , after which time the cells were treated with doxorubicin alone ( sigma ) at concentrations from 0.1 nm1,000 nm , loperamide alone at 10 m or a combination of increasing concentrations of doxorubicin ( 0.1 nm1,000 nm ) with 10 m of loperamide . the dose of 10 m was selected as it was near the ic50 value for most cell lines . a separate experiment using 25 m of loperamide instead of 10 m was also run for the cmt-12 cell line , since this cell line had a higher ic50 than the other three cell lines . control cells were treated with media alone at the same volume as used for the drugs . cells were incubated with the drugs for 72 hr before the alamar blue assay was run as described above . statistical analysis : all statistical analyses below were conducted separately for each of the 4 cell lines ( cmt-12 , cml-1 , ctac and d-17 ) . cell proliferation or cytotoxicity data ( cell counts ) were subjected to a 3-way anova . the main effects for this linear model were media ( 1% vs. 10% fbs ) , loperamide dose ( 0 , 0.01 , 0.1 , 1.0 , 3.2 , 10 , 32 and 100 m ) and exposure time ( 24 , 48 and 72 hr ) . residuals analysis suggested a box - cox power transformation of the cell count response , the results from which are reported . these data were subjected to a 2-way anova model using dose ( 0 , 10 and 30 m ) and exposure time ( 6 , 24 and 48 hr ) . the frequency ( % ) of cells in the g0/g1 phase of the cell cycle was subjected also to a 2-way anova model as the experimental design matched that of the apoptosis data . the synergistic effects of doxorubicin with loperamide ( chemosensitization ) were measured by a 2-way anova of the cell proliferation response subject to doxorubicin concentration ( 0 , 0.1 , 1.0 , 10 , 100 and 1,000 nm ) and loperamide concentration ( 0 and 10 m ) as main effects . residuals analysis suggested a weighted least - squares regression method , which was performed and reported as addressing the problem of heteroscedasticity . in addition , the test for a statistically significant ( p<0.05 ) interaction between doxorubicin and loperamide was equivalent to the bliss test for synergism . multiple comparisons among levels of the main effects in any of these analyses were conducted using the tukey - kramer hsd ( honestly significant difference ) test [ 10 , 22 ] . loperamide induced a dose - dependent cytotoxicity in canine cancer cell lines : loperamide produced anti - proliferative effects with increasing concentrations in all cell lines tested ( fig . 1.loperamide impaired cell viability following 72 hr of incubation in a dose dependent fashion in ctac , d-17 , cml-1 and cmt-12 canine cancer cell lines . viability data were garnered from experiments performed in triplicate and assessed by alamar blue assay . cell viability for both ctac and cmt-12 was significantly lower than control cells at concentrations 32 m ; cell viability for d-17 and cml-1 was significantly lower than control cells at concentrations 10 m . ) . cell lines had different sensitivities to the cytotoxic effects of loperamide , with the d-17 osteosarcoma cell line most sensitive ( table 1table 1.ic50 concentrations of loperamide in canine cancer cellsctacd-17cml-1cmt-12ic50 ( m ) 24 hr 20 0.88.7 0.519.4 0.6 27 0.5ic50 ( m ) 48 hr16.2 0.88.2 0.816.7 1.125.5 1.7ic50 ( m ) 72 hr19.3 0.57.2 0.314.8 1.125.8 0.3ic50 concentrations for 4 canine cancer cell lines after 24 , 48 and 72 hr of incubation with loperamide . there was no difference in ic50 based on time , as analyzed via anova with tukey post - hoc test . specifically , ctac and cmt-12 cell viability at concentrations 32 m were significantly lower compared to control cells . cell viability in both the d-17 and cml-1 cell lines was significantly lower at loperamide concentrations 10 m than compared to control cells . 2.morphological changes of the d-17 cell line after treating with 0 , 10 and 100 m of loperamide for 24 cell morphology changed from an attached spindle - shaped appearance ( control ) to detached and rounded with increasing doses of loperamide . this is a representative cell line , as all others showed similar changes . ) . there were no significant differences in the ic50 concentrations for any cell line based on timing ( 24 , 48 and 72 hr time points ) , indicating the loperamide exhibits its maximal effect by 24 hr . in addition , there was no effect of the fbs concentration in the media ( 1% vs. 10% ) on cell survival for either the ctac or the d-17 cell lines ( data not shown ) . however , there was a positive effect on cml-1 cell line survival with increasing fbs solution strength and negative effect on cmt-12 cell line survival ( data not shown ) . loperamide impaired cell viability following 72 hr of incubation in a dose dependent fashion in ctac , d-17 , cml-1 and cmt-12 canine cancer cell lines . viability data were garnered from experiments performed in triplicate and assessed by alamar blue assay . cell viability for both ctac and cmt-12 was significantly lower than control cells at concentrations 32 m ; cell viability for d-17 and cml-1 was significantly lower than control cells at concentrations 10 m . ic50 concentrations for 4 canine cancer cell lines after 24 , 48 and 72 hr of incubation with loperamide . there was no difference in ic50 based on time , as analyzed via anova with tukey post - hoc test . morphological changes of the d-17 cell line after treating with 0 , 10 and 100 m of loperamide for 24 hr . cell morphology changed from an attached spindle - shaped appearance ( control ) to detached and rounded with increasing doses of loperamide . loperamide induced a dose and time - dependent apoptosis : cells were stained with 7-aad and analyzed via flow cytometry to confirm that loperamide induced cell death . results in all cell lines showed a dose and time - dependent decrease in viable cells following treatment with loperamide ( table 2table 2.induction of apoptosis following incubation with loperamidectacd-17cml-1cmt-126 hr0 m2.6 0.86.1 1.71.9 0.59.7 1.510 m1 0.27.6 1.32.4 0.68.6 0.630 m2.8 0.55.8 0.23.5 1.610.8 3.224 hr0 m1.5 0.52.2 0.61.5 0.39.1 2.610 m3.2 0.92.4 11.4 0.36.2 1.730 m39.9 6.1 * 27.6 7.3 * 46.5 13.5 * 25.5 4.4 * 48 hr0 m4.1 1.61.1 0.21.8 0.57.1 0.710 m6.2 21.4 0.41.4 0.29 0.930 m92.7 0.1 * 57.2 4.6 * 86.1 3.5 * 72.8 2.3*percentage of cells undergoing apoptosis at three different time points and three different loperamide concentrations . an * indicates that there are significantly more apoptotic cells compared to the control ( 0 m of drug ) as analyzed via anova with tukey post - hoc test . ) . in the absence of loperamide , hr , there were no significant differences in percentage of apoptotic cells from baseline in any cell line ; however , following both 24 and 48 hr of exposure to 30 m , a significantly higher number of dead cells were detected . there was no difference between the 10 m group and the control group at any time point for any of the cell lines . percentage of cells undergoing apoptosis at three different time points and three different loperamide concentrations . an * indicates that there are significantly more apoptotic cells compared to the control ( 0 m of drug ) as analyzed via anova with tukey post - hoc test . loperamide induced a dose and time - dependent g0/g1 accumulation : a commercially available cell assay for live cells based on standard propidium iodide staining was used to assess cell cycle effects on all cell lines . loperamide caused a dose and time - dependent g0/g1 accumulation in viable cells ( table 3table 3.percentage of cells in g0/g1 following incubation with loperamidectacd-17cml-1cmt-126 hr0 m48.5 3.145.8 1.254.6 0.349.7 1.410 m50 244.5 0.555.8 1.251.5 230 m41.3 1.144.8 0.659.7 1.349 2.324 hr0 m38.1 3.352.6 1.550.6 0.358.7 110 m53.5 2.155.5 2.257.2 1.659.6 0.230 m56.8 0.8 * 63.4 3.7 * 70 1.9 * 78.2 2.4 * 48 hr0 m52.7 5.154.4 1.249.7 3.180.4 1.110 m51.2 1.660.7 1.850.3 1.877.6 230 m62.8 2.4 * 78.8 1.1 * 71.6 0.8 * 85.7 1.7*percentage of cells in g0/g1 at three different time points and three different loperamide concentrations . an * indicates that there are significantly more g0/g1 cells compared to the control ( 0 m of drug ) as analyzed via anova with tukey post - hoc test . , fig . 3fig . 3.loperamide caused an accumulation of cells in the g0/g1 phase as assessed by flow cytometry . representative flow cytometry histograms of d-17 cells are displayed for cells treated for 48 hr with loperamide ( 30 m ) ( b ) and control ( a ) . ) . there was no difference between the control and the 10 m concentrations at any time point . percentage of cells in g0/g1 at three different time points and three different loperamide concentrations . an * indicates that there are significantly more g0/g1 cells compared to the control ( 0 m of drug ) as analyzed via anova with tukey post - hoc test . loperamide caused an accumulation of cells in the g0/g1 phase as assessed by flow cytometry . representative flow cytometry histograms of d-17 cells are displayed for cells treated for 48 hr with loperamide ( 30 m ) ( b ) and control ( a ) . loperamide sensitized canine cancer cells to doxorubicin : four cell lines were screened to determine if loperamide increased the sensitivity of cancer cells to the chemotherapeutic drug doxorubicin in vitro . there was no evidence of synergism for the d-17 , cml-1 or cmt-12 cell lines with 10 m loperamide . however , there was synergism between the two drugs at 100 nm of doxorubicin and 10 m loperamide for the ctac cell line . due to the fact that the ic50 of the cmt-12 cell line was higher than the other three cell lines , the experiment was repeated using the same concentrations of doxorubicin , but with 25 m of loperamide , nearer to the ic50 . 4.doxorubicin alone is represented by the interrupted line , while the combination of doxorubicin and loperamide is represented by the non - interrupted line . loperamide concentration in a - c is 10 m , while it is 25 m in d. the curves are representative samples of experiments performed in triplicate . ) . doxorubicin alone is represented by the interrupted line , while the combination of doxorubicin and loperamide is represented by the non - interrupted line . loperamide concentration in a - c is 10 m , while it is 25 m in d. the curves are representative samples of experiments performed in triplicate . thus , the discovery of existing drugs with potentially expanded roles beyond their traditional purpose is attractive . here , loperamide , a commonly used anti - diarrheal in canine and human cancer patients , caused a significant dose - dependent cytotoxicity in 4 different canine cancer cell lines . the ic50 for loperamide ranged from 7.227 m , which correlates well with a study performed on a library of human cancer cell lines , showing ic50 concentrations ranging from 11.841.4 m . as seen in the human study , canine osteosarcoma cells were most sensitive to loperamide , while canine breast carcinoma cells were less sensitive ( 6 ) . cell death in the canine cancer cell lines investigated here was mediated in part by apoptosis , as demonstrated through 7-aad staining and flow cytometry , a technique shown to correlate well with other common apoptosis analyses , such as annexin - v and propidium iodide [ 15 , 25 ] . the mechanism behind apoptosis in these cell lines has not been elucidated , although in human cancer cell lines , loperamide has been shown to cause apoptosis via activation of the caspase 3 pathway . apoptotic canine cancer cells were identified at 24 hr , which was slightly later than with human cancer cells , as apoptosis was evident as early as 6 hr following loperamide exposure . this may be a reflection of different assays used or possibly decreased potency against canine cells ; it is possible that with higher doses of loperamide , a significant change in 7-aad staining would have been noted prior to 24 hr . interestingly , other opiate agonists , such as etorphine , morphine and buprenorphine , have also been shown to induce apoptosis in some human cell lines [ 7 , 8 , 24 ] . an unexpected finding was that unlike a g2/m arrest seen in human cancer cell lines , loperamide caused a dose and time - dependent g0/g1 accumulation in canine cancer cells . our group s initial intent was to consider using loperamide as a radiation sensitizer , given that human cancer cell lines are arrested in sensitive phases of the cell cycle [ 16 , 26 ] . it is still possible that loperamide , or a similar peripheral opiate agonist , might exert synergism with radiotherapy through another mechanism and may be worth exploring . it is unclear why the cells tested here demonstrated g0/g1 arrest rather than g2/m , however , it is possible that canine cancer cells may have altered cell cycle kinetics compared to human cancer cells . in addition to its negative effects on cell viability and its pro - apoptotic effect , loperamide also demonstrated synergism with doxorubicin in both the ctac and cmt-12 cell lines at concentrations near the ic50 when combined with doxorubicin . while it is interesting that both of these cell lines are carcinomas , further investigation is indicated to determine if similar effects are seen in other cell types . in a human breast cancer cell line stably transfected with the mdr-1 gene leading to doxorubicin resistance , loperamide was able to reverse this resistance due to high affinity for the p - glycoprotein pump , therefore leading to intracellular accumulation of the chemotherapeutic . while our laboratory has performed preliminary work investigating the immunohistochemical expression of p - glycoprotein in our selected cell lines , further work is needed to determine if there is a relationship between p - glycoprotein expression and loperamide effects . the apparent synergism of doxorubicin and loperamide in some cell lines may also be due to occupation of another shared efflux pump , such as mrp-1 , or through alternative mechanisms not related to drug efflux . it may be interesting in future studies to evaluate the effect of loperamide on doxorubicin - resistance canine cell lines compared to nave cancer cell lines to further elucidate the potential for adjuvant therapy . a possible limitation of the in vitro work performed here is that the concentrations of loperamide used are above what has safely been used in vivo . one study showed that after a typical dosage of 0.2 mg / kg , blood concentrations of 0.02 m were achieved . the maximally tolerated dose of loperamide is not known , but toxicities including vomiting , diarrhea and central nervous system depression have been reported at doses as low as 1.25 mg / kg . in dogs with mutations in their p - glycoprotein pumps ( abcb1 mutation ) , toxicity may be seen at doses well tolerated by normal dogs . however , it is certainly possible that the in vitro work done in this study is not a true reflection of the in vivo activity of this drug . it is also important to note that in this work , cells were only exposed to a single dose of loperamide . if loperamide is capable of reversing resistance , doses in the realm of 10 m may still be necessary , which would be difficult to achieve in dogs unless the target cancer cells are capable of accumulating drug . loperamide exerted negative effects on the cancer cell lines studied here as early as 24 hr following exposure ; given that loperamide may be administered more frequently , chronic dosing may have altered effects . it is also important to note that variations in drug metabolism and patient variability may lead to anti - neoplastic effects in a spontaneous tumor model with lower drug concentrations . despite these limitations , this study establishes a proof - of - principle in support of loperamide as an anti - cancer agent . further studies are needed to establish mechanisms of action of its activity as well as in vivo tolerability as part of multi - modality therapy . it is possible that drugs similar to loperamide will exert similar anticancer properties at clinically relevant doses . loperamide would be an attractive drug in the clinic , as it has minimal side effects , is an over - the - counter drug , and is inexpensive . in conclusion , results presented here loperamide negatively affected canine cancer cell viability with ic50 values similar to those reported in human cancer cell lines . loperamide also caused apoptosis in a dose - dependent fashion and induced a g0/g1 cell cycle arrest . in vitro chemosensitivity studies further work on the efficacy of loperamide or similar peripheral -opioid agonist in the management of cancer is warranted .

loperamide is a peripheral opiate agonist that can cause apoptosis and g2/m arrest in human cancer cell lines and may sensitize cells to chemotherapy . the objectives of this study were to investigate the effects of loperamide on viability , apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin . cell viability was assessed using alamar blue . cell death and cell cycle were studied using flow cytometry with 7-aminoactinomycin - d ( 7-aad ) and propidium iodide ( pi ) , respectively . loperamide decreased cell viability in a dose - dependent fashion and was most effective against canine osteosarcoma cells . in all cell lines , it induced a dose and time dependent apoptosis and resulted in accumulation in g0/g1 . when co - incubated with doxorubicin , loperamide induced a synergistic cell kill in canine carcinoma cells . investigation is warranted into the role of loperamide in the treatment of canine cancer .

MATERIALS AND METHODS RESULTS DISCUSSION

cell lines and culture conditions : four cell lines ( provided by the university of wisconsin - madison ) , ctac ( canine thyroid carcinoma , the ohio state university ) , d-17 ( canine osteosarcoma , american type culture collection ) , cml-1 ( canine melanoma , auburn university ) and cmt-12 ( canine mammary gland carcinoma , auburn university ) were used . all cell lines were grown under standard culture conditions with dulbecco s modified eagle medium ( dmem ; mediatech masassas , herndon , va , u.s.a . ) fresh dilutions of loperamide hydrochloride were made from the stock solutions in media supplemented with 10% fbs for each experiment such that the dmso concentration did not exceed 0.2% as per standard experimental procedures , which has previously been shown to have no effect on growth of these cell lines [ 9 , 11 ] . inhibition of cellular proliferation assay : the effect of loperamide on cellular proliferation was evaluated using a bioreductive fluorometric assay ( alamar blue , molecular probes , invitrogen , carlsbad , ca , u.s.a . ) number of cells plated per well was dependent on the rate of growth of cell lines in the flask . cell viability was determined at 24 , 48 and 72 hr following addition of loperamide . the ic50 was determined by nonlinear regression analysis fitting to a dose - response curve using a computer software program ( prism 4 , graphpad software , la jolla , ca , u.s.a . ) to complement cell viability assessment , a second set of cells for each cell line these cells were plated at various concentrations between 75,000200,000 cells per well depending on growth rate in the flask . the cells were observed with light microscopy and photographed at 6 , 24 and 48 hr after addition of loperamide . the cells were incubated for 1224 hr at 37c , at which time media were removed , and various concentrations of loperamide ( 0 , 10 , and 30 m ) diluted in 10% media were added . one hundred l of 7-aad , prepared in a supplemented buffer ( 0.1% bovine serum albumen ( bsa ) , 0.1% nan3 and 1.0% fbs ) , was added . cell cycle analysis : cells were grown and treated with loperamide for cell cycle analysis in the same manner as for apoptosis analysis ( 0 , 10 and 30 m ) and allowed to incubate for 6 , 24 and 48 hr . the samples were placed on ice and immediately analyzed using flow cytometry to detect propidium iodide fluorescence , using an excitation of 488 nm and emission detected through a 575/26 band pass filterand analyzed with flowjo software ( tree star , ashland , or , u.s.a . ) after plating , cells were allowed to adhere for 1224 hr , after which time the cells were treated with doxorubicin alone ( sigma ) at concentrations from 0.1 nm1,000 nm , loperamide alone at 10 m or a combination of increasing concentrations of doxorubicin ( 0.1 nm1,000 nm ) with 10 m of loperamide . the dose of 10 m was selected as it was near the ic50 value for most cell lines . a separate experiment using 25 m of loperamide instead of 10 m was also run for the cmt-12 cell line , since this cell line had a higher ic50 than the other three cell lines . cells were incubated with the drugs for 72 hr before the alamar blue assay was run as described above . the main effects for this linear model were media ( 1% vs. 10% fbs ) , loperamide dose ( 0 , 0.01 , 0.1 , 1.0 , 3.2 , 10 , 32 and 100 m ) and exposure time ( 24 , 48 and 72 hr ) . the frequency ( % ) of cells in the g0/g1 phase of the cell cycle was subjected also to a 2-way anova model as the experimental design matched that of the apoptosis data . the synergistic effects of doxorubicin with loperamide ( chemosensitization ) were measured by a 2-way anova of the cell proliferation response subject to doxorubicin concentration ( 0 , 0.1 , 1.0 , 10 , 100 and 1,000 nm ) and loperamide concentration ( 0 and 10 m ) as main effects . loperamide induced a dose - dependent cytotoxicity in canine cancer cell lines : loperamide produced anti - proliferative effects with increasing concentrations in all cell lines tested ( fig . 1.loperamide impaired cell viability following 72 hr of incubation in a dose dependent fashion in ctac , d-17 , cml-1 and cmt-12 canine cancer cell lines . cell viability for both ctac and cmt-12 was significantly lower than control cells at concentrations 32 m ; cell viability for d-17 and cml-1 was significantly lower than control cells at concentrations 10 m . ) cell lines had different sensitivities to the cytotoxic effects of loperamide , with the d-17 osteosarcoma cell line most sensitive ( table 1table 1.ic50 concentrations of loperamide in canine cancer cellsctacd-17cml-1cmt-12ic50 ( m ) 24 hr 20 0.88.7 0.519.4 0.6 27 0.5ic50 ( m ) 48 hr16.2 0.88.2 0.816.7 1.125.5 1.7ic50 ( m ) 72 hr19.3 0.57.2 0.314.8 1.125.8 0.3ic50 concentrations for 4 canine cancer cell lines after 24 , 48 and 72 hr of incubation with loperamide . cell viability in both the d-17 and cml-1 cell lines was significantly lower at loperamide concentrations 10 m than compared to control cells . 2.morphological changes of the d-17 cell line after treating with 0 , 10 and 100 m of loperamide for 24 cell morphology changed from an attached spindle - shaped appearance ( control ) to detached and rounded with increasing doses of loperamide . this is a representative cell line , as all others showed similar changes . ) there were no significant differences in the ic50 concentrations for any cell line based on timing ( 24 , 48 and 72 hr time points ) , indicating the loperamide exhibits its maximal effect by 24 hr . in addition , there was no effect of the fbs concentration in the media ( 1% vs. 10% ) on cell survival for either the ctac or the d-17 cell lines ( data not shown ) . loperamide impaired cell viability following 72 hr of incubation in a dose dependent fashion in ctac , d-17 , cml-1 and cmt-12 canine cancer cell lines . viability data were garnered from experiments performed in triplicate and assessed by alamar blue assay . ic50 concentrations for 4 canine cancer cell lines after 24 , 48 and 72 hr of incubation with loperamide . loperamide induced a dose and time - dependent apoptosis : cells were stained with 7-aad and analyzed via flow cytometry to confirm that loperamide induced cell death . results in all cell lines showed a dose and time - dependent decrease in viable cells following treatment with loperamide ( table 2table 2.induction of apoptosis following incubation with loperamidectacd-17cml-1cmt-126 hr0 m2.6 0.86.1 1.71.9 0.59.7 1.510 m1 0.27.6 1.32.4 0.68.6 0.630 m2.8 0.55.8 0.23.5 1.610.8 3.224 hr0 m1.5 0.52.2 0.61.5 0.39.1 2.610 m3.2 0.92.4 11.4 0.36.2 1.730 m39.9 6.1 * 27.6 7.3 * 46.5 13.5 * 25.5 4.4 * 48 hr0 m4.1 1.61.1 0.21.8 0.57.1 0.710 m6.2 21.4 0.41.4 0.29 0.930 m92.7 0.1 * 57.2 4.6 * 86.1 3.5 * 72.8 2.3*percentage of cells undergoing apoptosis at three different time points and three different loperamide concentrations . in the absence of loperamide , hr , there were no significant differences in percentage of apoptotic cells from baseline in any cell line ; however , following both 24 and 48 hr of exposure to 30 m , a significantly higher number of dead cells were detected . loperamide induced a dose and time - dependent g0/g1 accumulation : a commercially available cell assay for live cells based on standard propidium iodide staining was used to assess cell cycle effects on all cell lines . loperamide caused a dose and time - dependent g0/g1 accumulation in viable cells ( table 3table 3.percentage of cells in g0/g1 following incubation with loperamidectacd-17cml-1cmt-126 hr0 m48.5 3.145.8 1.254.6 0.349.7 1.410 m50 244.5 0.555.8 1.251.5 230 m41.3 1.144.8 0.659.7 1.349 2.324 hr0 m38.1 3.352.6 1.550.6 0.358.7 110 m53.5 2.155.5 2.257.2 1.659.6 0.230 m56.8 0.8 * 63.4 3.7 * 70 1.9 * 78.2 2.4 * 48 hr0 m52.7 5.154.4 1.249.7 3.180.4 1.110 m51.2 1.660.7 1.850.3 1.877.6 230 m62.8 2.4 * 78.8 1.1 * 71.6 0.8 * 85.7 1.7*percentage of cells in g0/g1 at three different time points and three different loperamide concentrations . 3.loperamide caused an accumulation of cells in the g0/g1 phase as assessed by flow cytometry . representative flow cytometry histograms of d-17 cells are displayed for cells treated for 48 hr with loperamide ( 30 m ) ( b ) and control ( a ) . ) percentage of cells in g0/g1 at three different time points and three different loperamide concentrations . loperamide caused an accumulation of cells in the g0/g1 phase as assessed by flow cytometry . representative flow cytometry histograms of d-17 cells are displayed for cells treated for 48 hr with loperamide ( 30 m ) ( b ) and control ( a ) . loperamide sensitized canine cancer cells to doxorubicin : four cell lines were screened to determine if loperamide increased the sensitivity of cancer cells to the chemotherapeutic drug doxorubicin in vitro . there was no evidence of synergism for the d-17 , cml-1 or cmt-12 cell lines with 10 m loperamide . due to the fact that the ic50 of the cmt-12 cell line was higher than the other three cell lines , the experiment was repeated using the same concentrations of doxorubicin , but with 25 m of loperamide , nearer to the ic50 . 4.doxorubicin alone is represented by the interrupted line , while the combination of doxorubicin and loperamide is represented by the non - interrupted line . loperamide concentration in a - c is 10 m , while it is 25 m in d. the curves are representative samples of experiments performed in triplicate . ) here , loperamide , a commonly used anti - diarrheal in canine and human cancer patients , caused a significant dose - dependent cytotoxicity in 4 different canine cancer cell lines . the ic50 for loperamide ranged from 7.227 m , which correlates well with a study performed on a library of human cancer cell lines , showing ic50 concentrations ranging from 11.841.4 m . as seen in the human study , canine osteosarcoma cells were most sensitive to loperamide , while canine breast carcinoma cells were less sensitive ( 6 ) . cell death in the canine cancer cell lines investigated here was mediated in part by apoptosis , as demonstrated through 7-aad staining and flow cytometry , a technique shown to correlate well with other common apoptosis analyses , such as annexin - v and propidium iodide [ 15 , 25 ] . the mechanism behind apoptosis in these cell lines has not been elucidated , although in human cancer cell lines , loperamide has been shown to cause apoptosis via activation of the caspase 3 pathway . apoptotic canine cancer cells were identified at 24 hr , which was slightly later than with human cancer cells , as apoptosis was evident as early as 6 hr following loperamide exposure . this may be a reflection of different assays used or possibly decreased potency against canine cells ; it is possible that with higher doses of loperamide , a significant change in 7-aad staining would have been noted prior to 24 hr . interestingly , other opiate agonists , such as etorphine , morphine and buprenorphine , have also been shown to induce apoptosis in some human cell lines [ 7 , 8 , 24 ] . an unexpected finding was that unlike a g2/m arrest seen in human cancer cell lines , loperamide caused a dose and time - dependent g0/g1 accumulation in canine cancer cells . our group s initial intent was to consider using loperamide as a radiation sensitizer , given that human cancer cell lines are arrested in sensitive phases of the cell cycle [ 16 , 26 ] . it is still possible that loperamide , or a similar peripheral opiate agonist , might exert synergism with radiotherapy through another mechanism and may be worth exploring . it is unclear why the cells tested here demonstrated g0/g1 arrest rather than g2/m , however , it is possible that canine cancer cells may have altered cell cycle kinetics compared to human cancer cells . in addition to its negative effects on cell viability and its pro - apoptotic effect , loperamide also demonstrated synergism with doxorubicin in both the ctac and cmt-12 cell lines at concentrations near the ic50 when combined with doxorubicin . while it is interesting that both of these cell lines are carcinomas , further investigation is indicated to determine if similar effects are seen in other cell types . in a human breast cancer cell line stably transfected with the mdr-1 gene leading to doxorubicin resistance , loperamide was able to reverse this resistance due to high affinity for the p - glycoprotein pump , therefore leading to intracellular accumulation of the chemotherapeutic . while our laboratory has performed preliminary work investigating the immunohistochemical expression of p - glycoprotein in our selected cell lines , further work is needed to determine if there is a relationship between p - glycoprotein expression and loperamide effects . the apparent synergism of doxorubicin and loperamide in some cell lines may also be due to occupation of another shared efflux pump , such as mrp-1 , or through alternative mechanisms not related to drug efflux . it may be interesting in future studies to evaluate the effect of loperamide on doxorubicin - resistance canine cell lines compared to nave cancer cell lines to further elucidate the potential for adjuvant therapy . a possible limitation of the in vitro work performed here is that the concentrations of loperamide used are above what has safely been used in vivo . the maximally tolerated dose of loperamide is not known , but toxicities including vomiting , diarrhea and central nervous system depression have been reported at doses as low as 1.25 mg / kg . however , it is certainly possible that the in vitro work done in this study is not a true reflection of the in vivo activity of this drug . if loperamide is capable of reversing resistance , doses in the realm of 10 m may still be necessary , which would be difficult to achieve in dogs unless the target cancer cells are capable of accumulating drug . loperamide exerted negative effects on the cancer cell lines studied here as early as 24 hr following exposure ; given that loperamide may be administered more frequently , chronic dosing may have altered effects . despite these limitations , this study establishes a proof - of - principle in support of loperamide as an anti - cancer agent . further studies are needed to establish mechanisms of action of its activity as well as in vivo tolerability as part of multi - modality therapy . in conclusion , results presented here loperamide negatively affected canine cancer cell viability with ic50 values similar to those reported in human cancer cell lines . loperamide also caused apoptosis in a dose - dependent fashion and induced a g0/g1 cell cycle arrest . in vitro chemosensitivity studies further work on the efficacy of loperamide or similar peripheral -opioid agonist in the management of cancer is warranted .

epidemiological studies have shown that cardiovascular diseases rarely affect women before menopause , suggesting that the decrease in the level of circulating estrogen ( 17 estradiol ) might be a risk factor for the development of hypertension . experimental studies have shown that estrogen plays an important role in the maintenance of the baroreceptor reflex . the bed nucleus of stria terminalis ( bst ) is a limbic forebrain structure that concentrate estrogen , and aromatase enzyme , which locally convert testosterone to estrogen . the bst is known to be influenced by circulating gonadal hormones altering immunoreactivity of the neuropeptides vasopressin , substance p , and cholecystokinin . the bst is also one of the major sites for integrating steroid hormones and olfactory information for sexual behavior . this finding suggests that gonadal steroid hormones may play an important role in the regulation of the bst function . microinjection of glutamate into the bst decreased arterial pressure ( ap ) and heart rate ( hr ) . a recent study has also indicated that the cholinergic system of the bst is involved in baroreflex activity and cardiovascular response . moreover , the gabaergic system of the bst mediates cardiovascular effect via the sympathetic system and vasopressin release . the areas within the bst that elicit cardiovascular responses contain high densities of estrogen receptors . in addition , the neurons within these areas that concentrate estrogen send axonal projections to medullary regions ; the caudal ventrolateral medulla ( cvlm ) , and the rostroventrolateral medulla ( rvlm ) , which are both directly involved in autonomic output to the heart and vasculature . therefore , it is hypothesized that estrogen can act on neurons within the bst to alter cardiovascular function . on the other hand , major outputs from the bst go to the rvlm and this medullary region may mediate the bst cardiovascular responses to the heart and vasculature . the rvlm contain many neurotransmitters and neuromodulators , for example gaba is one of the major inhibitory neurotransmitters . nitric oxide ( no ) in the rvlm increases the release of gaba and glutamate in conscious rats . the inhibition of gabaa receptor by microinjection of bicuculline into the rvlm caused a marked increase in mean arterial pressure ( map ) in normotensive ( but not hypertensive ) rats . this study was performed to find the contribution of the rvlm to cardiovascular responses elicited by glutamate microinjection into the bst . we studied ovariectomized ( ovx ) and ovx estrogen treated ( ovx+e ) anesthetized rats to assess the following : -the effect of circulatory estrogen on cardiovascular responses of the bst , -the effect of reversible ablation of the rvlm on the bst cardiovascular responses , and -the effect of blockade of the gaba system of rvlm on the bst cardiovascular responses . experiments were performed on 53 female wistar rats ( 200 - 250 g ) in accordance with the european communities council directive of 24 november 1986 ( 86/609/eec ) . the rats were anesthetized with equithesin ( 0.3 ml/100 g , intraperitoneally [ i.p . ] ) . ovariectomy was performed using sterile procedures , by making a 1 cm incision on both sides of the back to expose the ovaries . the ovaries were clamped and removed with the fallopian tubes being ligated , and the skin was then sutured . then , a silastic capsule ( internal diameter : 1.57 mm , outer diameter : 3.17 mm , length : 5.0 mm ) was implanted between the shoulder blades subcutaneously which either contained cholesterol ( for ovx rats , n=27 ) or 17 estradiol ( for ovx+e rats , n=26 ) . the estrogen containing capsule produced a plasma level estrogen concentration of approximately 30 pg / ml of blood which mimics the proestrus stage of the estrous cycle in rats . on the other hand , one study showed that the estrogen level is not detectable ( < 1 pg / ml ) in ovx animals implanted with a cholesterol capsule . for a period of 10 - 20 days the animals were housed under controlled condition with 12 h light / dark cycle with food and water available ad libitum . on the day of experiments , the animals were anesthetized again with equithesin ( i.p ) for surgical procedure and for the rest of the experiments with alpha chloralose ( 60 mg / kg i.v ) . the trachea was cannulated and the animals were artificially ventilated using a small rodent ventilator ( harvard apparatus inc . , u.s , model 683 ) with a mixture of room air and 95% o2 . body temperature was maintained at 37.00.2 using a heating pad controller ( model 73 ; yellow spring instrument , yellow spring , ohio ) . the femoral artery was cannulated with polyethylene catheter ( pe-50 , stoleting , usa ) filled with heparinized saline and connected to a statham p23xl pressure transducer and hr was monitored with a 7p4def grass tachograph ( usa ) triggered by the ap pulse . ap and hr were continuously recorded by a grass 79d polygraph . in some of the experiments ap and hr were continuously recorded by both a harvard polygraph and a computer program written in our laboratory . the animals were placed in prone position in a stereotaxic frame ( stoelting , usa ) and two bilateral small holes were drilled through the parietal bone over the bst and through the occipital bone over the rvlm . drug microinjection microinjection of the drugs were performed by two single barreled micropipettes with an internal diameter of 35 - 45 mm . micropipette tips were positioned in the bst and rvlm according to a stereotaxic atlas of the brain . the stereotaxic coordinates of the bst were explored -0.2 to -0.4 mm caudal , 2 mm lateral , and 5.5 - 7.5 mm ventral from the bregma . the rvlm was explored -11.6 to -12 caudal , 2 mm lateral , and 9.6 - 10.8 mm ventral from the bregma . the injection sites were 200 m apart , and 1 - 6 injections were made in each animal on both sides . microinjection was performed by a pressurized nitrogen pulse controlled by a picospritzer ( general valve , fairfield , nj ) . the injection volume was measured by direct observation of the fluid meniscus in the micropipette with a microscope fitted with an ocular micrometer . the injection volume of glutamate ( glu ) into the bst and rvlm were 20 and 50 nl , respectively . experimental groups the experiments were designed for studying the neuronal connectivity between bst and rvlm in relation to cardiovascular responses . the experiments were done on different groups of ovx and ovx+e female rats , as follows : -microinjection of saline into the bst ( the control group [ n=6 ] , 20 nl ; ovx [ n=3 ] , 13 injections ; and ovx+e [ n=3 ] , 14 injections ) -microinjection of l - glutamate into the bst ( 0.25 m/20 nl ; sigma , ovx [ n=24 ] , 81 injections ; and ovx+e [ n=23 ] , 76 injections ) . -to find the neuronal connectivity between the bst and the rvlm , l - glutamate was initially injected into the bst ( control ) , then after arterial pressure and hr returned to the baseline , reversible synaptic blocker cobalt chloride ( cocl2 5 mm/50 nl , sigma ) was injected into the rvlm . the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of cocl2 into the rvlm of the ovx ( n=6 , 23 injections ) and ovx+e ( n=4 , 19 injections ) rats . -to investigate the effect of inhibition of gabaa receptors of the rvlm on cardiovascular responses of the bst , first l - glutamate was injected into the bst ( control ) and after the arterial pressure and hr returned to the baseline , a gabaa antagonist , bicuculline ( 1 mm/50 nl , sigma ) was injected into the rvlm and the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of bicuculline into the rvlm of the ovx ( n=6 , 36 injections ) and ovx+e ( n=7 , 41 injections ) rats . -to find the effect of inhibition of gabab receptors of the rvlm on cardiovascular responses of the bst , first l - glutamate was injected into the bst ( control ) and after the ap and hr returned to the baseline , a gabab antagonist phaclophen ( 5 mm/50 nl , sigma ) was injected into the rvlm and the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of phaclophen into the rvlm of the ovx ( n=6 , 36 injections ) and ovx+e ( n=7 , 41 injections ) rats . at the end of each experiment , 20 and 50 nl pontamine sky blue ( sigma , usa ) were microinjected into the bst and the rvlm , respectively , then the animal was sacrificed by a high dose of the anesthetic , and perfused transcardially with100 ml of 0.9% saline followed by 100 ml of 10% saline formalin . the brain was removed and stored in 10% formalin for at least 24 hours . frozen serial transverse sections ( 50 m ) of the regions of the bst and rvlm were cut and stained with neutral red ( merk , germany ) . the injection sites were determined according to a rat brain atlas , under the light microscope ( nikon , japan ) ( figures 1 and 2 ) . schematic coronal sections of rat brain adopted from an atlas,23 show the injection of glutamate ( ) into the bst sites which decrease blood pressure and heart rate . ac : anterior commissure ; acp : anterior commissure posterior part ; bstm : bed nucleus stria terminals medial part ; ic : inferior colliculus ; fx : fornix ; ox : optic chiasma ; 3v : third ventricle schematic coronal sections of rat brain adopted from an atlas,23 show the injection sites of cobalt chloride ( ) , bicuculline ( ) , and phaclophen ( o ) into the rvlm . amb : ambiggus ; ion : inferior olive nucleus ; pyr : pyramid tract the results are expressed as meanstandard error of mean ( sem ) . all data were normal except for map after cocl2 injection in the rvlm , for which the wilcoxon analysis was used . the maximum changes of heart rate ( hr ) and the maximum changes of map ( map ) were compared between ovx , ovx+e , and saline groups using independent t test . cardiovascular responses of regional controls in the anaesthetized female ovx and ovx+e rats , microinjection of 20 nl of glutamate in the regions around the bst did not affect the pressure ( map=2.63.6 mmhg ) and the hr ( hr=-3.22.3 beats / min ; 43 injections ) . cardiovascular responses to vehicle microinjection into the bst to test whether cardiovascular response of bst was related to the volume of injection or mechanical torsion , 20 nl saline was microinjected into the bst of ovx and ovx+e animals . the changes in map ( map=0.70.6 mmhg ) and hr ( hr=0.570.2 beats / min ) were not significantly different from the pre - injection values . cardiovascular responses to glutamate microinjection into the bst to determine the effect of glutamate on the map and hr , it was microinjected into the bst ( 0.25 m/20 nl ) . the baseline mean ( se ) ap and hr were 113.3 ( 3.23 ) mmhg and 386.4 ( 7.6 ) bpm in ovx rats and 112.1 ( 3.98 ) mmhg and 339.9 ( 20.2 ) bpm in ovx+e rats , respectively . the changes of the hr in the ovx+e rats was lower than the ovx rats ( p<0.01 , figure 3 ) . microinjection of glutamate into the dorsolateral and ventral subnuclei of the bst complex in both ovx and ovx+e rats showed decreases in hr and map ( ovx : map : -25.70.4 mmhg and hr : -14.30.3 bpm ; ovx+e : map : -25.50.3 mmhg and hr ; -12.60.5 bpm ) . the changes were significantly different from the saline group ( p<0.01 ) and the pre - injection values ( p<0.01 ) . however , the magnitude of bradycardia and depressor response was not significantly different between ovx and ovx+e rats . this bar chart show the magnitude of blood pressure and heart rate in the ovx and ovx+e rats . * significant difference between ovx and ovx+e groups , t test , p<0.01 cardiovascular response elicited by glutamate injection into the bst after the injection of synaptic blocker in the rvlm to investigate the possibility that the bst cardiovascular responses were mediated by the projection to rvlm , glutamate was first injected into the bst of the ovx and ovx+e rats . the depressor and bradycardic responses were similar in the magnitude in the pervious experiments ( ovx : map : -23.85.97 mmhg and hr : -10.02.5 bpm ; ovx+e : map : -19.02.3 mmhg and hr : -10.06.7 bpm , p<0.01 ) , then cocl2 , was injected into the rvlm . microinjection of cocl2 into the rvlm of ovx and ovx+e rats had no significant effect on the baseline values of map and hr compared with the pre - injection values ( ovx : map : -0.20.2 mmhg and hr : -4.02.3 bpm ; ovx+e : map : -1.00.2 mmhg and hr : 0.80.1 bpm ) . re - stimulation of the bst , 10 minutes after microinjection of cocl2 into the cardiovascular site of rvlm significantly attenuated the depressor and bradycardic responses of the same site of the bst . the magnitude of depressor response during stimulation of bst 10 minutes after cocl2 microinjection into the rvlm was significantly different from the pre - injection values ( ovx : map : -6.32.1 mmhg and hr : -2.52.5 bpm [ p<0.01 ] ; ovx+e : map : -11.31.6.3 mmhg and hr : -6.24.2 bpm [ p<0.05 ] ) . the magnitude of depressor and bradycardic responses by re - stimulation of bst , 60 min after cocl2 microinjection into the rvlm ( ovx : map : -19.83 . 7 mmhg and hr : -10.04.5 bpm ; ovx+e : map : -18.92.5 mmhg and hr : -8.5.05.2 bpm ) did not yield different results from the control values indicating that the effect of cocl2 was abolished ( figures 4 and 5 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of cocl2 into the rvlm and re - stimulation of bst at 10 and 60 minutes after injection of cocl2 in rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of cocl2 ( 5 mm/50 nl ) into the rvlm in ovx and ovx+e rats . * significant different compared with pre - injection values ( control ) , paired t test , p<0.01 cardiovascular responses elicited by glutamate injection into the bst after the injection of bicuculline in the rvlm to test the possible inhibition of cardiovascular responses of the bst by gabaergic neurons of the rvlm , first glutamate was injected into the bst of the ovx and ovx+e rats , then a gabaa antagonist bicuculline , was injected into the rvlm . microinjection of bicuculline into the rvlm of the ovx and ovx+e rats led to a significant increase on the map with no significant effect on hr compared with their pre - injection value ( ovx : map : 30.45.2 mmhg and hr : 12.2.08.3 bpm ; ovx+e : map : 35.954.4 mmhg and hr : 9.26.11 bpm , p<0.01 ) . similar to pervious experiments , 10 minutes later , glutamate was injected into the bst of the ovx and ovx+e rats . the depressor and bradycardic responses caused by stimulation of the bst were similar to those of the pervious experiments ( ovx : map : -22.52.68 mmhg and hr : -12.22.1 bpm ; ovx+e : map : -28.13.7 mmhg and hr : -14.23.5 bpm , p<0.01 , t test ) . the magnitude of depressor response during stimulation of the bst 10 minutes after bicuculline microinjection into the rvlm were reduced to almost 50% of their control value ( ovx : map : -11.93.3 mmhg and hr : -10.02.5 bpm ; ovx+e : map : -16.32.4 mmhg and hr : -7.51.7 bpm , p<0.01 ) . 60 min after the microinjection of bicuculline into the rvlm , the magnitude of depressor and bradycardic responses by re - stimulation of the bst approximately returned to their control value ( ovx : map : -15.83.7 mmhg and hr : -9.04.5 bpm ; ovx+e : map : -23.03.6 mmhg and hr : -12.52.5 bpm , figures 6 and 7 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of bicuculline ( 1 mm/50 nl ) into the rvlm and re - stimulation of bst at 10 and 60 minutes after injection of bicuculline into the rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of bicuculline into the rvlm in ovx and ovx+e rats . * significant difference with pre - injection values ( control ) , paired t test , p<0.01 cardiovascular response elicited by glutamate injected into the bst after the injection of phaclophen in the rvlm to find the possible effects of gabab receptors of the rvlm on the cardiovascular responses of the bst , phaclophen , a gabab antagonist , was microinjected into the rvlm of the ovx and ovx+e rats . no significant difference was found in both map and hr compared with their pre - injection values ( ovx : map : 4.31.1 mmhg and hr : 3.32.1 bpm ; ovx+e : map : 6.91.4 mmhg and hr : 8.73.2 bpm ) . similar to pervious experiments , microinjection of glutamate into the bst elicited bradycardic and depressor responses ( ovx : map : -26.13.0 mmhg and hr : -16.93.1 bpm ; ovx+e : map : -26.82.62 mmhg and hr : -13.65 bpm , p<0.01 , t test ) . unlike the effect of bicuculline , microinjection of phaclophen did not alter the magnitude of depressor and bradycardic responses by re - stimulation of the bst , 10 , 20 , 40 , and 60 minutes after microinjection of phaclophen into the cardiovascular site of rvlm ( figures 8 and 9 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of phaclophen into the rvlm and re - stimulation of bst at 10 and 60 minutes after the injection of phaclophen into the rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of phaclophen ( 5 mm/50 nl ) into the rvlm in ovx and ovx+e rats . we found that estrogen decreased hr significantly , circulatory estrogen did not alter the cardiovascular depressor response evoked by bst stimulation , rvlm neurons in the medulla mediated the bst cardiovascular responses , and the depressor response of the bst was partly mediated by the activation of gaba neurons of the rvlm . circulating estrogen within the physiological range affects hr , since the baseline hr in the ovx+e rats was lower than in the ovx rats . our finding supports the previous report that estrogen directly affects the heart and alters cardiac output . estrogen facilitates parasympathetic activity of the heart and potentiates the bradycardic component of the barorereflex . the effect of estrogen on resting vascular tone was mediated by the estrogen beta receptor subtype , whereas its effect on the heart rate was mediated by the alpha subtype . the role of endogenous estrogen in the protection of the heart from ischemic heart disease has recently been suggested . estrogens also stimulate no generation and cause no dependent vasodilatation which may increase myocardial blood flow . microinjection of estrogen analogue in the rvlm reportedly produced a depressor effect through a decrease in sympathetic nerve activity , and activation of inducible nitric oxide synthase ( inos)-derived no in rats . we investigated the role of estrogen in the cardiovascular responses of the bst for the first time . microinjection of glutamate into the bst of ovx and ovx+e female anesthetized rats elicited depressor and bradycardic responses in both groups ( figures 3 - 9 ) . the magnitude of depressor and bradycardic responses in ovx and ovx+e rats were similar , suggesting that the circulatory level of estrogen did not affect the bst cardiovascular responses , even though the bst contains a high density of estrogen receptors that affect some physiological functions such as sexual behavior , social affiliation , sexual dimorphism , and masculinization of the bst neurons . the depressor and bradycardic responses to the bst stimulation by glutamate are similar to the findings of the pervious experiments in male anesthetized rat . the depressor effect is caused by the inhibition of sympathetic effect on the vasculature and heart . in other areas of the central nervous system , the regulatory effect of estrogen on the cardiovascular system has been shown . for example circulatory estrogen potentiates the bradycardic response to stimulation of nucleus tractus solitarius by microinjection of hypocretine-1 ; however , we found that the estrogen receptor of the bst had no significant effect on its cardiovascular effects . for the first time , our study showed that the decrease in map and hr in response to the bst stimulation was partly mediated by a pathway from the bst to the rvlm . this is based on the finding that microinjection of the reversible synaptic blocker cocl2 into the rvlm of ovx and ovx+e animals reduced the depressor and bradycardic responses elicited by the bst stimulation for approximately 20 minutes . the responses to re - stimulation of the bst 60 min after the injection of cocl2 into the rvlm returned to the control values , suggesting that previous bst stimulation of the same site did not produce neuronal damage . the injection of cocl2 into the rvlm disrupt temporary neuronal transmission because of reversible inactivation of calcium channel on presynaptic terminals . in another part of this study we showed that gabaergic neurons in the rvlm are involved in the cardiovascular responses elicited by activation of the bst . but injection of phaclophen , a gabab antagonist , did not alter the bst cardiovascular responses . the alternation of the bst responses by the injection of bicuculline suggests that the cardiovascular output neurons of the bst might stimulate the rvlm neurons . it is probable that the activation of gabaergic neuron of the rvlm in part might be under the influence of the bst neurons . a previous study showed that gaba neurons in the rvlm were located caudal to the end pole of the facial nucleus , which is the same site that we injected cocl2 and gaba antagonists . taken together , these data suggest that there is a direct projection from the bst to the rvlm . however , this possibility can not be considered that the bst innervate other forebrain and brainstem area , that all are involved in cardiovascular regulation and which in turn project to the rvlm . the rvlm is a critical site in the tonic and reflex control of blood pressure and monosynaptically connected to sympathetic preganglionic neurons in intermediolateral horn of the spinal cord . the spinal cord mediates the sympathetic effect of the forebrain and brain stem nuclei to vasculature and heart . we demonstrated that circulatory estrogen at the physiological level did not alter the depressor and bradycardic responses to the bst stimulation by glutamate . for the first time we showed that these responses are mediated partly by pathways that terminate at rvlm neurons . the present result also indicates that the gabaa receptors of the rvlm are in part involved in the bst cardiovascular responses .

background : the areas of the bed nucleus of the stria terminalis ( bst ) with a high density of estrogen receptors are involved in cardiovascular regulation and send axonal projections to the rostroventrolateral medulla ( rvlm ) . we aimed to find the contribution of the rvlm to cardiovascular responses elicited by glutamate microinjection into the bst . methods : experiments were done in -chloralose anesthetized ovariectomized ( ovx ) or ovx estrogen treated ( ovx+e ) female wistar rats . drugs were microinjected into the bst and rvlm . the average changes in mean arterial pressure ( map ) and heart rate ( hr ) were compared between the case and control groups using t test and with the pre - injection values using paired t test . results : unilateral microinjection of glutamate ( 0.25 m/50 nl ) into the bst decreased map and hr , in the ovx+e and ovx rats . these cardiovascular responses were reversibly attenuated 10 minutes after microinjection of synaptic blocker cobalt chloride ( cocl2 , 5 mm/50 nl ) into the ipsilateral rvlm . re - stimulation of the bst 60 min after cocl2 injection elicited cardiovascular responses that were not different from the control values . ipsilateral microinjection of gabaa antagonist bicuculline ( 1.0 mm/50 nl ) into the rvlm caused a 50% attenuation of glutamate induced depressor and bradycardic responses in both groups . ipsilateral microinjection of gabab antagonist , phaclophen ( 5.0 mm/50 nl ) , into the rvlm did not affect the depressor and bradycardic responses due to re - stimulation of the bst by glutamate . conclusion : the rvlm sympathetic premotor neurons contain gabaa receptors that mediate in part the sympathoinhibitory responses to stimulation of the bst in the ovx animals .

Introduction Materials and Methods Results Discussion Conclusion None

the bed nucleus of stria terminalis ( bst ) is a limbic forebrain structure that concentrate estrogen , and aromatase enzyme , which locally convert testosterone to estrogen . microinjection of glutamate into the bst decreased arterial pressure ( ap ) and heart rate ( hr ) . the areas within the bst that elicit cardiovascular responses contain high densities of estrogen receptors . in addition , the neurons within these areas that concentrate estrogen send axonal projections to medullary regions ; the caudal ventrolateral medulla ( cvlm ) , and the rostroventrolateral medulla ( rvlm ) , which are both directly involved in autonomic output to the heart and vasculature . on the other hand , major outputs from the bst go to the rvlm and this medullary region may mediate the bst cardiovascular responses to the heart and vasculature . the inhibition of gabaa receptor by microinjection of bicuculline into the rvlm caused a marked increase in mean arterial pressure ( map ) in normotensive ( but not hypertensive ) rats . this study was performed to find the contribution of the rvlm to cardiovascular responses elicited by glutamate microinjection into the bst . we studied ovariectomized ( ovx ) and ovx estrogen treated ( ovx+e ) anesthetized rats to assess the following : -the effect of circulatory estrogen on cardiovascular responses of the bst , -the effect of reversible ablation of the rvlm on the bst cardiovascular responses , and -the effect of blockade of the gaba system of rvlm on the bst cardiovascular responses . the animals were placed in prone position in a stereotaxic frame ( stoelting , usa ) and two bilateral small holes were drilled through the parietal bone over the bst and through the occipital bone over the rvlm . micropipette tips were positioned in the bst and rvlm according to a stereotaxic atlas of the brain . the injection volume of glutamate ( glu ) into the bst and rvlm were 20 and 50 nl , respectively . experimental groups the experiments were designed for studying the neuronal connectivity between bst and rvlm in relation to cardiovascular responses . the experiments were done on different groups of ovx and ovx+e female rats , as follows : -microinjection of saline into the bst ( the control group [ n=6 ] , 20 nl ; ovx [ n=3 ] , 13 injections ; and ovx+e [ n=3 ] , 14 injections ) -microinjection of l - glutamate into the bst ( 0.25 m/20 nl ; sigma , ovx [ n=24 ] , 81 injections ; and ovx+e [ n=23 ] , 76 injections ) . -to find the neuronal connectivity between the bst and the rvlm , l - glutamate was initially injected into the bst ( control ) , then after arterial pressure and hr returned to the baseline , reversible synaptic blocker cobalt chloride ( cocl2 5 mm/50 nl , sigma ) was injected into the rvlm . the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of cocl2 into the rvlm of the ovx ( n=6 , 23 injections ) and ovx+e ( n=4 , 19 injections ) rats . -to investigate the effect of inhibition of gabaa receptors of the rvlm on cardiovascular responses of the bst , first l - glutamate was injected into the bst ( control ) and after the arterial pressure and hr returned to the baseline , a gabaa antagonist , bicuculline ( 1 mm/50 nl , sigma ) was injected into the rvlm and the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of bicuculline into the rvlm of the ovx ( n=6 , 36 injections ) and ovx+e ( n=7 , 41 injections ) rats . -to find the effect of inhibition of gabab receptors of the rvlm on cardiovascular responses of the bst , first l - glutamate was injected into the bst ( control ) and after the ap and hr returned to the baseline , a gabab antagonist phaclophen ( 5 mm/50 nl , sigma ) was injected into the rvlm and the bst was re - stimulated at 10 , 20 , 40 , and 60 minutes after the injection of phaclophen into the rvlm of the ovx ( n=6 , 36 injections ) and ovx+e ( n=7 , 41 injections ) rats . at the end of each experiment , 20 and 50 nl pontamine sky blue ( sigma , usa ) were microinjected into the bst and the rvlm , respectively , then the animal was sacrificed by a high dose of the anesthetic , and perfused transcardially with100 ml of 0.9% saline followed by 100 ml of 10% saline formalin . schematic coronal sections of rat brain adopted from an atlas,23 show the injection of glutamate ( ) into the bst sites which decrease blood pressure and heart rate . ac : anterior commissure ; acp : anterior commissure posterior part ; bstm : bed nucleus stria terminals medial part ; ic : inferior colliculus ; fx : fornix ; ox : optic chiasma ; 3v : third ventricle schematic coronal sections of rat brain adopted from an atlas,23 show the injection sites of cobalt chloride ( ) , bicuculline ( ) , and phaclophen ( o ) into the rvlm . the maximum changes of heart rate ( hr ) and the maximum changes of map ( map ) were compared between ovx , ovx+e , and saline groups using independent t test . cardiovascular responses of regional controls in the anaesthetized female ovx and ovx+e rats , microinjection of 20 nl of glutamate in the regions around the bst did not affect the pressure ( map=2.63.6 mmhg ) and the hr ( hr=-3.22.3 beats / min ; 43 injections ) . cardiovascular responses to vehicle microinjection into the bst to test whether cardiovascular response of bst was related to the volume of injection or mechanical torsion , 20 nl saline was microinjected into the bst of ovx and ovx+e animals . the changes in map ( map=0.70.6 mmhg ) and hr ( hr=0.570.2 beats / min ) were not significantly different from the pre - injection values . cardiovascular responses to glutamate microinjection into the bst to determine the effect of glutamate on the map and hr , it was microinjected into the bst ( 0.25 m/20 nl ) . the changes of the hr in the ovx+e rats was lower than the ovx rats ( p<0.01 , figure 3 ) . microinjection of glutamate into the dorsolateral and ventral subnuclei of the bst complex in both ovx and ovx+e rats showed decreases in hr and map ( ovx : map : -25.70.4 mmhg and hr : -14.30.3 bpm ; ovx+e : map : -25.50.3 mmhg and hr ; -12.60.5 bpm ) . the changes were significantly different from the saline group ( p<0.01 ) and the pre - injection values ( p<0.01 ) . * significant difference between ovx and ovx+e groups , t test , p<0.01 cardiovascular response elicited by glutamate injection into the bst after the injection of synaptic blocker in the rvlm to investigate the possibility that the bst cardiovascular responses were mediated by the projection to rvlm , glutamate was first injected into the bst of the ovx and ovx+e rats . the depressor and bradycardic responses were similar in the magnitude in the pervious experiments ( ovx : map : -23.85.97 mmhg and hr : -10.02.5 bpm ; ovx+e : map : -19.02.3 mmhg and hr : -10.06.7 bpm , p<0.01 ) , then cocl2 , was injected into the rvlm . microinjection of cocl2 into the rvlm of ovx and ovx+e rats had no significant effect on the baseline values of map and hr compared with the pre - injection values ( ovx : map : -0.20.2 mmhg and hr : -4.02.3 bpm ; ovx+e : map : -1.00.2 mmhg and hr : 0.80.1 bpm ) . re - stimulation of the bst , 10 minutes after microinjection of cocl2 into the cardiovascular site of rvlm significantly attenuated the depressor and bradycardic responses of the same site of the bst . the magnitude of depressor response during stimulation of bst 10 minutes after cocl2 microinjection into the rvlm was significantly different from the pre - injection values ( ovx : map : -6.32.1 mmhg and hr : -2.52.5 bpm [ p<0.01 ] ; ovx+e : map : -11.31.6.3 mmhg and hr : -6.24.2 bpm [ p<0.05 ] ) . the magnitude of depressor and bradycardic responses by re - stimulation of bst , 60 min after cocl2 microinjection into the rvlm ( ovx : map : -19.83 . 7 mmhg and hr : -10.04.5 bpm ; ovx+e : map : -18.92.5 mmhg and hr : -8.5.05.2 bpm ) did not yield different results from the control values indicating that the effect of cocl2 was abolished ( figures 4 and 5 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of cocl2 into the rvlm and re - stimulation of bst at 10 and 60 minutes after injection of cocl2 in rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of cocl2 ( 5 mm/50 nl ) into the rvlm in ovx and ovx+e rats . * significant different compared with pre - injection values ( control ) , paired t test , p<0.01 cardiovascular responses elicited by glutamate injection into the bst after the injection of bicuculline in the rvlm to test the possible inhibition of cardiovascular responses of the bst by gabaergic neurons of the rvlm , first glutamate was injected into the bst of the ovx and ovx+e rats , then a gabaa antagonist bicuculline , was injected into the rvlm . microinjection of bicuculline into the rvlm of the ovx and ovx+e rats led to a significant increase on the map with no significant effect on hr compared with their pre - injection value ( ovx : map : 30.45.2 mmhg and hr : 12.2.08.3 bpm ; ovx+e : map : 35.954.4 mmhg and hr : 9.26.11 bpm , p<0.01 ) . similar to pervious experiments , 10 minutes later , glutamate was injected into the bst of the ovx and ovx+e rats . the depressor and bradycardic responses caused by stimulation of the bst were similar to those of the pervious experiments ( ovx : map : -22.52.68 mmhg and hr : -12.22.1 bpm ; ovx+e : map : -28.13.7 mmhg and hr : -14.23.5 bpm , p<0.01 , t test ) . the magnitude of depressor response during stimulation of the bst 10 minutes after bicuculline microinjection into the rvlm were reduced to almost 50% of their control value ( ovx : map : -11.93.3 mmhg and hr : -10.02.5 bpm ; ovx+e : map : -16.32.4 mmhg and hr : -7.51.7 bpm , p<0.01 ) . 60 min after the microinjection of bicuculline into the rvlm , the magnitude of depressor and bradycardic responses by re - stimulation of the bst approximately returned to their control value ( ovx : map : -15.83.7 mmhg and hr : -9.04.5 bpm ; ovx+e : map : -23.03.6 mmhg and hr : -12.52.5 bpm , figures 6 and 7 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of bicuculline ( 1 mm/50 nl ) into the rvlm and re - stimulation of bst at 10 and 60 minutes after injection of bicuculline into the rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of bicuculline into the rvlm in ovx and ovx+e rats . * significant difference with pre - injection values ( control ) , paired t test , p<0.01 cardiovascular response elicited by glutamate injected into the bst after the injection of phaclophen in the rvlm to find the possible effects of gabab receptors of the rvlm on the cardiovascular responses of the bst , phaclophen , a gabab antagonist , was microinjected into the rvlm of the ovx and ovx+e rats . no significant difference was found in both map and hr compared with their pre - injection values ( ovx : map : 4.31.1 mmhg and hr : 3.32.1 bpm ; ovx+e : map : 6.91.4 mmhg and hr : 8.73.2 bpm ) . similar to pervious experiments , microinjection of glutamate into the bst elicited bradycardic and depressor responses ( ovx : map : -26.13.0 mmhg and hr : -16.93.1 bpm ; ovx+e : map : -26.82.62 mmhg and hr : -13.65 bpm , p<0.01 , t test ) . unlike the effect of bicuculline , microinjection of phaclophen did not alter the magnitude of depressor and bradycardic responses by re - stimulation of the bst , 10 , 20 , 40 , and 60 minutes after microinjection of phaclophen into the cardiovascular site of rvlm ( figures 8 and 9 ) . this figure shows tracings of blood pressure and heart rate responses elicited by microinjection of glutamate into the bst before ( control ) and after injection of phaclophen into the rvlm and re - stimulation of bst at 10 and 60 minutes after the injection of phaclophen into the rvlm in ovx and ovx+e rats , arrows indicate the injection times . scale bar : 1 min this figure shows the cardiovascular effect of glutamate ( 0.25 m/20 nl ) injection into the bst before ( control ) and 10 , 20 , 40 , and 60 min after injection of phaclophen ( 5 mm/50 nl ) into the rvlm in ovx and ovx+e rats . we found that estrogen decreased hr significantly , circulatory estrogen did not alter the cardiovascular depressor response evoked by bst stimulation , rvlm neurons in the medulla mediated the bst cardiovascular responses , and the depressor response of the bst was partly mediated by the activation of gaba neurons of the rvlm . circulating estrogen within the physiological range affects hr , since the baseline hr in the ovx+e rats was lower than in the ovx rats . we investigated the role of estrogen in the cardiovascular responses of the bst for the first time . microinjection of glutamate into the bst of ovx and ovx+e female anesthetized rats elicited depressor and bradycardic responses in both groups ( figures 3 - 9 ) . the magnitude of depressor and bradycardic responses in ovx and ovx+e rats were similar , suggesting that the circulatory level of estrogen did not affect the bst cardiovascular responses , even though the bst contains a high density of estrogen receptors that affect some physiological functions such as sexual behavior , social affiliation , sexual dimorphism , and masculinization of the bst neurons . the depressor and bradycardic responses to the bst stimulation by glutamate are similar to the findings of the pervious experiments in male anesthetized rat . for example circulatory estrogen potentiates the bradycardic response to stimulation of nucleus tractus solitarius by microinjection of hypocretine-1 ; however , we found that the estrogen receptor of the bst had no significant effect on its cardiovascular effects . for the first time , our study showed that the decrease in map and hr in response to the bst stimulation was partly mediated by a pathway from the bst to the rvlm . this is based on the finding that microinjection of the reversible synaptic blocker cocl2 into the rvlm of ovx and ovx+e animals reduced the depressor and bradycardic responses elicited by the bst stimulation for approximately 20 minutes . the responses to re - stimulation of the bst 60 min after the injection of cocl2 into the rvlm returned to the control values , suggesting that previous bst stimulation of the same site did not produce neuronal damage . in another part of this study we showed that gabaergic neurons in the rvlm are involved in the cardiovascular responses elicited by activation of the bst . but injection of phaclophen , a gabab antagonist , did not alter the bst cardiovascular responses . however , this possibility can not be considered that the bst innervate other forebrain and brainstem area , that all are involved in cardiovascular regulation and which in turn project to the rvlm . we demonstrated that circulatory estrogen at the physiological level did not alter the depressor and bradycardic responses to the bst stimulation by glutamate . the present result also indicates that the gabaa receptors of the rvlm are in part involved in the bst cardiovascular responses .

in introductory biology courses , many students find that the adjustment to college expectations can be difficult . this can lead to lower success rates on exams , which can be immensely frustrating to both students and their professors . students may not realize how much they need to prepare due to a false level of confidence in their understanding . alternatively , a substantial percentage of students are frustrated with the perception that they study and prepare sufficiently to be successful on exams but still experience a lack of successful outcome . this can be due to students becoming familiar with material by reviewing notes or rereading text without actually developing reliable recall of the information , or it can be due to a study environment that , unlike a testing environment , provides cues that aid in information recall . evidence indicates conditions that create difficulty in retrieving information actually optimize long - term storage and recall ( bjork and bjork , 2011 ) . bjork 's studies point to two distinct events that must occur for students to experience success on classroom exams . students must first successfully store presented information through lecture , integration of material using familiar associations , and subsequent study of the material . instructors can aid students in their endeavors to learn and store presented information by sharing real - life analogies as a framework into which students can integrate the presented material . equally important to success on these exams is the practice of retrieval of the stored information . bjork asserts that frequent , low - stakes testing opportunities enable students to practice retrieval as a learning event and will strengthen retrieval efforts as students prepare for their exams ( bjork and bjork , 2011 ) . this investigation seeks to utilize bjork 's findings to increase success of community college students in the introductory ( majors ) biology classroom through the implementation of required quizzes using an online homework platform . although the effectiveness of online quizzing on increasing exam performance has been studied in courses such as undergraduate social psychology ( johnson , 2009 ) and exercise physiology ( dobson , 2008 ) , the effectiveness of this approach when utilized in an introductory ( majors ) biology course has not been well documented . we evaluate the efficacy of quizzing in increasing student success on exams in the context of the significant challenge inherent to the mastery of the content and vocabulary and rigor of the material in the majors biology classroom . additionally , optional online quizzing has been associated with increased achievement on tests in a college educational psychology classroom , but student participation was low , resulting in data gathered specifically from the self - selected population of those students choosing to take these optional quizzes ( johnson , 2006 ) . we introduce quiz taking as a required component of the lecture grade to decrease this self - selection factor . specifically , we have studied the efficacy of using frequent pre - exam quizzing to increase information retrieval by creating required pre - exam quizzes for those chapter topics that are known to present students with difficulty in learning and understanding . quiz questions were created that assessed understanding of the subject content to be covered on exams . questions were unique to the quizzes so as to not be a preview of upcoming exam questions . quiz questions were designed to match or exceed the level of difficulty of exam questions . desirable difficulties , as our intent was to challenge the students level of recall ( bjork and bjork , 2011 ) . data were gathered during two semesters fall 2011 and spring 2012to assess the reliability of our results . ten quizzes were given throughout the semester , targeting the chapter material that seems most difficult for students . quizzes were established as required in the course syllabus , worth 10% of the total lecture grade . three groups were created within the mb course , and students were randomly assigned to these groups . randomization was accomplished by assigning students to groups a , b , or c as they registered for mb ( across all sections ) . mb settings for pre - exam quizzes were selected to randomize the item sequence so students within a given group would not see quiz questions presented in the same order . quizzes were generally limited to a 15-min time limit ( 10 questions ) to reduce the chance that internet searches would be used to determine the correct answer . to reduce the chance that students would help each other , students were able to see whether an answer was correct only after the assignment was due , and if students answered incorrectly , the correct answer was shown only after the assignment due date . additionally , no hints were shown , no printing was allowed , item titles were hidden , and student access was limited ( no access between completion and due date ) . pre - exam quiz averages , exam scores , and percent of pre - exam quizzes taken were recorded for analysis . table 1 provides a comparison of the percentage of students completing required pre - exam quizzes prior to each of the exams given over the course of the fall 2011 and spring 2012 semesters . students who became inactive in the class during the course of the semester , either by formally withdrawing or by ceasing to take exams , were not included in this analysis . student compliance with the pre - exam quiz requirement was similar for the fall 2011 and spring 2012 semesters . compliance with the pre - exam quizzing requirement was high at the beginning of each semester , as evidenced by close to 80% of the students taking all quizzes prior to exam 1 . the percentage of students taking all pre - exam quizzes dropped as each semester progressed , with only 59% taking these quizzes prior to exam 4 . likewise , the percentage of students taking no pre - exam quizzes was low at the beginning of the semester ( just above 2% ) but increased throughout the semester , with close to 15% of the students taking 0% of quizzes prior to exam 4 . comparison of percent pre - exam quizzes taken prior to each exam for fall 2011 and spring 2012 average exam scores of students grouped by the percentage of pre - exam quizzes completed prior to each of the four exams were compared for the fall 2011 and spring 2012 semesters ( figures 1 and 2 , respectively ) . scores for exams 2 , 3 , and 4 are significantly lower for the 0% pre - exam quiz group compared with the 100% pre - exam quiz group . the group of students completing 100% of the quizzes , as well as the group completing 0% of the quizzes , maintained a consistent course average ( no significant difference ) throughout each semester analyzed . comparison of exam averages for quiz takers and non quiz takers for fall 2011 . comparison of exam averages for quiz takers and non quiz takers for spring 2012 . exam data were analyzed for both the fall 2011 and spring 2012 ( figures 1 and 2 , respectively ) semesters to determine whether exam averages of those taking 0% of the quizzes were significantly below the class average . the sample size for exam 1 ( fall : n = 5 ; spring : n = 4 ) for 0% quiz completers does not allow any significant analysis . however , for both semesters , the mean of the 0% pre - exam quiz completion group was significantly below the class average and the average for the 100% pre - exam quiz completion group for exams 2 , 3 , and 4 . class exam average remained fairly constant for each given exam in both semesters . to determine whether students of all abilities benefit from participation in quizzing , we grouped students by their averages on exams 1 and 2 . performance on these two exams allows us to identify the scholastic ability of students taking this course . student groups defined by the average of exams 1 and 2 figure 3 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 3 average than those students in the pass group who took 0% of pre - exam quizzes . the groups ( sample sizes ) shown in figure 3 are : 100% pass ( fall 2011 : n = 71 ; spring 2012 : n = 64 ) , 0% pass ( fall 2011 : n = 5 ; spring 2012 : n = 9 ) , 100% fail ( fall 2011 : n = 41 ; spring 2012 : n = 27 ) , and 0% fail ( fall 2011 : n = 7 ; spring 2012 : n = 11 ) . comparison of exam 3 averages for pass and fail groups between 100% quiz takers and 0% quiz takers . analysis of the fail group of students ( figure 3 ) shows that students taking 100% of the pre - exam quizzes in fall 2011 also had significantly higher exam 3 averages than students in the fail group taking 0% of the pre - exam quizzes . in spring 2012 , the 95% confidence intervals for the students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped . a higher exam 3 average for the group taking all pre - exam quizzes figure 4 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 4 average than those students in the pass group who took 0% of pre - exam quizzes . the groups ( sample sizes ) shown in figure 4 are : 100% pass ( fall 2011 : n = 68 ; spring 2012 : n = 56 ) , 0% pass ( fall 2011 : n = 8 ; spring 2012 : n = 15 ) , 100% fail ( fall 2011 : n = 34 ; spring 2012 : n = 22 ) , and 0% fail ( fall 2011 : n = 8 ; spring 2012 : n = 14 ) . comparison of exam 4 averages for pass and fail groups between 100% quiz takers and 0% quiz takers . the group of students who took 100% of the pre - exam quizzes in the fail group in fall 2011 also had significantly higher exam 4 averages than those students in the fail group who took 0% of the pre - exam quizzes ( figure 4 ) . in the spring 2012 semester , the 95% confidence intervals for students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped , but a higher exam 4 average for the group taking all pre - exam quizzes was still observed . in an effort to do an even more granular analysis of the benefit of quizzing using demonstrated performance on exams , we sorted students into three groups representing a more narrow distribution of exam score averages ( table 2 ) . these groups are composed of both fall 2011 and spring 2012 students , such that sample sizes might be large enough that statistically significant differences could be observed . the groups ( sample sizes ) shown in figure 5 are : 100% low ( n = 26 ) , 0% low ( n = 7 ) , 100% middle ( n = 92 ) , 0% middle ( n = 21 ) , 100% high ( n = 85 ) , and 0% high ( n = 4 ) . the groups ( sample sizes ) shown in figure 6 are : 100% low ( n = 19 ) , 0% low ( n = 9 ) , 100% middle ( n = 82 ) , 0% middle ( n = 26 ) , 100% high ( n = 79 ) , and 0% high ( n = 10 ) . comparison of exam 3 averages for low , middle , and high groups between 100% quiz takers and 0% quiz takers for fall 2011 and spring 2012 combined . comparison of exam 4 averages for low , middle , and high groups between 100% quiz takers and 0% quiz takers for fall 2011 and spring 2012 combined . figure 5 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . low - performing students taking 100% of pre - exam quizzes also had higher averages for exam 3 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . figure 6 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . as was seen for exam 3 , students with a low performance taking 100% of pre - exam quizzes also have higher averages for exam 4 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . table 1 provides a comparison of the percentage of students completing required pre - exam quizzes prior to each of the exams given over the course of the fall 2011 and spring 2012 semesters . students who became inactive in the class during the course of the semester , either by formally withdrawing or by ceasing to take exams , were not included in this analysis . student compliance with the pre - exam quiz requirement was similar for the fall 2011 and spring 2012 semesters . compliance with the pre - exam quizzing requirement was high at the beginning of each semester , as evidenced by close to 80% of the students taking all quizzes prior to exam 1 . the percentage of students taking all pre - exam quizzes dropped as each semester progressed , with only 59% taking these quizzes prior to exam 4 . likewise , the percentage of students taking no pre - exam quizzes was low at the beginning of the semester ( just above 2% ) but increased throughout the semester , with close to 15% of the students taking 0% of quizzes prior to exam 4 . comparison of percent pre - exam quizzes taken prior to each exam for fall 2011 and spring 2012 average exam scores of students grouped by the percentage of pre - exam quizzes completed prior to each of the four exams were compared for the fall 2011 and spring 2012 semesters ( figures 1 and 2 , respectively ) . scores for exams 2 , 3 , and 4 are significantly lower for the 0% pre - exam quiz group compared with the 100% pre - exam quiz group . the group of students completing 100% of the quizzes , as well as the group completing 0% of the quizzes , maintained a consistent course average ( no significant difference ) throughout each semester analyzed . comparison of exam averages for quiz takers and non quiz takers for fall 2011 . comparison of exam averages for quiz takers and non quiz takers for spring 2012 . exam data were analyzed for both the fall 2011 and spring 2012 ( figures 1 and 2 , respectively ) semesters to determine whether exam averages of those taking 0% of the quizzes were significantly below the class average . the sample size for exam 1 ( fall : n = 5 ; spring : n = 4 ) for 0% quiz completers does not allow any significant analysis . however , for both semesters , the mean of the 0% pre - exam quiz completion group was significantly below the class average and the average for the 100% pre - exam quiz completion group for exams 2 , 3 , and 4 . to determine whether students of all abilities benefit from participation in quizzing , we grouped students by their averages on exams 1 and 2 . performance on these two exams allows us to identify the scholastic ability of students taking this course . student groups defined by the average of exams 1 and 2 figure 3 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 3 average than those students in the pass group who took 0% of pre - exam quizzes . the groups ( sample sizes ) shown in figure 3 are : 100% pass ( fall 2011 : n = 71 ; spring 2012 : n = 64 ) , 0% pass ( fall 2011 : n = 5 ; spring 2012 : n = 9 ) , 100% fail ( fall 2011 : n = 41 ; spring 2012 : n = 27 ) , and 0% fail ( fall 2011 : n = 7 ; spring 2012 : n = 11 ) . comparison of exam 3 averages for pass and fail groups between 100% quiz takers and 0% quiz takers . analysis of the fail group of students ( figure 3 ) shows that students taking 100% of the pre - exam quizzes in fall 2011 also had significantly higher exam 3 averages than students in the fail group taking 0% of the pre - exam quizzes . in spring 2012 , the 95% confidence intervals for the students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped . a higher exam 3 average for the group taking all pre - exam quizzes figure 4 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 4 average than those students in the pass group who took 0% of pre - exam quizzes . the groups ( sample sizes ) shown in figure 4 are : 100% pass ( fall 2011 : n = 68 ; spring 2012 : n = 56 ) , 0% pass ( fall 2011 : n = 8 ; spring 2012 : n = 15 ) , 100% fail ( fall 2011 : n = 34 ; spring 2012 : n = 22 ) , and 0% fail ( fall 2011 : n = 8 ; spring 2012 : n = 14 ) . comparison of exam 4 averages for pass and fail groups between 100% quiz takers and 0% quiz takers . the group of students who took 100% of the pre - exam quizzes in the fail group in fall 2011 also had significantly higher exam 4 averages than those students in the fail group who took 0% of the pre - exam quizzes ( figure 4 ) . in the spring 2012 semester , the 95% confidence intervals for students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped , but a higher exam 4 average for the group taking all pre - exam quizzes was still observed . in an effort to do an even more granular analysis of the benefit of quizzing using demonstrated performance on exams , we sorted students into three groups representing a more narrow distribution of exam score averages ( table 2 ) . these groups are composed of both fall 2011 and spring 2012 students , such that sample sizes might be large enough that statistically significant differences could be observed . ( students took the same exams and the same quizzes each semester . ) the groups ( sample sizes ) shown in figure 5 are : 100% low ( n = 26 ) , 0% low ( n = 7 ) , 100% middle ( n = 92 ) , 0% middle ( n = 21 ) , 100% high ( n = 85 ) , and 0% high ( n = 4 ) . the groups ( sample sizes ) shown in figure 6 are : 100% low ( n = 19 ) , 0% low ( n = 9 ) , 100% middle ( n = 82 ) , 0% middle ( n = 26 ) , 100% high ( n = 79 ) , and 0% high ( n = 10 ) . comparison of exam 3 averages for low , middle , and high groups between 100% quiz takers and 0% quiz takers for fall 2011 and spring 2012 combined . comparison of exam 4 averages for low , middle , and high groups between 100% quiz takers and 0% quiz takers for fall 2011 and spring 2012 combined . figure 5 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . low - performing students taking 100% of pre - exam quizzes also had higher averages for exam 3 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . figure 6 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . as was seen for exam 3 , students with a low performance taking 100% of pre - exam quizzes also have higher averages for exam 4 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . the implication of this study is that frequent online quizzing is an effective approach in increasing students ability to retrieve information and subsequent performance in the introductory ( majors ) biology classroom . students who take pre - exam quizzes do significantly better than those who do not take pre - exam quizzes . the exam average gap between quiz takers and non quiz takers observed as the semester progressed is notable , as the exam average of the group of students not taking any of the required pre - exam quizzes falls significantly below the class average as the semester progresses . while we do observe that increased quiz scores are significantly correlated with increased exam scores ( unpublished data ) , what is particularly notable about our findings is that simply participating in pre - exam quizzing ( 100% quiz takers vs. 0% quiz takers ) is associated with increased exam scores . this supports bjork 's assertions that testing serves as a learning event and should not be utilized only as an assessment tool . most intriguing is the evidence that students of all abilities benefit from participation in pre - exam quizzing . that students who take quizzes have higher exam averages than those who did not take quizzes is not surprising ( figures 1 and 2 ) many would point to the fact that merely taking quizzes is an indicator of the level of scholastic strength and motivation in a given student . to determine whether this was the case , we grouped these students by ability ( as assessed by exam 1 and 2 average ) and then compared 100% quiz takers with 0% quiz takers within each group . this effort was made to mitigate the innate ability of the student as a confounding factor and to focus on the potential benefit participation in quizzing might give a student . in both fall 2011 and spring 2012 , students with a passing average on exams 1 and 2 were observed to significantly benefit from taking 100% of pre - exam quizzes as compared with those not taking these quizzes . additionally , we were quite encouraged to find that those students with a failing average taking 100% pre - exam quizzes showed significantly higher exam 3 and exam 4 averages than those taking no pre - exam quizzes in the fall 2011 semester . a similar trend of higher exam scores for students taking 100% of pre - exam quizzes was observed in spring 2012 , although significance was not established for that group ( figures 3 and 4 ) . this resulted in our redefining the pass / fail student groups into groups that were more narrowly defined ( high , middle , and low ) . analysis of these groups demonstrates not only that students with a high performance benefit from pre - exam quizzing , but also that those middle students also see significant benefit from taking pre - exam quizzes ( figures 5 and 6 ) . those students averaging below 60% on exams 1 and 2 also seemed to experience some benefit from the quiz - taking activity , although significance was not established , due to low sample size . the advantage to participating in pre - exam quizzing seems to give even the weakest students a letter grade advantage over those not taking pre - exam quizzes . the benefit seen by high or strong students taking 100% versus 0% of pre - exam quizzes seems particularly strong when compared with the benefit seen by middle / average students taking 100% versus 0% of pre - exam quizzes ( figures 5 and 6 ) . however , specific analysis of the gains in exam average with increased quiz - taking activity indicates that the benefit yielded to each of these two groups is not significantly different . in summary , in every group analyzed , students who took all of the offered pre - exam quizzes had higher exam scores than those taking none of the offered pre - exam quizzes . of particular interest is the fact that compliance in taking the required pre - exam quizzes declined throughout the semester , in spite of the required nature of these quizzes . desirable difficulties to students in the college classroom ( bjork and bjork , 2011 ) . the introduction of required pre - exam quizzes seems to create a significant enough difficulty for some students such that they reject the effort entirely . it should be noted that the difficulty of the quizzes was significant but not extreme ( on a scale of 15 , in which 1 is easy and 5 is most difficult , quizzes were assessed by mb to average around 2.8 ) . this may be due to the nature of the student population , as this study was done in a community college classroom , in which no selection criteria for admission are in place . this yields a notable variation in the student population from a motivation and preparation standpoint . current efforts focus on increasing student compliance by emphasizing the potential benefit of quizzing on exam scores to the students prior to each quiz period . surveying students regarding their experience with pre - exam quizzing indicates that most students found this strategy valuable , as 87.32% of students surveyed indicated that they plan to continue quizzing as part of their preparation for future classes ( n = 142 ) . particularly interesting is that 73.42% reported that taking the required quizzes caused them to go back and do additional studying of the material / additional preparation prior to the exam . pre - exam quizzing may act as an early warning system for students , showing them that they have the illusion of knowing the material but have not learned the material sufficiently for accurate recollection in a testing environment ( koriat and bjork , 2005 ) . in fact , surveyed students reported that pre - exam quizzing increased their understanding of the material ( 26.28% ) , but more commonly helped them to pinpoint how to study for exams ( 57.69% ) . while pre - exam quizzing is only one of many strategies that a student may utilize in preparing for exams , koriat and bjork 's research indicates that having all of the information present , such as in the conditions in which most students study ( reading the textbook and reviewing notes ) , is less effective in yielding maximum recall of the information and more likely to produce an illusion of competence . this leads us to discard the idea that students not participating in pre - exam quizzing could simply increase the time committed to reviewing their notes and the text and see the same results as those taking 100% of the pre - exam quizzes . overall , the benefit of pre - exam quizzing seems to be driven by the decision of the individual student to commit to taking all of the required quizzes . our analysis indicates that this benefit is not limited to only strong students who are typically successful on exams , but that students of all abilities see a benefit from taking online , pre - exam quizzes . pre - exam quizzing utilizing an online platform seems to be an effective way to increase student performance on introductory biology exams , and quiz averages serve to give students feedback as to potential exam performance .

required pre - exam quizzes were given via an online homework platform . students taking these quizzes had a significantly higher exam average than those who took none of the quizzes and had a significantly higher exam average than the class average . the benefit of quizzing is demonstrated to be significant for students of diverse academic abilities .

INTRODUCTION METHODS RESULTS Student Compliance with the Quiz Requirement Decreased over the Course of Each Semester That Quizzes Were Given Students Who Take Pre-Exam Quizzes Tend to be More Successful on Exams Students of All Abilities Benefit from Participating in Pre-Exam Quizzing DISCUSSION

this can lead to lower success rates on exams , which can be immensely frustrating to both students and their professors . alternatively , a substantial percentage of students are frustrated with the perception that they study and prepare sufficiently to be successful on exams but still experience a lack of successful outcome . this can be due to students becoming familiar with material by reviewing notes or rereading text without actually developing reliable recall of the information , or it can be due to a study environment that , unlike a testing environment , provides cues that aid in information recall . bjork 's studies point to two distinct events that must occur for students to experience success on classroom exams . students must first successfully store presented information through lecture , integration of material using familiar associations , and subsequent study of the material . equally important to success on these exams is the practice of retrieval of the stored information . bjork asserts that frequent , low - stakes testing opportunities enable students to practice retrieval as a learning event and will strengthen retrieval efforts as students prepare for their exams ( bjork and bjork , 2011 ) . this investigation seeks to utilize bjork 's findings to increase success of community college students in the introductory ( majors ) biology classroom through the implementation of required quizzes using an online homework platform . we evaluate the efficacy of quizzing in increasing student success on exams in the context of the significant challenge inherent to the mastery of the content and vocabulary and rigor of the material in the majors biology classroom . we introduce quiz taking as a required component of the lecture grade to decrease this self - selection factor . specifically , we have studied the efficacy of using frequent pre - exam quizzing to increase information retrieval by creating required pre - exam quizzes for those chapter topics that are known to present students with difficulty in learning and understanding . quiz questions were created that assessed understanding of the subject content to be covered on exams . questions were unique to the quizzes so as to not be a preview of upcoming exam questions . data were gathered during two semesters fall 2011 and spring 2012to assess the reliability of our results . ten quizzes were given throughout the semester , targeting the chapter material that seems most difficult for students . quizzes were established as required in the course syllabus , worth 10% of the total lecture grade . mb settings for pre - exam quizzes were selected to randomize the item sequence so students within a given group would not see quiz questions presented in the same order . quizzes were generally limited to a 15-min time limit ( 10 questions ) to reduce the chance that internet searches would be used to determine the correct answer . pre - exam quiz averages , exam scores , and percent of pre - exam quizzes taken were recorded for analysis . table 1 provides a comparison of the percentage of students completing required pre - exam quizzes prior to each of the exams given over the course of the fall 2011 and spring 2012 semesters . students who became inactive in the class during the course of the semester , either by formally withdrawing or by ceasing to take exams , were not included in this analysis . student compliance with the pre - exam quiz requirement was similar for the fall 2011 and spring 2012 semesters . compliance with the pre - exam quizzing requirement was high at the beginning of each semester , as evidenced by close to 80% of the students taking all quizzes prior to exam 1 . the percentage of students taking all pre - exam quizzes dropped as each semester progressed , with only 59% taking these quizzes prior to exam 4 . likewise , the percentage of students taking no pre - exam quizzes was low at the beginning of the semester ( just above 2% ) but increased throughout the semester , with close to 15% of the students taking 0% of quizzes prior to exam 4 . comparison of percent pre - exam quizzes taken prior to each exam for fall 2011 and spring 2012 average exam scores of students grouped by the percentage of pre - exam quizzes completed prior to each of the four exams were compared for the fall 2011 and spring 2012 semesters ( figures 1 and 2 , respectively ) . scores for exams 2 , 3 , and 4 are significantly lower for the 0% pre - exam quiz group compared with the 100% pre - exam quiz group . the group of students completing 100% of the quizzes , as well as the group completing 0% of the quizzes , maintained a consistent course average ( no significant difference ) throughout each semester analyzed . comparison of exam averages for quiz takers and non quiz takers for fall 2011 . exam data were analyzed for both the fall 2011 and spring 2012 ( figures 1 and 2 , respectively ) semesters to determine whether exam averages of those taking 0% of the quizzes were significantly below the class average . however , for both semesters , the mean of the 0% pre - exam quiz completion group was significantly below the class average and the average for the 100% pre - exam quiz completion group for exams 2 , 3 , and 4 . class exam average remained fairly constant for each given exam in both semesters . to determine whether students of all abilities benefit from participation in quizzing , we grouped students by their averages on exams 1 and 2 . performance on these two exams allows us to identify the scholastic ability of students taking this course . student groups defined by the average of exams 1 and 2 figure 3 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 3 average than those students in the pass group who took 0% of pre - exam quizzes . analysis of the fail group of students ( figure 3 ) shows that students taking 100% of the pre - exam quizzes in fall 2011 also had significantly higher exam 3 averages than students in the fail group taking 0% of the pre - exam quizzes . in spring 2012 , the 95% confidence intervals for the students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped . a higher exam 3 average for the group taking all pre - exam quizzes figure 4 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 4 average than those students in the pass group who took 0% of pre - exam quizzes . the group of students who took 100% of the pre - exam quizzes in the fail group in fall 2011 also had significantly higher exam 4 averages than those students in the fail group who took 0% of the pre - exam quizzes ( figure 4 ) . in the spring 2012 semester , the 95% confidence intervals for students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped , but a higher exam 4 average for the group taking all pre - exam quizzes was still observed . in an effort to do an even more granular analysis of the benefit of quizzing using demonstrated performance on exams , we sorted students into three groups representing a more narrow distribution of exam score averages ( table 2 ) . figure 5 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . low - performing students taking 100% of pre - exam quizzes also had higher averages for exam 3 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . figure 6 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . as was seen for exam 3 , students with a low performance taking 100% of pre - exam quizzes also have higher averages for exam 4 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . table 1 provides a comparison of the percentage of students completing required pre - exam quizzes prior to each of the exams given over the course of the fall 2011 and spring 2012 semesters . students who became inactive in the class during the course of the semester , either by formally withdrawing or by ceasing to take exams , were not included in this analysis . student compliance with the pre - exam quiz requirement was similar for the fall 2011 and spring 2012 semesters . compliance with the pre - exam quizzing requirement was high at the beginning of each semester , as evidenced by close to 80% of the students taking all quizzes prior to exam 1 . the percentage of students taking all pre - exam quizzes dropped as each semester progressed , with only 59% taking these quizzes prior to exam 4 . likewise , the percentage of students taking no pre - exam quizzes was low at the beginning of the semester ( just above 2% ) but increased throughout the semester , with close to 15% of the students taking 0% of quizzes prior to exam 4 . comparison of percent pre - exam quizzes taken prior to each exam for fall 2011 and spring 2012 average exam scores of students grouped by the percentage of pre - exam quizzes completed prior to each of the four exams were compared for the fall 2011 and spring 2012 semesters ( figures 1 and 2 , respectively ) . scores for exams 2 , 3 , and 4 are significantly lower for the 0% pre - exam quiz group compared with the 100% pre - exam quiz group . the group of students completing 100% of the quizzes , as well as the group completing 0% of the quizzes , maintained a consistent course average ( no significant difference ) throughout each semester analyzed . comparison of exam averages for quiz takers and non quiz takers for fall 2011 . exam data were analyzed for both the fall 2011 and spring 2012 ( figures 1 and 2 , respectively ) semesters to determine whether exam averages of those taking 0% of the quizzes were significantly below the class average . however , for both semesters , the mean of the 0% pre - exam quiz completion group was significantly below the class average and the average for the 100% pre - exam quiz completion group for exams 2 , 3 , and 4 . to determine whether students of all abilities benefit from participation in quizzing , we grouped students by their averages on exams 1 and 2 . performance on these two exams allows us to identify the scholastic ability of students taking this course . student groups defined by the average of exams 1 and 2 figure 3 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 3 average than those students in the pass group who took 0% of pre - exam quizzes . analysis of the fail group of students ( figure 3 ) shows that students taking 100% of the pre - exam quizzes in fall 2011 also had significantly higher exam 3 averages than students in the fail group taking 0% of the pre - exam quizzes . in spring 2012 , the 95% confidence intervals for the students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped . a higher exam 3 average for the group taking all pre - exam quizzes figure 4 shows that students taking 100% of pre - exam quizzes in the pass group had a significantly higher exam 4 average than those students in the pass group who took 0% of pre - exam quizzes . comparison of exam 4 averages for pass and fail groups between 100% quiz takers and 0% quiz takers . the group of students who took 100% of the pre - exam quizzes in the fail group in fall 2011 also had significantly higher exam 4 averages than those students in the fail group who took 0% of the pre - exam quizzes ( figure 4 ) . in the spring 2012 semester , the 95% confidence intervals for students in the fail group taking 100% of the pre - exam quizzes versus 0% pre - exam quiz takers overlapped , but a higher exam 4 average for the group taking all pre - exam quizzes was still observed . in an effort to do an even more granular analysis of the benefit of quizzing using demonstrated performance on exams , we sorted students into three groups representing a more narrow distribution of exam score averages ( table 2 ) . figure 5 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 3 average than those students in the same group who took 0% of pre - exam quizzes . low - performing students taking 100% of pre - exam quizzes also had higher averages for exam 3 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . figure 6 shows that students taking 100% of pre - exam quizzes in the high group of students had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . additionally , students in the middle group of students also had a significantly higher exam 4 average than those students in the same group who took 0% of pre - exam quizzes . as was seen for exam 3 , students with a low performance taking 100% of pre - exam quizzes also have higher averages for exam 4 than those taking 0% of pre - exam quizzes , but due to the small sample size of this group , this difference is not significant using 95% confidence intervals . the implication of this study is that frequent online quizzing is an effective approach in increasing students ability to retrieve information and subsequent performance in the introductory ( majors ) biology classroom . students who take pre - exam quizzes do significantly better than those who do not take pre - exam quizzes . the exam average gap between quiz takers and non quiz takers observed as the semester progressed is notable , as the exam average of the group of students not taking any of the required pre - exam quizzes falls significantly below the class average as the semester progresses . while we do observe that increased quiz scores are significantly correlated with increased exam scores ( unpublished data ) , what is particularly notable about our findings is that simply participating in pre - exam quizzing ( 100% quiz takers vs. 0% quiz takers ) is associated with increased exam scores . most intriguing is the evidence that students of all abilities benefit from participation in pre - exam quizzing . that students who take quizzes have higher exam averages than those who did not take quizzes is not surprising ( figures 1 and 2 ) many would point to the fact that merely taking quizzes is an indicator of the level of scholastic strength and motivation in a given student . this effort was made to mitigate the innate ability of the student as a confounding factor and to focus on the potential benefit participation in quizzing might give a student . in both fall 2011 and spring 2012 , students with a passing average on exams 1 and 2 were observed to significantly benefit from taking 100% of pre - exam quizzes as compared with those not taking these quizzes . additionally , we were quite encouraged to find that those students with a failing average taking 100% pre - exam quizzes showed significantly higher exam 3 and exam 4 averages than those taking no pre - exam quizzes in the fall 2011 semester . a similar trend of higher exam scores for students taking 100% of pre - exam quizzes was observed in spring 2012 , although significance was not established for that group ( figures 3 and 4 ) . analysis of these groups demonstrates not only that students with a high performance benefit from pre - exam quizzing , but also that those middle students also see significant benefit from taking pre - exam quizzes ( figures 5 and 6 ) . the advantage to participating in pre - exam quizzing seems to give even the weakest students a letter grade advantage over those not taking pre - exam quizzes . the benefit seen by high or strong students taking 100% versus 0% of pre - exam quizzes seems particularly strong when compared with the benefit seen by middle / average students taking 100% versus 0% of pre - exam quizzes ( figures 5 and 6 ) . however , specific analysis of the gains in exam average with increased quiz - taking activity indicates that the benefit yielded to each of these two groups is not significantly different . in summary , in every group analyzed , students who took all of the offered pre - exam quizzes had higher exam scores than those taking none of the offered pre - exam quizzes . of particular interest is the fact that compliance in taking the required pre - exam quizzes declined throughout the semester , in spite of the required nature of these quizzes . desirable difficulties to students in the college classroom ( bjork and bjork , 2011 ) . the introduction of required pre - exam quizzes seems to create a significant enough difficulty for some students such that they reject the effort entirely . it should be noted that the difficulty of the quizzes was significant but not extreme ( on a scale of 15 , in which 1 is easy and 5 is most difficult , quizzes were assessed by mb to average around 2.8 ) . this may be due to the nature of the student population , as this study was done in a community college classroom , in which no selection criteria for admission are in place . this yields a notable variation in the student population from a motivation and preparation standpoint . current efforts focus on increasing student compliance by emphasizing the potential benefit of quizzing on exam scores to the students prior to each quiz period . surveying students regarding their experience with pre - exam quizzing indicates that most students found this strategy valuable , as 87.32% of students surveyed indicated that they plan to continue quizzing as part of their preparation for future classes ( n = 142 ) . particularly interesting is that 73.42% reported that taking the required quizzes caused them to go back and do additional studying of the material / additional preparation prior to the exam . pre - exam quizzing may act as an early warning system for students , showing them that they have the illusion of knowing the material but have not learned the material sufficiently for accurate recollection in a testing environment ( koriat and bjork , 2005 ) . in fact , surveyed students reported that pre - exam quizzing increased their understanding of the material ( 26.28% ) , but more commonly helped them to pinpoint how to study for exams ( 57.69% ) . while pre - exam quizzing is only one of many strategies that a student may utilize in preparing for exams , koriat and bjork 's research indicates that having all of the information present , such as in the conditions in which most students study ( reading the textbook and reviewing notes ) , is less effective in yielding maximum recall of the information and more likely to produce an illusion of competence . this leads us to discard the idea that students not participating in pre - exam quizzing could simply increase the time committed to reviewing their notes and the text and see the same results as those taking 100% of the pre - exam quizzes . overall , the benefit of pre - exam quizzing seems to be driven by the decision of the individual student to commit to taking all of the required quizzes . our analysis indicates that this benefit is not limited to only strong students who are typically successful on exams , but that students of all abilities see a benefit from taking online , pre - exam quizzes . pre - exam quizzing utilizing an online platform seems to be an effective way to increase student performance on introductory biology exams , and quiz averages serve to give students feedback as to potential exam performance .

in his inaugural speech dr robert wah , president of the american medical association ( ama ) , named star trek 's iconic physician leonard bones ' mccoy as his favourite fictional character . in his view , mccoy embodied the qualities of an exceptional leader . he was willing to collaborate to solve problems and to question decisions from a scientific perspective while acting as an advocate for health . bones bridged the gaps between the extremes of logic and instinct , rules and regulation , scientific knowledge and human compassion.1 there has been considerable interest in how doctors are represented in fictional television24 and significant research concerning how medicine and medical devices are used in science fiction futures,57 yet little research to date examines doctors administering space medicine in science fiction . this is surprising as cultural images of medical professionals have long been considered vital to sustaining the power of institutional medicine.8 furthermore , the science fiction genre offers important spaces for valuable debates concerning medicine , technoscience and the ethical implications or social uses of biomedicine . we argue that the space doctor personalises societal fears and anxieties concerning diverse social issues , including gender and diversity , ethics of surveillance and authority as well as medical enhancement and posthuman futures . drawing on debates within medical sociology and cultural studies this article explores how the qualities , norms and ethics represented by doctors in space ( ships ) are intertwined with issues of gender equality , speciesism and posthuman ethics . analysis of fictional media stories here starts from the principle that the production of meaning or signification within the narrative is itself a specific practice rather than a mere reflection of reality . the focus of analysis is on the way that narrative discourses become a field in which political and cultural articulations are played out in an attempt to establish hegemonic formations.9 medical drama continues to occupy an extraordinary position in contemporary television with early series such as dr finlay 's casebook ( 19621971 ) and dr kildare ( 19611966 ) establishing the enduring role of the fictional doctor . around the same time indeed , some of science fiction 's most memorable characters have been medical doctors , and it could be argued that the character is now an essential ingredient of the genre . but what are the specificities of television science fiction rather than other forms of fictional drama ? perhaps the first , and most obvious , theme of science fiction is claims about futures . all fictional forms exist within an arena of cultural relationships that are historically specific , but science fiction , in both its utopian and dystopian forms , uses narratives to project visions of the future in order to disrupt the present to remind us that the future was not going to be what respectable people imagined.10 this leads to a second , and related theme , found in science fiction that presumes that these narrative futures are the result of scientific gadgetry and sociotechnical change . yet the important issue for analysis is not realism , but rather is a close reading of what the sign technology is attempting to constitute in the narrative in this case , what is the use of medical technoscience trying to signify ? the final theme , as argued by suvin , is the brechtian idea of estrangement ( verfremdungseffekt ) that distinguishes science fiction from myth and fantasy . myth and fantasy often see human relationships as fixed or supernaturally determined , whereas science fiction focuses on the variable future and problematises human relationships to explore where they may lead in the future . as a representation it estranges ( allowing ) us to recognise its subject , but at the same time making it seem unfamiliar.11 in this paper our analysis draws on these themes to argue that the figure of the doctor is discursively deployed to deliver medical care and frequently acts as a moral compass at the centre of the programme narrative a conduit through which societal anxieties about the futures of health , sociotechnical change , bioethics and medical science can be expressed and explored . star trek constitutes a self - contained subgenre within science fiction.12 the tremendous influence of star trek on the science fiction genre coupled with the global reach of the show means that we focus on this particular franchise in detail . the creator of the show gene roddenberry was a humanist , and it was originally intended to have a progressive political agenda , using the genre to tackle contemporary social issues in an enlightened way , though this aim was concealed from the networks at the time.13 the role of doctor has been a consistent feature throughout all the trek franchises with each recreation of the physician being distinctive and mirroring key aspects of the position of medicine in the era in which it was created . star trek : the original series ( st : tos 19661969 ) was leonard bones mccoy , an idealised general practitioner with a broad skill range , willing to carry out any procedure . while mccoy regularly used advanced medical technology he was also depicted as being uneasy with some aspects of space living and famously had a phobia about using the ship 's matter transporters . mccoy was frequently the moral centre of the original series and often argued with the unemotional and utilitarian vulcan science officer , mr spock . early in the series , mccoy describes himself as a simple country doctor , undoubtedly a reference to the classic w eugene smith photo essay for life magazine , country doctor published in 1948.14 this influential photo essay helped to mythologise the idea of the community - based physician . life researched a suitably attractive location and selected an appropriate doctor , ernest ceriani , who was chosen partly for his looks and youth.15 smith 's study painted an evocative picture of a hardworking , emotionally drained physician firmly embedded with his patients in the rituals of rural community life . like mccoy , dr ceriani is shown carrying out a wide range of activities making house calls , talking to patients and conducting operations in surgical gowns . both the life photos and st : tos 's dr mccoy can be read politically in different and somewhat opposing ways . at the time of life 's publication , there was considerable debate in the usa about the introduction of compulsory health insurance to increase the number of doctors serving local communities . this was vehemently opposed by the ama , and the political intentions of the photo essay were to provide a strong counterpoint to debates about us national healthcare or socialised medicine.15 the pictures contrast dr ceriani striding across agricultural fields to make house calls , with him dressed in a surgical gown in a hospital operating theatre . this juxtaposition implies that additional physicians , funded by compulsory insurance , were not needed politically smith , a lifelong liberal , was at variance with the magazine 's political intentions for the photo essay . the authentic depictions of medical failure and ceriani 's frozen exhausted stare undermine any message that implies america might not require more doctors . similarly , the fictional dr mccoy presents audiences with a figure that is both traditional and modern . he exhibits some distinctly premodern beliefs about natura medica and the natural healing powers of the body while being comfortable with 23rd - century medical technology . while mccoy almost always starts his diagnosis by using his tricorder , he is shown throughout st : tos using an older and more traditional diagnostic technique that of palpation , the method of feeling with fingers and hands during a physical examination . for example , in the deadly years ( st : tos ) a mysterious malady causes some crew members , including captain kirk , to undergo extreme ageing . during an examination , mccoy initially uses the tricorder on kirk 's body but concludes his diagnosis by making physical contact using fingers to manipulate his patient 's joints . it is only after this material connection that he finds kirk is suffering from advanced arthritis . in , the enemy within ( st : tos ) , the futuristic technology of the matter transporter provides a plot device to explore good and evil in the human mind . a malfunction in the transporter causes kirk to split into two ( evil kirk characterised by hostility , lust and violence and good kirk who embodies compassion , love and tenderness ) . when good kirk , expresses revulsion that his evil doppelganger came from within himself it is mccoy who dispenses simple psychological advice about the human condition . the role of mccoy in st : tos has to be understood in the context of other popular medical drama which emerged during this period . the portrayal of fictional doctors was being established in television series such as dr finlay 's casebook ( 19621971 ) , dr kildare , ( 19611966 ) ben casey ( 19611966 ) and marcus welby md ( 19691976 ) . story themes emphasised the dedicated doctor willing to move beyond their professional boundaries to help their patients , motivated not by financial reward but by a noble calling.16 each series centred on the medical hero who was concerned with the lives of their patients , sometimes to their own personal detriment . many received an official stamp of approval from the ama advisory committee or the american academy of family physicians . at this time medicine was becoming more reliant on high - technology science , which dramatically improved treatment and raised its cost , with severe consequences for patient - doctor relations in the usa : as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 in other areas of the mass media and academic medical sociology1821 the flaws in the medical establishment were being very clearly illuminated with critical accounts of medical negligence and lawsuits , rising healthcare costs , and patronising doctor - patient relations . television audiences were captivated by these nostalgic cultural representations which embodied the type of doctors they desired rather than ones who bore a resemblance to their actual healthcare professionals . the television doctors were infallible , had endless time to spend with few patients and were not financially motivated.the more rushed real - life doctors become , the more leisurely the pace of their fictional counterparts . and the same went for money : as american medicine became increasingly profit orientated , with tales of impecunious patients being turned away from casualty , american medical dramas depicted a medical practice where fees were almost never discussed , and patients never rejected because of their inability to pay.22 the more rushed real - life doctors become , the more leisurely the pace of their fictional counterparts . and the same went for money : as american medicine became increasingly profit orientated , with tales of impecunious patients being turned away from casualty , american medical dramas depicted a medical practice where fees were almost never discussed , and patients never rejected because of their inability to pay.22 setting an entire series in space facilitates significant creative freedom regarding representing the positive aspects of medicine where doctors have infinite time to care for patients and are untroubled by concerns about funding or payment . turow argues that this utopian view of healthcare began a trajectory of medical drama in which audiences assume that healthcare is a limitless resource.4 in st : tos mccoy never needs to engage with financing problems . his role depicts a future in which futuristic technoscience and gadgetry has a medical role that complements the more traditional and idealistic functions of the physician . mccoy still has time to provide reassuring psychological advice to his patients and carry out physical examinations manually . also , it is the futuristic technology in st : tos that often provides the defamiliarisation effect in the narrative , such as transporters splitting characters into evil and good dyads . mccoy then comes to stabilise and provides a counterpoint to narrative disruption by using time - honoured traditional medical techniques those of the simple country doctor. themes of estrangement and the problematising of human relationships dominate storylines involving female physicians in science fiction television . these self - confident female characters provide a crucial counterpoint to the first male - dominated medical dramas and to a wider media responsible for the symbolic annihilation of women.23 as a strong leading character in british series space 1999 ( 19751977 ) dr helena russell conforms to the theme of dedicated and independent physician . as with dr janet fraiser , chief medical officer in stargate sg1 ( 19972007 ) , dr russell could overrule the typical hierarchy of the military setting on the basis of medical authority and defended the ideals of medical ethics against the demands of military necessity . significantly , within star trek : the next generation ( st : tng ) there is a gender - integrated crew . however , the two lead female characters are both healthcare related : dr beverly crusher , the chief medical officer and deanna troi , the ship 's therapist . this shift from st : tos , where the most senior female was iconic communication officer nyota uhura , is significant and reflects more general debates which featured in the 1980s media concerning gender equality . these characters clearly represent some sort of progress regarding positive depictions of women in primetime television . however , as with fraiser and russell , both mainly reproduce traits ( culturally constructed ) which tend to be attached to women in fictional media.24 they possess skills associated with a constructed both are at times preoccupied with concerns about their personal lives.25 thus deanna troi is an extreme example of this constructed femininity with female medical abilities she is a half - human , half - betazoid empath who can telepathically read emotions and judge whether an individual is attempting deception or subterfuge . at a time when debates were circulating concerning gender equality , dr crusher 's character appeared to be designed as the fantasy woman . she is the most senior female officer in the enterprise crew , leads a medical team and is a close confidante of the captain . she is in all respects a superwoman.26 like mccoy before , crusher 's character has had an undoubted positive influence with material consequences beyond the show . one female medical student recalled i think dr crusher had a real impact on my formative years a woman physician who was strong , smart , and respected , who the guys went to when they did n't have the answers.27 the role of female physician also provides opportunities to explore the ways in which science fiction television facilitates disruptions along the lines of gender and sexuality that would appear to challenge the heteronormativity of television drama and the media of the time . the ability for science fiction to offer provocative possibilities concerning sexuality and female power is coupled with the potential to disrupt traditional assumptions concerning gender , sexuality and bodies . however , such depictions are often limited by the prevailing sociocultural mores . in a ground - breaking episode which explores the complexities of sexuality , crusher falls in love with a trill ambassador , odan ( the host , st : tng ) . after odan is fatally injured he temporarily transfers his identity to a male starfleet officer 's body , william t riker . crusher is a close friend of riker and initially struggles with this transition but eventually continues a sexual relationship . the ethical consequences of riker providing a body , used for sexual purposes by his close colleague , were only hinted at in the narrative . however , when a woman arrives as odan 's permanent host , crusher rejects her saying perhaps it is a human failing ; but we are not accustomed to these kinds of changes. this exploration of transgender issues and ultimate rejection by crusher on the grounds of universalism drew mixed responses from it seems to imply that sexual relations with someone as they go through a gender transition are impossible , even in the 23rd century.28 discussions concerning the fluid and unstable nature of identity were often reflected in the representations of doctors in science fiction that followed , with gender and sexuality being common themes . in particular , debates about intersectionality opened up the possibilities of subject positions being multiple and relational.29 for example , doctor phlox from star trek : enterprise ( st : ent 20012005 ) is the alien ( denobulan ) chief medical officer on enterprise he is part of an interspecies medical exchange. as well as being a fully trained doctor he holds six degrees in interspecies veterinary medicine and is an advocate of a species - spanning approach to healthcare . he maintains a menagerie of plants and animals in the sick bay which he uses to prepare therapies to complement his traditional pharmaceuticals . hence , as a multicultural physician of the future he is relaxed with technology and is able to draw on alternative healing methodsdr . phlox reflects the swelling backlash against failed technologies that presently lead so many to experiment with non - traditional or remedies.26 in this prequel to st : tos his attitudes are contrasted with those of his fellow crew members and he maintains an open and positive stance on diversity ( species and cultures ) . in a night in sickbay ( st : ent ) , the primary focus is on captain archer 's companion animal , a dog named a subplot involves increasing tensions between captain archer and his female vulcan first officer , t'pol . during a long night with archer and the doctor together in sickbay , phlox suggests that this conflict may be due to sexual tension : for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) during a delicate procedure to transplant a pituitary gland from a calrissian chameleon into porthos , archer asks phlox whether his expertise on sexual matters was based on professional or personal experience . phlox explains that he has two wives , who in turn each have two other husbands beside himself . his family unit consists of a total of 720 possible relationships , 42 of which have romantic possibilities , and 31 children ( archer sounds very complicated , phlox very . why else be polygamous ) . the character of phlox thus represents a complex and fluid chain of equivalences.30 his own sociotechnical background and non - species specific training , his use of alternative and traditional therapies and his non - monogamous group relationship and polyamory all represent floating signifiers which ascribe a diverse fluidity to his subject position . but his status as an alien outsider , visually marked as different , on board the enterprise means this estrangement is narratively present , but effectively neutralised . the doctor as different is a theme also explored with the representations of two other star trek physicians : dr julian bashir on star trek : deep space nine ( st : ds9 19931999 ) and the artificial intelligence ( ai ) doctor known simply as the emergency medical hologram ( emh ) on star trek : voyager ( st : voy 19952001 ) . in st : ds9 issues concerning the psychosocial implications of genetic engineering and enhancement are examined . we discover that dr bashir has been genetically engineered as a young boy because he had learning difficulties . thus bashir embodies the mentally superior and physically enhanced doctor that is in some respects posthuman ( simultaneously using and also being a product himself of genetic modification technology ) . within the star trek united federation of planets , human genetic engineering is illegal , and bashir has kept his abilities secret throughout most of his adult life . in addition to the satirical opportunities that the ai doctor affords ( eg , expressing unhappiness with his working conditions and an ai arrogance ) he also reverberates with the changing medical environment of the period reflecting the increasing use and reliance upon intelligent medical devices ' and debates that speak to the deskilling of medical practice . a constant theme throughout the series franchise , continued here , concerns what it means to be human . again we can see an estrangement within the narrative discourse as the ai doctor challenges and disrupts the taken - for - granted assumptions that humans are morally superior . this is brought into focus when the emh discovers he has been denied access to his own memory files on the orders of captain janeway . it emerges that the emh has developed a dangerous feedback loop between his cognitive and ethical subroutines caused by an internal cognitive conflict where he chooses to save a friend rather than another member of the crew . portions of his memory are removed in order to rectify the perilous logic loop. it is the artificial non - organic emh who challenges the actions of janeway as unethical , reminding her that despite being a machine he can still feel violated:emh : how would you like it if i operated on you without your consent or without your knowledge ? . you 'd feel as violated as i do right now ( st : voy latent image ) . emh : how would you like it if i operated on you without your consent or without your knowledge ? . you 'd feel as violated as i do right now ( st : voy latent image ) . thus this character is able to provoke reflection on what it means to be human , the nature of individuality and to what extent the crew are willing to cross ethical boundaries to ensure the preservation of their artificial crew member . the emh emulates human characteristics , yet he is treated like a machine when his programming causes him to feel unresolvable guilt. the moral and ethical role of the physician at the heart of these futuristic programmes appears remarkably stable . the various trek outings share a set of positive characteristics about the future of healthcare including equality of care and an altruistic concern for the welfare of patients regardless of race or species ( indeed the starfleet federation have an explicit policy of not restricting access to its medical supplies , st : tng the mind 's eye ) . yet , the delivery of medical care is consistently represented as taking place within a quasi - military context , although one that is dedicated to exploration . nevertheless , the star trek series consistently represents a utopian federation , a vision of the evolution of an idealised united nations that shares some characteristics with the utopian culture of iain m banks 's novels . in contrast , the reimagining of battlestar galactica ( bsg , 20032009 ) introduces a far bleaker , more complex vision of the moral uncertainties of a overarching storylines concern the elements of democracy which can be sacrificed in the struggle to win a war . these cylons are sentient enemies that look human and share our biology but are focused on annihilating the human race . other.31 in bsg , the doctor , chief medical officer major sherman cottle , is firmly embedded in a military state where national security is in constant jeopardy , civilians are under martial law , and many of his colleagues are experiencing burnout and paranoia . dr cottle displays a healthy disdain for authority and is almost always seen smoking a cigarette , even during patient consultations . the show raises important questions concerning biopolitics , race , gender and torture.3235 here dr cottle embodies the considerable pressures faced by medics in combat zones . his medical role , and the relative autonomy it allows him serves to accelerate the plot at crucial points in the story arc , not least faking the death of a hybrid human - cylon infant to protect the child ( bsg downloaded ) . like previous science fiction doctors , he upholds the hippocratic oath in treating cylons and humans equally , actively disapproving of the grave sexual assault of cylon prisoner sharron which challenges his colleagues ' view that a machine ca n't be raped ( bsg resurrection ship ) . indeed issues of the precarious position of rights over reproduction and body politics within war are a strong recurring theme in the series.33 where fear of overpopulation is evident in star trek,5 the converse is the case in bsg where there are just 50 000 human souls left prompting the traditionally liberal president roslin to criminalise abortion . it emerges that cottle has long been performing abortions on request without asking a lot of questions ( bsg the captain 's hand ) . cottle also disagrees ethically with the use of alien blood to treat the dying president roslin 's breast cancer , saying i do n't like what you 're doing , i think it 's unnatural and damn dangerous maybe it 's just her time ( bsg epiphanies ) . the doctor here simultaneously expresses fears about the dangers of both tampering with nature and of the hybridisation that might result from mixing human and alien blood . there are obvious references here to the frankenstein frame , the governing myth for debates about life creation and biotechnology for over two centuries.36 doctor cottle 's administration of medicine and healthcare can , therefore , be seen as signifying an ethical resistance to the all - encompassing needs of the military in times of war although a resistance that is largely ineffectual . this contrasts strongly with the universe of st : tng where deanna troi 's empathic abilities are frequently used by captain picard to uncover hidden motivations of potential enemies and also members of his own crew to literally get inside the minds of friends and foes alike . issues of surveillance and privacy and the ethical repercussions of using troi 's psychological powers in this way are simply not featured as ethically problematic in these earlier series yet in our post 9/11 era the militarisation of psychology is an issue of considerable debate.37 star trek constitutes a self - contained subgenre within science fiction.12 the tremendous influence of star trek on the science fiction genre coupled with the global reach of the show means that we focus on this particular franchise in detail . the creator of the show gene roddenberry was a humanist , and it was originally intended to have a progressive political agenda , using the genre to tackle contemporary social issues in an enlightened way , though this aim was concealed from the networks at the time.13 the role of doctor has been a consistent feature throughout all the trek franchises with each recreation of the physician being distinctive and mirroring key aspects of the position of medicine in the era in which it was created . the first and best known of the regular trek doctors from star trek : the original series ( st : tos 19661969 ) was leonard bones mccoy , an idealised general practitioner with a broad skill range , willing to carry out any procedure . while mccoy regularly used advanced medical technology he was also depicted as being uneasy with some aspects of space living and famously had a phobia about using the ship 's matter transporters . mccoy was frequently the moral centre of the original series and often argued with the unemotional and utilitarian vulcan science officer , mr spock . early in the series , mccoy describes himself as a simple country doctor , undoubtedly a reference to the classic w eugene smith photo essay for life magazine , country doctor published in 1948.14 this influential photo essay helped to mythologise the idea of the community - based physician . life researched a suitably attractive location and selected an appropriate doctor , ernest ceriani , who was chosen partly for his looks and youth.15 smith 's study painted an evocative picture of a hardworking , emotionally drained physician firmly embedded with his patients in the rituals of rural community life . like mccoy , dr ceriani is shown carrying out a wide range of activities making house calls , talking to patients and conducting operations in surgical gowns . both the life photos and st : tos 's dr mccoy can be read politically in different and somewhat opposing ways . at the time of life 's publication , there was considerable debate in the usa about the introduction of compulsory health insurance to increase the number of doctors serving local communities . this was vehemently opposed by the ama , and the political intentions of the photo essay were to provide a strong counterpoint to debates about us national healthcare or socialised medicine.15 the pictures contrast dr ceriani striding across agricultural fields to make house calls , with him dressed in a surgical gown in a hospital operating theatre . this juxtaposition implies that additional physicians , funded by compulsory insurance , were not needed yet politically smith , a lifelong liberal , was at variance with the magazine 's political intentions for the photo essay . the authentic depictions of medical failure and ceriani 's frozen exhausted stare undermine any message that implies america might not require more doctors . similarly , the fictional dr mccoy presents audiences with a figure that is both traditional and modern . he exhibits some distinctly premodern beliefs about natura medica and the natural healing powers of the body while being comfortable with 23rd - century medical technology . while mccoy almost always starts his diagnosis by using his tricorder , he is shown throughout st : tos using an older and more traditional diagnostic technique that of palpation , the method of feeling with fingers and hands during a physical examination . for example , in the deadly years ( st : tos ) a mysterious malady causes some crew members , including captain kirk , to undergo extreme ageing . during an examination , mccoy initially uses the tricorder on kirk 's body but concludes his diagnosis by making physical contact using fingers to manipulate his patient 's joints . it is only after this material connection that he finds kirk is suffering from advanced arthritis . in , the enemy within ( st : tos ) , the futuristic technology of the matter transporter provides a plot device to explore good and evil in the human mind . a malfunction in the transporter causes kirk to split into two ( evil kirk characterised by hostility , lust and violence and good kirk who embodies compassion , love and tenderness ) . when good kirk , expresses revulsion that his evil doppelganger came from within himself it is mccoy who dispenses simple psychological advice about the human condition . the role of mccoy in st : tos has to be understood in the context of other popular medical drama which emerged during this period . the portrayal of fictional doctors was being established in television series such as dr finlay 's casebook ( 19621971 ) , dr kildare , ( 19611966 ) ben casey ( 19611966 ) and marcus welby md ( 19691976 ) . story themes emphasised the dedicated doctor willing to move beyond their professional boundaries to help their patients , motivated not by financial reward but by a noble calling.16 each series centred on the medical hero who was concerned with the lives of their patients , sometimes to their own personal detriment . many received an official stamp of approval from the ama advisory committee or the american academy of family physicians . at this time medicine was becoming more reliant on high - technology science , which dramatically improved treatment and raised its cost , with severe consequences for patient - doctor relations in the usa : as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 in other areas of the mass media and academic medical sociology1821 the flaws in the medical establishment were being very clearly illuminated with critical accounts of medical negligence and lawsuits , rising healthcare costs , and patronising doctor - patient relations . television audiences were captivated by these nostalgic cultural representations which embodied the type of doctors they desired rather than ones who bore a resemblance to their actual healthcare professionals . the television doctors were infallible , had endless time to spend with few patients and were not financially motivated.the more rushed real - life doctors become , the more leisurely the pace of their fictional counterparts . and the same went for money : as american medicine became increasingly profit orientated , with tales of impecunious patients being turned away from casualty , american medical dramas depicted a medical practice where fees were almost never discussed , and patients never rejected because of their inability to pay.22 the more rushed real - life doctors become , the more leisurely the pace of their fictional counterparts . and the same went for money : as american medicine became increasingly profit orientated , with tales of impecunious patients being turned away from casualty , american medical dramas depicted a medical practice where fees were almost never discussed , and patients never rejected because of their inability to pay.22 setting an entire series in space facilitates significant creative freedom regarding representing the positive aspects of medicine where doctors have infinite time to care for patients and are untroubled by concerns about funding or payment . turow argues that this utopian view of healthcare began a trajectory of medical drama in which audiences assume that healthcare is a limitless resource.4 in st : tos mccoy never needs to engage with financing problems . his role depicts a future in which futuristic technoscience and gadgetry has a medical role that complements the more traditional and idealistic functions of the physician . mccoy still has time to provide reassuring psychological advice to his patients and carry out physical examinations manually . also , it is the futuristic technology in st : tos that often provides the defamiliarisation effect in the narrative , such as transporters splitting characters into evil and good dyads . mccoy then comes to stabilise and provides a counterpoint to narrative disruption by using time - honoured traditional medical techniques those of the simple country doctor. themes of estrangement and the problematising of human relationships dominate storylines involving female physicians in science fiction television . these self - confident female characters provide a crucial counterpoint to the first male - dominated medical dramas and to a wider media responsible for the symbolic annihilation of women.23 as a strong leading character in british series space 1999 ( 19751977 ) dr helena russell conforms to the theme of dedicated and independent physician . as with dr janet fraiser , chief medical officer in stargate sg1 ( 19972007 ) , dr russell could overrule the typical hierarchy of the military setting on the basis of medical authority and defended the ideals of medical ethics against the demands of military necessity . significantly , within star trek : the next generation ( st : tng ) there is a gender - integrated crew . however , the two lead female characters are both healthcare related : dr beverly crusher , the chief medical officer and deanna troi , the ship 's therapist . this shift from st : tos , where the most senior female was iconic communication officer nyota uhura , is significant and reflects more general debates which featured in the 1980s media concerning gender equality . these characters clearly represent some sort of progress regarding positive depictions of women in primetime television . however , as with fraiser and russell , both mainly reproduce traits ( culturally constructed ) which tend to be attached to women in fictional media.24 they possess skills associated with a constructed both are at times preoccupied with concerns about their personal lives.25 thus deanna troi is an extreme example of this constructed femininity with female medical abilities she is a half - human , half - betazoid empath who can telepathically read emotions and judge whether an individual is attempting deception or subterfuge . at a time when debates were circulating concerning gender equality , dr crusher 's character appeared to be designed as the fantasy woman . she is the most senior female officer in the enterprise crew , leads a medical team and is a close confidante of the captain . she is in all respects a superwoman.26 like mccoy before , crusher 's character has had an undoubted positive influence with material consequences beyond the show . one female medical student recalled i think dr crusher had a real impact on my formative years a woman physician who was strong , smart , and respected , who the guys went to when they did n't have the answers.27 the role of female physician also provides opportunities to explore the ways in which science fiction television facilitates disruptions along the lines of gender and sexuality that would appear to challenge the heteronormativity of television drama and the media of the time . the ability for science fiction to offer provocative possibilities concerning sexuality and female power is coupled with the potential to disrupt traditional assumptions concerning gender , sexuality and bodies . however , such depictions are often limited by the prevailing sociocultural mores . in a ground - breaking episode which explores the complexities of sexuality , crusher falls in love with a trill ambassador , odan ( the host , st : tng ) . after odan is fatally injured he temporarily transfers his identity to a male starfleet officer 's body , william t riker . crusher is a close friend of riker and initially struggles with this transition but eventually continues a sexual relationship . the ethical consequences of riker providing a body , used for sexual purposes by his close colleague , were only hinted at in the narrative . however , when a woman arrives as odan 's permanent host , crusher rejects her saying perhaps it is a human failing ; but we are not accustomed to these kinds of changes. this exploration of transgender issues and ultimate rejection by crusher on the grounds of universalism drew mixed responses from gaylaxians who felt the episode embodied the ideological limitations of the show . it seems to imply that sexual relations with someone as they go through a gender transition are impossible , even in the 23rd century.28 discussions concerning the fluid and unstable nature of identity were often reflected in the representations of doctors in science fiction that followed , with gender and sexuality being common themes . in particular , debates about intersectionality opened up the possibilities of subject positions being multiple and relational.29 for example , doctor phlox from star trek : enterprise ( st : ent 20012005 ) is the alien ( denobulan ) chief medical officer on enterprise he is part of an interspecies medical exchange. as well as being a fully trained doctor he holds six degrees in interspecies veterinary medicine and is an advocate of a species - spanning approach to healthcare . he maintains a menagerie of plants and animals in the sick bay which he uses to prepare therapies to complement his traditional pharmaceuticals . hence , as a multicultural physician of the future he is relaxed with technology and is able to draw on alternative healing methodsdr . phlox reflects the swelling backlash against failed technologies that presently lead so many to experiment with non - traditional or remedies.26 in this prequel to st : tos his attitudes are contrasted with those of his fellow crew members and he maintains an open and positive stance on diversity ( species and cultures ) . in a night in sickbay ( st : ent ) , the primary focus is on captain archer 's companion animal , a dog named a subplot involves increasing tensions between captain archer and his female vulcan first officer , t'pol . during a long night with archer and the doctor together in sickbay , phlox suggests that this conflict may be due to sexual tension : for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) during a delicate procedure to transplant a pituitary gland from a calrissian chameleon into porthos , archer asks phlox whether his expertise on sexual matters was based on professional or personal experience . phlox explains that he has two wives , who in turn each have two other husbands beside himself . his family unit consists of a total of 720 possible relationships , 42 of which have romantic possibilities , and 31 children ( archer sounds very complicated , phlox very . why else be polygamous ) . the character of phlox thus represents a complex and fluid chain of equivalences.30 his own sociotechnical background and non - species specific training , his use of alternative and traditional therapies and his non - monogamous group relationship and polyamory all represent floating signifiers which ascribe a diverse fluidity to his subject position . but his status as an alien outsider , visually marked as different , on board the enterprise means this estrangement is narratively present , but effectively neutralised . the doctor as different is a theme also explored with the representations of two other star trek physicians : dr julian bashir on star trek : deep space nine ( st : ds9 19931999 ) and the artificial intelligence ( ai ) doctor known simply as the emergency medical hologram ( emh ) on star trek : voyager ( st : voy 19952001 ) . in st : ds9 issues concerning the psychosocial implications of genetic engineering and enhancement are examined . we discover that dr bashir has been genetically engineered as a young boy because he had learning difficulties . thus bashir embodies the mentally superior and physically enhanced doctor that is in some respects posthuman ( simultaneously using and also being a product himself of genetic modification technology ) . within the star trek united federation of planets , human genetic engineering is illegal , and bashir has kept his abilities secret throughout most of his adult life . in addition to the satirical opportunities that the ai doctor affords ( eg , expressing unhappiness with his working conditions and an ai arrogance ) he also reverberates with the changing medical environment of the period reflecting the increasing use and reliance upon intelligent medical devices ' and debates that speak to the deskilling of medical practice . a constant theme throughout the series franchise , continued here , concerns what it means to be human . again we can see an estrangement within the narrative discourse as the ai doctor challenges and disrupts the taken - for - granted assumptions that humans are morally superior . this is brought into focus when the emh discovers he has been denied access to his own memory files on the orders of captain janeway . it emerges that the emh has developed a dangerous feedback loop between his cognitive and ethical subroutines caused by an internal cognitive conflict where he chooses to save a friend rather than another member of the crew . portions of his memory are removed in order to rectify the perilous logic loop. it is the artificial non - organic emh who challenges the actions of janeway as unethical , reminding her that despite being a machine he can still feel violated:emh : how would you like it if i operated on you without your consent or without your knowledge ? janeway : if the operation saved my life ? . you 'd feel as violated as i do right now ( st : voy latent image ) . emh : how would you like it if i operated on you without your consent or without your knowledge ? you 'd feel as violated as i do right now ( st : voy latent image ) . thus this character is able to provoke reflection on what it means to be human , the nature of individuality and to what extent the crew are willing to cross ethical boundaries to ensure the preservation of their artificial crew member . the emh emulates human characteristics , yet he is treated like a machine when his programming causes him to feel unresolvable the moral and ethical role of the physician at the heart of these futuristic programmes appears remarkably stable . the various trek outings share a set of positive characteristics about the future of healthcare including equality of care and an altruistic concern for the welfare of patients regardless of race or species ( indeed the starfleet federation have an explicit policy of not restricting access to its medical supplies , st : tng the mind 's eye ) . yet , the delivery of medical care is consistently represented as taking place within a quasi - military context , although one that is dedicated to exploration . nevertheless , the star trek series consistently represents a utopian federation , a vision of the evolution of an idealised united nations that shares some characteristics with the utopian culture of iain m banks 's novels . in contrast , the reimagining of battlestar galactica ( bsg , 20032009 ) introduces a far bleaker , more complex vision of the moral uncertainties of a overarching storylines concern the elements of democracy which can be sacrificed in the struggle to win a war . these cylons are sentient enemies that look human and share our biology but are focused on annihilating the human race . other.31 in bsg , the doctor , chief medical officer major sherman cottle , is firmly embedded in a military state where national security is in constant jeopardy , civilians are under martial law , and many of his colleagues are experiencing burnout and paranoia . dr cottle displays a healthy disdain for authority and is almost always seen smoking a cigarette , even during patient consultations . the show raises important questions concerning biopolitics , race , gender and torture.3235 here dr cottle embodies the considerable pressures faced by medics in combat zones . his medical role , and the relative autonomy it allows him serves to accelerate the plot at crucial points in the story arc , not least faking the death of a hybrid human - cylon infant to protect the child ( bsg downloaded ) . like previous science fiction doctors , he upholds the hippocratic oath in treating cylons and humans equally , actively disapproving of the grave sexual assault of cylon prisoner sharron which challenges his colleagues ' view that a machine ca n't be raped ( bsg resurrection ship ) . indeed issues of the precarious position of rights over reproduction and body politics within war are a strong recurring theme in the series.33 where fear of overpopulation is evident in star trek,5 the converse is the case in bsg where there are just 50 000 human souls left prompting the traditionally liberal president roslin to criminalise abortion . it emerges that cottle has long been performing abortions on request without asking a lot of questions ( bsg the captain 's hand ) . cottle also disagrees ethically with the use of alien blood to treat the dying president roslin 's breast cancer , saying i do n't like what you 're doing , i think it 's unnatural and damn dangerous maybe it 's just her time ( bsg epiphanies ) . the doctor here simultaneously expresses fears about the dangers of both tampering with nature and of the hybridisation that might result from mixing human and alien blood . there are obvious references here to the frankenstein frame , the governing myth for debates about life creation and biotechnology for over two centuries.36 doctor cottle 's administration of medicine and healthcare can , therefore , be seen as signifying an ethical resistance to the all - encompassing needs of the military in times of war although a resistance that is largely ineffectual . this contrasts strongly with the universe of st : tng where deanna troi 's empathic abilities are frequently used by captain picard to uncover hidden motivations of potential enemies and also members of his own crew to literally get inside the minds of friends and foes alike . issues of surveillance and privacy and the ethical repercussions of using troi 's psychological powers in this way are simply not featured as ethically problematic in these earlier series yet in our post 9/11 era the militarisation of psychology is an issue of considerable debate.37 we might argue that doctors in space ( ships ) perform essential narrative articulations in an attempt to establish hegemonic formations . indeed , they function as an ethical point of narrative stability they effectively became a collective moral gadgets available to these futuristic medics , the role of the doctor continues to be the reassuring figure of a family physician , the general practitioner who is firmly embedded in their community and who performs a task of delivering healthcare and can act as confidante and advisor . mccoy speaks to a nostalgia for this fantasy physician , at ease with the technoscience of the future but capable of understanding the human condition and the psychosocial mccoy represents the doctor we would all want to have at our bedside and ( re)appeared at a time of increasing concerns about the funding of healthcare . his nostalgic presence functions to stabilise the potential narrative disruptions brought about by futuristic technoscience and gadgets. we can see this too with dr crusher whose very presence reflected wider critiques concerning professional women in television ( and medicine ) and who carries the significant burden of representing multiple idealised identities ( parent , physician and flawless professional ) . yet she can not step far beyond her ideological narrative constraints when it comes to issues of alternative sexualities . the setting of science fiction television in a utopian or dystopian future always refracts and focuses concerns about our present it makes manifest our collective anxieties , transforming and projecting them onto monstrous and alien bodies.39 the figure of the doctor represents different tensions about the delivery of posthuman healthcare : the genetically enhanced human , the alien physician and the ai non - human professional . difference is signified either visually ( phlox the alien ) or referenced internally ( part human hybrid bashir , non - organic emh ) . but this difference , in turn , is part of a discursive stabilisation of the narrative . for example , if it were human members of the starfleet crew that were widely engaged in polyamorous activity as the norm , then this would challenge our social definitions of appropriate sexual behaviour . the alternative sexualities and lifestyles of phlox are permissible because he does not resemble his viewers and thus can be neatly othered. dr cottle represents a different posthuman future an apocalyptic one where humans may cease to exist at all . as well as symbolising concerns about the future of humanity , he also presents us with a discourse of hope , an idealised ( possibly unrealistic ) view of the tensions that the caring health sciences must navigate in a world engaged in a war on terror. at the time of writing , it is being reported that we may be on the brink of a new period of both local and global insecurity . the delivery of healthcare is still in flux with debates continuing concerning funding , rationing and the threat to human identity through novel technologies . how this might influence and reflect the doctors that will be presented to future sci - fi audiences remains in question , but the science fiction doctor facilitates the personalisation of technological change , a useful conduit through which public anxieties concerning health , bioethics and morality are expressed and explored .

there has been considerable interest in images of medicine in popular science fiction and in representations of doctors in television fiction . surprisingly little attention has been paid to doctors administering space medicine in science fiction . this article redresses this gap . we analyse the evolving figure of the doctor in different popular science fiction television series . building upon debates within medical sociology , cultural studies and media studies we argue that the figure of the doctor is discursively deployed to act as the moral compass at the centre of the programme narrative . our analysis highlights that the qualities , norms and ethics represented by doctors in space ( ships ) are intertwined with issues of gender equality , speciesism and posthuman ethics . we explore the signifying practices and political articulations that are played out through these cultural imaginaries . for example , the ways in which the simple country doctor is deployed to help establish hegemonic formations concerning potentially destabilising technoscientific futures involving alternative sexualities , or military dystopia . doctors mostly function to provide the ethical point of narrative stability within a world in flux , referencing a nostalgia for the traditional , attentive , humanistic family physician . the science fiction doctor facilitates the personalisation of technological change and thus becomes a useful conduit through which societal fears and anxieties concerning medicine , bioethics and morality in a post 9/11 world can be expressed and explored .

Introduction A simple country doctor (in space) The female physician: empathy, sexualities, gender and bodies Beyond human: identity, difference and diversity Universality of health care and the aliens amongst us: all of this has happened before, and it will happen again Conclusion

bones bridged the gaps between the extremes of logic and instinct , rules and regulation , scientific knowledge and human compassion.1 there has been considerable interest in how doctors are represented in fictional television24 and significant research concerning how medicine and medical devices are used in science fiction futures,57 yet little research to date examines doctors administering space medicine in science fiction . this is surprising as cultural images of medical professionals have long been considered vital to sustaining the power of institutional medicine.8 furthermore , the science fiction genre offers important spaces for valuable debates concerning medicine , technoscience and the ethical implications or social uses of biomedicine . we argue that the space doctor personalises societal fears and anxieties concerning diverse social issues , including gender and diversity , ethics of surveillance and authority as well as medical enhancement and posthuman futures . drawing on debates within medical sociology and cultural studies this article explores how the qualities , norms and ethics represented by doctors in space ( ships ) are intertwined with issues of gender equality , speciesism and posthuman ethics . the focus of analysis is on the way that narrative discourses become a field in which political and cultural articulations are played out in an attempt to establish hegemonic formations.9 medical drama continues to occupy an extraordinary position in contemporary television with early series such as dr finlay 's casebook ( 19621971 ) and dr kildare ( 19611966 ) establishing the enduring role of the fictional doctor . around the same time indeed , some of science fiction 's most memorable characters have been medical doctors , and it could be argued that the character is now an essential ingredient of the genre . all fictional forms exist within an arena of cultural relationships that are historically specific , but science fiction , in both its utopian and dystopian forms , uses narratives to project visions of the future in order to disrupt the present to remind us that the future was not going to be what respectable people imagined.10 this leads to a second , and related theme , found in science fiction that presumes that these narrative futures are the result of scientific gadgetry and sociotechnical change . as a representation it estranges ( allowing ) us to recognise its subject , but at the same time making it seem unfamiliar.11 in this paper our analysis draws on these themes to argue that the figure of the doctor is discursively deployed to deliver medical care and frequently acts as a moral compass at the centre of the programme narrative a conduit through which societal anxieties about the futures of health , sociotechnical change , bioethics and medical science can be expressed and explored . star trek constitutes a self - contained subgenre within science fiction.12 the tremendous influence of star trek on the science fiction genre coupled with the global reach of the show means that we focus on this particular franchise in detail . the creator of the show gene roddenberry was a humanist , and it was originally intended to have a progressive political agenda , using the genre to tackle contemporary social issues in an enlightened way , though this aim was concealed from the networks at the time.13 the role of doctor has been a consistent feature throughout all the trek franchises with each recreation of the physician being distinctive and mirroring key aspects of the position of medicine in the era in which it was created . mccoy was frequently the moral centre of the original series and often argued with the unemotional and utilitarian vulcan science officer , mr spock . early in the series , mccoy describes himself as a simple country doctor , undoubtedly a reference to the classic w eugene smith photo essay for life magazine , country doctor published in 1948.14 this influential photo essay helped to mythologise the idea of the community - based physician . both the life photos and st : tos 's dr mccoy can be read politically in different and somewhat opposing ways . at the time of life 's publication , there was considerable debate in the usa about the introduction of compulsory health insurance to increase the number of doctors serving local communities . this was vehemently opposed by the ama , and the political intentions of the photo essay were to provide a strong counterpoint to debates about us national healthcare or socialised medicine.15 the pictures contrast dr ceriani striding across agricultural fields to make house calls , with him dressed in a surgical gown in a hospital operating theatre . the portrayal of fictional doctors was being established in television series such as dr finlay 's casebook ( 19621971 ) , dr kildare , ( 19611966 ) ben casey ( 19611966 ) and marcus welby md ( 19691976 ) . at this time medicine was becoming more reliant on high - technology science , which dramatically improved treatment and raised its cost , with severe consequences for patient - doctor relations in the usa : as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 in other areas of the mass media and academic medical sociology1821 the flaws in the medical establishment were being very clearly illuminated with critical accounts of medical negligence and lawsuits , rising healthcare costs , and patronising doctor - patient relations . and the same went for money : as american medicine became increasingly profit orientated , with tales of impecunious patients being turned away from casualty , american medical dramas depicted a medical practice where fees were almost never discussed , and patients never rejected because of their inability to pay.22 setting an entire series in space facilitates significant creative freedom regarding representing the positive aspects of medicine where doctors have infinite time to care for patients and are untroubled by concerns about funding or payment . his role depicts a future in which futuristic technoscience and gadgetry has a medical role that complements the more traditional and idealistic functions of the physician . mccoy then comes to stabilise and provides a counterpoint to narrative disruption by using time - honoured traditional medical techniques those of the simple country doctor. themes of estrangement and the problematising of human relationships dominate storylines involving female physicians in science fiction television . at a time when debates were circulating concerning gender equality , dr crusher 's character appeared to be designed as the fantasy woman . one female medical student recalled i think dr crusher had a real impact on my formative years a woman physician who was strong , smart , and respected , who the guys went to when they did n't have the answers.27 the role of female physician also provides opportunities to explore the ways in which science fiction television facilitates disruptions along the lines of gender and sexuality that would appear to challenge the heteronormativity of television drama and the media of the time . this exploration of transgender issues and ultimate rejection by crusher on the grounds of universalism drew mixed responses from it seems to imply that sexual relations with someone as they go through a gender transition are impossible , even in the 23rd century.28 discussions concerning the fluid and unstable nature of identity were often reflected in the representations of doctors in science fiction that followed , with gender and sexuality being common themes . in particular , debates about intersectionality opened up the possibilities of subject positions being multiple and relational.29 for example , doctor phlox from star trek : enterprise ( st : ent 20012005 ) is the alien ( denobulan ) chief medical officer on enterprise he is part of an interspecies medical exchange. the doctor as different is a theme also explored with the representations of two other star trek physicians : dr julian bashir on star trek : deep space nine ( st : ds9 19931999 ) and the artificial intelligence ( ai ) doctor known simply as the emergency medical hologram ( emh ) on star trek : voyager ( st : voy 19952001 ) . in addition to the satirical opportunities that the ai doctor affords ( eg , expressing unhappiness with his working conditions and an ai arrogance ) he also reverberates with the changing medical environment of the period reflecting the increasing use and reliance upon intelligent medical devices ' and debates that speak to the deskilling of medical practice . it emerges that the emh has developed a dangerous feedback loop between his cognitive and ethical subroutines caused by an internal cognitive conflict where he chooses to save a friend rather than another member of the crew . the moral and ethical role of the physician at the heart of these futuristic programmes appears remarkably stable . yet , the delivery of medical care is consistently represented as taking place within a quasi - military context , although one that is dedicated to exploration . nevertheless , the star trek series consistently represents a utopian federation , a vision of the evolution of an idealised united nations that shares some characteristics with the utopian culture of iain m banks 's novels . in contrast , the reimagining of battlestar galactica ( bsg , 20032009 ) introduces a far bleaker , more complex vision of the moral uncertainties of a overarching storylines concern the elements of democracy which can be sacrificed in the struggle to win a war . other.31 in bsg , the doctor , chief medical officer major sherman cottle , is firmly embedded in a military state where national security is in constant jeopardy , civilians are under martial law , and many of his colleagues are experiencing burnout and paranoia . like previous science fiction doctors , he upholds the hippocratic oath in treating cylons and humans equally , actively disapproving of the grave sexual assault of cylon prisoner sharron which challenges his colleagues ' view that a machine ca n't be raped ( bsg resurrection ship ) . indeed issues of the precarious position of rights over reproduction and body politics within war are a strong recurring theme in the series.33 where fear of overpopulation is evident in star trek,5 the converse is the case in bsg where there are just 50 000 human souls left prompting the traditionally liberal president roslin to criminalise abortion . the doctor here simultaneously expresses fears about the dangers of both tampering with nature and of the hybridisation that might result from mixing human and alien blood . there are obvious references here to the frankenstein frame , the governing myth for debates about life creation and biotechnology for over two centuries.36 doctor cottle 's administration of medicine and healthcare can , therefore , be seen as signifying an ethical resistance to the all - encompassing needs of the military in times of war although a resistance that is largely ineffectual . issues of surveillance and privacy and the ethical repercussions of using troi 's psychological powers in this way are simply not featured as ethically problematic in these earlier series yet in our post 9/11 era the militarisation of psychology is an issue of considerable debate.37 star trek constitutes a self - contained subgenre within science fiction.12 the tremendous influence of star trek on the science fiction genre coupled with the global reach of the show means that we focus on this particular franchise in detail . the creator of the show gene roddenberry was a humanist , and it was originally intended to have a progressive political agenda , using the genre to tackle contemporary social issues in an enlightened way , though this aim was concealed from the networks at the time.13 the role of doctor has been a consistent feature throughout all the trek franchises with each recreation of the physician being distinctive and mirroring key aspects of the position of medicine in the era in which it was created . mccoy was frequently the moral centre of the original series and often argued with the unemotional and utilitarian vulcan science officer , mr spock . early in the series , mccoy describes himself as a simple country doctor , undoubtedly a reference to the classic w eugene smith photo essay for life magazine , country doctor published in 1948.14 this influential photo essay helped to mythologise the idea of the community - based physician . both the life photos and st : tos 's dr mccoy can be read politically in different and somewhat opposing ways . at the time of life 's publication , there was considerable debate in the usa about the introduction of compulsory health insurance to increase the number of doctors serving local communities . this was vehemently opposed by the ama , and the political intentions of the photo essay were to provide a strong counterpoint to debates about us national healthcare or socialised medicine.15 the pictures contrast dr ceriani striding across agricultural fields to make house calls , with him dressed in a surgical gown in a hospital operating theatre . at this time medicine was becoming more reliant on high - technology science , which dramatically improved treatment and raised its cost , with severe consequences for patient - doctor relations in the usa : as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 as physicians ' incomes rapidly increased , and the profession fought to preserve traditional fee - for - service medicine , many viewed the profession as avaricious and uninterested in public health.17 in other areas of the mass media and academic medical sociology1821 the flaws in the medical establishment were being very clearly illuminated with critical accounts of medical negligence and lawsuits , rising healthcare costs , and patronising doctor - patient relations . mccoy then comes to stabilise and provides a counterpoint to narrative disruption by using time - honoured traditional medical techniques those of the simple country doctor. themes of estrangement and the problematising of human relationships dominate storylines involving female physicians in science fiction television . at a time when debates were circulating concerning gender equality , dr crusher 's character appeared to be designed as the fantasy woman . one female medical student recalled i think dr crusher had a real impact on my formative years a woman physician who was strong , smart , and respected , who the guys went to when they did n't have the answers.27 the role of female physician also provides opportunities to explore the ways in which science fiction television facilitates disruptions along the lines of gender and sexuality that would appear to challenge the heteronormativity of television drama and the media of the time . it seems to imply that sexual relations with someone as they go through a gender transition are impossible , even in the 23rd century.28 discussions concerning the fluid and unstable nature of identity were often reflected in the representations of doctors in science fiction that followed , with gender and sexuality being common themes . during a long night with archer and the doctor together in sickbay , phlox suggests that this conflict may be due to sexual tension : for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) for the past few months i 've noticed increasing friction between you and the sub - commander , you must understand that i am trained to observe these things when one person believes their sexual attraction toward another is inappropriate , they often exhibit unexpected behaviour ( st : ent ) during a delicate procedure to transplant a pituitary gland from a calrissian chameleon into porthos , archer asks phlox whether his expertise on sexual matters was based on professional or personal experience . the doctor as different is a theme also explored with the representations of two other star trek physicians : dr julian bashir on star trek : deep space nine ( st : ds9 19931999 ) and the artificial intelligence ( ai ) doctor known simply as the emergency medical hologram ( emh ) on star trek : voyager ( st : voy 19952001 ) . it emerges that the emh has developed a dangerous feedback loop between his cognitive and ethical subroutines caused by an internal cognitive conflict where he chooses to save a friend rather than another member of the crew . the emh emulates human characteristics , yet he is treated like a machine when his programming causes him to feel unresolvable the moral and ethical role of the physician at the heart of these futuristic programmes appears remarkably stable . yet , the delivery of medical care is consistently represented as taking place within a quasi - military context , although one that is dedicated to exploration . in contrast , the reimagining of battlestar galactica ( bsg , 20032009 ) introduces a far bleaker , more complex vision of the moral uncertainties of a overarching storylines concern the elements of democracy which can be sacrificed in the struggle to win a war . other.31 in bsg , the doctor , chief medical officer major sherman cottle , is firmly embedded in a military state where national security is in constant jeopardy , civilians are under martial law , and many of his colleagues are experiencing burnout and paranoia . like previous science fiction doctors , he upholds the hippocratic oath in treating cylons and humans equally , actively disapproving of the grave sexual assault of cylon prisoner sharron which challenges his colleagues ' view that a machine ca n't be raped ( bsg resurrection ship ) . the doctor here simultaneously expresses fears about the dangers of both tampering with nature and of the hybridisation that might result from mixing human and alien blood . there are obvious references here to the frankenstein frame , the governing myth for debates about life creation and biotechnology for over two centuries.36 doctor cottle 's administration of medicine and healthcare can , therefore , be seen as signifying an ethical resistance to the all - encompassing needs of the military in times of war although a resistance that is largely ineffectual . issues of surveillance and privacy and the ethical repercussions of using troi 's psychological powers in this way are simply not featured as ethically problematic in these earlier series yet in our post 9/11 era the militarisation of psychology is an issue of considerable debate.37 we might argue that doctors in space ( ships ) perform essential narrative articulations in an attempt to establish hegemonic formations . indeed , they function as an ethical point of narrative stability they effectively became a collective moral gadgets available to these futuristic medics , the role of the doctor continues to be the reassuring figure of a family physician , the general practitioner who is firmly embedded in their community and who performs a task of delivering healthcare and can act as confidante and advisor . mccoy speaks to a nostalgia for this fantasy physician , at ease with the technoscience of the future but capable of understanding the human condition and the psychosocial mccoy represents the doctor we would all want to have at our bedside and ( re)appeared at a time of increasing concerns about the funding of healthcare . yet she can not step far beyond her ideological narrative constraints when it comes to issues of alternative sexualities . the setting of science fiction television in a utopian or dystopian future always refracts and focuses concerns about our present it makes manifest our collective anxieties , transforming and projecting them onto monstrous and alien bodies.39 the figure of the doctor represents different tensions about the delivery of posthuman healthcare : the genetically enhanced human , the alien physician and the ai non - human professional . for example , if it were human members of the starfleet crew that were widely engaged in polyamorous activity as the norm , then this would challenge our social definitions of appropriate sexual behaviour . the alternative sexualities and lifestyles of phlox are permissible because he does not resemble his viewers and thus can be neatly othered. as well as symbolising concerns about the future of humanity , he also presents us with a discourse of hope , an idealised ( possibly unrealistic ) view of the tensions that the caring health sciences must navigate in a world engaged in a war on terror. how this might influence and reflect the doctors that will be presented to future sci - fi audiences remains in question , but the science fiction doctor facilitates the personalisation of technological change , a useful conduit through which public anxieties concerning health , bioethics and morality are expressed and explored .

for determination of the demographics and practice patterns of diabetes care providers , an internet - based survey was performed of the membership of the american diabetes association , the pediatric endocrine society , the american academy of pediatrics , the endocrine society , and the american association of clinical endocrinologists using software provided by surveygizmo 3.0 . participants who indicated that they do not currently treat patients with diabetes were excluded from the analysis . in addition , the american association of diabetes educators posted a link to the survey on its web site . one e - mail reminder notice was sent to members in each of the five organizations that communicated directly with their members . since many providers of diabetes care belong to multiple organizations , the survey was set with a browser cookie to prevent duplicate responses . there were a total of 1,267 responses to the survey . owing to survey distribution to multiple diabetes - focused organizations with overlapping membership , the total number of surveys sent out and , thus , the total response rate are unknown . we used the standards - of - care economic model to build a theoretical model to estimate the resources necessary for providers to consistently deliver the current standards of care to diabetic patients in the u.s . ; the objectives were also to evaluate provider costs to meet those standards and to assess patient outcomes specified in diabetes care guidelines relative to current reimbursement for these services . three sets of national standards ( american diabetes association , american association of clinical endocrinologists , and the endocrine society ) for diabetes care were integrated to produce the consolidated set of 28 standards of optimal care ( 1113 ) ( table 1 ) . a matrix of providers rendering the care needed to achieve the standards was developed , including physicians ( adult and pediatric , general , and specialty care ) , certified diabetes educators ( cdes ) , registered dietitians ( rds ) , registered nurses , physician assistants , medical assistants , eye care professionals , mental health professionals , podiatrists , clinical laboratory personnel , and smoking cessation professionals . six clinical vignettes three patients with type 1 diabetes and three with type 2 diabetes ( table 2)were developed representing a broad spectrum of patients . three independent panels of four to seven diabetes care professionals ( physicians , dietitians , nurses , and diabetes educators ) were convened , and with a facilitator s guidance , the minimum and maximum time ( in minutes ) needed to achieve that standard of care for patients with those specific characteristics over 1 year was estimated . a separate expert panel was convened to estimate the time needed to start or continue to follow patients using continuous subcutaneous insulin infusion ( csii ) or the subset of type 1 diabetic patients using a continuous glucose - monitoring system ( cgm ) throughout 1 year . panel meetings were conducted by a facilitator , who gained group consensus for the inputs and assured that the panel had not overestimated total time . consolidated standards of optimal diabetes care from the american association of clinical endocrinologists , the american diabetes association , and the endocrine society time estimates based on baseline case and best case were determined for each activity , where baseline case was the amount of time required assuming a mix of patient complications , nonoptimal patient / caretaker adherence , and possible administrative delays ( e.g. , delays in scheduling , paperwork , etc . ) and best case was the amount of time required to provide standard - based care assuming optimal patient / caretaker adherence , no patient complications , and no administrative delays . provider costs were calculated by multiplying provider time estimates by the average wage and overhead amounts for each provider type . salary and indirect ( overhead ) rates were based on data from nationally representative sources ( 14,15 ) . sums of total provider costs were used to determine cost per activity , and total activity costs were used to determine a per - visit cost . next , the expert panels timing estimates were mapped to current procedural terminology ( cpt ) and healthcare common procedure coding system billing codes and the associated medicare national average payment rate ( adjusted as necessary for non - medicare - applicable vignettes ) to compute the reimbursement ( based on the expected payer mix for the patient characteristics ) that would be collected by providers as a result of activity performance . total reimbursement was finalized by repeating these revenue calculations in a fashion similar to that used for the provider costs . the total reimbursement amount was compared with the total provider costs and any differences noted as a reimbursement gap . provider cost , reimbursement , and gap amounts were calculated on a per - patient , per - year basis using the median number of diabetic patients seen per week for adult and pediatric practices ( as found in the provider survey ) , multiplied by the average number of patients seen per year based on national estimates from the medical group management association ( 14 ) . the survey showed that practice or employment arrangements were as follows : group practice in 32.2% ( 24 physicians 15% ; 5 or more physicians 17.2% ) ; solo practice office , 13.9% ; and hospital setting , 41.4% ( university teaching hospital , 30.3% ; community teaching hospital , 7.1% ; and community nonteaching hospital , 4.0% ) . the remaining 12.5% reported working as staff at an hmo or other private plan , being employed by a diabetes manufacturer , working at an accredited or recognized diabetes education program , or working as staff at a community nonteaching hospital . the results of the baseline model of the standards - of - care economic model show that provider costs exceed reimbursements for all scenarios ( table 3 ) , whereas best case provider time estimates reimbursement exceeds costs in five of the six scenarios . sensitivity analyses showed that the model is highly sensitive to assumptions about provider reimbursement , particularly assumptions regarding the level of office visit code reimbursed . payers often limit the amount of diabetes education and nutrition therapy allowed , and two of the type 1 diabetic patient vignettes are sensitive to assumptions about the amount of diabetes education and nutrition therapy reimbursed . provider costs exceed reimbursement for csii and cgm services for both adult and pediatric patients when baseline case time estimates are used ( table 4 ) . baseline and best case reimbursement gaps for evaluation and management services with different reimbursement assumptions * baseline case and best case reimbursement gaps for initiation of csii or cgm with different reimbursement assumptions * the annual gap between provider cost and reimbursement for a typical adult and pediatric diabetes practice was calculated based on the number of diabetic patients seen per year . this estimate is based both on the median number of diabetic patients seen per week for adult and pediatric practices and the mean number of patients seen per year based on the provider survey and data obtained from the medical group management association . depending on the number and case mix of patients seen by diabetes care providers , the costs of treating diabetic patients in an adult practice would exceed reimbursement by > 750,000 usd / year . for a pediatric practice , costs would exceed reimbursement by > 471,000 usd / year . these gaps are increased for patients using intensive management technologies such as csii and cgm . an adult diabetologist s practice would require a 19% increase in overall reimbursement in order to break even in the baseline case scenario ; the individual diabetologist would require a 63% increase . the reason for the higher increase in individual reimbursement compared with the overall practice is the significant disparity between the cost versus reimbursement for evaluation and management services of providing cognitive services to patients with diabetes . this disparity is much smaller for such services such as ophthalmologic evaluation , podiatric care , diabetes education , and nutritional services , which are all considered part of the practice costs . this modeling study demonstrates that compensation for optimal diabetes care is inadequate and inconsistent with national standards . even using conservative assumptions regarding reimbursement , the model results suggest that provider costs greatly exceed reimbursement for most patients for standards - based care . indeed , the three scenarios for type 2 diabetes while representing a spectrum of age , weight , and comorbidities are relatively straightforward cases commonly seen in a primary care setting , suggesting that the reimbursement gap would be even larger for more complex cases . first , it uses time estimates based on input from experienced diabetes providers , which might be substantially different with another panel of experts or of primary care providers ( pcps ) . the vast majority of people with diabetes receive their health care from pcps and not diabetes specialists ; yet , their care is no less governed by the standards of care than is care from specialists . while magnitude of the misalignment of incentives to provide guideline - driven care is likely to be less for a pcp because of the diversity of illness that they see , it will still be substantial given the prevalence of diabetes in the u.s . in addition , the model assumes that all standards - based services included in the model will be covered by all payers . finally , it assumes that providers will receive full reimbursement for the services provided and that they can collect the full patient copayment / coinsurance amounts from all patients . indeed , the sensitivity analyses show that the model is sensitive to assumptions about coverage of services and level of reimbursement , and the gap between provider costs and reimbursement would increase significantly under differing assumptions . the modeling study confirms general perceptions of an untenable situation in which providers are financially unable to meet the established standards of diabetes care that would prevent or delay costly diabetes complications . the panels time estimates were high and were guided by the stated goal of what it would take to produce guideline - driven care . we believe that these time estimates are likely to represent the large amount of unreimbursed care that is generally provided to patients with diabetes . the dwg recommends the following changes , which are arrayed across three areas of provider engagement : care management , payment reform , and workforce supply . we recognize that some of the care management and payment reform options may not be appropriate or possible in some practice settings at this time . diabetes is unique among chronic diseases in that , by definition , it requires a high level of engagement and never - ending self - management by patients and ( often ) their family members . improved glycemic control and thereby reduced complications can be enhanced with greater provider focus on care management . in fact , enhanced provider / patient communication leads to greater adherence among patients with diabetes ( 16 ) . among the strategies to accomplish this are the following : increasing the use of shared decision making with providers discussing the standards of care , the treatment options , and their recommendations with patients to maximize patient engagement in self - management of diabetes . this heightened understanding increases a patient s motivation and sense of empowerment to reach treatment goals that are their own rather than those of their providers ( 17,18 ) . creating strong teams to implement the shared decision - making approach and promoting the use of the core team explicitly to patients . the core team includes a physician ( or nurse practitioner or physicians assistant ) , a nurse , a dietitian , and a cde . other team members that can assist with care include a podiatrist , a pharmacist , and a psychologist or social worker . each of these provider types manages aspects of the standards of care such as glucose monitoring , diabetes self - management education , nutrition therapy , and psychosocial assessment and care ( 19 ) . leveraging existing health information technology more fully to assist patients in diabetes self - management and track blood glucose values and overall performance ( 2025 ) . the use of electronic prescribing has increased dramatically in recent years . by mid-2012 , 48% of physicians in the u.s . are using e - prescribing systems an increase from 7% in 2008 ( 2628 ) ; participating in patient registries or locally based databases to track and trend goal achievement . recent emphasis on coordinated care models that are currently being piloted and adopted provides an opportunity for registries to be designed and implemented in a more coordinated and comprehensive fashion . accountable care organizations and patient - centered medical homes ( pcmhs ) require the collection and sharing of data on their patient populations ( 2931 ) . consideration of a broad spectrum of payment solutions is necessary to fully address provider barriers . first , much of the care delivered to diabetes patients is not described by existing cpt codes that determine coverage and payment , is considered by payers to be included or bundled into existing cpt codes , or is described by existing cpt codes that are not covered or reimbursed by payers . second , in cases where appropriate codes exist , the associated payments are often insufficient . both of these problems are exacerbated by the large amount of non face - to - face care delivered to patients with diabetes . better aligning payment with desired patient outcomes can both improve outcomes and lower costs through decreasing emergency department and inpatient hospital use . for example , contracting with third - party payers for pediatric and adolescent diabetes intensive case - management services has been an effective strategy , since it allows for intensive education and immediate access to the diabetes care team for crisis management ( 32 ) . the solutions include the following : reviewing and revising billing codes in the current fee - for - service system to more appropriately describe the work being performed and ensuring that the coding results in adequate payment . testing and implementing new payment models that reward providers for supplying optimal care to patients with diabetes . payment models that hold promise for diabetes include the following : the episode - of - care payment model that allows for reimbursement of multiple services at one time covering different providers and different types of care . unlike fee - for - service models , it creates efficiencies by encouraging provider teams to work with a set amount of funding for each care episode and to tailor that experience to the patient s needs . several pilot programs of diabetes episode - of - care payments , including the prometheus payment model for diabetes , are under way , but there are no published results at this time ( 33 ) . the patient - management - fee model that provides a monthly per - patient payment for all care . it would facilitate extensive care coordination , education , and training services and cover the various patient - management activities required to achieve optimal patient outcomes , including between - visit care via phone or e - mail and excluding acute services such as episodes of ketoacidosis . the payment would account for the multidisciplinary team required for optimal care , allowing for education services from diabetes educators , nutritionists , and dietitians as appropriate . the diabetes - focused pcmh option that both encourages care coordination and aligns reimbursement incentives while incorporating guideline - directed quality measures that benefit the patient . initial evidence suggests that such a model has potential benefits for patients , providers , and payers ( 30,34,35 ) . it would place value on the services necessary to provide optimal diabetes care ( e.g. , between - visit care and patient education ) . these payment models could be applied to a system whereby there are shared ( i.e. , group ) medical appointments . some studies of shared medical appointments have shown improvement surrogate end points of a1c and cardiovascular risk in patients with diabetes ( 36,37 ) . the current supply of diabetes specialists , including both physician and nonphysician providers , is inadequate to meet the demands of today and certainly will fall short of future needs , including many of the care management and innovative payment recommendations . wait times for appointments range from 3 to 9 months , and many practices are closed to accepting new diabetic patients ( 38 ) . expanding the workforce is a necessary investment if we are to have a chance at resolving the barriers to optimal diabetes care . the solutions include the following : forgiving educational loans to make diabetes care an attractive choice for new medical professionals . such programs , similar to the national health service corps and state - supported programs , could be implemented by state or federal agencies , private sector organizations , and educational institutions through funding from nonprofit foundations and trusts ( 3941 ) . this could counteract the reputation of diabetes care as an underpaid professional endeavor that dissuades providers from seeking to enter it as they face looming debt repayment . for physicians , these programs could operate in a manner similar to existing , successful loan - forgiveness programs for pcps and physicians working in rural and underserved areas ( 42 ) . offering similar types of loan - forgiveness programs and other financial assistance to potential cdes and to rds specializing in diabetes patient care is equally critical ( 43 ) . realigning financial incentives to allow for more time with diabetic patients and for the provision of non face - to - face care . this will project a more positive image for those who work to keep diabetic patients healthy . encouraging diabetes - centric professional societies to promote the positive attributes of working with diabetic patients to medical , nursing , pharmacy , and nutrition students . better educating pcps including nurse practitioners and physicians assistants on the current standards of care , the principles of proactive management , and the need for timely referral to specialist . this training approach acts as a force multiplier , thereby mitigating some of the specialist work force supply issues . in summary , the dwg has found that delivering high - quality , guideline - based diabetes care is unrealistic given the current care and payment paradigms and proposes alternative approaches that may mitigate the increasing medical and financial burdens of this epidemic chronic illness . diabetes is unique among chronic diseases in that , by definition , it requires a high level of engagement and never - ending self - management by patients and ( often ) their family members . improved glycemic control and thereby reduced complications can be enhanced with greater provider focus on care management . in fact , enhanced provider / patient communication leads to greater adherence among patients with diabetes ( 16 ) . among the strategies to accomplish this are the following : increasing the use of shared decision making with providers discussing the standards of care , the treatment options , and their recommendations with patients to maximize patient engagement in self - management of diabetes . this heightened understanding increases a patient s motivation and sense of empowerment to reach treatment goals that are their own rather than those of their providers ( 17,18 ) . creating strong teams to implement the shared decision - making approach and promoting the use of the core team explicitly to patients . the core team includes a physician ( or nurse practitioner or physicians assistant ) , a nurse , a dietitian , and a cde . other team members that can assist with care include a podiatrist , a pharmacist , and a psychologist or social worker . each of these provider types manages aspects of the standards of care such as glucose monitoring , diabetes self - management education , nutrition therapy , and psychosocial assessment and care ( 19 ) . leveraging existing health information technology more fully to assist patients in diabetes self - management and track blood glucose values and overall performance ( 2025 ) . the use of electronic prescribing has increased dramatically in recent years . by mid-2012 , 48% of physicians in the u.s . are using e - prescribing systems an increase from 7% in 2008 ( 2628 ) ; participating in patient registries or locally based databases to track and trend goal achievement . recent emphasis on coordinated care models that are currently being piloted and adopted provides an opportunity for registries to be designed and implemented in a more coordinated and comprehensive fashion . accountable care organizations and patient - centered medical homes ( pcmhs ) require the collection and sharing of data on their patient populations ( 2931 ) . consideration of a broad spectrum of payment solutions is necessary to fully address provider barriers . first , much of the care delivered to diabetes patients is not described by existing cpt codes that determine coverage and payment , is considered by payers to be included or bundled into existing cpt codes , or is described by existing cpt codes that are not covered or reimbursed by payers . second , in cases where appropriate codes exist , the associated payments are often insufficient . both of these problems are exacerbated by the large amount of non face - to - face care delivered to patients with diabetes . better aligning payment with desired patient outcomes can both improve outcomes and lower costs through decreasing emergency department and inpatient hospital use . for example , contracting with third - party payers for pediatric and adolescent diabetes intensive case - management services has been an effective strategy , since it allows for intensive education and immediate access to the diabetes care team for crisis management ( 32 ) . the solutions include the following : reviewing and revising billing codes in the current fee - for - service system to more appropriately describe the work being performed and ensuring that the coding results in adequate payment . testing and implementing new payment models that reward providers for supplying optimal care to patients with diabetes . payment models that hold promise for diabetes include the following : the episode - of - care payment model that allows for reimbursement of multiple services at one time covering different providers and different types of care . unlike fee - for - service models , it creates efficiencies by encouraging provider teams to work with a set amount of funding for each care episode and to tailor that experience to the patient s needs . several pilot programs of diabetes episode - of - care payments , including the prometheus payment model for diabetes , are under way , but there are no published results at this time ( 33 ) . the patient - management - fee model that provides a monthly per - patient payment for all care . it would facilitate extensive care coordination , education , and training services and cover the various patient - management activities required to achieve optimal patient outcomes , including between - visit care via phone or e - mail and excluding acute services such as episodes of ketoacidosis . the payment would account for the multidisciplinary team required for optimal care , allowing for education services from diabetes educators , nutritionists , and dietitians as appropriate . the diabetes - focused pcmh option that both encourages care coordination and aligns reimbursement incentives while incorporating guideline - directed quality measures that benefit the patient . initial evidence suggests that such a model has potential benefits for patients , providers , and payers ( 30,34,35 ) . it would place value on the services necessary to provide optimal diabetes care ( e.g. , between - visit care and patient education ) . these payment models could be applied to a system whereby there are shared ( i.e. , group ) medical appointments . some studies of shared medical appointments have shown improvement surrogate end points of a1c and cardiovascular risk in patients with diabetes ( 36,37 ) . the current supply of diabetes specialists , including both physician and nonphysician providers , is inadequate to meet the demands of today and certainly will fall short of future needs , including many of the care management and innovative payment recommendations . wait times for appointments range from 3 to 9 months , and many practices are closed to accepting new diabetic patients ( 38 ) . expanding the workforce is a necessary investment if we are to have a chance at resolving the barriers to optimal diabetes care . the solutions include the following : forgiving educational loans to make diabetes care an attractive choice for new medical professionals . such programs , similar to the national health service corps and state - supported programs , could be implemented by state or federal agencies , private sector organizations , and educational institutions through funding from nonprofit foundations and trusts ( 3941 ) . this could counteract the reputation of diabetes care as an underpaid professional endeavor that dissuades providers from seeking to enter it as they face looming debt repayment . for physicians , these programs could operate in a manner similar to existing , successful loan - forgiveness programs for pcps and physicians working in rural and underserved areas ( 42 ) . offering similar types of loan - forgiveness programs and other financial assistance to potential cdes and to rds specializing in diabetes patient care is equally critical ( 43 ) . realigning financial incentives to allow for more time with diabetic patients and for the provision of non face - to - face care . this will project a more positive image for those who work to keep diabetic patients healthy . encouraging diabetes - centric professional societies to promote the positive attributes of working with diabetic patients to medical , nursing , pharmacy , and nutrition students . better educating pcps including nurse practitioners and physicians assistants on the current standards of care , the principles of proactive management , and the need for timely referral to specialist . this training approach acts as a force multiplier , thereby mitigating some of the specialist work force supply issues . in summary , the dwg has found that delivering high - quality , guideline - based diabetes care is unrealistic given the current care and payment paradigms and proposes alternative approaches that may mitigate the increasing medical and financial burdens of this epidemic chronic illness .

the cost of diabetes , driven primarily by the cost of preventable diabetes complications , will continue to increase with the epidemic rise in its prevalence in the u.s . the diabetes working group ( dwg ) , a consortium of professional organizations and individuals , was created to examine the barriers to better diabetes care and to recommend mitigating solutions . we consolidated three sets of guidelines promulgated by national professional organizations into 29 standards of optimal care and empanelled independent groups of diabetes care professionals to estimate the minimum and maximum time needed to achieve those standards of care for each of six clinical vignettes representing typical patients seen by diabetes care providers . we used a standards - of - care economic model to compare provider costs with reimbursement and calculated reimbursement gaps . the reimbursement gap was calculated using the maximum and minimum provider cost estimate ( reflecting the baseline- and best - case provider time estimates from the panels ) . the cost of guideline - driven care greatly exceeded reimbursement in almost all vignettes , resulting in estimated provider losses of 470,000750,000 usd / year depending on the case mix . such losses dissuade providers of diabetes care from using best practices as recommended by national diabetes organizations . the dwg recommendations include enhancements in care management , workforce supply , and payment reform .

METHODS RESULTS CONCLUSIONS AND RECOMMENDATIONS Care management Payment reform Workforce supply

for determination of the demographics and practice patterns of diabetes care providers , an internet - based survey was performed of the membership of the american diabetes association , the pediatric endocrine society , the american academy of pediatrics , the endocrine society , and the american association of clinical endocrinologists using software provided by surveygizmo 3.0 . participants who indicated that they do not currently treat patients with diabetes were excluded from the analysis . in addition , the american association of diabetes educators posted a link to the survey on its web site . one e - mail reminder notice was sent to members in each of the five organizations that communicated directly with their members . since many providers of diabetes care belong to multiple organizations , the survey was set with a browser cookie to prevent duplicate responses . we used the standards - of - care economic model to build a theoretical model to estimate the resources necessary for providers to consistently deliver the current standards of care to diabetic patients in the u.s . ; the objectives were also to evaluate provider costs to meet those standards and to assess patient outcomes specified in diabetes care guidelines relative to current reimbursement for these services . three sets of national standards ( american diabetes association , american association of clinical endocrinologists , and the endocrine society ) for diabetes care were integrated to produce the consolidated set of 28 standards of optimal care ( 1113 ) ( table 1 ) . a matrix of providers rendering the care needed to achieve the standards was developed , including physicians ( adult and pediatric , general , and specialty care ) , certified diabetes educators ( cdes ) , registered dietitians ( rds ) , registered nurses , physician assistants , medical assistants , eye care professionals , mental health professionals , podiatrists , clinical laboratory personnel , and smoking cessation professionals . six clinical vignettes three patients with type 1 diabetes and three with type 2 diabetes ( table 2)were developed representing a broad spectrum of patients . three independent panels of four to seven diabetes care professionals ( physicians , dietitians , nurses , and diabetes educators ) were convened , and with a facilitator s guidance , the minimum and maximum time ( in minutes ) needed to achieve that standard of care for patients with those specific characteristics over 1 year was estimated . a separate expert panel was convened to estimate the time needed to start or continue to follow patients using continuous subcutaneous insulin infusion ( csii ) or the subset of type 1 diabetic patients using a continuous glucose - monitoring system ( cgm ) throughout 1 year . panel meetings were conducted by a facilitator , who gained group consensus for the inputs and assured that the panel had not overestimated total time . consolidated standards of optimal diabetes care from the american association of clinical endocrinologists , the american diabetes association , and the endocrine society time estimates based on baseline case and best case were determined for each activity , where baseline case was the amount of time required assuming a mix of patient complications , nonoptimal patient / caretaker adherence , and possible administrative delays ( e.g. , delays in scheduling , paperwork , etc . ) and best case was the amount of time required to provide standard - based care assuming optimal patient / caretaker adherence , no patient complications , and no administrative delays . provider costs were calculated by multiplying provider time estimates by the average wage and overhead amounts for each provider type . sums of total provider costs were used to determine cost per activity , and total activity costs were used to determine a per - visit cost . next , the expert panels timing estimates were mapped to current procedural terminology ( cpt ) and healthcare common procedure coding system billing codes and the associated medicare national average payment rate ( adjusted as necessary for non - medicare - applicable vignettes ) to compute the reimbursement ( based on the expected payer mix for the patient characteristics ) that would be collected by providers as a result of activity performance . total reimbursement was finalized by repeating these revenue calculations in a fashion similar to that used for the provider costs . the total reimbursement amount was compared with the total provider costs and any differences noted as a reimbursement gap . provider cost , reimbursement , and gap amounts were calculated on a per - patient , per - year basis using the median number of diabetic patients seen per week for adult and pediatric practices ( as found in the provider survey ) , multiplied by the average number of patients seen per year based on national estimates from the medical group management association ( 14 ) . the remaining 12.5% reported working as staff at an hmo or other private plan , being employed by a diabetes manufacturer , working at an accredited or recognized diabetes education program , or working as staff at a community nonteaching hospital . the results of the baseline model of the standards - of - care economic model show that provider costs exceed reimbursements for all scenarios ( table 3 ) , whereas best case provider time estimates reimbursement exceeds costs in five of the six scenarios . payers often limit the amount of diabetes education and nutrition therapy allowed , and two of the type 1 diabetic patient vignettes are sensitive to assumptions about the amount of diabetes education and nutrition therapy reimbursed . provider costs exceed reimbursement for csii and cgm services for both adult and pediatric patients when baseline case time estimates are used ( table 4 ) . baseline and best case reimbursement gaps for evaluation and management services with different reimbursement assumptions * baseline case and best case reimbursement gaps for initiation of csii or cgm with different reimbursement assumptions * the annual gap between provider cost and reimbursement for a typical adult and pediatric diabetes practice was calculated based on the number of diabetic patients seen per year . this estimate is based both on the median number of diabetic patients seen per week for adult and pediatric practices and the mean number of patients seen per year based on the provider survey and data obtained from the medical group management association . depending on the number and case mix of patients seen by diabetes care providers , the costs of treating diabetic patients in an adult practice would exceed reimbursement by > 750,000 usd / year . for a pediatric practice , costs would exceed reimbursement by > 471,000 usd / year . these gaps are increased for patients using intensive management technologies such as csii and cgm . an adult diabetologist s practice would require a 19% increase in overall reimbursement in order to break even in the baseline case scenario ; the individual diabetologist would require a 63% increase . the reason for the higher increase in individual reimbursement compared with the overall practice is the significant disparity between the cost versus reimbursement for evaluation and management services of providing cognitive services to patients with diabetes . this disparity is much smaller for such services such as ophthalmologic evaluation , podiatric care , diabetes education , and nutritional services , which are all considered part of the practice costs . this modeling study demonstrates that compensation for optimal diabetes care is inadequate and inconsistent with national standards . even using conservative assumptions regarding reimbursement , the model results suggest that provider costs greatly exceed reimbursement for most patients for standards - based care . indeed , the three scenarios for type 2 diabetes while representing a spectrum of age , weight , and comorbidities are relatively straightforward cases commonly seen in a primary care setting , suggesting that the reimbursement gap would be even larger for more complex cases . first , it uses time estimates based on input from experienced diabetes providers , which might be substantially different with another panel of experts or of primary care providers ( pcps ) . the vast majority of people with diabetes receive their health care from pcps and not diabetes specialists ; yet , their care is no less governed by the standards of care than is care from specialists . while magnitude of the misalignment of incentives to provide guideline - driven care is likely to be less for a pcp because of the diversity of illness that they see , it will still be substantial given the prevalence of diabetes in the u.s . in addition , the model assumes that all standards - based services included in the model will be covered by all payers . finally , it assumes that providers will receive full reimbursement for the services provided and that they can collect the full patient copayment / coinsurance amounts from all patients . indeed , the sensitivity analyses show that the model is sensitive to assumptions about coverage of services and level of reimbursement , and the gap between provider costs and reimbursement would increase significantly under differing assumptions . the modeling study confirms general perceptions of an untenable situation in which providers are financially unable to meet the established standards of diabetes care that would prevent or delay costly diabetes complications . the panels time estimates were high and were guided by the stated goal of what it would take to produce guideline - driven care . we believe that these time estimates are likely to represent the large amount of unreimbursed care that is generally provided to patients with diabetes . the dwg recommends the following changes , which are arrayed across three areas of provider engagement : care management , payment reform , and workforce supply . we recognize that some of the care management and payment reform options may not be appropriate or possible in some practice settings at this time . diabetes is unique among chronic diseases in that , by definition , it requires a high level of engagement and never - ending self - management by patients and ( often ) their family members . improved glycemic control and thereby reduced complications can be enhanced with greater provider focus on care management . in fact , enhanced provider / patient communication leads to greater adherence among patients with diabetes ( 16 ) . among the strategies to accomplish this are the following : increasing the use of shared decision making with providers discussing the standards of care , the treatment options , and their recommendations with patients to maximize patient engagement in self - management of diabetes . this heightened understanding increases a patient s motivation and sense of empowerment to reach treatment goals that are their own rather than those of their providers ( 17,18 ) . the core team includes a physician ( or nurse practitioner or physicians assistant ) , a nurse , a dietitian , and a cde . other team members that can assist with care include a podiatrist , a pharmacist , and a psychologist or social worker . each of these provider types manages aspects of the standards of care such as glucose monitoring , diabetes self - management education , nutrition therapy , and psychosocial assessment and care ( 19 ) . by mid-2012 , 48% of physicians in the u.s . are using e - prescribing systems an increase from 7% in 2008 ( 2628 ) ; participating in patient registries or locally based databases to track and trend goal achievement . accountable care organizations and patient - centered medical homes ( pcmhs ) require the collection and sharing of data on their patient populations ( 2931 ) . first , much of the care delivered to diabetes patients is not described by existing cpt codes that determine coverage and payment , is considered by payers to be included or bundled into existing cpt codes , or is described by existing cpt codes that are not covered or reimbursed by payers . both of these problems are exacerbated by the large amount of non face - to - face care delivered to patients with diabetes . for example , contracting with third - party payers for pediatric and adolescent diabetes intensive case - management services has been an effective strategy , since it allows for intensive education and immediate access to the diabetes care team for crisis management ( 32 ) . the solutions include the following : reviewing and revising billing codes in the current fee - for - service system to more appropriately describe the work being performed and ensuring that the coding results in adequate payment . testing and implementing new payment models that reward providers for supplying optimal care to patients with diabetes . payment models that hold promise for diabetes include the following : the episode - of - care payment model that allows for reimbursement of multiple services at one time covering different providers and different types of care . unlike fee - for - service models , it creates efficiencies by encouraging provider teams to work with a set amount of funding for each care episode and to tailor that experience to the patient s needs . several pilot programs of diabetes episode - of - care payments , including the prometheus payment model for diabetes , are under way , but there are no published results at this time ( 33 ) . it would facilitate extensive care coordination , education , and training services and cover the various patient - management activities required to achieve optimal patient outcomes , including between - visit care via phone or e - mail and excluding acute services such as episodes of ketoacidosis . the payment would account for the multidisciplinary team required for optimal care , allowing for education services from diabetes educators , nutritionists , and dietitians as appropriate . the diabetes - focused pcmh option that both encourages care coordination and aligns reimbursement incentives while incorporating guideline - directed quality measures that benefit the patient . initial evidence suggests that such a model has potential benefits for patients , providers , and payers ( 30,34,35 ) . it would place value on the services necessary to provide optimal diabetes care ( e.g. , between - visit care and patient education ) . the current supply of diabetes specialists , including both physician and nonphysician providers , is inadequate to meet the demands of today and certainly will fall short of future needs , including many of the care management and innovative payment recommendations . wait times for appointments range from 3 to 9 months , and many practices are closed to accepting new diabetic patients ( 38 ) . expanding the workforce is a necessary investment if we are to have a chance at resolving the barriers to optimal diabetes care . the solutions include the following : forgiving educational loans to make diabetes care an attractive choice for new medical professionals . such programs , similar to the national health service corps and state - supported programs , could be implemented by state or federal agencies , private sector organizations , and educational institutions through funding from nonprofit foundations and trusts ( 3941 ) . this could counteract the reputation of diabetes care as an underpaid professional endeavor that dissuades providers from seeking to enter it as they face looming debt repayment . offering similar types of loan - forgiveness programs and other financial assistance to potential cdes and to rds specializing in diabetes patient care is equally critical ( 43 ) . encouraging diabetes - centric professional societies to promote the positive attributes of working with diabetic patients to medical , nursing , pharmacy , and nutrition students . better educating pcps including nurse practitioners and physicians assistants on the current standards of care , the principles of proactive management , and the need for timely referral to specialist . in summary , the dwg has found that delivering high - quality , guideline - based diabetes care is unrealistic given the current care and payment paradigms and proposes alternative approaches that may mitigate the increasing medical and financial burdens of this epidemic chronic illness . diabetes is unique among chronic diseases in that , by definition , it requires a high level of engagement and never - ending self - management by patients and ( often ) their family members . improved glycemic control and thereby reduced complications can be enhanced with greater provider focus on care management . among the strategies to accomplish this are the following : increasing the use of shared decision making with providers discussing the standards of care , the treatment options , and their recommendations with patients to maximize patient engagement in self - management of diabetes . this heightened understanding increases a patient s motivation and sense of empowerment to reach treatment goals that are their own rather than those of their providers ( 17,18 ) . the core team includes a physician ( or nurse practitioner or physicians assistant ) , a nurse , a dietitian , and a cde . other team members that can assist with care include a podiatrist , a pharmacist , and a psychologist or social worker . each of these provider types manages aspects of the standards of care such as glucose monitoring , diabetes self - management education , nutrition therapy , and psychosocial assessment and care ( 19 ) . leveraging existing health information technology more fully to assist patients in diabetes self - management and track blood glucose values and overall performance ( 2025 ) . by mid-2012 , 48% of physicians in the u.s . recent emphasis on coordinated care models that are currently being piloted and adopted provides an opportunity for registries to be designed and implemented in a more coordinated and comprehensive fashion . accountable care organizations and patient - centered medical homes ( pcmhs ) require the collection and sharing of data on their patient populations ( 2931 ) . first , much of the care delivered to diabetes patients is not described by existing cpt codes that determine coverage and payment , is considered by payers to be included or bundled into existing cpt codes , or is described by existing cpt codes that are not covered or reimbursed by payers . both of these problems are exacerbated by the large amount of non face - to - face care delivered to patients with diabetes . for example , contracting with third - party payers for pediatric and adolescent diabetes intensive case - management services has been an effective strategy , since it allows for intensive education and immediate access to the diabetes care team for crisis management ( 32 ) . the solutions include the following : reviewing and revising billing codes in the current fee - for - service system to more appropriately describe the work being performed and ensuring that the coding results in adequate payment . testing and implementing new payment models that reward providers for supplying optimal care to patients with diabetes . payment models that hold promise for diabetes include the following : the episode - of - care payment model that allows for reimbursement of multiple services at one time covering different providers and different types of care . unlike fee - for - service models , it creates efficiencies by encouraging provider teams to work with a set amount of funding for each care episode and to tailor that experience to the patient s needs . several pilot programs of diabetes episode - of - care payments , including the prometheus payment model for diabetes , are under way , but there are no published results at this time ( 33 ) . the patient - management - fee model that provides a monthly per - patient payment for all care . it would facilitate extensive care coordination , education , and training services and cover the various patient - management activities required to achieve optimal patient outcomes , including between - visit care via phone or e - mail and excluding acute services such as episodes of ketoacidosis . the payment would account for the multidisciplinary team required for optimal care , allowing for education services from diabetes educators , nutritionists , and dietitians as appropriate . the diabetes - focused pcmh option that both encourages care coordination and aligns reimbursement incentives while incorporating guideline - directed quality measures that benefit the patient . initial evidence suggests that such a model has potential benefits for patients , providers , and payers ( 30,34,35 ) . it would place value on the services necessary to provide optimal diabetes care ( e.g. , between - visit care and patient education ) . the current supply of diabetes specialists , including both physician and nonphysician providers , is inadequate to meet the demands of today and certainly will fall short of future needs , including many of the care management and innovative payment recommendations . wait times for appointments range from 3 to 9 months , and many practices are closed to accepting new diabetic patients ( 38 ) . expanding the workforce is a necessary investment if we are to have a chance at resolving the barriers to optimal diabetes care . the solutions include the following : forgiving educational loans to make diabetes care an attractive choice for new medical professionals . such programs , similar to the national health service corps and state - supported programs , could be implemented by state or federal agencies , private sector organizations , and educational institutions through funding from nonprofit foundations and trusts ( 3941 ) . this could counteract the reputation of diabetes care as an underpaid professional endeavor that dissuades providers from seeking to enter it as they face looming debt repayment . offering similar types of loan - forgiveness programs and other financial assistance to potential cdes and to rds specializing in diabetes patient care is equally critical ( 43 ) . this will project a more positive image for those who work to keep diabetic patients healthy . encouraging diabetes - centric professional societies to promote the positive attributes of working with diabetic patients to medical , nursing , pharmacy , and nutrition students . better educating pcps including nurse practitioners and physicians assistants on the current standards of care , the principles of proactive management , and the need for timely referral to specialist . in summary , the dwg has found that delivering high - quality , guideline - based diabetes care is unrealistic given the current care and payment paradigms and proposes alternative approaches that may mitigate the increasing medical and financial burdens of this epidemic chronic illness .

many electronic properties of molecules and materials are determined by and understood through the energetics of the valence electrons , which are often probed experimentally using photoemission spectroscopy ( pes ) . via measurement of the kinetic energy of photoemitted electrons , pes provides direct experimental access to the electron ejection energies , the smallest of which is the ionization potential ( ip ) . calculating pes data from first principles is a long - standing challenge to modern electronic structure methods . a state - of - the - art method for obtaining ionization spectra theoretically is many - body perturbation theory ( mbpt ) , which calculates quasi - particle excitation energies via solving the dyson equation , typically within the gw approximation ( where g is the green function and w is the dynamically screened coulomb potential ) . moreover , in particular for gas - phase computations that are at the center of this work , results can be sensitive to details of the specific gw scheme employed and can also be challenging to converge . density functional theory ( dft ) , in which the ground - state electron density , rather than the many - electron wave function , is the fundamental quantity , is a computationally efficient first principles method for calculating the electronic structure of many - electron systems . sham ( ks ) scheme , in which the original many - electron problem is mapped uniquely into a fictitious noninteracting electron system yielding the same electron density . this mapping leads to effective single - particle equations that provide a significant conceptual and computational simplification of the original many - electron problem . however , due to the fictitious nature of the noninteracting electrons , the correspondence of ks eigenvalues with ionization energies measured in an experiment is not at all straightforward . it can be shown that for the exact ks potential , the energy of the highest occupied molecular orbital ( homo ) equals the negative of the ip , a result known as the ip theorem . lower - lying eigenvalues do not strictly correspond to electron removal energies . for outer - valence electrons , however , exact dft eigenvalues may still serve as a useful and even quantitative approximation to electron removal energies . we note that in general the simulation of photoemission spectra also requires that the photoionization cross - section is addressed . its calculation is outside the scope of the present manuscript , which focuses on the correct description of the energetics . our comparison between the calculated density of states and the photoelectron spectra concentrates , therefore , on peak positions rather than peak intensities . we note , however , that for gas - phase ultraviolet photoemission spectroscopy ( ups)the standard choice for probing molecular outer - valence states angle - dependent cross - section effects are irrelevant , owing to orientational averaging , and orbital - dependent cross - section effects are relatively weak . the ks mapping scheme relies on an exchange - correlation energy - functional of the density , the exact form of which is generally unknown and must be approximated . common approximate exchange - correlation functionals used within the ks scheme are the local density approximation ( lda ) and the generalized gradient approximation ( gga ) . in the former , one assumes that at each point in space the exchange - correlation energy per particle is given by its value for a homogeneous electron gas . in the latter , information on deviations from homogeneity unfortunately , for gas - phase molecules , ks eigenvalues obtained through the use of these approximations are not in good agreement with experimental ionization spectra . first , the ip theorem is grossly disobeyed and the negative of the homo energy usually underestimates the ip severely . even if this difference is accounted for by rigidly shifting the theoretical eigenvalue spectrum , the calculated eigenvalue spectrum may still exhibit qualitative failures , notably an erroneous ordering of the electronic levels ( see , e.g. , refs ( 33) ) . these two drawbacks can be traced back to two different ( yet not unrelated ) deficiencies of the above - described approximations : lack of a derivative discontinuity ( dd ) and orbital self - interaction errors ( sie ) . jump in the ks potential , when approaching the integer electron number either from above or from below jump helps to account for the discontinuity of the chemical potential at integer electron numbers , i.e. , for the fact that the electron removal energy is not the same as the electron insertion energy . part of the discontinuity in the chemical potential is accounted for by the ks kinetic energy term . but the kinetic energy contribution to the discontinuity in the chemical potential is generally insufficient , and the remaining discontinuity must be provided by the dd . because the electron ion and hartree energies are continuous in the electron density , the remaining discontinuity can only be attained by a jump in the exchange - correlation potential . however , in standard lda or gga functionals , the exchange - correlation potential explicitly depends on the electron density and does not incorporate any orbital dependence ( in contrast to , e.g. , fock exchange ) . as a result , calculations based on these approximations can not exhibit any dd in the exchange - correlation part of the potential . instead , they approximately average over it , and as a consequence the ks - homo energy is strongly underbinding with respect to the true ionization potential . electron repulsion term ( hartree potential ) in the ks equation means that each electron is repelled from the total charge in the system , including a spurious repulsion from itself . because ks theory is , in principle , exact , whatever error one makes in the hartree term must then be completely canceled out by the exact exchange - correlation term . for strongly localized orbitals , self - interaction may be significant and spuriously destabilize electron energies dft , i.e. , functionals that contain exact exchange based expressions employed using a nonlocal fock operator . we emphasize that while such functionals do not fall within the ks scheme , they are still very much within the dft framework through the generalized kohn sham ( gks ) scheme . in the ks scheme , many - body effects are incorporated entirely in a multiplicative potential ( which is the sum of the hartree and exchange - correlation potentials ) . in contrast , in the gks scheme , many - body effects are incorporated in a combination of a multiplicative potential and a nonlocal operator ( nonmultiplicative potential ) . generally , in the gks scheme , the additional nonlocal operator can mitigate the need for a dd in the multiplicative potential . most hybrid functionals in everyday use are of the global type , i.e. , they contain a fixed fraction of fock exchange . in practice , one typically observes that homo energies extracted from such hybrid functionals are closer to experimental ionization energies than those obtained from lda or gga but still significantly underestimate those observables . spectral distortions ( including the possibility of an erroneous ordering of the electronic levels ) are often mitigated , as the fraction of fock exchange reduces the sie . however , the quantitative details of the eigenvalue spectrum typically still depend on the specific choice of the approximate hybrid functional . one reason for the failure of conventional hybrid functionals to obey the ip theorem is the presence of only a fraction of exact exchange . because of this , they do not yield the correct 1/r asymptotic potential that should be felt by an electron at large distances from the molecule , which is especially relevant for describing the ionization process . however , when using the full fock exchange to correct for that , the delicate balance between exchange and correlation is disrupted , which is highly detrimental especially for short - range electron electron interactions that govern chemical bonding . a promising strategy for tackling that problem is offered by the more recent class of range - separated hybrid ( rsh ) dft functionals , pioneered by savin and co - workers . in these functionals , the interelectron coulomb repulsion term is separated into long - range ( lr ) and short - range ( sr ) components via a range - separation parameter . the lr term is mapped using full fock exchange , thereby establishing the correct asymptotic potential . the sr termed is ( typically ) mapped using a gga approach , maintaining the compatibility between the exchange and correlation expressions . in this approach , one still needs to determine the range - separation parameter , . both formal considerations and practical simulations show that aiming at accurate results for a broad range of systems , one typically needs significantly different values of . this is taken care of by using optimally tuned rsh ( ot - rsh ) functionals . there , instead of using one and the same range - separation parameter for all systems , is tuned for each system such that physically motivated tuning conditions are fulfilled without introducing any empirical parameters . in particular , it has been shown for gas phase molecules that insisting on the homo energy being equal to the negative of the ip ( i.e. , fulfilling the ip theorem ) for the neutral and anion species yields highly accurate homo energies and homo lumo gaps when compared to experimentally measured fundamental gaps or to the results of gw calculations . more recently , some of us have shown that in addition to the ip and the fundamental gap , the entire higher - lying part of the valence - electron spectrum of gas - phase molecules and molecular solids can be accurately described by the eigenvalues of ot - rsh ground - state dft calculations . in particular , it has been suggested that a more general ot - rsh functional that introduces a fraction of fock exchange in the sr and simultaneously maintains the full fock exchange in the lr allows for a more flexible treatment of differently localized molecular orbitals , resulting in an accurate description of more complex organic molecules relevant for applications in organic electronics . in the present contribution , we further investigate the capabilities of such more general ot - rsh functionals for predicting outer - valence electron spectra of organic molecules in the gas phase . first , we study the prototypical aromatic building blocks benzene , pyridine , and pyrimidine ( see figure 1a ) . this choice is motivated by the fact that for such simple systems , existing high - level experimental pes data can serve as useful benchmarks for theory . moreover , the nitrogen heteroatoms in the azabenzenes can be expected to result in differently localized molecular orbitals in the higher - lying part of the valence electron system and , in particular , close - lying and states . for their accurate description , the ot - rsh approach would need to attain a quantitatively satisfactory balance of self - interaction errors for both . moreover , these prototypical systems were recently identified as a challenge for both the gw and the ot - rsh methods . here , we perform both ot - rsh and gw calculations for these systems and find that they overall yield similar ( to 0.2 ev ) eigenvalue energies , both being highly accurate compared to pes experiments . we suggest that a simultaneous reliable prediction of both and orbital energies is indeed within the realm of the ot - rsh functional applied here . still , we identify one specific molecular orbital that is peculiar to ring - type molecules , which in the ot - rsh calculations displays a significant deviation from experimental results and gw calculations . we analyze the origin of this discrepancy by further computing the spectra of the same systems using conventional hybrid calculations , as well as explicitly self - interaction corrected ( sic ) calculations . ( b ) more complex representative systems terpyrimidinethiol and copper phthalocyanine . with the obtained overall very encouraging results at hand , we proceed toward larger and more complex systems , here chosen to be terpyrimidinethiol and copper phthalocyanine ( 3n - thiol and cupc , see figure 1b ) . these molecules , which also contain n as a heteroatom , are interesting for novel applications in organic electronics but at the same time challenging to assess theoretically due to pronounced differences in sie among the high - lying orbitals in the valence electron spectrum . through a comparison to gw calculations and/or pes experiments , we show that ot - rsh can provide accurate valence - electron spectra also for these more complex organic systems , with an optimal choice for the short - range fock exchange that is guided by conventional hybrid functional calculations . our results clearly demonstrate that ot - rsh functionals are a highly promising , state - of - the - art approach for predicting ionization spectra of gas - phase organic molecules . as mentioned in the introduction , we examine a generalized rsh form , which allows for different amounts of fock exchange in the short range and in the long range . we use the range - partitioning expression of yanai et al . , given by1here , r is the interelectron coordinate and , , and are adjustable parameters . naturally , this partition is not unique , but the choice of the error function is computationally convenient when using a gaussian basis for expanding the wave functions of finite systems . equation 1 defines the range - separation procedure , where the coulomb operator , 1/r , in the exchange - part of the xc potential is replaced by two complementary terms , which are treated differently . as suggested by the hf and gga delimiters in the equation , the first term is treated using hartree fock exchange , the second term is treated using gga semilocal exchange . specifically , using the perdew burke ernzerhof ( pbe ) form for the gga exchange - correlation leads to the following expression for the exchange - correlation energy2where the superscripts lr and sr denote that the full coulomb repulsion , 1/r , has been substituted by the lr and sr coulomb repulsions , erf(r)/r and erfc(r)/r , respectively . yanai et al . viewed , , and as semiempirical parameters and determined universal , we wish to determine these parameters based on the satisfaction of inherent constraints on the exchange - correlation density functional , without recourse to experimental data . from eq 2 it is clear that for full long - range fock exchange , which guarantees the correct asymptotic potential , one condition is that + = 1 ( note that the fit to thermochemistry data performed by yanai et al . leads to + = 0.65 , i.e. , to a potential that is not asymptotically correct and will , therefore , run into problems when trying to associate eigenvalues with ionization energies or electron affinities ) . this is because in the former limit , the sr behavior is expected to be gga - like ; in the latter limit , the sr behavior is expected to resemble that of a conventional hybrid functional . for example , in the pbe0 hybrid functional , which is based on the pbe semilocal functional , a global exact - exchange fraction of 25% is used . in a rsh functional , the sr fock exchange fraction is indeed expected to be somewhat smaller than in a conventional hybrid functional , because the lr fock exchange is 100% in the former but only a finite fraction in the latter . the remaining parameter , , controls the range - separation . as mentioned in the introduction , we do not seek a universal value for . instead , we rely on a nonempirical tuning procedure , where is adjusted on a per - system basis . one possibility to determine the optimal gamma , , is to choose it such that the ip theorem is satisfied:3h(n ) is the homo energy of the n - electron system and ip(n ) is the ionization potential of the n - electron system , determined from total energy difference of the n- and ( n 1)-electron systems . in general , as emphasized by the superscript notation , both h(n ) and ip(n ) display a strong dependence . it is often useful to invoke the ip theorem not only for the molecule in its neutral state but also in certain charged states . the condition which then needs to be fulfilled is that the target function j , given by4is minimized . in eq 4 , i can in principle adopt any integer number , and one can observe the effect of adding further terms on the residual j value ( vide infra ) . in practice , only values of i that are close to zero are of interest to avoid information from highly charged radical species a point elaborated below . in particular , including the anion has been found to be highly beneficial for the prediction of fundamental gaps and the energies of charge - transfer excited states . note that when restricting i to 0 in eq 4 , which we do here if the electron affinity is negative and the molecule does not bind an extra electron , then eq 3 is recovered . in agreement with previous work , it was repeatedly found that the optimal strongly changes from one system to the other , showing that our per system tuning approach is indeed necessary . the above - mentioned optimal - tuning of can , in principle , be performed for any choice of the sr fock exchange parameter , . in fact , various strategies can be employed to determine . these are discussed below in detail , along with their pros and cons , in the context of specific computational results . all pbe calculations ( within the ks scheme ) and pbe0 and rsh calculations ( within the gks scheme ) presented in this article were obtained within the qchem and nwchem codes , using cc - pvtz basis functions . all geometries were optimized using the pbe functional . as the above - described tuning scheme is based on components taken from well - established density functionals ( cf . eq 2 ) and range - separation is available in many different electronic structure codes , optimal tuning of via eq 4 is straightforward to perform . moreover , the tuning strategy is computationally efficient , as it relies on a series of standard total - energy dft calculations . we note , however , that when charging the gas - phase molecule as part of the above - explained tuning procedure , the configuration of the cation and/or anion must be identified carefully , as it may affect the results of the calculation . one of the molecules of figure 1 , which is discussed extensively in the results section . when tuning using the above - described approach , it is important to ensure that the character of the homo ( related to the left - hand term in eq 3 ) corresponds to the hole density , defined as the charge - density difference of the neutral and charged states ( and , consequently , related to the right - hand term in eq 3 ) . for pyridine , the self - consistent solution of the gks equation with the rsh functional was found to lead to two different doublet configurations of the cation , depending on the initial guess used in the procedure . these two configurations correspond to two qualitatively different hole densities ( see figure 2 , left part ) . as shown in the right part of figure 2 , the reason for the different hole densities is that the two cation configurations possess two different lumo orbitals ; i.e. , the two cationic ground states represent two different ionization processes . loss is , in one case , from a orbital and , in the other case , from a orbital . these two cationic configurations are energetically close , which is consistent with the observation that the homo and the homo1 of pyridine are very close in energy ( vide infra ) . importantly , however , only the hole density depicted in figure 2b which is associated with the configuration lower in energy , i.e. , the true ground state predicted for the cation corresponds to the homo of pyridine ( see figure 2c ) . therefore , one has to ensure that the cationic state shown in figure 2b is indeed the one entering the tuning procedure , in order to retain consistency for the orbital energies and total energies required in eq 3 . charge - density difference between neutral and cation ( left ) and lumo of cation ( right ) , obtained using two different doublet configurations ( a and b ) of the pyridine cation . the configuration denoted by b is the energetically more stable one . in the charge - density difference plots , red ( blue ) regions denote areas of electron density depletion ( accumulation ) as a consequence of the ionization process . ( c ) homo of the neutral pyridine molecule . for our analysis of the ot - rsh results , we also performed comparative gw calculations , as well as self - interaction - corrected calculations and ks - pbe0 calculations ( the latter are defined and explained below ) . our gw calculations are based on a standard g0w0 scheme , where quasi - particle energies are computed via a first - order correction to dft eigenvalues , with no self - consistent update of the starting wave functions . the starting quasi - particle wave function for the g0w0 corrections was obtained from the pbe functional . the static dielectric function is computed within the random - phase approximation and extended to finite frequency via the generalized plasmon - pole ( gpp ) model of hybertsen and louie . our g0w0 calculations were performed using the berkeleygw package , which employs a plane - wave basis set to compute the dielectric function and self - energy , using a pbe starting point . dft - pbe calculations were performed within the quantum espresso package , which is compatible with berkeleygw . the nuclei and core electrons were described by troullier martins relativistic norm - conserving pseudopotentials , which are part of the quantum espresso pseudopotential library . here , one , four , five , and six electrons were explicitly considered as valence electrons for h , c , n , and s , respectively , with cutoff radii ( in bohr ) of 1.3 , 0.5 , 1.0 , and 1.7 , respectively . we used a plane - wave basis cutoff of 80 ry for benzene and 120 ry for pyridine , pyrimidine , and 3n - thiol . these values lead to a total dft energy converged to < 1 mev / atom . to avoid spurious interactions with periodic images , we used a supercell with lattice vectors twice the size necessary to contain 99% of the charge density and , when computing the gw self - energy , the coulomb interaction was truncated at distances larger than half of the unit cell size . the supercell dimensions , in atomic units , were 35 39 24 ; 30 20 32 , 19 30 30 ; and 64 26 15.5 for benzene , pyridine , pyrimidine , and 3n - thiol , respectively . our static dielectric function and self - energy were constructed from 4914 , 5515 , 5071 , and 3598 unoccupied states , respectively , for benzene , pyridine , pyrimidine , and 3n - thiol . for the former three prototypical small molecules , this energy range corresponds to 90 ev above the vacuum energy , while for 3n - thiol , it corresponds to 50 ev above the vacuum energy . fewer states were included for 3n - thiol due to the greater computational expense associated with this rather large system . a static remainder approach was applied to the self - energy to approximately complete the unoccupied subspace . the plane - wave cutoff for the dielectric function was 30 ry for pyridine and pyrimidine and 24 ry for 3n - thiol and benzene . we find that these parameters converge the homo energies of the prototypical small molecules to less than 0.1 ev . on the basis of the convergence behavior of these molecules and the residual differences that we find for gw and ot - rsh homo energies ( vide infra ) , we extrapolate the errors associated with eigenvalues of the 3n - thiol calculation to be less than 0.2 ev . all sic calculations were based on the seminal sic concept of perdew and zunger . however , we constructed a spatially local , multiplicative exchange - correlation potential identical for all orbitals in the system , which ensures that the sic remains within the ks realm . this is based on the generalized optimized effective potential ( oep ) equation , which extends the original oep equation to the case of unitarily variant functionals . unlike the kli approximation to the standard oep equation , which can introduce significant deviations for the sic , the generalized kli approximation used here has been shown to be an excellent approximation to the generalized oep . the additional degree of freedom arising from the variance inherent to our procedure can be used to construct a set of orbitals that minimize the total sic energy of the system , where we applied a complex - valued energy minimizing unitary orbital transformation . for additional insights , we also used the pbe0 functional in conjunction with a local multiplicative potential , constructed in contrast to traditional gks schemes via the kli approximation for the exact exchange part of the functional . all sic and pbe0 calculations were performed with the bayreuth version of the parsec real - space code , where we employed a grid - spacing of 0.2 bohr and troullier martins norm - conserving pseudopotentials . finally , for a meaningful comparison between the results of different functionals and/or computational approaches , we tested explicitly that eigenvalues obtained from different codes and basis - set expansions ( gaussian , planewave , real - space ) do not differ by more than 0.1 ev for the same underlying functional . furthermore , we verified by visual inspection that eigenvalues calculated from different methods correspond to the same molecular orbitals . we start our analysis by considering the prototypical aromatic gas - phase molecules of figure 1a . the computed peak positions in the eigenvalue spectra ( neglecting differences of the photoionization cross sections ) should correspond to those in the photoemission spectra ( in arbitrary units , denoted as a.u . or arb . the computed spectra are obtained from ot - rsh eigenvalues with sr fock exchange fractions of = 0 and = 0.2 for ( a ) benzene , ( b ) pyridine , and ( c ) pyrimidine . the results are compared to gas - phase pes spectra from refs ( 97) and to the results of our gw calculations . importantly , the spectra are plotted on an absolute energy scale ; i.e. , the calculated eigenvalue spectra shown in figure 3 have not been shifted so as to align the theoretical data with experimental results . to facilitate a visual comparison with the experimental spectra , we broadened the calculated eigenvalue spectra via convolution with a gaussian ( with a standard deviation of 0.4 ev for benzene and 0.2 ev for pyridine and pyrimidine ) . for all three systems , we find an overall excellent agreement between ot - rsh , our gw calculations , and experimental results . in particular , the homo energies calculated from the ot - rsh approach correspond very well to the first peak in the experimental spectrum , indicating the success of the tuning procedure . in addition , the shape of the experimental spectra at higher binding energies is very well reproduced by the ot - rsh spectra . note that the comparison extends over a relatively large energy range of 8 ev below the first ip ( much more than the 3 ev in previous work on somewhat larger aromatic - based organic molecules ) . the agreement over such a wide energy range is remarkable , considering that the correspondence between dft eigenvalues and photoemission energies is expected , on general theoretical grounds , to deteriorate for lower - lying states . interestingly , the shape of the ot - rsh spectra for these molecules is only mildly sensitive to the choice of ( which is not always the case see below ) . valence electron spectra for ( a ) benzene , ( b ) pyridine , ( c ) pyrimidine , as obtained from experiment ( benzene and pyridine , ref ( 97 ) ; pyrimidine , refs ( 99 ) and ( 98 ) , reference i and ii in the figure ) , compared with simulated data obtained from gw and ot - rsh calculations ( with two different amounts of sr fock exchange , ) , broadened with gaussians of widths 0.4 , 0.2 , and 0.2 ev , respectively , to facilitate comparison with experimental results . despite the overall agreement , there is one particular feature which does not agree with experimental results for each of the three molecules : for benzene , the pronounced intensity in the ot - rsh spectra around 13 ev is in contrast to the very low intensity seen in the experiment . for pyridine and pyrimidine , mismatches occur around 13 ev and 15 ev , respectively . these discrepancies , discussed in more detail below , are observed for both considered sr choices ( = 0 and = 0.2 ) . the main deviation between our gw calculations and experiments is found for the benzene molecule , where the feature that in the experiment occurs around 11.5 ev is shifted to higher binding energies . a similar discrepancy ( as well as some additional ones for the other rings ) has previously been pointed out by marom et al . a precise quantitative comparison between theory and experiment beyond the above statements is complicated by several aspects : ( i ) the peaks in the experiments are not well separated ; i.e. , to determine peak maxima and , correspondingly , vertical ionization energies , one would need to perform extensive fitting . ( ii ) while important information on orbital symmetry and localization can be obtained directly from experimental results , it is common practice to provide a detailed assignment of orbital shape and ordering based on comparisons to theory , as was done in , e.g. , ref ( 97 ) . ( iii ) there are also some deviations between different experimental reports , e.g. , for pyrimidine , where the two experimental spectra from refs ( 98 ) and ( 99 ) ( shown in figure 3c ) appear to be shifted with respect to each other by 0.2 ev . therefore , in the following , we choose to compare theoretical data only to the original experimental spectra and avoid the extraction of vertical ionization series from the experiments . nevertheless , in the interest of putting our ot - rsh results for these prototypical systems into perspective with previous literature findings , we adopt the extracted experimental values for the ionization energies reported by marom et al . we can then perform a straightforward statistical analysis of mean absolute differences ( mads ) between theory and experiment . this yields mads of 0.1 ev for our gw calculations and mads of 0.2 ev for the ot - rsh ( = 0.2 ) calculations . ref ( 62 ) can be understood to suggest that the accuracy of the ot - rsh method falls short of that of gw . in fact , the mads of the ot - rsh calculations differ from those of our gw calculations by an extent which is marginal and on par with the experimental resolution . furthermore , the mad between ot - rsh and gw results is 0.2 ev , i.e. , in the same range . similar conclusions hold for the 3,4,9,10-perylene - tetracarboxylic - dianhydride ( ptcda ) and 1,4,5,8-naphtalene - tetracarboxylic - dianhydride ( ntcda ) molecules discussed in detail in refs ( 58 ) and ( 62 ) . for a deeper analysis of the remaining differences between theory and experiment and their origin , we turn to a detailed comparison between the ot - rsh and gw eigenvalues , given in figure 4 . for each eigenvalue valence eigenvalue spectra of ( a ) benzene , ( b ) pyridine , and ( c ) pyrimidine , obtained from gw , ot - rsh , and the nonoptimally tuned lc- pbe and lc-pbe0 rsh functionals . the gw spectra shown were obtained from literature data ( scanned in with corresponding color coding from ref ( 61 ) ) with two different starting points , and from our own work . the ot - rsh data were obtained from two different choices for the amount of sr fock exchange , . it has been recently implied that the satisfactory agreement between ot - rsh and gw results , reported in ref ( 58 ) , worsens if the comparison is made to a gw calculation based on a hybrid - functional dft starting point , rather than on a pbe starting point . to examine this , the first two columns in figure 4 show pbe- and pbe0-based g0w0 valence - electron energies computed by marom et al . for the small aromatic systems . our pbe - based g0w0 calculations , based on the scheme presented in ref ( 14 ) and already shown in figure 3 to agree well with experiment results , are given in the third column in figure 4 . they are followed by our two ( = 0 and = 0.2 ) ot - rsh results . as in figure 3 , all eigenvalues are shown on an absolute energy scale , with no shifting of the computed data . for all three prototypical rings , our pbe - based g0w0 calculations , within a plasmon - pole approximation , are closer to the pbe0-based full frequency g0w0 calculations of marom et al . than to their pbe - based ones . specifically , the mad between our pbe - based g0w0 results and the literature ones ( averaged over outer valence orbitals of all three molecules ) is 0.56 ev . the mad is reduced to only 0.22 ev upon comparison with the g0w0 results based on pbe0 . the difference stems from the fact that these two non - self - consistent , , we use a plasmon - pole approximation , pseudopotentials , and a plane - wave basis , an approach that yields good agreement with measured ips for small gas - phase molecules ; ref ( 61 ) reports all - electron calculations , with a fully frequency - dependent dielectric function and a finite localized basis . indeed , the outcome of a gw calculation can depend on more than just the starting point , and more specifically , plasmon - pole models have been seen , in other classes of systems such as simple oxides , to enhance the magnitude of gw corrections . a detailed discussion of the relative accuracy and precision and the pros and cons of different g0w0 approaches for molecular systems is well outside the scope of this manuscript and will be taken up elsewhere . here , the only salient point is that the similarity between the second and third columns of figure 4 validates the comparison made here and in ref ( 58 ) , to the pbe - based gw methodology of ref ( 14 ) ( simply referred to as gw hereafter ) . generally , differences of 0.10.2 ev , owing to numerical precision , are expected in present - day gw approaches . as mentioned above , uncertainties of similar magnitude are expected in many of the experiments relevant in the present context . beyond numerical precision , owing to the physical approximations inherent in any choice of dft functional , as well as in a g0w0 or gw approximation to the self energy , energy differences smaller than 0.10.2 ev are beyond the accuracy of these approaches . turning to the detailed comparison between ot - rsh and our gw calculation , given in figure 4 , first , the ot - rsh homo energies for all three systems are in very good ( < 0.15 ev ) agreement with the respective lowest quasi - hole energy computed from gw . excellent agreement of ot - rsh homo values with reference theoretical or experimental ips has been previously reported for a variety of systems see , e.g. , refs ( 52 ) , ( 57 ) , and ( 78 ) . nevertheless , this finding is still not trivial , because the homo of benzene consists of degenerate orbitals , whereas the homo of pyridine and pyrimidine is of character ( n , to be precise ) . we emphasize the importance of the tuning procedure for obtaining this level of accuracy for the homo energy . for comparison , both are pbe - based rsh functionals just like the one used here , with ( , ) pairs of ( = 0 , = 0.4 bohr ) and ( = 0.2 , = 0.2 bohr ) , respectively ; i.e. , they employ a fixed nontuned value of . the outer - valence energies obtained from these two functionals are shown in the two right - most columns of figure 4 . for the lc-pbe functional , the fixed- value is much larger than the optimally tuned one at = 0 ( cf . figure 3 ; = 0.287 bohr , = 0.312 bohr , and = 0.353 bohr for benzene , pyridine , and pyrimidine , respectively ) , and consequently , the lc-pbe spectrum is too low in energy . complementarily , the lc-pbe0 fixed- value is smaller than that obtained with optimal tuning at = 0.2 ( cf . figure 3 ; = 0.238 bohr , = 0.235 bohr , and = 0.278 bohr for benzene , pyridine , and pyrimidine , respectively ) , and the spectrum is too high in energy . even for benzene , where this is a smaller effect , the lc-pbe0 are shifted by 0.35 ev from our gw data and by 0.25 ev from the ot - rsh ( = 0.2 ) data . for pyrimidine , the effect is more pronounced and the shifts amount to 0.55 ev and 0.6 ev , respectively . this observation is fully consistent with the discussion of ref ( 32 ) ( especially figure 6 therein ) and underscores once again the importance of optimal tuning . with the optimally tuned functional , the predicted deeper - lying parts of the spectra ( with one exception , mentioned previously and discussed in more detail below ) are in overall excellent agreement with our gw calculations in spite of the large energy range and the fact that for all systems both and orbitals are present . increasing from 0 to 0.2 slightly improves the agreement between ot - rsh and gw , but the effect is overall minor for this kind of system ; shifts in orbital energy amount to up to 0.2 ev , as shown in the supporting information ( si ) ) . one may question the accuracy of the ot - rsh functional for describing states with higher binding - energies because of the absence of piecewise linearity ( discussed in more detail below ) for highly charged species of the studied system . however , it should be kept in mind that a highly charged radical is much less stable and undergoes considerable charge density relaxation and orbital reordering with respect to the neutral or cationic molecule . as shown above for the case of the pyridine molecule , even for the ( only singly charged ) cation , it can be difficult to obtain a meaningful ground state , and this issue can be expected to be more severe for more highly charged molecules . therefore , while indeed piecewise linearity in principle should be observed with the exact functional also for highly charged species , we believe that other issues are more relevant for describing accurately the excited states of the singly charged cation , which are the ones essential for describing the photoemission process . the one discrepancy between the gw and ot - rsh data ( vide supra ) involves a particular ring - shaped orbital ( orange - colored in figure 4 ) , which is specific to cyclic compounds . in the ot - rsh , this orbital appears significantly below its gw position ( by 0.4 ev for benzene and pyridine and an even worse 0.7 ev for pyrimidine , almost independently of ) . for all three molecules , this orbital is primarily responsible for the remaining disagreement between the ot - rsh calculations and the experimental spectra in figure 3 . to understand the ot - rsh results in more detail , and to explore the possible origins of this remaining discrepancy , we performed additional dft calculations with several different functionals . of all dft functionals studied here , the ot - rsh one is the only one capable of obeying the ionization potential theorem of eq 3 and , consequently , providing homo energies that are close to the experimental ip . therefore , in the following comparison , all energies are reported relative to the homo energy of the respective calculation ( which is set to zero ) . the resulting shifted eigenvalue spectra for the three prototypical aromatic molecules are shown in figure 5 , with the original homo energies shown on top . ( b ) pyridine , and ( c ) pyrimidine obtained from different theoretical schemes : a semilocal functional ( pbe ) , a conventional hybrid functional ( pbe0 ) in both the kohn sham ( ks ) and generalized kohn sham ( gks ) scheme , a self - interaction - corrected ( sic ) calculation , with and without additional stretching of the energy axis , gw calculations based on a pbe starting point , and optimally tuned range - separated hybrid ( ot - rsh ) calculations with two different short - range exchange parameters , = 0 and = 0.2 . see text for further details on the computational approaches . the absolute homo energy , in electronvolts , is given at the top of each column . first , we compare our ot - rsh results to those obtained with their parent semilocal functional pbe and those obtained from pbe0 , the most popular conventional hybrid functional based on pbe . in the latter functional , semilocal pbe exchange is mixed with 25% nonlocal fock exchange for the entire interaction range . this leads to an orbital - dependent functional for the exchange - correlation energy , which can then be used within dft in two different ways . if one wishes to remain within the ks framework , one must take the variational derivative of the exchange - correlation energy with respect to the density , so as to determine the multiplicative ks potential . this can be achieved for an implicit density - functional by solving the ( integro - differential ) optimized effective potential ( oep ) equation . here , we solve this equation within the kli approximation ( for details , see the computational details section ) and refer to the result as pbe0 . this is the almost universal practice with hybrid functionals , which ( as explained in the introduction ) is rigorously justified within the generalized kohn a first observation is that the shifted pbe0 eigenvalues are virtually identical to the shifted pbe ones for all systems . . one notable difference between the pbe and pbe0 results is that the shift needed to align the pbe0 homo energy to the experimental ip energy is substantially smaller than in pbe , albeit still significant . while the amount of shift needed for alignment of the homo with experimental results is essentially the same for pbe0 and pbe0 , the pbe0 eigenvalues are overall stretched (= energy rescaled ) with respect to the pbe0 data as seen most clearly for the case of benzene . for pyridine and pyrimidine , some additional orbital reordering is found , especially in the upper part of the valence band , due to mitigation of self - interaction errors , as discussed below . such a stretching of the energy scale has been observed previously ( see , e.g. , refs ( 53 ) , ( 112 ) , and ( 113 ) ) : krzdrfer and kmmel have rationalized it by arguing that , if the differences in the shapes of the ks and gks orbitals are ignored , then the difference between gks and corresponding ks eigenvalue can be viewed in terms of first - order perturbation theory involving the difference between fock and semilocal exchange . they suggested that this first - order correction mimics successfully the first - order correction between ks values and ionization energies , which is known to dominate for outer - valence electrons . indeed , the shifted pbe0 eigenvalues are in much better agreement with gw than the pbe0 ones . this underscores the beneficial effect of the nonlocal potential operator that is inherent to the gks scheme but absent in the ks schemes . as a next step , the role played by the self - interaction error ( sie ) in the description of these systems shall be assessed , also in order to understand whether orbital sie considerations may explain the discrepancy found above for the ring - shaped orbital . in order to examine that , we have performed sic calculations within the ks scheme ( for details , see the computational details section ) , which results in sie - free orbitals and eigenvalues . generally speaking , the shifted sic spectra are quite similar ( but not identical ) to the shifted pbe0 spectra for all systems , although some eigenvalue differences between the two spectra ( up to 0.3 ev for benzene and 0.4 ev for pyridine and pyrimidine ) are found . this leads to the above - mentioned orbital reordering found for pyridine and pyrimidine once the sie is mitigated . we furthermore find that sic modifies pbe eigenvalues differently for and orbitals , by an average of 0.2 ev , which in particular leads to a reordering of the close - lying homo1 and homo2 of pyrimidine . notably , these differences in the sic corrections for and pbe orbital energies are rather similar for all three systems , meaning that the relative differences of sies in the and orbitals of benzene are similar to the ones in pyridine and pyrimidine . we further note that , for these particular systems , the magnitude of the sic modifications is moderate , suggesting a moderate sie in these systems . comparison of the sic data to our gw calculations reveals immediately that , just like the pbe0 data , they would benefit from further stretching . this is fully consistent with the fact that both sic and pbe0 spectra arise from a ks , rather than a gks , calculation . we can simulate the effect of such stretching on the sic spectrum by multiplying the shifted sic eigenvalues with a constant ( a procedure that is , in fact , often performed heuristically in the comparison of ks data with experimental spectra see , e.g. , refs ( 114 ) and ( 115 ) ) . for each sic spectrum , we chose this stretching factor such that the lowest - energy pbe0 and sic eigenvalues agree . for all three prototypical systems , the resulting stretched sic eigenvalue spectra are in overall better agreement with the pbe0 spectra than the regular ( unstretched ) sic spectra and , furthermore , are in very good agreement with the ot - rsh data and with gw . for a few of the pyridine and pyrimidine orbitals , we find some remaining deviations of the stretched sic from the pbe0 and ot - rsh results , which is , however , expected as the three approaches are very different . this agreement shows that a ( range - separated or conventional ) hybrid can be highly effective in the quantitative mitigation of sies , even though it is not rigorously self - interaction free because only a fraction of exact exchange is employed in the short range . indeed , similar observations have been made for many other molecular systems , often by comparison to experimental results , but also by comparison to sic . ( orange - colored ) orbital by ot - rsh is due to a general lack of balance in the description of and orbitals owing to the sie . instead , we attribute the incorrect description of this highly delocalized orbital to the absence of beyond - pbe ( and possibly nonlocal ) correlation . this is supported by the observation that this orbital is also overbound in the shifted pbe0 data , albeit by a lesser amount of 0.2 to 0.35 ev . as semilocal exchange is known to mimic static correlation , it stands to reason that further removal of some of it , as in the ot - rsh scheme , would worsen the situation . from this point of view , the error in this orbital is then yet one more issue of compatibility between exact exchange and correlation , especially a static one . however , one can not rule out that even if the correlation issue is overcome , an error would still remain , owing to further dynamic relaxation which is not captured in any eigenvalue - based and thus static description . indications for such effects have been reported in the literature for small metal clusters , and the response of a very delocalized orbital may be overcoming this drawback , e.g. , by examining more advanced correlation functionals is , therefore , a challenge for future work . encouraged by the overall success of the ot - rsh approach for the prototypical aromatic rings , we now turn to examining the performance of the ot - rsh method for more complex organic molecules : terpyrimidinethiol ( 3n - thiol ) and copper phthalocyanine ( cupc)see figure 1b . pyrimidinethiols such as 3n - thiol are known to form self - assembled monolayers ( sams ) , and they were shown to display a number of interesting phenomena ; e.g. , they exhibit diode - type current voltage characteristics in molecular - scale electronic devices . in the context of 3n - thiol sams , some of us have predicted theoretically that the electronic structure is significantly altered due to collective electrostatic effects in the sam , leading to a localization of the frontier molecular orbitals and a concomitant pronounced reduction in the homo 3n - thiols were also suggested as sams to strongly reduce or enhance the work function of an underlying metal . these appealing findings render 3n - thiols an interesting candidate for applications in organic and molecular electronics . 3n - thiols are also interesting from a methodological perspective , as they pose a serious challenge to semilocal ks schemes : by means of sic calculations analogous to the ones performed here , it was shown that close - lying and orbitals in the outer - valence region of 3n - thiol have markedly different sies and , as a consequence , are wrongly ordered in ( semi)local dft functionals such as pbe . ernezerhof ( hse ) sr hybrid functional to a large extent reproduces the sic corrections to the pbe outer - valence eigenvalues . in hse , the sr components are the same as in pbe0 , but in the lr there is no nonlocal exchange . from the photoemission spectroscopy point of view , the behavior of hse is known to be similar to that of conventional hybrid functionals . the above - mentioned findings are supported by the data in figure 6a , which shows the shifted eigenvalue spectra of 3n - thiol , i.e. , aligning all homo levels to zero , starting with the literature data for pbe , sic , and hse . here , we focus on the range of 3 ev below the homo , which we have previously identified for molecules of a similar size as a useful range of accuracy for the rsh - derived eigenvalues . generally , pbe tends to produce orbitals that are underbound owing to significant sie . because the homo obtained from pbe is of nature , shifting according to its energy results in orbitals that appear to be overbound . the sic calculations ( as above , the spectrum is shown both as calculated and stretched ) correct the sie and , as discussed above , strongly shift the orbitals down in energy with respect to the ones , leading to extensive reordering of the eigenvalue spectrum . this effect also applies to the hse spectrum , which needs no stretching as it originates from a gks calculation . we additionally performed pbe0 calculations , also shown in figure 6a , and found an overall very good agreement with the hse spectrum and with the stretched sic with an average deviation of the shifted eigenvalues around 0.1 ev . this is consistent with the above - mentioned findings , namely that hse is comparable to conventional hybrid functionals as far as photoemission spectra are concerned . ( a ) shifted eigenvalue spectra of 3n - thiol as obtained from different theoretical schemes ( see text for details ) . pbe , sic , and hse values were taken from ref ( 68 ) . for the ot - rsh calculations , the optimal value was 0.217 and 0.187 ( in bohr ) for = 0 and = 0.15 , respectively . the absolute homo energy , in electronvolts , is given at the top of each column . ( b ) unshifted eigenvalue spectra of the same system , as calculated from hse , pbe0 , ot - rsh calculations with the two different values , and gw . to the best of our knowledge , an experimental pes spectrum of 3n - thiol is not available in the literature . therefore , we performed reference gw calculations for which the ( again shifted ) eigenvalue spectrum is also given in figure 6a . as seen above for the prototypical aromatic rings , the agreement of the shifted gw spectrum with the shifted hybrid functional ( pbe0 or hse ) spectrum is very good . some discrepancy is observed between 2.0 to 2.5 ev , but in this context it should be kept in mind that here the plotted energy range is much smaller than for the aromatic rings in figures 4 and 5 . in fact , in the present case , the errors do not exceed 0.2 ev , which is within the expected level of agreement between the two methods ( vide supra ) . molecules that the partial correction of sie and a stretching of the spectrum are the essential effects of a hybrid functional . despite the excellent agreement of the shifted conventional hybrid ( and stretched sic ) data with the gw results , this can be gleaned even from figure 6a , where the homo eigenvalue obtained from each method is given at the top of each column , but is made obvious by figure 6b , which shows unshifted spectra on an absolute energy scale . this discrepancy is removed by the ot - rsh results , also shown in figure 6a and b ( again with two different choices of sr exact exchange = 0 and = 0.15the motivation for this particular selection of values is discussed below ) . as with the simpler systems , the ips deduced from our computed ot - rsh and gw eigenvalues are very close in energy ( 0.2 ev ) for both choices of . consequently , the ot - rsh scheme produces quantitatively meaningful spectra also on an absolute energy scale . this is in sharp contrast to all other dft methods presented in figure 6 . a remaining and important issue is the best choice for the short - range fock - exchange fraction , . in contrast to the case of the small aromatic rings , where different choices for have had no qualitative effect and only a small quantitative effect , for the 3n - thiol system the differences between the ot - rsh spectra for the two choices of are more pronounced . as shown in figure 6 , these differences include qualitative deviations , e.g. , a reordering of the homo3 and homo4 orbitals . in particular , we observe that , while the position of the orbitals is similar in both spectra , the orbitals are systematically underbound in the = 0 calculation , in comparison to the = 0.15 or the gw calculations . the different dependence of and orbitals on is underscored in figure 7 , which shows the dependence of orbital eigenvalues on in the entire range of = 0 to 0.2 , with the range - separation parameter , , optimized individually for each choice of . we relate the underbinding of orbitals at small to the fact that the spatial extent of the and orbitals within the molecular plane is significantly different ; i.e. , the orbitals are clearly more localized than states and , therefore , should suffer from a larger sie . the finding that the agreement with our gw calculations is significantly improved for = 0.15 underscores that , as suggested in ref ( 58 ) and discussed in the introduction , a fraction of fock exchange in the sr together with full fock exchange in the lr allows for a mitigation of sie and therefore a balanced treatment of differently localized molecular orbitals , while retaining the correct absolute position of the energy levels . in principle , a similar statement applies for the small aromatic rings as well . however , there the difference in degree of localization is not the same and , perhaps more importantly , the energy differences between individual eigenvalues are much larger . therefore , the practical importance of is smaller in the prototypical small ( hetero)cycles , as shown in figure 4 and in the si . eigenvalues of 3n - thiol , obtained from optimally tuned range - separated hybrid ( ot - rsh ) calculations , as a function of the short - range exchange fraction , , with the optimal value of the range - separation parameter , ( in bohr ) determined for each choice of . these considerations raise the question of whether an optimal can be chosen a priori . srebro and autschbach have suggested that it can be achieved by tuning . specifically , they sought the value of that minimizes the curvature of the ideally piecewise - linear total energy versus particle number curve for the addition and removal of one charge from the neutral molecule . this was done while simultaneously determining the optimal for each choice of obtained from eq 4 ( with i restricted to 0 and 1 ) . as discussed in ref ( 58 ) , some of the present authors successfully employed a similar approach to determine an optimal value of also for the purpose of obtaining an outer - valence eigenvalue spectra , but only if it also involved the removal of a second electron . recently , stein et al . established a rigorous quantitative equality between deviations from piecewise linearity and deviations from the ip theorem . therefore , one can equivalently seek directly by minimizing the target function j from eq 4 without an explicit consideration of fractional densities . indeed , if this procedure is applied to ptcda and ntcda , the same optimal value is found ( see si ) . therefore , we discuss these two tuning procedures ( by piecewise linearity and by the ip theorem ) together in the following . indeed , in cases where an optimal can be clearly identified , several properties , including the eigenvalue spectrum , have been found to be predicted satisfactorily however , in the case of 3n - thiol , we could not employ that strategy : with i restricted to 0 and 1 , a similar minimal value of the target function j of eq 4 is obtained across a large range of values : the residual j only changed by 0.01 ev from = 0.00 to = 0.60 , and no minimum of j occurred . removal of the second electron resulted in orbital reordering ( see si for more details ) . in the absence of viable alternatives , a possible strategy would be to resort to the generally recommended value of = 0.2 , which indeed has been shown to yield very good results , for the prototypes studied above as well as for other systems . this would also be the case here ; i.e. , the mad between the absolute eigenenergies from our gw calculations and ot - rsh with = 0.2 , for the states shown in figure 7 , is 0.1 ev . however , as the 3n - thiol results do depend more sensitively on , it is still interesting , if possible , to determine an optimal value from additional considerations . in this context , a useful practical observation is that the shifted results of the conventional hybrid functional pbe0 are in good agreement with experimental and/or theoretical reference data for the systems discussed here , as well as for many other cases . therefore , a pragmatic approach would be to simply tune so as to obtain agreement between splitting of eigenvalues in the ot - rsh and in the pbe0 spectra . specifically , one should seek a balance in the relative description of delocalized and localized states . the easiest way to achieve that is to tune such that the energy difference between the homo and homo1 states is the same in the ot - rsh and the pbe0 calculations . this allows us to obtain the same useful level of sie mitigation as in a conventional hybrid functional , without the need for spectral shifting . it is this approach which has led to the value of = 0.15 for which the data shown in figure 6 have been obtained . comparing our unshifted gw and ot - rsh ( = 0.15 ) calculations of figure 6b , one , indeed one should , however , note that the eigenvalues clustered there are very close in energy and that this reordering does not involve deviations greater than 0.1 ev between corresponding eigenvalues of the two methods . overall , the mad between the gw and ot - rsh ( = 0.15 ) calculations is a satisfying 0.1 ev , with the largest deviation being 0.2 ev for the homo . for comparison , with ot - rsh ( = 0 ) the mad is 0.25 ev , with the largest deviation being 0.45 ev , where the reduced level of agreement is mostly due to the less accurate description of the orbitals . encouraged by this further success , we now turn to an even more complex system copper phthalocyanine ( cupc see figure 1b ) . in molecular - solid form , cupc is a highly stable organic semiconductor with a broad range of applications , including light - emitting diodes , solar cells , gas sensors , and thin - film transistors . owing to these applications , there is considerable interest in investigating its electronic structure ( see ref ( 69 ) and additional references therein ) . in the present context , cupc mainly serves as a test case that poses several additional challenges for the ot - rsh method : first , it is an open shell molecule ( s = 1/2 ) . second , the interaction between the d orbitals of the copper atom and the s and p orbitals of the embedding macrocycle result in a highly nontrivial set of localized and delocalized orbitals an issue elaborated below . for the cupc molecule , marom et al . have previously established that : ( 1 ) semilocal functionals , such as pbe , result in eigenvalue spectra that are strongly distorted by severe sie ; ( 2 ) these errors are mitigated substantially , though not completely , by the use of hybrid functionals such as pbe0 or hse ; ( 3 ) severe sie distortions at the dft level are partly carried over to gw calculations building on the dft densities . to better understand the above claims , consider figure 8a , which provides a scheme containing the computed frontier eigenvalues and orbitals . we illustrate the severe sie ( claim 1 ) by considering several selected frontier orbitals . whereas the a1u orbital and the doubly degenerate eg orbitals are highly delocalized on the macrocycle , the spin - split b1 g orbitals are quite strongly localized around the copper atom . configuration 1 ( left side of figure 8a ) was obtained from the ot - rsh calculations discussed below but is equivalent to the one obtained from the gw calculations of ref ( 69 ) . it identifies the delocalized a1u and eg orbitals as the homo and lumo , respectively , and places the spin - split b1g and b1g orbitals as homo1 and lumo+1 , respectively . pbe calculations , however , predict configuration 2 ( right side of figure 8a ) , in which the spin - split b1g and b1g are identified as homo and lumo , respectively . clearly , the localized b1g orbital is spuriously pushed to higher energies by the sie , the effect being strong enough to make the b1g orbital become the homo . this forces the unoccupied b1g orbital to be spuriously shifted to lower energies in order to maintain the spin - splitting symmetry , to the point of it becoming the lumo . pbe0 or hse strongly mitigate this error ( claim 2 ) and yield configuration 1 . ( a ) schematic diagram of selected frontier eigenvalues and orbitals for the cupc molecule , as obtained from an ot - rsh calculation ( configuration 1 , left ) and from a pbe calculation ( configuration 2 , right ) . ( b ) ot - rsh charge - density differences between neutral and cation ( left ) and lumo of cation ( right ) , obtained from the open - shell singlet configuration of the cupc cation , corresponding to ionization of netrual configuration 1 ( in green ) , and from the closed - shell singlet configuration of the cupc cation , corresponding to ionization of configuration 2 ( in red ) . in the charge density difference plots , green or red ( blue ) regions denote areas of electron density depletion ( accumulation ) as a consequence of the ionization process . to understand the manifestation of the sie ( and its mitigation ) in the simulated photoelectron data , spectra computed with different computational methods importantly , in this figure we compare occupied - state eigenvalues to the gas - phase photoelectron spectrum as before , but further compare unoccupied - state eigenvalues to experimental inverse photoemission spectroscopy ( ipes ) . in ipes , photons are emitted from a sample due to its irradiation with fixed - energy electrons , and the energy distribution of the emitted photons is measured , yielding experimental information on virtual states . cupc is the first system in this article for which such comparison is possible , because for the small aromatic rings the virtual states are unbound and because for 3n - thiol no ( regular or inverse ) experimental pes data are available . in fact , gas - phase inverse photoemission spectroscopy is nonexistent in general . therefore , the comparison is to experimental data obtained from thin cupc films . due to polarization effects , the electron affinity of a film is much smaller than that of an isolated molecule , and the computed empty state energies and the measured inverse photoemission spectroscopy data can be compared only up to a rigid shift . therefore , to allow comparison to experimental results without modifying the computational gas - phase data , the experimental ipes spectra were shifted so as to align the lowest - energy peak with the gw spectrum shown at the topmost computed spectrum in figure 9 . simulated dft and gw spectra ( see text for details ) , obtained from computed energy levels ( shown as sticks ) by broadening via convolution with a 0.15-ev - wide gaussian . pbe , pbe0 , and gw data were taken from ref ( 69 ) . theoretical data are compared to the experimental gas phase photoemission data of evangelista et al . and to the thin film inverse photoemission data of ( a ) murdey et al . the experimental inverse photoemission spectra were shifted so as to align the lumo peak with the computed gw@pbe0 lumo peak . on the ot - rsh data eigenvalues corresponding to the eg , a1u , and b1 g orbitals are designated by the same color scheme as in figure 8 . as discussed above , it is well - known that for either pbe or pbe0 the homo and lumo do not correspond to the ionization potential or the electron affinity , respectively ( see , e.g. , refs ( 26 ) and ( 32 ) ) . this causes an uncontrolled rigid shift of the simulated photoemission curve . to facilitate a meaningful comparison also for these functionals , the pbe and pbe0 spectra of figure 9 the ionization potential is computed as the total energy difference between the neutral species and the cation , and the filled - state eigenvalue spectrum is rigidly shifted such that the homo energy coincides with the computed ionization potential . an equivalent procedure is applied to the unoccupied - state eigenvalue spectrum , which is rigidly shifted such that the lumo energy coincides with the computed electron affinity . comparison of the pbe- and pbe0-based simulated spectra in figure 9 ( taken from ref ( 69 ) ) with the experimental data reveals that whereas the shifted pbe spectrum is in poor agreement with experiment for both filled and empty states , the shifted pbe0 spectrum provides for a much improved agreement with experimental results . this highlights both the severe sie in the pbe calculation and its mitigation by the pbe0 calculation . better agreement yet , including an accurate placement of the spin - split filled b1g and empty b1g orbitals , is afforded by gw calculations of marom et al . it is in significantly better agreement with experimental results than their gw calculations based on a pbe starting point ( at least with the particular flavor of gw used in ref ( 69 ) , which , as discussed earlier , is different from the one we used above for benzene , pyridine , and pyrimidine ; we refrained from conducting such gw calculations also for cupc , because of the computational cost involved ) . this improved description of the electronic structure of cupc by the pbe0 based gw calculations substantiates the third above claim , i.e. , the possible carry - over of severe sie to the gw calculation . it is intriguing to determine how well the ot - rsh approach performs in this more complicated scenario . as for the case of pyridine ( see figure 2 ) , it is important to choose the correct cationic configuration for the tuning procedure . open shell singlet , with a single spin in the b1 g orbital and in the a1u orbital , which corresponds to removal of an electron from the a1u homo of configuration 1 in figure 8a . the other is a closed - shell singlet cation , which corresponds to removal of an electron from the b1 g homo of configuration 2 in figure 8a . importantly , only for the open - shell singlet configuration ( which is lower in total energy ) is the lumo consistent with the character of the neutral homo , and with the hole density , as shown in figure 8b . just like in the pyridine example of figure 2 , all tuning must be performed solely with this configuration . we note that multiple stable configurations are well - known to occur in metal - phthalocyanines , which is why we strongly stress the need for correct identification and usage of compatible neutral and ionic configurations in the tuning procedure . having ascertained the validity of our tuning procedure , we turn to the spectra simulated using the ot - rsh method , also shown in figure 9 . as for the case of 3n - thiol , the obtained spectra ( second and fourth theoretical curves in the figure ) are strongly -dependent . this is shown explicitly in figure 10 , which shows the dependence of orbital eigenvalues on , with the range - separation parameter , , again optimized individually for each choice of . it is particulary striking that the orbitals most sensitive to are the highly localized spin - split b1 g pair , which is fully consistent with our above discussion on the relation between orbital localization and sensitivity to short - range fock exchange . unfortunately , here direct tuning of based on eq 4 , even with i = 0 , 1 , 1 , was not useful because the minimal value of the target function j was very small and very weakly dependent on within the numerical accuracy of our work ( see si ) . thus , while the procedure of ref ( 58 ) did not fail , in the sense of providing an incorrect value for , it did not succeed either . therefore , we determined as for the case of 3n - thiol , i.e. , based on the success of the shifted pbe0 spectrum . as discussed above , a crucial aspect in the theoretical description of the cupc spectrum is the energy separation between the delocalized a1u orbital and the filled localized b1 g orbital of figure 8 . therefore , we tune the value of so as to agree with the pbe0-calculated separation between the eigenvalues for the a1u and b1 g orbitals . this yields = 0.17 , which is again close to the default value of 0.2 , further supporting the latter as a useful choice even without any -tuning . = 0.17 was then used to calculate the second theoretical curve in figure 9 . it yields excellent agreement with prior gw@pbe0 calculations ( shown in the figure ) and the experimental data in both the top valence ( i.e. , occupied ) states and the bottom conduction ( i.e. , unoccupied ) states . in particular , the ot - rsh alignment of the a1u , b1 g , and eg states of figure 8 agrees very well with the prior gw calculations . as orbitals above and below these frontier ones tend to be clustered for these larger molecule , we do not discuss them individually . however , it is clear that the agreement between gw and ot - rsh gradually deteriorates as one goes further down or up in the valence or conduction states , respectively . in particular , it appears that agreement of the lower valence states with the gw results could be further assisted by some stretching of the energy scale an observation consistent with the generally expected differences between nonlocal exchange and self - energy operators , as well as with our general guideline of restricting our attention to states within a few electronvolts of the frontier orbitals . nevertheless , in the absence of detailed orbital information , at the level of the given experimental resolution , the ot - rsh results can be viewed as agreeing with experimental results as well as the gw ones , possibly even relatively deep into the valence band . eigenvalues of cupc , obtained from optimally tuned range - separated hybrid ( ot - rsh ) calculations , as a function of the short - range exchange fraction , , with the optimal value of the range - separation parameter , ( in bohr ) , determined for different choices of . as an additional observation , we note that the homo and lumo eigenvalues obtained from the ot - rsh ( = 0.17 ) calculation ( as is , without any rigid shifting of the data ) are in excellent agreement with the lowest quasi - hole and quasi - electron excitations , respectively , obtained from gw@pbe0 . this is fully consistent with the work of stein et al . , who suggested that ot - rsh gaps can be identified with quasi - particle gaps an observation that has since been confirmed for a large variety of systems by ourselves , as well as by several other groups . a related interesting observation is that the ot - rsh ( = 0 ) data are rather similar to the gw@pbe calculations from ref ( 69 ) ( again , for that particular flavor of gw ) : both are far better than the pbe data , but in both spectra , the a1u b1 g and eg this highlights yet again the positive role of sr exchange in mitigating sies where they are significant . in conclusion , we examined the performance of optimally tuned range - separated hybrid functionals for predicting the photoemission spectra of several challenging prototypical ( benzene , pyridine , and pyrimidine ) and complex ( 3n - thiol and cupc ) ( hetero)cyclic organic molecules . overall , the resulting spectra agree very well ( typically within 0.10.2 ev ) with experimental data and our gw calculations , even for states several electronvolts away from the frontier orbitals . we performed several additional hybrid dft calculations in both the ks and gks schemes and found that the inclusion of a nonlocal operator strongly benefits the calculated spectrum . moreover , our sic calculations confirmed that self - interaction errors can be efficiently mitigated in ot - rsh functionals , which shows that , with a pbe0-based optimal choice of the short - range fraction of fock exchange , the ot - rsh method can offer an excellent balance in the description of localized and delocalized states . the sole exception found in the studied systems is a high - symmetry orbital , particular to small aromatic rings . we conclude that the ot - rsh method is a highly accurate dft method for outer - valence pes prediction for such systems , and its accuracy is comparable to state - of - the - art gw schemes . this success comes at the price of increased computational and conceptual cost that is inherent to the parameter tuning . while this increase in effort is notable compared to standard , nontuned dft calculations , it is not overwhelming and still lower than the cost of a gw calculation .

density functional theory with optimally tuned range - separated hybrid ( ot - rsh ) functionals has been recently suggested [ refaely - abramson et al . phys . rev . lett.2012 , 109 , 226405 ] as a nonempirical approach to predict the outer - valence electronic structure of molecules with the same accuracy as many - body perturbation theory . here , we provide a quantitative evaluation of the ot - rsh approach by examining its performance in predicting the outer - valence electron spectra of several prototypical gas - phase molecules , from aromatic rings ( benzene , pyridine , and pyrimidine ) to more complex organic systems ( terpyrimidinethiol and copper phthalocyanine ) . for a range up to several electronvolts away from the frontier orbital energies , we find that the outer - valence electronic structure obtained from the ot - rsh method agrees very well ( typically within 0.10.2 ev ) with both experimental photoemission and theoretical many - body perturbation theory data in the gw approximation . in particular , we find that with new strategies for an optimal choice of the short - range fraction of fock exchange , the ot - rsh approach offers a balanced description of localized and delocalized states . we discuss in detail the sole exception found a high - symmetry orbital , particular to small aromatic rings , which is relatively deep inside the valence state manifold . overall , the ot - rsh method is an accurate dft - based method for outer - valence electronic structure prediction for such systems and is of essentially the same level of accuracy as contemporary gw approaches , at a reduced computational cost .

Introduction Theoretical and Methodological Details Prototypical Aromatic RingsResults and Discussion More Complex Aromatic HeterocyclesResults and Discussion Conclusions

a state - of - the - art method for obtaining ionization spectra theoretically is many - body perturbation theory ( mbpt ) , which calculates quasi - particle excitation energies via solving the dyson equation , typically within the gw approximation ( where g is the green function and w is the dynamically screened coulomb potential ) . density functional theory ( dft ) , in which the ground - state electron density , rather than the many - electron wave function , is the fundamental quantity , is a computationally efficient first principles method for calculating the electronic structure of many - electron systems . however , when using the full fock exchange to correct for that , the delicate balance between exchange and correlation is disrupted , which is highly detrimental especially for short - range electron electron interactions that govern chemical bonding . this is taken care of by using optimally tuned rsh ( ot - rsh ) functionals . more recently , some of us have shown that in addition to the ip and the fundamental gap , the entire higher - lying part of the valence - electron spectrum of gas - phase molecules and molecular solids can be accurately described by the eigenvalues of ot - rsh ground - state dft calculations . in particular , it has been suggested that a more general ot - rsh functional that introduces a fraction of fock exchange in the sr and simultaneously maintains the full fock exchange in the lr allows for a more flexible treatment of differently localized molecular orbitals , resulting in an accurate description of more complex organic molecules relevant for applications in organic electronics . in the present contribution , we further investigate the capabilities of such more general ot - rsh functionals for predicting outer - valence electron spectra of organic molecules in the gas phase . first , we study the prototypical aromatic building blocks benzene , pyridine , and pyrimidine ( see figure 1a ) . moreover , the nitrogen heteroatoms in the azabenzenes can be expected to result in differently localized molecular orbitals in the higher - lying part of the valence electron system and , in particular , close - lying and states . here , we perform both ot - rsh and gw calculations for these systems and find that they overall yield similar ( to 0.2 ev ) eigenvalue energies , both being highly accurate compared to pes experiments . still , we identify one specific molecular orbital that is peculiar to ring - type molecules , which in the ot - rsh calculations displays a significant deviation from experimental results and gw calculations . with the obtained overall very encouraging results at hand , we proceed toward larger and more complex systems , here chosen to be terpyrimidinethiol and copper phthalocyanine ( 3n - thiol and cupc , see figure 1b ) . these molecules , which also contain n as a heteroatom , are interesting for novel applications in organic electronics but at the same time challenging to assess theoretically due to pronounced differences in sie among the high - lying orbitals in the valence electron spectrum . through a comparison to gw calculations and/or pes experiments , we show that ot - rsh can provide accurate valence - electron spectra also for these more complex organic systems , with an optimal choice for the short - range fock exchange that is guided by conventional hybrid functional calculations . as mentioned in the introduction , we examine a generalized rsh form , which allows for different amounts of fock exchange in the short range and in the long range . from eq 2 it is clear that for full long - range fock exchange , which guarantees the correct asymptotic potential , one condition is that + = 1 ( note that the fit to thermochemistry data performed by yanai et al . on the basis of the convergence behavior of these molecules and the residual differences that we find for gw and ot - rsh homo energies ( vide infra ) , we extrapolate the errors associated with eigenvalues of the 3n - thiol calculation to be less than 0.2 ev . the computed spectra are obtained from ot - rsh eigenvalues with sr fock exchange fractions of = 0 and = 0.2 for ( a ) benzene , ( b ) pyridine , and ( c ) pyrimidine . in particular , the homo energies calculated from the ot - rsh approach correspond very well to the first peak in the experimental spectrum , indicating the success of the tuning procedure . in addition , the shape of the experimental spectra at higher binding energies is very well reproduced by the ot - rsh spectra . interestingly , the shape of the ot - rsh spectra for these molecules is only mildly sensitive to the choice of ( which is not always the case see below ) . valence electron spectra for ( a ) benzene , ( b ) pyridine , ( c ) pyrimidine , as obtained from experiment ( benzene and pyridine , ref ( 97 ) ; pyrimidine , refs ( 99 ) and ( 98 ) , reference i and ii in the figure ) , compared with simulated data obtained from gw and ot - rsh calculations ( with two different amounts of sr fock exchange , ) , broadened with gaussians of widths 0.4 , 0.2 , and 0.2 ev , respectively , to facilitate comparison with experimental results . despite the overall agreement , there is one particular feature which does not agree with experimental results for each of the three molecules : for benzene , the pronounced intensity in the ot - rsh spectra around 13 ev is in contrast to the very low intensity seen in the experiment . ref ( 62 ) can be understood to suggest that the accuracy of the ot - rsh method falls short of that of gw . in fact , the mads of the ot - rsh calculations differ from those of our gw calculations by an extent which is marginal and on par with the experimental resolution . for a deeper analysis of the remaining differences between theory and experiment and their origin , we turn to a detailed comparison between the ot - rsh and gw eigenvalues , given in figure 4 . for each eigenvalue valence eigenvalue spectra of ( a ) benzene , ( b ) pyridine , and ( c ) pyrimidine , obtained from gw , ot - rsh , and the nonoptimally tuned lc- pbe and lc-pbe0 rsh functionals . the ot - rsh data were obtained from two different choices for the amount of sr fock exchange , . the difference stems from the fact that these two non - self - consistent , , we use a plasmon - pole approximation , pseudopotentials , and a plane - wave basis , an approach that yields good agreement with measured ips for small gas - phase molecules ; ref ( 61 ) reports all - electron calculations , with a fully frequency - dependent dielectric function and a finite localized basis . turning to the detailed comparison between ot - rsh and our gw calculation , given in figure 4 , first , the ot - rsh homo energies for all three systems are in very good ( < 0.15 ev ) agreement with the respective lowest quasi - hole energy computed from gw . figure 3 ; = 0.287 bohr , = 0.312 bohr , and = 0.353 bohr for benzene , pyridine , and pyrimidine , respectively ) , and consequently , the lc-pbe spectrum is too low in energy . even for benzene , where this is a smaller effect , the lc-pbe0 are shifted by 0.35 ev from our gw data and by 0.25 ev from the ot - rsh ( = 0.2 ) data . one may question the accuracy of the ot - rsh functional for describing states with higher binding - energies because of the absence of piecewise linearity ( discussed in more detail below ) for highly charged species of the studied system . to understand the ot - rsh results in more detail , and to explore the possible origins of this remaining discrepancy , we performed additional dft calculations with several different functionals . ( b ) pyridine , and ( c ) pyrimidine obtained from different theoretical schemes : a semilocal functional ( pbe ) , a conventional hybrid functional ( pbe0 ) in both the kohn sham ( ks ) and generalized kohn sham ( gks ) scheme , a self - interaction - corrected ( sic ) calculation , with and without additional stretching of the energy axis , gw calculations based on a pbe starting point , and optimally tuned range - separated hybrid ( ot - rsh ) calculations with two different short - range exchange parameters , = 0 and = 0.2 . while the amount of shift needed for alignment of the homo with experimental results is essentially the same for pbe0 and pbe0 , the pbe0 eigenvalues are overall stretched (= energy rescaled ) with respect to the pbe0 data as seen most clearly for the case of benzene . they suggested that this first - order correction mimics successfully the first - order correction between ks values and ionization energies , which is known to dominate for outer - valence electrons . for a few of the pyridine and pyrimidine orbitals , we find some remaining deviations of the stretched sic from the pbe0 and ot - rsh results , which is , however , expected as the three approaches are very different . encouraged by the overall success of the ot - rsh approach for the prototypical aromatic rings , we now turn to examining the performance of the ot - rsh method for more complex organic molecules : terpyrimidinethiol ( 3n - thiol ) and copper phthalocyanine ( cupc)see figure 1b . 3n - thiols are also interesting from a methodological perspective , as they pose a serious challenge to semilocal ks schemes : by means of sic calculations analogous to the ones performed here , it was shown that close - lying and orbitals in the outer - valence region of 3n - thiol have markedly different sies and , as a consequence , are wrongly ordered in ( semi)local dft functionals such as pbe . ( b ) unshifted eigenvalue spectra of the same system , as calculated from hse , pbe0 , ot - rsh calculations with the two different values , and gw . in fact , in the present case , the errors do not exceed 0.2 ev , which is within the expected level of agreement between the two methods ( vide supra ) . despite the excellent agreement of the shifted conventional hybrid ( and stretched sic ) data with the gw results , this can be gleaned even from figure 6a , where the homo eigenvalue obtained from each method is given at the top of each column , but is made obvious by figure 6b , which shows unshifted spectra on an absolute energy scale . as with the simpler systems , the ips deduced from our computed ot - rsh and gw eigenvalues are very close in energy ( 0.2 ev ) for both choices of . in contrast to the case of the small aromatic rings , where different choices for have had no qualitative effect and only a small quantitative effect , for the 3n - thiol system the differences between the ot - rsh spectra for the two choices of are more pronounced . in particular , we observe that , while the position of the orbitals is similar in both spectra , the orbitals are systematically underbound in the = 0 calculation , in comparison to the = 0.15 or the gw calculations . the finding that the agreement with our gw calculations is significantly improved for = 0.15 underscores that , as suggested in ref ( 58 ) and discussed in the introduction , a fraction of fock exchange in the sr together with full fock exchange in the lr allows for a mitigation of sie and therefore a balanced treatment of differently localized molecular orbitals , while retaining the correct absolute position of the energy levels . eigenvalues of 3n - thiol , obtained from optimally tuned range - separated hybrid ( ot - rsh ) calculations , as a function of the short - range exchange fraction , , with the optimal value of the range - separation parameter , ( in bohr ) determined for each choice of . as discussed in ref ( 58 ) , some of the present authors successfully employed a similar approach to determine an optimal value of also for the purpose of obtaining an outer - valence eigenvalue spectra , but only if it also involved the removal of a second electron . the easiest way to achieve that is to tune such that the energy difference between the homo and homo1 states is the same in the ot - rsh and the pbe0 calculations . overall , the mad between the gw and ot - rsh ( = 0.15 ) calculations is a satisfying 0.1 ev , with the largest deviation being 0.2 ev for the homo . for comparison , with ot - rsh ( = 0 ) the mad is 0.25 ev , with the largest deviation being 0.45 ev , where the reduced level of agreement is mostly due to the less accurate description of the orbitals . in the present context , cupc mainly serves as a test case that poses several additional challenges for the ot - rsh method : first , it is an open shell molecule ( s = 1/2 ) . configuration 1 ( left side of figure 8a ) was obtained from the ot - rsh calculations discussed below but is equivalent to the one obtained from the gw calculations of ref ( 69 ) . ( b ) ot - rsh charge - density differences between neutral and cation ( left ) and lumo of cation ( right ) , obtained from the open - shell singlet configuration of the cupc cation , corresponding to ionization of netrual configuration 1 ( in green ) , and from the closed - shell singlet configuration of the cupc cation , corresponding to ionization of configuration 2 ( in red ) . it is in significantly better agreement with experimental results than their gw calculations based on a pbe starting point ( at least with the particular flavor of gw used in ref ( 69 ) , which , as discussed earlier , is different from the one we used above for benzene , pyridine , and pyrimidine ; we refrained from conducting such gw calculations also for cupc , because of the computational cost involved ) . it is particulary striking that the orbitals most sensitive to are the highly localized spin - split b1 g pair , which is fully consistent with our above discussion on the relation between orbital localization and sensitivity to short - range fock exchange . in particular , the ot - rsh alignment of the a1u , b1 g , and eg states of figure 8 agrees very well with the prior gw calculations . in particular , it appears that agreement of the lower valence states with the gw results could be further assisted by some stretching of the energy scale an observation consistent with the generally expected differences between nonlocal exchange and self - energy operators , as well as with our general guideline of restricting our attention to states within a few electronvolts of the frontier orbitals . nevertheless , in the absence of detailed orbital information , at the level of the given experimental resolution , the ot - rsh results can be viewed as agreeing with experimental results as well as the gw ones , possibly even relatively deep into the valence band . eigenvalues of cupc , obtained from optimally tuned range - separated hybrid ( ot - rsh ) calculations , as a function of the short - range exchange fraction , , with the optimal value of the range - separation parameter , ( in bohr ) , determined for different choices of . as an additional observation , we note that the homo and lumo eigenvalues obtained from the ot - rsh ( = 0.17 ) calculation ( as is , without any rigid shifting of the data ) are in excellent agreement with the lowest quasi - hole and quasi - electron excitations , respectively , obtained from gw@pbe0 . in conclusion , we examined the performance of optimally tuned range - separated hybrid functionals for predicting the photoemission spectra of several challenging prototypical ( benzene , pyridine , and pyrimidine ) and complex ( 3n - thiol and cupc ) ( hetero)cyclic organic molecules . overall , the resulting spectra agree very well ( typically within 0.10.2 ev ) with experimental data and our gw calculations , even for states several electronvolts away from the frontier orbitals . moreover , our sic calculations confirmed that self - interaction errors can be efficiently mitigated in ot - rsh functionals , which shows that , with a pbe0-based optimal choice of the short - range fraction of fock exchange , the ot - rsh method can offer an excellent balance in the description of localized and delocalized states . the sole exception found in the studied systems is a high - symmetry orbital , particular to small aromatic rings . we conclude that the ot - rsh method is a highly accurate dft method for outer - valence pes prediction for such systems , and its accuracy is comparable to state - of - the - art gw schemes .

intestinal inflammatory diseases are a serious problem in human as well as veterinary medicine . the etiology of these diseases is often multifactorial and the underlying molecular mechanisms are poorly understood . the current medicinal therapies for inflammatory gut diseases involve treatment with nonsteroidal anti - inflammatory drugs , antibiotics , corticosteroids , and immunosuppressant , but the application of these drugs is limited due to their toxicity and side effects . therefore , there is an increased interest in finding an alternative treatment with fewer side effects . there is evidence supporting the therapeutic usefulness of oral administration of various plant extracts in inflammatory diseases of the gut . experimental data obtained in mouse and rat models of colitis suggest that the beneficial effects of the plant extracts could be mediated by their effects on mucosal cytokines production or / and action [ 39 ] . increased levels of proinflammatory cytokines [ il-1 , il-6 , il-8 , tnf , il-12 , and ifn ] were found in inflamed intestinal mucosa in various animal models and humans [ 1013 ] , as well as in farm animals [ 14 , 15 ] . thyme ( thymus vulgaris l. ) and oregano ( origanum vulgare l. ) are aromatic plants of the mediterranean flora commonly used as spices and for medicinal purposes . like other various thymus species , furthermore , thyme possesses antimicrobial , antifungal , antioxidative , and antiviral properties [ 1619 ] . the essential oil derived from thyme ( t. vulgaris l. ) is a mixture of monoterpenes and one of the main compounds of this oil is a natural terpenoid thymol . thymol exhibits multiple biological activities including anti - inflammatory , immunomodulating , antioxidant , antibacterial [ 24 , 25 ] , antifungal , and free radical scavenging properties . oregano is recognized for its potential therapeutic role because of its diaphoretic , carminative , antispasmodic , antiseptic , and tonic properties . oregano , ( origanum syriacum l. ) similar to thyme , evinces antioxidant and antimicrobial activities and some reports deal with its antimutagenic and anticarcinogenic effects . origanum essential oil is obtained by steam distillation of o. vulgare and its major compounds are carvacrol and thymol . origanum essential oil is known to possess antimicrobial , antifungal , and antioxidant activities [ 31 , 32 ] . the aim of our study was to examine possible beneficial effects of thyme and oregano essential oils on intestinal inflammation . the results of our preliminary experiment suggested that the administration of thyme oil in combination with oregano oil could be more effective in improvement of trinitrobenzene sulphonic acid ( tnbs)-induced colitis than the separate administration of these essential oils . in the present study , we evaluated further the effect of administration of three different doses of thyme and oregano oil combination on tnbs - induced colitis in mice . male 7-week - old balb / c mice weighing 1825 g were purchased from velaz ( prague , czech republic ) . the animals were maintained under standard conditions of temperature ( 211c ) , relative humidity ( 5510% ) , and 12 hours/12 hours light / dark cycle . all animal experimentations were reviewed and approved by the ethical committee of the institute of animal physiology . after a period of adaptation , weight - matched animals were randomized into five groups : group a ( 20.20.971 g ) , group b ( 20.60.75 g ) , and group c ( 20.710.844 g ) , mice with tnbs - induced colitis treated with three different doses ( see table 1 ) of thyme and oregano oil combination ; group d ( 21.031.025 g ) , mice with tnbs - induced colitis ; group e ( 18.741.189 g ) , sham - treated mice . 4 ) and oregano aromatic oil ( origani aetheroleum ) were purchased from calendula , ( nov l'ubova , slovakia ; thyme aromatic oil : lot 5 - 015 - 003 - 10 - 04 ; oregano aromatic oil : lot 5 - 027 - 007 - 10 - 04 ) . p - cymene and 24% of thymol , and the oregano aromatic oil contained about 55% of carvacrol . 2,4,6-trinitrobenzene sulphonic acid ( tnbs ) was purchased from fluka chemie ( buchs , switzerland ) . the thyme oil and oregano oil were mixed with the diet at concentrations as shown in table 1 . both aromatic oils were suspended in edible soya oil ( brlio , hamm , germany ) and added to powdery commercial rodent diet ( diet for laboratory mice and rats spf , m1 ; frantisek machal , ricmanice , czech republic ) . in the tnbs and sham groups , edible soya oil was mixed with the powdery rodent diet at a concentration of 1% ( wt / wt ) . diets were fed ad libitum throughout the experiment , starting 6 days before administration of tnbs . the mice were anesthetized with ketamine and xylazine , and colitis was induced by intrarectal administration of 120 mg / kg of the hapten reagent tnbs ( fluka chemie ) in 50% ethanol , and they were then kept in a vertical position for 30 seconds . the total injection volume was 30 l . development of colitis was assessed daily by measurement of body weight . the colons were removed , cut longitudinally , and cleared of fecal material with gentle spray of 0.9% saline solution . the extent of mucosal damage was assessed using the colon macroscopic scoring system adapted from wallace et al . . ulceration : ( 1 ) focal hyperemia , no ulcer ; ( 2 ) ulceration , no hyperemia / bowel wall thickening ; ( 3 ) ulceration , inflammation at one site ; ( 4 ) ulceration , inflammation at 2 or more sites ; ( 5 ) major injury > 1 cm ; 610 major damage > 2 cm . adhesion : ( 1 ) minor ( colon easily separated from other tissue ) ; ( 2 ) major . colon tissues were fixed in 4% formalin in 0.1 m phosphate buffer , dehydrated with increasing concentrations of ethanol , embedded in paraffin , and sectioned . sections ( 46 m thick ) were mounted on slides , cleared , hydrated , and stained with hematoxylin and eosin . the slides were examined and photographed with an olympus bx51 microscope ( olympus , japan ) . strips of colonic tissue ( 1530 mg from segments most intensively affected by the inflammation ) were cut out , immersed in liquid nitrogen , and kept at 70c until the cytokine measurement . total rna was isolated from the mouse colon ( about 15 mg of tissue for each sample ) with trizol reagent ( invitrogen life technologies , karlsruhe , germany ) according to the manufacturer 's instructions . total rna preparations were then cleaned and dnase i was treated with rneasy micro kit ( qiagen , hilden , germany ) according to the manufacturer 's protocol . in order to quantify total rna extracted from each sample , optical density at 260 nm was measured . the rna ( 0.75 g from each sample ) was reverse transcribed at 42c for 1 hour in 30 l containing 300 units of superscript ii rnase hreverse transcriptase ( invitrogen life technologies ) , 7 m anchored oligo dt13vn , 50 mm tris - hcl ph 8.3 , 3 mm mgcl2 , 75 mm kcl , 10 mm dtt , 500 m dntps ( datp , dttp , dctp , dgtp ) , 60 units rnase out ( recombinant ribonuclease inhibitor , invitrogen life technologies ) , and 0.75 g acetylated bsa . the reaction was terminated by heating at 95c for 5 minutes . to check for the presence of genomic dna contamination in the rna preparations , reverse transcriptase negative control ( no reverse transcriptase in the reaction ) was carried out in parallel , using rna pool prepared from aliquots of all rna samples . the pool of colon rna obtained from aliquots of all samples served as standard rna . the relative standard curve was generated using mx 3000p 2.0 software ( stratagene , la jolla , calif ) . pcr reactions were carried out in a 20 l final volume in duplicates using sybrgreen i as a fluorescent detection dye . the reactions contained 0.8 l of cdna ( corresponding to 20 ng of sample total rna ) , one unit of platinum taq dna polymerase ( invitrogen life technologies ) , sybrgreen i in final dilution of 1 : 25000 ( sigma - aldrich , munich , germany ) , 30 nm rox ( passive reference dye for correction of non - pcr - related fluctuations in fluorescence signal , stratagene ) , 0.2 mm dntps ( datp , dttp , dctp , dgtp ) , 50 mm kcl , 10 mm tris - hcl ph 8.3 , 2.5 mm mgcl2(except for the -actin reaction , where 1.5 mm mgcl2 was used ) , forward and reverse primers in final concentration of 0.25 m ( for il-1 , il-12b , tnf , ifn- , hprt , and sdha ) , or 0.5 m ( for il-6 , il-10 , gm - csf , and -actin ; see table 2 for full gene names ) . oligonucleotide primers used in the experiment were designed in our previous work , and their sequences are shown in table 2 . pcr amplification was performed in the real - time pcr system mx 3000p ( stratagene ) . after an initial step at 95c for 2 minutes ( dna denaturation and hot - start dna polymerase activation ) , 40 cycles with the following thermocycling conditions were carried out : 94c for 30 seconds , specific annealing temperature for 30 seconds , 72c for 30 seconds , and specific temperature at which the fluorescence was acquired ( acquiring temperature ) for 30 seconds . measurement of fluorescence at an elevated temperature ( acquiring temperature , a few degrees of celsius below the melting temperature of the specific pcr product ) enables elimination of the fluorescence signal produced by incidental short nonspecific pcr products . amplification specificity was then checked by generation of a melting curve using 41 cycles with temperature increments of 1c ( starting with 55c ) and a fluorescence measurement in each cycle . specific annealing and acquiring temperatures are shown in table 2 . to ensure the correctness of the quantification firstly , expression stability of several housekeeping genes was tested using genorm software . subsequently , the normalization factor for each sample was calculated ( by genorm software ) as the geometric means of the relative amounts of the three most stable housekeeping genes hprt , sdha , and -actin ( see table 2 for full gene names ) . finally , the relative amount of cytokine mrna in each sample was divided by the normalization factor of the sample . colon tissue samples were homogenized in ice - cold pbs containing protease inhibitor cocktail for use with mammalian cell and tissue extracts ( p8340 , sigma - aldrich ) , and the homogenates were then centrifuged at 12000 xg at 4c for 15 minutes . total protein amounts in the tissue supernatants were determined using bradford protein assay with bsa employed as the standard . il-1 and il-6 amounts were determined using an elisa kit , according to the manufacturer 's recommendation ( pierce - endogen , rockford , ill , usa ) . the kruskal - wallis test and the mann - whitney u the chi - square test was used to assess differences in mortality rate . the student t test the kruskal - wallis test was used for the comparison of differences in cytokine expression between groups and the mann - whitney u test was used to compare differences between the group of untreated colitic animals and other groups of animals . as shown in figure 1 , administration of tnbs caused a dramatic decrease in body weight ( almost 20% after 3 days ) ; body weight was recovered gradually from day 4 but not fully to the initial weight in day 7 . mice receiving 50% ethanol without tnbs ( control sham group ) showed only slight and transient loss of body weight . in mice of group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) , body weight was recovered gradually from day 3 when it became higher than the body weight of untreated colitic animals ( group d ) ; on day 7 , the body weight of animals in group b reached a level near to that of the control mice ( sham group ) . the body weight of mice in group a ( colitic animals treated with 0.4% thyme and 0.2% oregano oils ) and group c ( colitic animals treated with 0.1% thyme and 0.05% oregano oils ) did not differ significantly from that of untreated colitic animals ( group d ) . the mortality rate of mice with tnbs - induced colitis ( group d ) was 53.3% , while that of the control sham group was 0% ( see figure 2 ) . the mortality rate in group a ( colitic animals treated with 0.4% thyme and 0.2% oregano oils ) was 50% and in group c ( colitic animals treated with 0.1% thyme and 0.05% oregano oils ) was 62.5% , which are comparable to that found in group d. treatment with the combination of 0.2% thyme and 0.1% oregano oils ( group b ) decreased the mortality rate to 33.3% ( which was still not significantly different from that of mice with tnbs - induced colitis ) . macroscopic damage scores of mice in group d were significantly higher than those of mice in the control sham group . treatment with the combination of 0.2% thyme and 0.1% oregano oils ( group b ) significantly lowered the macroscopic damage scores in comparison to untreated colitic animals ( group d ) . animals in groups a and c showed no significant changes in macroscopic damage scores compared with mice in group d ( see figure 3 ) . representative samples of colon after hematoxylin and eosin stainings are shown in figure 4 . there is no evident histological modification in sham mice ( e ) . in mice with tnbs - induced colitis ( d ) , there is a wide range of histopathological changes including necrosis of epithelium , destruction of glands , and infiltration of inflammatory cells in the mucosa and submucosa ( up to 50% of colon section ) . the samples from mice treated with 0.2% thyme and 0.1% oregano oils ( b ) show intermediary histopathological changes ( up to 30% of colon section ) . relative amounts of il-1 , il-6 , gm - csf , and tnf mrnas ( for full gene names , see table 2 ) were significantly higher in animals with tnbs - induced colitis ( group d ) than in the control sham - treated animals ( see figure 5 ) . treatment of the colitic animals with the combination of thyme and oregano oils significantly lowered the amount of il-1 mrna , using all three tested doses of the aromatic oils ( see figure 5 ) . the amount of il-6 mrna in group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) was significantly lower than that in the group d , whereas the decrease of il-6 mrna level in groups a and c ( colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils ) was not statistically significant ( see figure 5 ) . a similar effect was found for gm - csf and tnf but the difference between group b and group d did not reach statistical significance ( p=.064 and p=.053 , resp . ; see figure 5 ) . we found no significant changes in mrna levels of two other cytokines ( il-10 and ifn , data were not shown ) . as shown in figure 6 , the amounts of il-1 and il-6 proteins were significantly higher in the animals with tnbs - induced colitis ( group d ) than in the control sham group . the concentrations of these proteins were significantly reduced in group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) compared to group d. the levels of il-1 and il-6 proteins in groups a and c ( colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils ) did not differ significantly from those found in group d ( see figure 6 ) . various aromatic plants and their products have been reported to have health benefit properties . in this study , we examined whether dietary supplementation with a combination of thyme essential oil and oregano essential oil could have a protective effect in intestinal inflammation . we applied three doses of thyme and oregano essential oil combination ( 0.4% thyme and 0.2% oregano oils , 0.2% thyme and 0.1% oregano oils , 0.1% thyme and 0.05% oregano oils ) to mice with tnbs - induced colitis , and found that administration of the medium dose decreased the mortality rate from 53% to 33% , significantly accelerated the body weight gain recovery , and significantly reduced the macroscopic damage of the colonic tissue . in animals fed with the two other doses of the essential oils , we did not find a significant improvement in the mortality rate , body weight gain recovery , or colonic tissue damage . examining the expression of cytokines , we found significantly elevated mrna levels of proinflammatory cytokines il-1 , il-6 , gm - csf , and tnf in mice with tnbs - induced colitis . this finding is in agreement with other studies showing increased expression of proinflammatory cytokines in mouse experimental models of colitis [ 47 ] . treatment of the colitic animals with the medium dose of aromatic oils ( 0.2% thyme and 0.1% oregano oils ) decreased the mrna amounts of il-1 , il-6 , gm - csf , and tnf ( in gm - csf and tnf , the difference did not reach statistical significance ) . other two doses ( 0.4% thyme and 0.2% oregano oils ; 0.1% thyme and 0.05% oregano oils ) were less effective and the difference in cytokine mrna level between animals treated with these doses of the aromatic oils and untreated colitic animals was significant only in the case of il-1. the most sensitive indicators of the effectiveness of the thyme and oregano oil treatment were mrna levels of proinflammatory cytokines il-1 and il-6 in our experiment . similarly , kwon et al . demonstrated that plant flavonoid rutin administered in diet to mice with dextran sulfate sodium ( dss)-induced colitis significantly suppressed mrna levels of il-1 and il-6 , whereas the effect on mrna level of gm - csf was less marked . increase of il-1 and il-6 mrna levels in inflamed intestinal tissue has been well documented [ 1214 ] , and there are data indicating that il-1 can stimulate production of il-6 in intestinal epithelial cells [ 37 , 38 ] . reported data about the expression of tnf are somewhat contradictory , showing either no change or an increase of tnf levels in intestinal inflammation . sugimoto et al . showed that elevated levels of tnf mrna in mice with tnbs - induced colitis can be suppressed by curcumin treatment , but kwon et al . found no significant changes in tnf mrna levels in mice with dss - induced colitis ( treated or untreated with plant flavonoid rutin ) . examination of il-1 and il-6 protein expression in our experiment confirmed the best efficacy of the medium dose of thyme and oregano oil combination in the suppression of colitis . the cytokine protein level in animals treated with the medium dose , but not in animals treated with the other two doses , was significantly lower than in the untreated colitic animals . the low effectiveness in attenuating the colitis found after administration of the highest dose of thyme and oregano oil combination could be connected with possible cytotoxic effects of higher concentrations of these oils . in our preliminary experiments with intact mice , we found negative effects of higher concentrations of thyme essential oil ( 1% ) and oregano essential oil ( 0.5% ) on body weight and food intake . moreover , the cytotoxic effect of higher concentrations of these oils has been demonstrated in intestinal cells and lymphocytes [ 39 , 40 ] . thus , the lack of the dose dependency found in this study could be due to a combination of positive and negative effects of the highest concentration of thyme and oregano essential oil combination . on the other hand , too low doses of essential oils can be insufficient to reduce the intensity of inflammation . a similar phenomenon was found in the study examining the protective effects of curcumin on tnbs - induced colitis in mice , where the medium tested dose was more effective at improving body weight gain than the lower or higher doses of curcumin . to explain the lack of the dose dependency , authors speculated about curcumin toxicity or about the influence on the food intake . thus , the use of optimal doses is essential for good efficacy of essential oils ( or their components ) in attenuating inflammation . further experiments are needed to establish the most efficient concentration of thyme and oregano essential oil combination . in our preliminary experiments ( using similar experimental conditions as in the present study ) , we administered thyme ( 0.5% , 0.25% , 0.12% ) or oregano ( 0.4 , 0.2% , 0.1% ) essential oil alone in diet to mice with tnbs - induced colitis and we found no significant positive effect . furthermore , we observed negative effects on body weight and food intake with oregano at 0.4% concentration . however , we observed a better recovery of body weight after treatment with a combination of these essential oils in preliminary tests . the results of the present study confirm the positive effects of the combined administration of appropriate concentrations of thyme and oregano oils on tnbs - induced colitis . it is known that oregano essential oil possesses strong antimicrobial activity , which is ascribed to carvacrol , the main component of this oil . thymol , one of the major compounds of thyme essential oil , has anti - inflammatory activity . thus , it seems that major components of these oils can complement one another , having synergic effects on inflammation so that their combination could exhibit positive preventive or therapeutic effects . a similar phenomenon was found in another study where the effect of peppermint oil and caraway oil on postinflammatory visceral hyperalgesia was examined ( using a rat model of tnbs - induced colitis ) . neither peppermint nor caraway oil administered individually had a significant effect on postinflammatory visceral hyperalgesia , but combined treatment with these essential oils significantly reduced the visceromotor response . mechanisms mediating the suppressive effects of thyme and oregano oils on colitis are unclear , and we can only speculate that there are several potential manners of action . one possibility could be the influence of the essential oils on nuclear factor b ( nf-b ) , a pleiotropic transcription factor which can activate expression of genes involved in immune and inflammation responses such as proinflammatory cytokines . several studies have demonstrated an inhibitory effect of various plant extracts or their components on nf-b activation . suppression of the nf-b inhibitory protein ( ib ) degradation in colonic epithelial cells and macrophages was demonstrated after the administration of curcumin and zerumbone [ 4 , 44 , 45 ] . reduced activation of nf-b was also shown after treatment with black tea polyphenol theaflavin in raw 264.7 cells . on the other hand , il-1 ( such as other proinflammatory cytokines ) is a potent inducer of nf-b and it has been shown that extract of thymus pulegioides can inhibit activation of nf-b by il-1 in human hepatoma cells . in conclusion , the present data indicate that dietary administration of a combination of thyme and oregano essential oils in appropriate concentrations can reduce the production of proinflammatory cytokines and attenuate the degree of colonic tissue injury , and thereby ameliorate tnbs - induced colitis in mice . we suggest that the combination of thyme and oregano essential oils has potential value as an additional or supporting treatment in gastrointestinal inflammations . our results indicate that some essential oils could have positive effects on tnbs - induced colitis but seemingly in rather narrow range of concentrations , thus limiting their therapeutic / preventive potential .

we examined the anti - inflammatory effects of the combination of thyme and oregano essential oil dietary administered at three concentrations ( 0.4% thyme and 0.2% oregano oils ; 0.2% thyme and 0.1% oregano oils ; 0.1% thyme and 0.05% oregano oils ) on mice with tnbs - induced colitis . treatment of colitic animals with the essential oils decreased the mrna levels of pro - inflammatory cytokines il-1 , il-6 , gm - csf , and tnf , especially after application of the medium dose . the medium dose of the essential oils significantly lowered the amount of il-1 and il-6 proteins too . moreover , administration of the medium dose decreased the mortality rate , accelerated the body weight gain recovery , and reduced the macroscopic damage of the colonic tissue . our results indicate that combined treatment with appropriate concentrations of thyme and oregano essential oils can reduce the production of proinflammatory cytokines , and thereby attenuate tnbs - induced colitis in mice .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION

the current medicinal therapies for inflammatory gut diseases involve treatment with nonsteroidal anti - inflammatory drugs , antibiotics , corticosteroids , and immunosuppressant , but the application of these drugs is limited due to their toxicity and side effects . there is evidence supporting the therapeutic usefulness of oral administration of various plant extracts in inflammatory diseases of the gut . experimental data obtained in mouse and rat models of colitis suggest that the beneficial effects of the plant extracts could be mediated by their effects on mucosal cytokines production or / and action [ 39 ] . increased levels of proinflammatory cytokines [ il-1 , il-6 , il-8 , tnf , il-12 , and ifn ] were found in inflamed intestinal mucosa in various animal models and humans [ 1013 ] , as well as in farm animals [ 14 , 15 ] . the essential oil derived from thyme ( t. vulgaris l. ) is a mixture of monoterpenes and one of the main compounds of this oil is a natural terpenoid thymol . thymol exhibits multiple biological activities including anti - inflammatory , immunomodulating , antioxidant , antibacterial [ 24 , 25 ] , antifungal , and free radical scavenging properties . origanum essential oil is known to possess antimicrobial , antifungal , and antioxidant activities [ 31 , 32 ] . the aim of our study was to examine possible beneficial effects of thyme and oregano essential oils on intestinal inflammation . the results of our preliminary experiment suggested that the administration of thyme oil in combination with oregano oil could be more effective in improvement of trinitrobenzene sulphonic acid ( tnbs)-induced colitis than the separate administration of these essential oils . in the present study , we evaluated further the effect of administration of three different doses of thyme and oregano oil combination on tnbs - induced colitis in mice . after a period of adaptation , weight - matched animals were randomized into five groups : group a ( 20.20.971 g ) , group b ( 20.60.75 g ) , and group c ( 20.710.844 g ) , mice with tnbs - induced colitis treated with three different doses ( see table 1 ) of thyme and oregano oil combination ; group d ( 21.031.025 g ) , mice with tnbs - induced colitis ; group e ( 18.741.189 g ) , sham - treated mice . the thyme oil and oregano oil were mixed with the diet at concentrations as shown in table 1 . the mice were anesthetized with ketamine and xylazine , and colitis was induced by intrarectal administration of 120 mg / kg of the hapten reagent tnbs ( fluka chemie ) in 50% ethanol , and they were then kept in a vertical position for 30 seconds . colon tissues were fixed in 4% formalin in 0.1 m phosphate buffer , dehydrated with increasing concentrations of ethanol , embedded in paraffin , and sectioned . strips of colonic tissue ( 1530 mg from segments most intensively affected by the inflammation ) were cut out , immersed in liquid nitrogen , and kept at 70c until the cytokine measurement . the rna ( 0.75 g from each sample ) was reverse transcribed at 42c for 1 hour in 30 l containing 300 units of superscript ii rnase hreverse transcriptase ( invitrogen life technologies ) , 7 m anchored oligo dt13vn , 50 mm tris - hcl ph 8.3 , 3 mm mgcl2 , 75 mm kcl , 10 mm dtt , 500 m dntps ( datp , dttp , dctp , dgtp ) , 60 units rnase out ( recombinant ribonuclease inhibitor , invitrogen life technologies ) , and 0.75 g acetylated bsa . the reactions contained 0.8 l of cdna ( corresponding to 20 ng of sample total rna ) , one unit of platinum taq dna polymerase ( invitrogen life technologies ) , sybrgreen i in final dilution of 1 : 25000 ( sigma - aldrich , munich , germany ) , 30 nm rox ( passive reference dye for correction of non - pcr - related fluctuations in fluorescence signal , stratagene ) , 0.2 mm dntps ( datp , dttp , dctp , dgtp ) , 50 mm kcl , 10 mm tris - hcl ph 8.3 , 2.5 mm mgcl2(except for the -actin reaction , where 1.5 mm mgcl2 was used ) , forward and reverse primers in final concentration of 0.25 m ( for il-1 , il-12b , tnf , ifn- , hprt , and sdha ) , or 0.5 m ( for il-6 , il-10 , gm - csf , and -actin ; see table 2 for full gene names ) . after an initial step at 95c for 2 minutes ( dna denaturation and hot - start dna polymerase activation ) , 40 cycles with the following thermocycling conditions were carried out : 94c for 30 seconds , specific annealing temperature for 30 seconds , 72c for 30 seconds , and specific temperature at which the fluorescence was acquired ( acquiring temperature ) for 30 seconds . subsequently , the normalization factor for each sample was calculated ( by genorm software ) as the geometric means of the relative amounts of the three most stable housekeeping genes hprt , sdha , and -actin ( see table 2 for full gene names ) . finally , the relative amount of cytokine mrna in each sample was divided by the normalization factor of the sample . il-1 and il-6 amounts were determined using an elisa kit , according to the manufacturer 's recommendation ( pierce - endogen , rockford , ill , usa ) . as shown in figure 1 , administration of tnbs caused a dramatic decrease in body weight ( almost 20% after 3 days ) ; body weight was recovered gradually from day 4 but not fully to the initial weight in day 7 . in mice of group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) , body weight was recovered gradually from day 3 when it became higher than the body weight of untreated colitic animals ( group d ) ; on day 7 , the body weight of animals in group b reached a level near to that of the control mice ( sham group ) . the body weight of mice in group a ( colitic animals treated with 0.4% thyme and 0.2% oregano oils ) and group c ( colitic animals treated with 0.1% thyme and 0.05% oregano oils ) did not differ significantly from that of untreated colitic animals ( group d ) . the mortality rate of mice with tnbs - induced colitis ( group d ) was 53.3% , while that of the control sham group was 0% ( see figure 2 ) . the mortality rate in group a ( colitic animals treated with 0.4% thyme and 0.2% oregano oils ) was 50% and in group c ( colitic animals treated with 0.1% thyme and 0.05% oregano oils ) was 62.5% , which are comparable to that found in group d. treatment with the combination of 0.2% thyme and 0.1% oregano oils ( group b ) decreased the mortality rate to 33.3% ( which was still not significantly different from that of mice with tnbs - induced colitis ) . treatment with the combination of 0.2% thyme and 0.1% oregano oils ( group b ) significantly lowered the macroscopic damage scores in comparison to untreated colitic animals ( group d ) . in mice with tnbs - induced colitis ( d ) , there is a wide range of histopathological changes including necrosis of epithelium , destruction of glands , and infiltration of inflammatory cells in the mucosa and submucosa ( up to 50% of colon section ) . the samples from mice treated with 0.2% thyme and 0.1% oregano oils ( b ) show intermediary histopathological changes ( up to 30% of colon section ) . relative amounts of il-1 , il-6 , gm - csf , and tnf mrnas ( for full gene names , see table 2 ) were significantly higher in animals with tnbs - induced colitis ( group d ) than in the control sham - treated animals ( see figure 5 ) . treatment of the colitic animals with the combination of thyme and oregano oils significantly lowered the amount of il-1 mrna , using all three tested doses of the aromatic oils ( see figure 5 ) . the amount of il-6 mrna in group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) was significantly lower than that in the group d , whereas the decrease of il-6 mrna level in groups a and c ( colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils ) was not statistically significant ( see figure 5 ) . a similar effect was found for gm - csf and tnf but the difference between group b and group d did not reach statistical significance ( p=.064 and p=.053 , resp . we found no significant changes in mrna levels of two other cytokines ( il-10 and ifn , data were not shown ) . as shown in figure 6 , the amounts of il-1 and il-6 proteins were significantly higher in the animals with tnbs - induced colitis ( group d ) than in the control sham group . the concentrations of these proteins were significantly reduced in group b ( colitic animals treated with 0.2% thyme and 0.1% oregano oils ) compared to group d. the levels of il-1 and il-6 proteins in groups a and c ( colitic animals treated with 0.4% thyme and 0.2% oregano oils or with 0.1% thyme and 0.05% oregano oils ) did not differ significantly from those found in group d ( see figure 6 ) . in this study , we examined whether dietary supplementation with a combination of thyme essential oil and oregano essential oil could have a protective effect in intestinal inflammation . we applied three doses of thyme and oregano essential oil combination ( 0.4% thyme and 0.2% oregano oils , 0.2% thyme and 0.1% oregano oils , 0.1% thyme and 0.05% oregano oils ) to mice with tnbs - induced colitis , and found that administration of the medium dose decreased the mortality rate from 53% to 33% , significantly accelerated the body weight gain recovery , and significantly reduced the macroscopic damage of the colonic tissue . in animals fed with the two other doses of the essential oils , we did not find a significant improvement in the mortality rate , body weight gain recovery , or colonic tissue damage . examining the expression of cytokines , we found significantly elevated mrna levels of proinflammatory cytokines il-1 , il-6 , gm - csf , and tnf in mice with tnbs - induced colitis . this finding is in agreement with other studies showing increased expression of proinflammatory cytokines in mouse experimental models of colitis [ 47 ] . treatment of the colitic animals with the medium dose of aromatic oils ( 0.2% thyme and 0.1% oregano oils ) decreased the mrna amounts of il-1 , il-6 , gm - csf , and tnf ( in gm - csf and tnf , the difference did not reach statistical significance ) . other two doses ( 0.4% thyme and 0.2% oregano oils ; 0.1% thyme and 0.05% oregano oils ) were less effective and the difference in cytokine mrna level between animals treated with these doses of the aromatic oils and untreated colitic animals was significant only in the case of il-1. the most sensitive indicators of the effectiveness of the thyme and oregano oil treatment were mrna levels of proinflammatory cytokines il-1 and il-6 in our experiment . demonstrated that plant flavonoid rutin administered in diet to mice with dextran sulfate sodium ( dss)-induced colitis significantly suppressed mrna levels of il-1 and il-6 , whereas the effect on mrna level of gm - csf was less marked . increase of il-1 and il-6 mrna levels in inflamed intestinal tissue has been well documented [ 1214 ] , and there are data indicating that il-1 can stimulate production of il-6 in intestinal epithelial cells [ 37 , 38 ] . showed that elevated levels of tnf mrna in mice with tnbs - induced colitis can be suppressed by curcumin treatment , but kwon et al . found no significant changes in tnf mrna levels in mice with dss - induced colitis ( treated or untreated with plant flavonoid rutin ) . examination of il-1 and il-6 protein expression in our experiment confirmed the best efficacy of the medium dose of thyme and oregano oil combination in the suppression of colitis . the cytokine protein level in animals treated with the medium dose , but not in animals treated with the other two doses , was significantly lower than in the untreated colitic animals . the low effectiveness in attenuating the colitis found after administration of the highest dose of thyme and oregano oil combination could be connected with possible cytotoxic effects of higher concentrations of these oils . in our preliminary experiments with intact mice , we found negative effects of higher concentrations of thyme essential oil ( 1% ) and oregano essential oil ( 0.5% ) on body weight and food intake . moreover , the cytotoxic effect of higher concentrations of these oils has been demonstrated in intestinal cells and lymphocytes [ 39 , 40 ] . thus , the lack of the dose dependency found in this study could be due to a combination of positive and negative effects of the highest concentration of thyme and oregano essential oil combination . on the other hand , too low doses of essential oils can be insufficient to reduce the intensity of inflammation . a similar phenomenon was found in the study examining the protective effects of curcumin on tnbs - induced colitis in mice , where the medium tested dose was more effective at improving body weight gain than the lower or higher doses of curcumin . further experiments are needed to establish the most efficient concentration of thyme and oregano essential oil combination . in our preliminary experiments ( using similar experimental conditions as in the present study ) , we administered thyme ( 0.5% , 0.25% , 0.12% ) or oregano ( 0.4 , 0.2% , 0.1% ) essential oil alone in diet to mice with tnbs - induced colitis and we found no significant positive effect . furthermore , we observed negative effects on body weight and food intake with oregano at 0.4% concentration . however , we observed a better recovery of body weight after treatment with a combination of these essential oils in preliminary tests . the results of the present study confirm the positive effects of the combined administration of appropriate concentrations of thyme and oregano oils on tnbs - induced colitis . thymol , one of the major compounds of thyme essential oil , has anti - inflammatory activity . a similar phenomenon was found in another study where the effect of peppermint oil and caraway oil on postinflammatory visceral hyperalgesia was examined ( using a rat model of tnbs - induced colitis ) . neither peppermint nor caraway oil administered individually had a significant effect on postinflammatory visceral hyperalgesia , but combined treatment with these essential oils significantly reduced the visceromotor response . mechanisms mediating the suppressive effects of thyme and oregano oils on colitis are unclear , and we can only speculate that there are several potential manners of action . one possibility could be the influence of the essential oils on nuclear factor b ( nf-b ) , a pleiotropic transcription factor which can activate expression of genes involved in immune and inflammation responses such as proinflammatory cytokines . suppression of the nf-b inhibitory protein ( ib ) degradation in colonic epithelial cells and macrophages was demonstrated after the administration of curcumin and zerumbone [ 4 , 44 , 45 ] . in conclusion , the present data indicate that dietary administration of a combination of thyme and oregano essential oils in appropriate concentrations can reduce the production of proinflammatory cytokines and attenuate the degree of colonic tissue injury , and thereby ameliorate tnbs - induced colitis in mice . we suggest that the combination of thyme and oregano essential oils has potential value as an additional or supporting treatment in gastrointestinal inflammations . our results indicate that some essential oils could have positive effects on tnbs - induced colitis but seemingly in rather narrow range of concentrations , thus limiting their therapeutic / preventive potential .

vestibular neuritis is defined as a sudden unilateral partial failure of the vestibular nerve that impairs the forwarding of vestibular information from the labyrinth . because normal vestibular function depends on continuous bilateral input , a unilateral failure leads to an immediate direction - specific imbalance of the bilateral vestibular tone . the patient suffers from vertigo , horizontal nystagmus and postural instability with a tendency toward ipsilesional falls . most patients experience a quick recovery that seems to be independent from the function of the affected vestibular nerve but is caused by a compensatory adaptation of central vestibular information processing ( strupp et al . , 1998 ) . the great majority of our current knowledge about the cortical processing of vestibular information in the human brain is derived from brain activation studies in healthy subjects that used pet and fmri during various kinds of vestibular stimulation methods , such as vestibular evoked myogenic potentials ( miyamoto et al . , 2007 ; janzen et al . , 2008 ; , 2001 ; stephan et al . , 2005 ; stephan et al . , 2009 ) and caloric irrigation ( suzuki et al . , 2001 ; fasold et al . , 2002 ; dieterich et al . , 2003 ; marcelli et al . , although still debated , some authors have suggested that there is no primary vestibular cortex that obtains projections exclusively from vestibular afferents ( guldin and grsser , 1998 ) . instead , several separate and distinct cortical areas have been identified that integrate vestibular , visual and somatosensory information ( faugier - grimaud and ventre , 1989 ; fredrickson et al . , 1966 ; guldin and grsser , 1998 ; odkvist et al . , 1974 ) . these multimodel areas include the temporo - parietal vestibular cortex , retroinsular areas , the superior temporal gyrus and the inferior parietal lobule ( for review see lackner and dizio , 2005 ; dieterich and brandt , 2008 ) . it was shown that unilateral vestibular stimulation increases the activity in these multimodal areas , while the activity in the visual cortex , the somatosensory cortex and the default mode network decreased ( klingner et al . , 2013b ) . however , a pet study showed that areas responsible for multisensory integration revealed an increased glucose metabolism , while a decreased metabolism was found in the visual cortex , the somatosensory cortex and parts of the auditory cortex ( bense et al . , 2004 ) . a further study showed that vestibular failure suppresses cortical visual motion processing ( deutschlander et al . , 2002 ) . these studies support the view that vertigo and dizziness following an acute vestibular neuritis originate from the conflicting information between sensory sources and an altered activity state of different sensory sources . because of the known balance between the task - positive and task - negative networks ( default mode network ; dmn ) such an impaired balance might explain findings such as difficulties with reading , arithmetic and concentration ( hanes and mccollum , 2006 ) . we further hypothesize that such an impaired balance between these networks should be measurable by an altered coupling . based on studies which demonstrated a tight coupling between behavioral changes and changes of the functional connectivity ( hampson et al . , 2006 ; albert et al . , 2009 ; barnes et al , 2009 ; lewis et al . , 2009 ; zhu et al . , 2011 ) , we hypothesize that symptoms following unilateral vestibular neuritis are reflected in changes in the functional connectivity between the involved networks . the identification of these networks and their disturbed connectedness are important pieces of information that can be used to deduce the pathophysiologic mechanism that underlies symptoms following vestibular neuritis . however , no analyses of the relevant functional connectivity that further investigate these assumptions are currently available for this disease . here we investigate the effects of unilateral vestibular neuritis on the functional connectivity between the default mode network and other particular task positive networks in the brain . the present study investigated this hypothesis by employing functional magnetic resonance imaging ( fmri ) in the resting state in a longitudinal design . all patients were investigated in the early stage of vestibular neuritis but after remission of nystagmus during visual fixation . the study population comprised 14 patients ( mean age 51.1 10.4 years , range 3267 years , 6 females , 8 males ) and 28 age and gender matched controls ( 12 females , 16 males ) without any history of neurological or psychiatric diseases . patients were diagnosed with left ( 7 ) or right ( 7 ) sided vestibular neuritis via ( 1 ) clinical criteria , ( 2 ) caloric hyporesponsiveness with at least 25% canal paresis of the affected side and ( 3 ) normal conventional brain imaging ( mri ) . all patients received an initial fmri scan at the early stage ( mean : 4.9 1.9 days after onset of symptoms ) . no patient received any sedative medication within 24 h prior to the fmri session . the patients were recalled at intervals of several months for clinical examination in the outpatient clinic of the department of otorhinolaryngology . a follow - up fmri was performed when both the physical examination revealed no pathologic findings such as nystagmus or abnormalities in posture or gaze and the patient reported subjective complete remission of symptoms ( mean interval to follow - up scan 12 4.6 months ) . all subjects were right - handed according to the edinburgh handedness inventory , with a laterality quotient greater than + 80 ( oldfield , 1971 ) . the study was approved by the local ethics committee and all subjects provided their written informed consent according to the declaration of helsinki . images were acquired on a 3.0-tesla mr scanner ( trio , siemens , erlangen , germany ) to obtain 203 echo - planar t2 * weighted image ( epi ) volumes and 192 transaxial t1 weighted structural images . a functional - image volume comprised 40 transaxial slices that included the whole cerebrum and cerebellum ( voxel size = 3 3 3 mm , repetition time = 3 s , te 35 ms ) . the high - resolution t1 weighted structural images exhibited a voxel size of 1 1 1 mm to allow for precise anatomical localization and normalization . the data analysis was performed on a workstation using matlab ( mathworks , natick , ma , usa ) with the gift toolbox ( http://icatb.sourceforge.net/ ) and spm8 software ( wellcome department of cognitive neurology , london , uk ; http://www.fil.ion.ucl.ac.uk/spm ) . for each subject , all images were realigned to the first volume using six - parameter rigid - body transformation to correct for motion artifacts ( friston et al . , 1995 ) . the images were co - registered with the subject 's corresponding anatomical ( t1-weighted ) images , normalized to the montreal neurological institute ( mni ) standard brain ( evans et al . , 1993 ) to report mni coordinates and smoothed using a 6-mm full - width - at - half - maximum gaussian kernel . several sources of variance were removed from the data by linear regression : ( 1 ) six parameters obtained by rigid body correction of head motion , ( 2 ) signals from a ventricular region of interest and ( 3 ) signals from a region centered in the white matter ( weissenbacher et al . , 2009 ) . all signal intensity time courses were band - pass filtered ( 0.01 < f < 0.1 ) to reduce the effect of low - frequency drift and high - frequency noise . statistical analysis was performed by an independent component analysis ( ica ) with the pre - processed images ( realigned , coregistered , normalized , and smoothed ) . after preprocessing , single subject data are combined together , followed by the independent component analysis , and finally individual subject maps and time courses are reconstructed . this analysis was carried out using group - ica toolbox gift ( calhoun et al . the number of independent components ( ics ) was estimated for each subject and ranged from 30 to 39 . to ensure that all the ics were present in each individual , we used the minimum number of components that were determined in a single dataset ( 30 ) . these 30 components were estimated using the infomax algorithm implemented in the gift software ( http://icatb.sourceforge.net/ ) ( calhoun et al . the chosen number of components provides a reasonable trade - off between preserving relevant variance in the data while easing the burden of interpretation ( calhoun et al . , 2001a ) . next , a voxel - wise random effects analysis was performed on the component image to obtain consistent group activation patterns . the resulting group statistical maps were corrected for multiple comparisons at a significance level of p < 0.005 ( bonferroni - corrected ) . the overlap between each of these group maps and the area of the cerebral spinal fluid was estimated . if we detected an overlap of more than 50% , the group activation map was designated as an artifact and excluded from further analysis . the remaining group activation maps were now used to identify ics that represent functional networks which were further investigated . we aimed to identify the following networks : default mode network , somatosensory network , vestibular network , motor network , fronto parietal network ( fpc ) , occipital network , cerebellar network . the selection of the ics was based on prior anatomical and functional knowledge and our hypothesis . we used the anatomy toolbox to identify the somatosensory cortex , the motor cortex , the visual cortex and the cerebellum ( eickhoff et al . , 2005 ) . the spatial locations of these areas were determined at a probability of at least 50% ( by using the anatomy toolbox ) . the selection of the ic that best represents the auditory / vestibular / insular cortex , default mode network and the fronto parietal cortex ( not included in the anatomy toolbox ) was based on metaanalytic results ( laird et al . , 2009 ; the average time course over all voxels that were associated with the respective rsn was estimated . if one voxel was associated with two rsns , it was excluded from the estimation of the connectivity . then , we estimated differences in the functional connectivity between different networks between the first measurement ( acute state of vestibular neuritis ) , the follow - up measurement as well as between the first measurement and healthy controls as follows:1.the pearson 's correlation coefficients were computed between a selected pair of brain regions for each subject.2.each correlation coefficient was converted to a z - value by the fisher r - to - z transformation ( z = 0.5 log[(1 + r ) / ( 1 r ) ] ) to improve the normality of the correlation coefficients.3.the z - value matrices were compared between the measurements ( patient acute vs. patient follow up , patient acute vs. healthy subject ) . to reduce the variance we age- and the corresponding two z - value matrices of the matched healthy subjects were averaged prior to the statistical testing . the pearson 's correlation coefficients were computed between a selected pair of brain regions for each subject . each correlation coefficient was converted to a z - value by the fisher r - to - z transformation ( z = 0.5 log[(1 + r ) / ( 1 r ) ] ) to improve the normality of the correlation coefficients . the z - value matrices were compared between the measurements ( patient acute vs. patient follow up , patient acute vs. healthy subject ) . to reduce the variance we age- and the corresponding two z - value matrices of the matched healthy subjects were averaged prior to the statistical testing . these values were tested for significant differences between the dmn and the other selected rsns with a paired t - test ( acute patient vs. follow up and acute patient vs. healthy controls ) . findings were considered to be significant at p < 0.05 ( bonferroni - corrected ) . the current method for estimating the functional connectivity among spatially independent resting state networks was previously used and validated ( jafri et al . vestibular testing was performed using infrared videonystagmography ( vng ; hortmann video cng analyser , gn otometrics ) and a thermal stimulation unit ( variotherm , atmos medizintechnik gmbh & co. kg , lenzkirch , germany ) . in all subjects , vestibular testing was performed on the day of the initial mri and during the follow - up visit . goggles were placed securely on the patient 's head to eliminate light from entering in . saccadic eye movements , smooth pursuit , optokinetic nystagmus , and any spontaneous and positional nystagmus were recorded . the gaze test was performed by recording eye movements as the patient gazed 30 eccentrically in four directions ( right , left , up , and down ) . each position was held for at least 20 s to allow for adequate recording of eye movements . spontaneous nystagmus was examined while the patient was in the supine position and her / his eyes were in the neutral position . positional and positioning ( dix hallpike ) tests were performed to determine if the vestibular system responded normally and symmetrically to changes in head position . findings for spontaneous and positional nystagmus were considered abnormal if the slow phase velocity ( spv ) was > 5/s . , vestibular paresis was defined as more than 25% asymmetry between the right - sided and the left - sided responses . this asymmetry was calculated with jongkees ' formula from the slow - phase velocity ( spv ) : [ ( r 33 + r 44 ) ( l 30 + l 44 ) ] / ( r 30 + r 44 + l to investigate the variability between different functional systems , we first performed an ica to identify important functional systems . the selection of the ics based on prior anatomical and functional knowledge and our hypothesis ( see materials and methods section for details ) . the corresponding ic maps were corrected for multiple comparisons at a significance level of p < 0.05 ( bonferroni - corrected ) . the spatial maps of these eight resting state networks ( rsns ) are illustrated in fig . the following networks were identified : default mode network ( 130 cm ) , left and right frontoparietal network ( 142 , 88 cm ) , occipital cortex ( 127 cm ) , cerebellar cortex ( 85 cm ) , auditory / vestibular / insular cortices ( 92 cm ) , primary somatosensory cortex ( 64 cm ) , primary and secondary motor cortex ( 93 cm ) . first , we tested whether there was an overall disturbed connectivity between the dmn and all other investigated networks . the resulting seven r - values were transformed to z - values and were then averaged for each subject . we found a decreased inter - network connectivity ( dmn vs. all other selected ics ) in the first measurement ( acute state of vestibular neuritis ) compared to the second measurement ( after complete clinical remission of symptoms ) ( fig . 1 , p < 0.05 , bonferroni - corrected ) . we further tested each of the selected networks whether the connectivity was decreased to the dmn . decreased functional connectivity was found between the dmn and multiple other networks namely the somatosensory cortex , the auditory / vestibular / insular cortex , the motor cortex , the occipital cortex , the lfpc and the rfpc in the first measurement ( acute state ) compared to the follow - up measurement ( after complete clinical remission of symptoms ) ( fig . 1 , p < 0.05 , bonferroni - corrected ) . by comparing the first measurement ( acute state ) with the group of healthy control subjects , the same areas ( except the occipital cortex ) showed a decreased connectedness to the dmn ( fig . the caloric testing results demonstrated a vestibular paresis in all subjects , with an averaged hyporesponsiveness of the affected side of 53.2% ( 25.0% ) compared to the unaffected side . all subjects showed reduced asymmetry of the caloric responsiveness in the follow - up investigation ( 20.8% 24.1% ) compared to the acute / subacute stage . the severity of the impaired vestibular function measured by the caloric test was tested for correlations with changes of the functional connectivity but did not reveal any significant results . in the present study , we used fmri to investigate the effects of an acute unilateral vestibular deficit on the functional connectivity between different brain networks . multiple studies have demonstrated a tight correlation between behavioral performance and the strength of the functional connectivity , suggesting altered information transfer of the corresponding networks ( lowe et al . , 2002 ; sorg et al . , 2008 ; abutalebi et al . , 2009 ; albert et al . , 2009 ; mintzopoulos et al . , 2009 ) . based on these results , our current findings of reduced internetwork functional connectivity of the dmn indicate that disturbed cerebral information transfer of the dmn is connected to the underlying symptoms in the early stage of a vestibular deficit . to draw conclusions from these results with respect to the underlying mechanisms of how an acute vestibular deficit may lead to the associated clinical symptoms , it is necessary to consider the physiologic function of those brain areas that are involved in the measured reduced functional connectivity . it is generally agreed that the brain is composed of two spatially distinct functional networks : the default - mode and task - positive networks ( corbetta et al . , 2002 ; during the performance of attention - demanding tasks , prefrontal and parietal structures that comprise the task - positive network are characterized by increased activity ; in contrast , the default mode network , including the posterior cingulate and medial prefrontal cortices , is characterized by decreased activity . during wakeful rest , the opposite pattern emerges , with the default mode network becoming more active and the task - positive network less active ( fox et al . , 2005 ; fox et al . , the default mode network has been hypothesized to generate spontaneous thoughts during mind - wandering and to be an essential component of creativity ( broyd et al . , 2009 ) . after an acute vestibular deficit , we found that the connectivity between these two networks ( task - positive and default mode networks ) was reduced . we suggest the following mechanism to explain this finding : in the case of an acute vestibular deficit , the imbalance in vestibular tone causes information to diverge from both sides and also a spontaneous nystagmus in the absence of visual fixation . in the resting condition , the diverging vestibular information and also the spontaneous eye movements ( nystagmus ) are in conflict with information from other sensory modalities . the attempt to integrate this conflicting information requires significantly greater capacity for the processing of information about spatial orientation and brings sensory information processing to our attention , which is normally an automated process that does not require attentional demand . these mechanisms are reflected by increased activity within brain areas responsible for the processing of vestibular information and the integration of multisensory information ( bense et al . , 2004 ) . the sustained increased activity in parts of the task - positive network and the attentional demand reduce the activity within the default mode network . it was shown in healthy subjects that such an activity decrease of the default network is associated with improved performance ( mckiernan et al . , 2003 ; eichele et al . , 2008 ; singh and fawcett , 2008 ; daselaar et al . , 2009 ) . this physiologic mechanism is useful for a short period of time to allocate attentional resources away from intrinsic thoughts and toward difficult extrinsic tasks . this mechanism is also important because the human brain is generally not very efficient at conducting multiple attention - demanding tasks simultaneously . however , in the case of a vestibular neuritis , the attentional demand of diverging sensory information is long - lasting , which greatly limits the ability to focus and process other extrinsic tasks for a long period of time . the default mode network compensates by decreasing the amount of information that is received from the task - positive network , leading to a decreased connectedness . this involvement of the default mode network in this disease is further supported by findings of difficulties with cognitive skills such as reading , arithmetic and concentration suggesting a decreased ability to engage the task positive networks ( hanes and mccollum , 2006 ) . in summary , we hypothesize that the measured disconnection of the default mode network is a physiologic mechanism that compensates for the imbalance between both networks by disconnecting the default mode network from the increased amount of information from the task - positive network . it might also be an expression of a sustained , increased utilization of the available processing capacity . this could be investigated in further studies by comparing a resting state scan with open ( without nystagmus ) vs. closed ( nystagmus ) eyes in patients and healthy controls . the question about the behavioral implications of such a reduced engagement of the default mode network remains . as outlined above , the default mode network is thought to generate spontaneous introspective thoughts during mind wandering . studies that investigated diseases in which patients particularly indulge in their own thoughts ( e.g. , schizophrenia , depression ) reported predominant hyper - connectivity / activity of the dmn ( broyd et al . , 2009 ; karbasforoushan and woodward , 2012 ; whitfield - gabrieli and ford , 2012 ) . if hyper - connectivity / activity of the dmn is associated with increased introspective thoughts , it could be hypothesized that a decreased connectivity / activity of the dmn ( as found in the current study ) is associated with a reduced generation of spontaneous introspective thoughts . we can tentatively speculate whether this inverse effect could modify clinical symptoms ( e.g. , are depressive symptoms reduced during the acute phase of vestibular neuritis by decreasing the pathologically increased connectivity of the dmn ? ) . a further point that needs to be discussed is the lack of a significant correlation between changes in the strength of the caloric hyporesponsiveness and changes of the connectivity between the analyzed networks . correlations between the resting - state functional connectivity and individual task performance have been found for multiple motor , sensory , and cognitive functions as well as during learning ( hampson et al . , 2006 ; albert et al . , 2009 ; lewis et al . , 2009 ; zhu et al . , 2011 ; klingner et al . , 2013a ) . therefore it is reasonable to assume , that also behavioral changes due to an improvement of caloric function are associated with changes of the functional connectivity . however , the question arises whether the caloric function is a suitable parameter for behavioral impairment . recent studies suggest that it is not and have shown a lack of correlation with the patient 's perceived disability ( mandala and nuti , 2009 ) or with the functional outcome or the duration of symptoms ( shupak et al . , 2008 ) . this might explain the lack of correlation between improvement of caloric function and changes in functional connectivity in the current study . however , it is also possible that caloric function is strongly correlated to the functional connectivity between networks other than the dmn ( which are not analyzed in the current study ) . particularly one might hypothesize that the connectivity between sensory brain areas of different modalities ( e.g. , between the visual and vestibular ) is more associated with changes in caloric function due to their adjustment of diverging information . in respect to the functional importance of the dmn one might further hypothesize that disturbed connectivity to the dmn it further suggests that the time course and severity of the clinical symptoms are mainly dependent on the success of utilizing central mechanisms to adapt and compensate for the diverging sensory information and are less dependent on the strength of the caloric hyporesponsiveness . although the mechanisms underlying the emergence and adaptation of motor symptoms due to vestibular neuritis are much better understood than those of dizziness , the question of whether there is a connection between motor symptoms and dizziness remains . it would be interesting to determine whether the same mechanism of a disconnected default mode network could be found in patients without motor symptoms , such as phobic postural vertigo . therefore , more studies are needed to elucidate the underlying mechanisms of the feeling of dizziness , which are difficult to access but nonetheless significantly affect patients ' quality of life . we hypothesize that the disturbed connectivity is caused by a sustained utilization of processing capacity by diverging sensory information . the reduced connectivity between task - positive and task - negative networks might be related to long lasting symptoms like dizziness or cognitive deficits .

vestibular neuritis is defined as a sudden unilateral partial failure of the vestibular nerve that impairs the forwarding of vestibular information from the labyrinth . the patient suffers from vertigo , horizontal nystagmus and postural instability with a tendency toward ipsilesional falls . although vestibular neuritis is a common disease , the central mechanisms to compensate for the loss of precise vestibular information remain poorly understood . it was hypothesized that symptoms following acute vestibular neuritis originate from difficulties in the processing of diverging sensory information between the responsible brain networks . accordingly an altered resting activity was shown in multiple brain areas of the task - positive network . because of the known balance between the task - positive and task - negative networks ( default mode network ; dmn ) we hypothesize that also the dmn is involved.here , we employ functional magnetic resonance imaging ( fmri ) in the resting state to investigate changes in the functional connectivity between the dmn and task - positive networks , in a longitudinal design combined with measurements of caloric function . we demonstrate an initially disturbed connectedness of the dmn after vestibular neuritis . we hypothesize that the disturbed connectivity between the default mode network and particular parts of the task - positive network might be related to a sustained utilization of processing capacity by diverging sensory information . the current results provide some insights into mechanisms of central compensation following an acute vestibular deficit and the importance of the dmn in this disease .

Introduction Materials and methods Results Discussion Conclusion

vestibular neuritis is defined as a sudden unilateral partial failure of the vestibular nerve that impairs the forwarding of vestibular information from the labyrinth . because normal vestibular function depends on continuous bilateral input , a unilateral failure leads to an immediate direction - specific imbalance of the bilateral vestibular tone . the patient suffers from vertigo , horizontal nystagmus and postural instability with a tendency toward ipsilesional falls . most patients experience a quick recovery that seems to be independent from the function of the affected vestibular nerve but is caused by a compensatory adaptation of central vestibular information processing ( strupp et al . the great majority of our current knowledge about the cortical processing of vestibular information in the human brain is derived from brain activation studies in healthy subjects that used pet and fmri during various kinds of vestibular stimulation methods , such as vestibular evoked myogenic potentials ( miyamoto et al . , 2002 ; dieterich et al . , 2003 ; marcelli et al . instead , several separate and distinct cortical areas have been identified that integrate vestibular , visual and somatosensory information ( faugier - grimaud and ventre , 1989 ; fredrickson et al . , 1974 ) . these multimodel areas include the temporo - parietal vestibular cortex , retroinsular areas , the superior temporal gyrus and the inferior parietal lobule ( for review see lackner and dizio , 2005 ; dieterich and brandt , 2008 ) . it was shown that unilateral vestibular stimulation increases the activity in these multimodal areas , while the activity in the visual cortex , the somatosensory cortex and the default mode network decreased ( klingner et al . however , a pet study showed that areas responsible for multisensory integration revealed an increased glucose metabolism , while a decreased metabolism was found in the visual cortex , the somatosensory cortex and parts of the auditory cortex ( bense et al . , 2004 ) . , 2002 ) . these studies support the view that vertigo and dizziness following an acute vestibular neuritis originate from the conflicting information between sensory sources and an altered activity state of different sensory sources . because of the known balance between the task - positive and task - negative networks ( default mode network ; dmn ) such an impaired balance might explain findings such as difficulties with reading , arithmetic and concentration ( hanes and mccollum , 2006 ) . we further hypothesize that such an impaired balance between these networks should be measurable by an altered coupling . based on studies which demonstrated a tight coupling between behavioral changes and changes of the functional connectivity ( hampson et al . , 2011 ) , we hypothesize that symptoms following unilateral vestibular neuritis are reflected in changes in the functional connectivity between the involved networks . the identification of these networks and their disturbed connectedness are important pieces of information that can be used to deduce the pathophysiologic mechanism that underlies symptoms following vestibular neuritis . however , no analyses of the relevant functional connectivity that further investigate these assumptions are currently available for this disease . here we investigate the effects of unilateral vestibular neuritis on the functional connectivity between the default mode network and other particular task positive networks in the brain . the present study investigated this hypothesis by employing functional magnetic resonance imaging ( fmri ) in the resting state in a longitudinal design . all patients were investigated in the early stage of vestibular neuritis but after remission of nystagmus during visual fixation . patients were diagnosed with left ( 7 ) or right ( 7 ) sided vestibular neuritis via ( 1 ) clinical criteria , ( 2 ) caloric hyporesponsiveness with at least 25% canal paresis of the affected side and ( 3 ) normal conventional brain imaging ( mri ) . the patients were recalled at intervals of several months for clinical examination in the outpatient clinic of the department of otorhinolaryngology . a follow - up fmri was performed when both the physical examination revealed no pathologic findings such as nystagmus or abnormalities in posture or gaze and the patient reported subjective complete remission of symptoms ( mean interval to follow - up scan 12 4.6 months ) . all subjects were right - handed according to the edinburgh handedness inventory , with a laterality quotient greater than + 80 ( oldfield , 1971 ) . the high - resolution t1 weighted structural images exhibited a voxel size of 1 1 1 mm to allow for precise anatomical localization and normalization . for each subject , all images were realigned to the first volume using six - parameter rigid - body transformation to correct for motion artifacts ( friston et al . , 1995 ) . the images were co - registered with the subject 's corresponding anatomical ( t1-weighted ) images , normalized to the montreal neurological institute ( mni ) standard brain ( evans et al . , 1993 ) to report mni coordinates and smoothed using a 6-mm full - width - at - half - maximum gaussian kernel . several sources of variance were removed from the data by linear regression : ( 1 ) six parameters obtained by rigid body correction of head motion , ( 2 ) signals from a ventricular region of interest and ( 3 ) signals from a region centered in the white matter ( weissenbacher et al . , 2009 ) . to ensure that all the ics were present in each individual , we used the minimum number of components that were determined in a single dataset ( 30 ) . these 30 components were estimated using the infomax algorithm implemented in the gift software ( http://icatb.sourceforge.net/ ) ( calhoun et al . the chosen number of components provides a reasonable trade - off between preserving relevant variance in the data while easing the burden of interpretation ( calhoun et al . , 2001a ) . the overlap between each of these group maps and the area of the cerebral spinal fluid was estimated . if we detected an overlap of more than 50% , the group activation map was designated as an artifact and excluded from further analysis . we aimed to identify the following networks : default mode network , somatosensory network , vestibular network , motor network , fronto parietal network ( fpc ) , occipital network , cerebellar network . the selection of the ics was based on prior anatomical and functional knowledge and our hypothesis . we used the anatomy toolbox to identify the somatosensory cortex , the motor cortex , the visual cortex and the cerebellum ( eickhoff et al . , 2005 ) . the selection of the ic that best represents the auditory / vestibular / insular cortex , default mode network and the fronto parietal cortex ( not included in the anatomy toolbox ) was based on metaanalytic results ( laird et al . if one voxel was associated with two rsns , it was excluded from the estimation of the connectivity . then , we estimated differences in the functional connectivity between different networks between the first measurement ( acute state of vestibular neuritis ) , the follow - up measurement as well as between the first measurement and healthy controls as follows:1.the pearson 's correlation coefficients were computed between a selected pair of brain regions for each subject.2.each correlation coefficient was converted to a z - value by the fisher r - to - z transformation ( z = 0.5 log[(1 + r ) / ( 1 r ) ] ) to improve the normality of the correlation coefficients.3.the z - value matrices were compared between the measurements ( patient acute vs. patient follow up , patient acute vs. healthy subject ) . to reduce the variance we age- and the corresponding two z - value matrices of the matched healthy subjects were averaged prior to the statistical testing . each correlation coefficient was converted to a z - value by the fisher r - to - z transformation ( z = 0.5 log[(1 + r ) / ( 1 r ) ] ) to improve the normality of the correlation coefficients . the z - value matrices were compared between the measurements ( patient acute vs. patient follow up , patient acute vs. healthy subject ) . to reduce the variance we age- and the corresponding two z - value matrices of the matched healthy subjects were averaged prior to the statistical testing . these values were tested for significant differences between the dmn and the other selected rsns with a paired t - test ( acute patient vs. follow up and acute patient vs. healthy controls ) . findings were considered to be significant at p < 0.05 ( bonferroni - corrected ) . the current method for estimating the functional connectivity among spatially independent resting state networks was previously used and validated ( jafri et al . in all subjects , vestibular testing was performed on the day of the initial mri and during the follow - up visit . goggles were placed securely on the patient 's head to eliminate light from entering in . the gaze test was performed by recording eye movements as the patient gazed 30 eccentrically in four directions ( right , left , up , and down ) . spontaneous nystagmus was examined while the patient was in the supine position and her / his eyes were in the neutral position . positional and positioning ( dix hallpike ) tests were performed to determine if the vestibular system responded normally and symmetrically to changes in head position . , vestibular paresis was defined as more than 25% asymmetry between the right - sided and the left - sided responses . this asymmetry was calculated with jongkees ' formula from the slow - phase velocity ( spv ) : [ ( r 33 + r 44 ) ( l 30 + l 44 ) ] / ( r 30 + r 44 + l to investigate the variability between different functional systems , we first performed an ica to identify important functional systems . the selection of the ics based on prior anatomical and functional knowledge and our hypothesis ( see materials and methods section for details ) . the spatial maps of these eight resting state networks ( rsns ) are illustrated in fig . the following networks were identified : default mode network ( 130 cm ) , left and right frontoparietal network ( 142 , 88 cm ) , occipital cortex ( 127 cm ) , cerebellar cortex ( 85 cm ) , auditory / vestibular / insular cortices ( 92 cm ) , primary somatosensory cortex ( 64 cm ) , primary and secondary motor cortex ( 93 cm ) . first , we tested whether there was an overall disturbed connectivity between the dmn and all other investigated networks . we found a decreased inter - network connectivity ( dmn vs. all other selected ics ) in the first measurement ( acute state of vestibular neuritis ) compared to the second measurement ( after complete clinical remission of symptoms ) ( fig . we further tested each of the selected networks whether the connectivity was decreased to the dmn . decreased functional connectivity was found between the dmn and multiple other networks namely the somatosensory cortex , the auditory / vestibular / insular cortex , the motor cortex , the occipital cortex , the lfpc and the rfpc in the first measurement ( acute state ) compared to the follow - up measurement ( after complete clinical remission of symptoms ) ( fig . 1 , p < 0.05 , bonferroni - corrected ) . by comparing the first measurement ( acute state ) with the group of healthy control subjects , the same areas ( except the occipital cortex ) showed a decreased connectedness to the dmn ( fig . the caloric testing results demonstrated a vestibular paresis in all subjects , with an averaged hyporesponsiveness of the affected side of 53.2% ( 25.0% ) compared to the unaffected side . all subjects showed reduced asymmetry of the caloric responsiveness in the follow - up investigation ( 20.8% 24.1% ) compared to the acute / subacute stage . the severity of the impaired vestibular function measured by the caloric test was tested for correlations with changes of the functional connectivity but did not reveal any significant results . in the present study , we used fmri to investigate the effects of an acute unilateral vestibular deficit on the functional connectivity between different brain networks . multiple studies have demonstrated a tight correlation between behavioral performance and the strength of the functional connectivity , suggesting altered information transfer of the corresponding networks ( lowe et al . based on these results , our current findings of reduced internetwork functional connectivity of the dmn indicate that disturbed cerebral information transfer of the dmn is connected to the underlying symptoms in the early stage of a vestibular deficit . to draw conclusions from these results with respect to the underlying mechanisms of how an acute vestibular deficit may lead to the associated clinical symptoms , it is necessary to consider the physiologic function of those brain areas that are involved in the measured reduced functional connectivity . it is generally agreed that the brain is composed of two spatially distinct functional networks : the default - mode and task - positive networks ( corbetta et al . , 2002 ; during the performance of attention - demanding tasks , prefrontal and parietal structures that comprise the task - positive network are characterized by increased activity ; in contrast , the default mode network , including the posterior cingulate and medial prefrontal cortices , is characterized by decreased activity . during wakeful rest , the opposite pattern emerges , with the default mode network becoming more active and the task - positive network less active ( fox et al . , 2005 ; fox et al . , the default mode network has been hypothesized to generate spontaneous thoughts during mind - wandering and to be an essential component of creativity ( broyd et al . after an acute vestibular deficit , we found that the connectivity between these two networks ( task - positive and default mode networks ) was reduced . we suggest the following mechanism to explain this finding : in the case of an acute vestibular deficit , the imbalance in vestibular tone causes information to diverge from both sides and also a spontaneous nystagmus in the absence of visual fixation . in the resting condition , the diverging vestibular information and also the spontaneous eye movements ( nystagmus ) are in conflict with information from other sensory modalities . the attempt to integrate this conflicting information requires significantly greater capacity for the processing of information about spatial orientation and brings sensory information processing to our attention , which is normally an automated process that does not require attentional demand . these mechanisms are reflected by increased activity within brain areas responsible for the processing of vestibular information and the integration of multisensory information ( bense et al . the sustained increased activity in parts of the task - positive network and the attentional demand reduce the activity within the default mode network . it was shown in healthy subjects that such an activity decrease of the default network is associated with improved performance ( mckiernan et al . however , in the case of a vestibular neuritis , the attentional demand of diverging sensory information is long - lasting , which greatly limits the ability to focus and process other extrinsic tasks for a long period of time . the default mode network compensates by decreasing the amount of information that is received from the task - positive network , leading to a decreased connectedness . this involvement of the default mode network in this disease is further supported by findings of difficulties with cognitive skills such as reading , arithmetic and concentration suggesting a decreased ability to engage the task positive networks ( hanes and mccollum , 2006 ) . in summary , we hypothesize that the measured disconnection of the default mode network is a physiologic mechanism that compensates for the imbalance between both networks by disconnecting the default mode network from the increased amount of information from the task - positive network . it might also be an expression of a sustained , increased utilization of the available processing capacity . this could be investigated in further studies by comparing a resting state scan with open ( without nystagmus ) vs. closed ( nystagmus ) eyes in patients and healthy controls . the question about the behavioral implications of such a reduced engagement of the default mode network remains . as outlined above , the default mode network is thought to generate spontaneous introspective thoughts during mind wandering . , schizophrenia , depression ) reported predominant hyper - connectivity / activity of the dmn ( broyd et al . , 2009 ; karbasforoushan and woodward , 2012 ; whitfield - gabrieli and ford , 2012 ) . if hyper - connectivity / activity of the dmn is associated with increased introspective thoughts , it could be hypothesized that a decreased connectivity / activity of the dmn ( as found in the current study ) is associated with a reduced generation of spontaneous introspective thoughts . we can tentatively speculate whether this inverse effect could modify clinical symptoms ( e.g. , are depressive symptoms reduced during the acute phase of vestibular neuritis by decreasing the pathologically increased connectivity of the dmn ? ) a further point that needs to be discussed is the lack of a significant correlation between changes in the strength of the caloric hyporesponsiveness and changes of the connectivity between the analyzed networks . correlations between the resting - state functional connectivity and individual task performance have been found for multiple motor , sensory , and cognitive functions as well as during learning ( hampson et al . , 2006 ; albert et al . , 2011 ; klingner et al . , 2013a ) . therefore it is reasonable to assume , that also behavioral changes due to an improvement of caloric function are associated with changes of the functional connectivity . however , the question arises whether the caloric function is a suitable parameter for behavioral impairment . recent studies suggest that it is not and have shown a lack of correlation with the patient 's perceived disability ( mandala and nuti , 2009 ) or with the functional outcome or the duration of symptoms ( shupak et al . this might explain the lack of correlation between improvement of caloric function and changes in functional connectivity in the current study . however , it is also possible that caloric function is strongly correlated to the functional connectivity between networks other than the dmn ( which are not analyzed in the current study ) . particularly one might hypothesize that the connectivity between sensory brain areas of different modalities ( e.g. , between the visual and vestibular ) is more associated with changes in caloric function due to their adjustment of diverging information . in respect to the functional importance of the dmn one might further hypothesize that disturbed connectivity to the dmn it further suggests that the time course and severity of the clinical symptoms are mainly dependent on the success of utilizing central mechanisms to adapt and compensate for the diverging sensory information and are less dependent on the strength of the caloric hyporesponsiveness . although the mechanisms underlying the emergence and adaptation of motor symptoms due to vestibular neuritis are much better understood than those of dizziness , the question of whether there is a connection between motor symptoms and dizziness remains . it would be interesting to determine whether the same mechanism of a disconnected default mode network could be found in patients without motor symptoms , such as phobic postural vertigo . therefore , more studies are needed to elucidate the underlying mechanisms of the feeling of dizziness , which are difficult to access but nonetheless significantly affect patients ' quality of life . we hypothesize that the disturbed connectivity is caused by a sustained utilization of processing capacity by diverging sensory information . the reduced connectivity between task - positive and task - negative networks might be related to long lasting symptoms like dizziness or cognitive deficits .

the main aim of a dentist is to remove the irreversibly demineralised superficial tissue and to remove the highly infected biomass of carious dentin during the carious process . new restorative materials are difficult to adhesion on this heavily infected dentin tissue , if it is retained at the cavity floor . thus , long - term prognosis of the final restoration is adversely affected [ 1 , 2 ] . however , the weight of experimental evidence might suggest that the bacteria in the biofilm are what matters , although some authors accept that this statement is controversial . it appears that lesion progression can be arrested either by simple removal of the biofilm or by the sealing of bacteria within the cavity and restoration of the tooth so that regular removal of the biofilm is possible . the discussion as to how much tissue must be removed in order to arrest the caries process is not new . in 1859 , tomes wrote it is better that a layer of discolored dentine should be allowed to remain for the protection of the pulp rather than run the risk of sacrificing the tooth , but in 1908 , black disagreed claiming ... it is better that it is removed of all decayed dentine overlaying . currently , physical criterion , used most commonly by dental practitioners to guide clinical excavation of this infected , demineralised that dentin is hardness / texture of the tissue , although some dentists may take into account its color and may use caries detector dyes [ 68 ] . all of these criteria suffer from the subjectivity inherent between dentists in clinical practice , which is likely to result in variations in the quality and quantity of dentin removed during operative intervention . however , at the chairside they have limited clues which include the subjective criteria of color and relative hardness of the carious dentin . in addition , when caries detection dyes , which generally consist of 1% acid red in propylene glycol [ 9 , 11 ] , are used in clinical situations , the dentin that becomes stained light pink should not be removed to prevent excessive dentin removal . however , the degree of light - pink dentin staining in deep carious layers is difficult to evaluate objectively by visual inspection alone . consequently , when removing demineralised dentin , it is not always easy to know at what point to stop excavation because there is an apparent lack of objective clinical markers . these variations may have clinical implication including differences in the size of the cavities and produced the pulpal health beneath prepared cavities and the strength of the remaining tooth structure . it seems sensible , therefore , to develop an objective marker for excavationable carious dentin . therefore , to ensure minimal tooth substance removal during clinical restorative treatment based on the concept of minimal intervention dentistry , an important need to assess the effect of some new objective caries detection techniques to aid in differentiating heavily infected dentin from affected dentin during excavation . in recent years , new techniques such as electrical conductivity measurement ( ecm ) and laser fluorescence ( lf ) devices have been developed for objective caries detection [ 1214 ] . the principle of the use of lf is based upon the principle of the laser fluorescence emitted from carious surfaces which is greater than that emitted from sound surfaces . as the tooth demineralised in caries process , the loss of mineral leads to increased porosity in the tooth structure . although the performance and reproducibility of these devices have been investigated on occlusal and proximal surfaces in vitro , and in vivo , there was limited information about the validity of lf [ 19 , 20 ] , and no information about the validity of ecm in detection of the residual caries after the excavation . furthermore , no previous studies have reported the comparison of the sensitivity , specificity , and intra- and interobserver agreement of ecm , lf , and cdd in detecting of residual caries . to compare these different diagnostic methods is also important for the objective identification of residual caries after carious dentin removal with conventional bur excavation . the main aim of this study was to compare histologically the efficiency of ecm , lf , and cdd in detection of residual dentinal caries in excavated cavities . also , other aim was to assess the accuracy , sensitivity , specificity , and inter- and intraobserver accuracy of these diagnostic methods in detecting of residual caries in cavity preparation . the protocol of this investigation was approved by the ethics committee of the faculty of dentistry , selcuk university ( process no . several private dental offices provided fifty freshly extracted human molar teeth with coronal dentin that include active caries and without developmental defects , restoration , plaque , cracking , and discoloration were used in this study . in each office , the teeth were extracted for periodontal , orthodontic , or prosthodontic reasons and were obtained from patients who consented to their use for research . teeth having radiographically d3 , caries limited to the outer half of the dentin were selected . immediately after extraction teeth were stored in saline solution without any antibacterial substance at 8c [ 2224 ] until further processing . before using , all soft periodontal tissue and extrinsic deposits on teeth enamel were removed by grinding the sample teeth perpendicularly to the carious surface using a slow - speed diamond saw under continuous water cooling until a flat surface was formed and the lesion in dentin was exposed . after that , caries dentin was mechanically removed with conventional round steel burs ( iso # 012 ; ela , engelskirchen , germany ) in a contra - angle speed- reducing hand piece ( 400 rpm ) . the hardness of dentine was checked with a dental explorer ( jensen , germany ) . this was repeated until either a leathery - hard texture was reached or a sharp scratching sound was heard in all teeth when each checked with a dental explorer [ 20 , 25 ] . the process of caries removal in all groups has been carried out by the same examiner ( a.r.c . ) . then , the cavity inspection for the successful removal of caries was carried out by three independent observers by direct visual examination using the criteria of kidd et al . . after completion of caries removal , digital images of the teeth were taken from the surfaces under investigation with a digital camera ( figures 1(a ) , 1(b ) , 1(c ) and figures 2(a ) , 2(b ) , 2(c ) ) . these images were stored on a computer ; the area to be evaluated was indicated on photographs with two black dots by using a pen to identify the location for the second measurements and to ensure that the same spots were analyzed each time ( figures 1(b ) and 2(b ) ) . before each measurement , photographs with black dot of each tooth were seen to ensure that the same spots were analyzed each time . all cavities were assessed by using laser fluorescence device ( lf , diagnodent pen , kavo , biberach , germany ) , electronic caries monitor ( ecm iv ; lode diagnostics , glaningen , the netherlands ) , caries detector dye ( cdd ; quadrant caritest , cavex , usa ) , and histological examination by three independent observers blindly . the examinations were carried out as follows : lfpen ( diagnodent pen , kavo , biberach , germany ) was used for the laser fluorescence measurements . each tooth was retrieved from the saline solution , wiped with a paper tissue , dried in air for approximately 10 seconds , and then measured by lfpen accordance with the manufacturer 's instructions . the fluorescence of a sound spot on smooth surface of the tooth was measured in order to provide a baseline reading for each tooth ( secondary calibration ) and again after every 10th tooth . lf values were carefully measured with the apex of the tip in contact with the surface of the carious dentin of the cavity floor of the approximal or occlusal cavities . the highest lf readings from the marked two lesion area were recorded for each sample . some studies reported that the lf cut - off point for dentin caries was from 18 to 30 [ 14 , 20 , 26 , 27 ] . in this study , it was already stated that the borderline was set at value of about 30 for operative intervention . the cut - off points were determined as follows ; 0 to 29 = no caries , 30 and over = dentinal caries ( residual caries ) . the status of the two marked areas on proximal or occlusal sites on each tooth was assessed by three observers . three measurements were done in each surface site and the mean value was calculated . after an interval of 2 weeks , same procedure was repeated by the same operator , under identical conditions , without access to the data from the previous session . for measurements with ecm ( ecm iv ; lode diagnostics , glaningen , the netherlands ) both , the tooth and reference electrode were held in the same hand without direct contact with each other . a measurement was made in accordance with the manufacturer 's instructions by touching the approximal or the occlusal site indicated on the black dots with the instrument probe . after visual , lf , and ecm examinations , the presence or absence of residual caries was also detected using a cdd . infected demineralized dentin will show definite dye stains , whereas noninfected dentin will not take up much of the dye [ 28 , 29 ] . the dye was applied to the approximal or occlusal cavities using a small brush and removed after 10 seconds by a 5-second water spray . the cavities were then examined under a light microscope ( 2 magnification , olympus bx 50 , japan ) for any dentin site stained by the dye . all observers were calibrated to the color of stained dentin on marked sites on each tooth as follow as 0 = white ( sound ) and 1 = blue ( carious ) . after all the examinations had been completed , color photographs of the caries removal surfaces were taken to assist the subsequent histological examination . prior to the histological examination , the specimens ' roots were separated from the coronal part and each tooth was prepared for histological examination . teeth were hemisectioned in a mesial - distal direction through the center of the marked lesion area with a high - speed drill and fine diamond bur . speed was set at 800 rpm and a moderate weight ( 100 g ) was chosen to guide the diamond - coated saw blade during the cutting procedure . one half of each tooth was processed for histological evaluations ( figures 1 and 2 ) . subsequently , specimens were dried in alcohol solvents of increasing concentration and embedded in technovit 9100 new ( heraeus kulzer , wehrheim , germany ) . serial sections of 200 m were prepared and polished to a final thickness of approximately 150 m . the specimens were immersed in water and wet sections were viewed under a polarized light microscope ( axiovert 135 , carl zeiss , jena , germany ) at 10 magnification for the presence or absence of residual caries . the existence or absence of residual caries the histologic validation was carried out by two observers one month after the diagnostic assessment . in cases of disagreement , the status of demineralized zone was then determined by majority opinion [ 20 , 30 ] . specificities and sensitivities were calculated for each diagnostic method using the histologic gold standard . intra- and interobserver accuracy values were assessed using kappa statistics [ 31 , 32 ] . the classification of kappa values was performed using criteria proposed in previous report [ 35 , 36 ] : kappa value 1 = perfect agreement , kappa values above 0.75 = excellent agreement , kappa values from 0.4 to 0.75 = good agreement and kappa values below 0.4 = marginal agreement . accuracy was calculated by the following formula in cross - tabs : the number of true positive values + the number of true negative values / total number . the histological examination of 50 teeth ( 100 test areas ) revealed that 12 ( 12% ) test sites had residual caries . for three observers , table 1 although the lf had the highest sensitivity value ( 0.85 ) , it had the lowest specificity value ( 0.50 ) . the lowest sensitivity ( 0.47 ) and the highest specificity ( 0.74 ) values were observed in ecm . the specificity and sensitivity values of cdd were similar ( 0.55 and 0.60 , resp . ) . table 2 shows the intra- and interobserver accuracy values of three observers for all diagnostic methods . the average intra- and interobserver accuracy values of cdd were 1.00 ( perfect agreement ) and 0.92 ( excellent agreement ) , respectively . the average intra- and interaccuracy values of lf were 0.50 ( good agreement ) , and 0.48 ( good agreement ) respectively . the lowest average interobserver accuracy value ( 0.36 , marginal agreement ) was observed in ecm . however , the average intraobserver accuracy value of ecm was 0.86 ( excellent agreement ) . the average accuracy value was 0.53 ( min = 0.52 , max = 0.56 ) for cdd , 0.51 ( min = 0.36 max = 0.62 ) for ecm , and 0.81 ( min = 0.76 , max = 0.86 ) for lf . there was no significant correlation between the gold standard and cdd ( p > .05 for all observers ) and between the gold standard and ecm ( p > .05 for observer 1 , p > .05 for observers 2 and 3 ) . therefore , there was a significant difference between the measurement of gold standard and cdd or ecm . there was found significant correlation between the gold standard and lf ( p = .001 for all observers ) . during caries removal in a clinical situation , visual inspection of the color of carious tissue , a caries detector dye , and detection with an excavator are used . whereas a significant correlation has been found between dentin hardness and level of bacterial infection , carious regions can easily be overlooked and deciding whether excavation is complete or not is often difficult . therefore , an objective and accurate technique to aid the clinician in differentiating heavily infected dentin from affected dentin during excavation is still needed . consequently , this present study aimed to investigate the efficiency of different diagnostic methods in detection of residual dentinal caries in excavated cavities . the objective identification of this infected / affective dentin carious boundary is also important for the dental researcher who wishes to determine the remaining dentin carious after caries excavation . some authors maintained that the simple histological analysis of a hemisected , carious lesion is difficult to interpret and it is not possible to distinguish the histological boundary in question [ 1 , 2 ] . however , using histopathological and microhardness evaluation , mendes et al . showed that the best lf performance was obtained at d3 , caries limited to the outer half of the dentin threshold . further , yazici et al . stated that lf agreed better than the caries detector dye with the histological evaluation in assessing teeth residual caries . however , using histological and scanning electron microscope analyses , gurbuz et al . stated that lf abilities to detect the residual caries were low after hand excavation and chemomechanical caries removal , and therefore , it is advisable to test the residual caries with an additional diagnostic tool such as visual tactile examination . thus , in the present study , we aimed to compare histologically the effectiveness of different diagnostic methods , such as ecm , lf , and cdd , on identification of residual caries . contrarily , some studies documenting the diagnosis of caries using the lf [ 14 , 19 , 35 ] reported that histological evaluation as a gold standard is subjective , for example , in regard to enamel and dentin caries . iwami et al . , using the rates of caries detection as a gold standard clearly , have showed the relationship between lf values of ground dentin surfaces and the rates of bacterial detection . also , they reported that there are some limitations in using lf for evaluating caries removal . the results of this present study showed that lf is advisable to test the residual caries but it should be improved to prevent the overexcavation . previous studies on lf [ 12 , 13 , 36 , 37 ] have established some cut - off points for lf in the diagnosis of dentin caries , and the manufacturer also states cut - off points in their literature on the clinical use of lf , but these cut - off points were different from each other . according to a clinical study of lussi et al . the cut - off values advised for clinical use of lf in dentin caries were 30 for operative intervention . they suggested that the border - line reading for operative intervention reduces the sensitivity of the device but increases its specificity and that a higher setting of this trigger for operative intervention also represents a safety for initially carious cases . 's in vitro study showed that no bacteria were detected at lf cut - off value of less than 15.6 . in their study , this value was considered a cut - off point for the complete removal of carious dentin . also , they stated that this cut - off value was obtained from the results of a limited number of specimens and can not be generalized . however , kidd and fejerskov reviewed that there is little evidence related with infected dentine that must be removed prior to sealing the tooth . in light of these studies , we determined 30 as of the cut - off points of lf in detection of residual dentin carious in order to prevent overexcavation . caries detecting dyes have been used to differentiate clinically infected from affected dentine during caries removal . the use of these dyes , however , does not provide a completely objective method for assessment of caries removal . pitts reported that the more superficial zone of infected dentine was an irreversibly damaged , bacterially infected layer that would never remineralize . the deepest affected dentine was shown to harden as a result of remineralization . fusayama 's group suggested the dye staining front coincided with the bacterial invasion of the dentine . however , several studies have reported that the dye does not discretely discriminate the bacterially infected from softened affected tissues [ 6 , 39 , 40 ] . also , banerjee et al . reported that the use of dyes is not routinely advocated in lesions extending into the middle third of dentin or deeper due to the increased risk of unnecessary and often avoidable pulpal involvement during cavity preparation . consequently , its injudicious use may lead to overpreparation of the tissues , encouraging excess removal at the enamel - dentine junction as well as unnecessary removal of dentine over the pulpal surface . previous studies [ 19 , 20 ] , which were assessment of residual caries in excavated cavity , reported that the sensitivity values were 0.40 and 0.65 , and the specificity values were 0.1 and 0.17 for ccd . in this present study , intra- and interobserver agreements were significantly higher than the other methods but average sensitivity value was 0.55 and specificity value was 0.60 for cdd . the average specificity value of cdd was similar with lf , but average sensitivity value of cdd was the lowest than lf . consistently with other studies , the results of this present study showed that cdd should be used with caution to test the residual caries in d3 level dentin carious and the use of cdd can result in overexcavation or incomplete removal of the carious lesion . therefore , restoration applying minimal intervention dentistry is needed for the least amount of enamel and dentin having to be removed . recently , new diagnostic devices , such as the lf and ecm , were developed for objective detection of caries before removal and have been reported as useful devices in objective evaluation of occlusal caries in vitro . lussi et al . have reported that carious dentin may be evaluated by the diagnodent during removal of caries as well as by caries detector dyes . however , in the literature , there are few studies which measured the reliability and validity of laser fluorescence in assessment of residual caries after caries removal [ 19 , 20 , 30 ] . it has been observed that lf does not adequately measure small mineral changes [ 12 , 13 ] . recently , several studies reported that the lf readings reflect changes in the organic matrix rather than in the inorganic content of the teeth [ 12 , 13 , 21 ] . it has been shown that the lf readings were useful to facilitate accurate removal of the caries - infected dentin . also , in present study , was compared the effectiveness of different diagnostic methods to identification of residual caries in d3 level carious lesions . ecm has been proposed for caries lesion detection and its measurement depends on the permeability changes due to demineralization of the tissues . in in vitro and in vivo studies the diagnostic performance of ecm has been evaluated but no information is available about the accuracy of ecm in assessment of residual caries in literature . in this study , we aimed also to investigate the accuracy of ecm and other some diagnostic methods in detection of residual caries . in the result of present study , we found that the specificity of ecm was higher ( 0.73 ) than other two diagnostic methods but its sensitivity was the lowest . in the literature , the range of specificity value of ecm is from 0.56 to 0.98 [ 16 , 43 , 44 ] . the specificity value of ecm in present study is in accordance with the literature [ 16 , 43 , 44 ] . it has been reported that the range of sensitivity value is from 0.39 to 0.97 for dentinal caries . in this study , ecm device can prevent the overexcavation with high specificity but its low sensitivity value can cause to remain carious lesions under the restoration . the use of ecm can be advised in the scanning examinations because of its higher specificity . electrical measurements are showing early promise as a technique for the clinical detection of caries in clinical practice . previously , usefulness of diagnodent for the detection of residual caries during excavation has been reported by lussi et al . . also , hossain et al . recommended a combination of hardness testing by an explorer and diagnodent for the assessment of carious dentin removal . the distribution of diagnodent values in primary teeth exhibits a difference in an unimportant degree with respect to that of permanent teeth . the best performance of diagnodent is said to be in dentinal caries which is emerged by opening the fissure and to predict the extension of caries lesions , mainly at dentinal caries threshold in primary teeth . gurbuz et al . reported that diagnodent and visual tactile examination can detect the remaining dentin as sound after bur excavation , but that diagnodent abilities to detect residual caries were low after hand excavation and chemomechanical caries removal and , therefore , it is advisable to test the residual caries with an additional diagnostic tool such as visual tactile examination . differently , in their study , diagnodent readings have been used to observe the condition of the dentin after removal of the caries - infected dentin tissues that were stained with caries detecting dye . however , lennon et al . and yazici et al . reported that lf had greater sensitivities than cdd in assessment of residual caries . in this study , similar to previous studies , we found that the highest average sensitivity was shown in the measurements of lf ( 0.85 ) for detection of residual caries . in the study of yazici et al . , the specificity value ( 0.86 ) was higher than sensitivity value ( 0.63 ) of lf . contrarily , in the study of lennon et al . , the sensitivity value ( 0.88 ) was higher than specificity value ( 0.70 ) . , in our study , the sensitivity value ( 0.85 ) was higher than specificity value ( 0.50 ) . in lf investigations on occlusal surface , some researchers reported that the sensitivity value was higher than specificity value [ 14 , 33 , 47 , 48 ] . lf , which has been high sensitivity , is a valuable adjunct to clinical examination to detect the residual caries in the cavity after excavation . according to these results , lf can prevent to remain of carious lesions under the restoration . an ideal diagnostic method should offer , among other characteristics , high sensitivity and high specificity . likewise , an increase in sensitivity will be accompanied by decrease in specificity ( increase in the false - positive diagnosis ) . considering that rise in the false - positive proportion can be dangerous as it can lead to overtreatment , a technique that offers high specificity even at the expense of a slight reduction in sensitivity seems to be more appropriate . in this present study , for lf , lower specificity values at d3 level caries when compared to sensitivity were found . deep lesions contain more protein because of bacterial deposits and would therefore be more affected by denaturing . also , in this study , the teeth with deep dentin caries were used ; higher readings may be therefore . this finding supports the proposal by shi et al . and hibst and paulus that increased readings of lf reflects , the increased organic material in a deep lesion . the storage temperature and storage medium were shown to influence lf readings [ 50 , 51 ] . hibst and paulus reported that lf readings dramatically increased after storage of the teeth in 10% neutral - buffered formalin instead of saturated thymol saline . they suggested that formalin denatured the proteins , changing the structure and quality of one or more organic components , resulting in increased fluorescence . lussi et al . demonstrated that the fluorescence values and hence the cut - off values decreased when the teeth were stored in thymol , chloramines , or formalin but remained stable when they were frozen during storage and were defrosted for fluorescence measurements . in this study , the teeth were stored in saline solution without any antibacterial substance at 8c . the cause of low specificity value of lf may be that teeth were kept in saline storage medium and at 8c . the sensitivity values of lf were higher than other diagnostic tests and , therefore , it is advisable to detect residual caries in cavity , but the specificity values of lf were low ; therefore , in addition to lf , residual dentin in deep dentin region must be evaluated as well as according to its hardness , color , and so forth . the intraobserver reproducibility values of lf ranged from 0.75 to 0.94 for smooth and occlusal surfaces in the literature . in this study for lf , yazici et al . found that intraobserver agreement was 0.93 and interobserver agreement was 0.61 for lf . the lower reproducibility may be resulted from the experience with the use of lf device of observers . therefore , the experience is an important factor in use of lf device and the measurements of lf have technical susceptibility to errors . repeated measurements can be advised to prevent the variability in measurements . in this study , ecm had the highest intraobserver agreement value ( 0.86 ) but its interobserver agreement value was the lowest . the intraobserver agreement value was 1 and interobserver agreement value was 0.92 for cdd but the accuracy values of these devices were significantly lower than lf in this study . furthermore , there was a significant difference between the measurements of ecm , cdd , and gold standard . lf can not detect the remaining dentin as sound ( low specificity ) ; therefore the use of lf could lead to over - preparation cavities . however , lf abilities to detect residual caries ( sensitivity ) were high and therefore it could be effective to evaluate the residual caries . for detection of residual caries , lf should be used with an additional diagnostic tool and it should be more improved to avoid excessive removal of the affected dentin .

the aim of this study was to investigate the efficiency of different diagnostic methods in detection of residual dentinal caries in excavated cavities . fifty extracted molar with deep dentinal carious lesions were excavated using a slow - speed handpiece . all cavities were assessed by laser fluorescence(lf ) device , electronic caries monitor(ecm ) , and caries detector dye(cdd ) by three independent observers blindly . the measurements were repeated after two weeks . specimens containing dentin slices 150 m in thickness were prepared for histological analyses . the existence and absence of carious dentin was determined using a lightmicroscope . the average intraobserver accuracy was 1.00 ( perfect agreement ) for cdd , 0.86 ( excellent agreement ) for ecm , and 0.50 ( good agreement ) for lf . the average interobserver accuracy values were 0.92 ( excellent agreement ) , ( 0.36 marginal agreement ) and 0.48 ( good agreement ) , for cdd , ecm , and lf , respectively . the average specificity was 0.60 for cdd , 73% for ecm , and 0.50 for lf . the average sensitivity was 0.55 for cdd , 0.85 for lf , and 0.47 for ecm . the average accuracy values were 0.53 , 0.51 , and 0.81 for cdd , ecm , and lf , respectively . lf had the greatest sensitivity and accuracy values of any of the methods tested . as a conclusion , lf device is appeared to most reliable method in detection of remain caries in cavity . however , because of its technical sensitivity it may susceptible to variations in measurements . to pay attention to the rule of usage and repeated measurements can minimize such variations in clinical practice . it was concluded that lf is an improvement on the currently available aids for residual caries detection .

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

the main aim of a dentist is to remove the irreversibly demineralised superficial tissue and to remove the highly infected biomass of carious dentin during the carious process . however , the weight of experimental evidence might suggest that the bacteria in the biofilm are what matters , although some authors accept that this statement is controversial . it appears that lesion progression can be arrested either by simple removal of the biofilm or by the sealing of bacteria within the cavity and restoration of the tooth so that regular removal of the biofilm is possible . in 1859 , tomes wrote it is better that a layer of discolored dentine should be allowed to remain for the protection of the pulp rather than run the risk of sacrificing the tooth , but in 1908 , black disagreed claiming ... it is better that it is removed of all decayed dentine overlaying . currently , physical criterion , used most commonly by dental practitioners to guide clinical excavation of this infected , demineralised that dentin is hardness / texture of the tissue , although some dentists may take into account its color and may use caries detector dyes [ 68 ] . all of these criteria suffer from the subjectivity inherent between dentists in clinical practice , which is likely to result in variations in the quality and quantity of dentin removed during operative intervention . however , at the chairside they have limited clues which include the subjective criteria of color and relative hardness of the carious dentin . in addition , when caries detection dyes , which generally consist of 1% acid red in propylene glycol [ 9 , 11 ] , are used in clinical situations , the dentin that becomes stained light pink should not be removed to prevent excessive dentin removal . however , the degree of light - pink dentin staining in deep carious layers is difficult to evaluate objectively by visual inspection alone . these variations may have clinical implication including differences in the size of the cavities and produced the pulpal health beneath prepared cavities and the strength of the remaining tooth structure . it seems sensible , therefore , to develop an objective marker for excavationable carious dentin . therefore , to ensure minimal tooth substance removal during clinical restorative treatment based on the concept of minimal intervention dentistry , an important need to assess the effect of some new objective caries detection techniques to aid in differentiating heavily infected dentin from affected dentin during excavation . in recent years , new techniques such as electrical conductivity measurement ( ecm ) and laser fluorescence ( lf ) devices have been developed for objective caries detection [ 1214 ] . the principle of the use of lf is based upon the principle of the laser fluorescence emitted from carious surfaces which is greater than that emitted from sound surfaces . although the performance and reproducibility of these devices have been investigated on occlusal and proximal surfaces in vitro , and in vivo , there was limited information about the validity of lf [ 19 , 20 ] , and no information about the validity of ecm in detection of the residual caries after the excavation . furthermore , no previous studies have reported the comparison of the sensitivity , specificity , and intra- and interobserver agreement of ecm , lf , and cdd in detecting of residual caries . to compare these different diagnostic methods is also important for the objective identification of residual caries after carious dentin removal with conventional bur excavation . the main aim of this study was to compare histologically the efficiency of ecm , lf , and cdd in detection of residual dentinal caries in excavated cavities . also , other aim was to assess the accuracy , sensitivity , specificity , and inter- and intraobserver accuracy of these diagnostic methods in detecting of residual caries in cavity preparation . the protocol of this investigation was approved by the ethics committee of the faculty of dentistry , selcuk university ( process no . several private dental offices provided fifty freshly extracted human molar teeth with coronal dentin that include active caries and without developmental defects , restoration , plaque , cracking , and discoloration were used in this study . teeth having radiographically d3 , caries limited to the outer half of the dentin were selected . before using , all soft periodontal tissue and extrinsic deposits on teeth enamel were removed by grinding the sample teeth perpendicularly to the carious surface using a slow - speed diamond saw under continuous water cooling until a flat surface was formed and the lesion in dentin was exposed . then , the cavity inspection for the successful removal of caries was carried out by three independent observers by direct visual examination using the criteria of kidd et al . after completion of caries removal , digital images of the teeth were taken from the surfaces under investigation with a digital camera ( figures 1(a ) , 1(b ) , 1(c ) and figures 2(a ) , 2(b ) , 2(c ) ) . these images were stored on a computer ; the area to be evaluated was indicated on photographs with two black dots by using a pen to identify the location for the second measurements and to ensure that the same spots were analyzed each time ( figures 1(b ) and 2(b ) ) . all cavities were assessed by using laser fluorescence device ( lf , diagnodent pen , kavo , biberach , germany ) , electronic caries monitor ( ecm iv ; lode diagnostics , glaningen , the netherlands ) , caries detector dye ( cdd ; quadrant caritest , cavex , usa ) , and histological examination by three independent observers blindly . the fluorescence of a sound spot on smooth surface of the tooth was measured in order to provide a baseline reading for each tooth ( secondary calibration ) and again after every 10th tooth . lf values were carefully measured with the apex of the tip in contact with the surface of the carious dentin of the cavity floor of the approximal or occlusal cavities . in this study , it was already stated that the borderline was set at value of about 30 for operative intervention . the cut - off points were determined as follows ; 0 to 29 = no caries , 30 and over = dentinal caries ( residual caries ) . the status of the two marked areas on proximal or occlusal sites on each tooth was assessed by three observers . three measurements were done in each surface site and the mean value was calculated . after an interval of 2 weeks , same procedure was repeated by the same operator , under identical conditions , without access to the data from the previous session . a measurement was made in accordance with the manufacturer 's instructions by touching the approximal or the occlusal site indicated on the black dots with the instrument probe . after visual , lf , and ecm examinations , the presence or absence of residual caries was also detected using a cdd . the dye was applied to the approximal or occlusal cavities using a small brush and removed after 10 seconds by a 5-second water spray . the cavities were then examined under a light microscope ( 2 magnification , olympus bx 50 , japan ) for any dentin site stained by the dye . all observers were calibrated to the color of stained dentin on marked sites on each tooth as follow as 0 = white ( sound ) and 1 = blue ( carious ) . prior to the histological examination , the specimens ' roots were separated from the coronal part and each tooth was prepared for histological examination . teeth were hemisectioned in a mesial - distal direction through the center of the marked lesion area with a high - speed drill and fine diamond bur . one half of each tooth was processed for histological evaluations ( figures 1 and 2 ) . serial sections of 200 m were prepared and polished to a final thickness of approximately 150 m . the specimens were immersed in water and wet sections were viewed under a polarized light microscope ( axiovert 135 , carl zeiss , jena , germany ) at 10 magnification for the presence or absence of residual caries . the existence or absence of residual caries the histologic validation was carried out by two observers one month after the diagnostic assessment . intra- and interobserver accuracy values were assessed using kappa statistics [ 31 , 32 ] . the classification of kappa values was performed using criteria proposed in previous report [ 35 , 36 ] : kappa value 1 = perfect agreement , kappa values above 0.75 = excellent agreement , kappa values from 0.4 to 0.75 = good agreement and kappa values below 0.4 = marginal agreement . accuracy was calculated by the following formula in cross - tabs : the number of true positive values + the number of true negative values / total number . for three observers , table 1 although the lf had the highest sensitivity value ( 0.85 ) , it had the lowest specificity value ( 0.50 ) . the lowest sensitivity ( 0.47 ) and the highest specificity ( 0.74 ) values were observed in ecm . the specificity and sensitivity values of cdd were similar ( 0.55 and 0.60 , resp . ) table 2 shows the intra- and interobserver accuracy values of three observers for all diagnostic methods . the average intra- and interobserver accuracy values of cdd were 1.00 ( perfect agreement ) and 0.92 ( excellent agreement ) , respectively . the average intra- and interaccuracy values of lf were 0.50 ( good agreement ) , and 0.48 ( good agreement ) respectively . the lowest average interobserver accuracy value ( 0.36 , marginal agreement ) was observed in ecm . however , the average intraobserver accuracy value of ecm was 0.86 ( excellent agreement ) . the average accuracy value was 0.53 ( min = 0.52 , max = 0.56 ) for cdd , 0.51 ( min = 0.36 max = 0.62 ) for ecm , and 0.81 ( min = 0.76 , max = 0.86 ) for lf . there was no significant correlation between the gold standard and cdd ( p > .05 for all observers ) and between the gold standard and ecm ( p > .05 for observer 1 , p > .05 for observers 2 and 3 ) . during caries removal in a clinical situation , visual inspection of the color of carious tissue , a caries detector dye , and detection with an excavator are used . consequently , this present study aimed to investigate the efficiency of different diagnostic methods in detection of residual dentinal caries in excavated cavities . showed that the best lf performance was obtained at d3 , caries limited to the outer half of the dentin threshold . stated that lf agreed better than the caries detector dye with the histological evaluation in assessing teeth residual caries . however , using histological and scanning electron microscope analyses , gurbuz et al . stated that lf abilities to detect the residual caries were low after hand excavation and chemomechanical caries removal , and therefore , it is advisable to test the residual caries with an additional diagnostic tool such as visual tactile examination . thus , in the present study , we aimed to compare histologically the effectiveness of different diagnostic methods , such as ecm , lf , and cdd , on identification of residual caries . contrarily , some studies documenting the diagnosis of caries using the lf [ 14 , 19 , 35 ] reported that histological evaluation as a gold standard is subjective , for example , in regard to enamel and dentin caries . , using the rates of caries detection as a gold standard clearly , have showed the relationship between lf values of ground dentin surfaces and the rates of bacterial detection . the results of this present study showed that lf is advisable to test the residual caries but it should be improved to prevent the overexcavation . previous studies on lf [ 12 , 13 , 36 , 37 ] have established some cut - off points for lf in the diagnosis of dentin caries , and the manufacturer also states cut - off points in their literature on the clinical use of lf , but these cut - off points were different from each other . they suggested that the border - line reading for operative intervention reduces the sensitivity of the device but increases its specificity and that a higher setting of this trigger for operative intervention also represents a safety for initially carious cases . in their study , this value was considered a cut - off point for the complete removal of carious dentin . however , kidd and fejerskov reviewed that there is little evidence related with infected dentine that must be removed prior to sealing the tooth . in light of these studies , we determined 30 as of the cut - off points of lf in detection of residual dentin carious in order to prevent overexcavation . the use of these dyes , however , does not provide a completely objective method for assessment of caries removal . however , several studies have reported that the dye does not discretely discriminate the bacterially infected from softened affected tissues [ 6 , 39 , 40 ] . previous studies [ 19 , 20 ] , which were assessment of residual caries in excavated cavity , reported that the sensitivity values were 0.40 and 0.65 , and the specificity values were 0.1 and 0.17 for ccd . in this present study , intra- and interobserver agreements were significantly higher than the other methods but average sensitivity value was 0.55 and specificity value was 0.60 for cdd . the average specificity value of cdd was similar with lf , but average sensitivity value of cdd was the lowest than lf . consistently with other studies , the results of this present study showed that cdd should be used with caution to test the residual caries in d3 level dentin carious and the use of cdd can result in overexcavation or incomplete removal of the carious lesion . recently , new diagnostic devices , such as the lf and ecm , were developed for objective detection of caries before removal and have been reported as useful devices in objective evaluation of occlusal caries in vitro . have reported that carious dentin may be evaluated by the diagnodent during removal of caries as well as by caries detector dyes . however , in the literature , there are few studies which measured the reliability and validity of laser fluorescence in assessment of residual caries after caries removal [ 19 , 20 , 30 ] . it has been observed that lf does not adequately measure small mineral changes [ 12 , 13 ] . also , in present study , was compared the effectiveness of different diagnostic methods to identification of residual caries in d3 level carious lesions . ecm has been proposed for caries lesion detection and its measurement depends on the permeability changes due to demineralization of the tissues . in in vitro and in vivo studies the diagnostic performance of ecm has been evaluated but no information is available about the accuracy of ecm in assessment of residual caries in literature . in this study , we aimed also to investigate the accuracy of ecm and other some diagnostic methods in detection of residual caries . in the result of present study , we found that the specificity of ecm was higher ( 0.73 ) than other two diagnostic methods but its sensitivity was the lowest . in this study , ecm device can prevent the overexcavation with high specificity but its low sensitivity value can cause to remain carious lesions under the restoration . the use of ecm can be advised in the scanning examinations because of its higher specificity . electrical measurements are showing early promise as a technique for the clinical detection of caries in clinical practice . previously , usefulness of diagnodent for the detection of residual caries during excavation has been reported by lussi et al . recommended a combination of hardness testing by an explorer and diagnodent for the assessment of carious dentin removal . the best performance of diagnodent is said to be in dentinal caries which is emerged by opening the fissure and to predict the extension of caries lesions , mainly at dentinal caries threshold in primary teeth . reported that diagnodent and visual tactile examination can detect the remaining dentin as sound after bur excavation , but that diagnodent abilities to detect residual caries were low after hand excavation and chemomechanical caries removal and , therefore , it is advisable to test the residual caries with an additional diagnostic tool such as visual tactile examination . differently , in their study , diagnodent readings have been used to observe the condition of the dentin after removal of the caries - infected dentin tissues that were stained with caries detecting dye . reported that lf had greater sensitivities than cdd in assessment of residual caries . in this study , similar to previous studies , we found that the highest average sensitivity was shown in the measurements of lf ( 0.85 ) for detection of residual caries . , the specificity value ( 0.86 ) was higher than sensitivity value ( 0.63 ) of lf . , the sensitivity value ( 0.88 ) was higher than specificity value ( 0.70 ) . , in our study , the sensitivity value ( 0.85 ) was higher than specificity value ( 0.50 ) . lf , which has been high sensitivity , is a valuable adjunct to clinical examination to detect the residual caries in the cavity after excavation . according to these results , lf can prevent to remain of carious lesions under the restoration . in this present study , for lf , lower specificity values at d3 level caries when compared to sensitivity were found . deep lesions contain more protein because of bacterial deposits and would therefore be more affected by denaturing . also , in this study , the teeth with deep dentin caries were used ; higher readings may be therefore . hibst and paulus reported that lf readings dramatically increased after storage of the teeth in 10% neutral - buffered formalin instead of saturated thymol saline . the sensitivity values of lf were higher than other diagnostic tests and , therefore , it is advisable to detect residual caries in cavity , but the specificity values of lf were low ; therefore , in addition to lf , residual dentin in deep dentin region must be evaluated as well as according to its hardness , color , and so forth . the intraobserver reproducibility values of lf ranged from 0.75 to 0.94 for smooth and occlusal surfaces in the literature . in this study for lf , yazici et al . found that intraobserver agreement was 0.93 and interobserver agreement was 0.61 for lf . the lower reproducibility may be resulted from the experience with the use of lf device of observers . therefore , the experience is an important factor in use of lf device and the measurements of lf have technical susceptibility to errors . repeated measurements can be advised to prevent the variability in measurements . in this study , ecm had the highest intraobserver agreement value ( 0.86 ) but its interobserver agreement value was the lowest . the intraobserver agreement value was 1 and interobserver agreement value was 0.92 for cdd but the accuracy values of these devices were significantly lower than lf in this study . furthermore , there was a significant difference between the measurements of ecm , cdd , and gold standard . however , lf abilities to detect residual caries ( sensitivity ) were high and therefore it could be effective to evaluate the residual caries . for detection of residual caries , lf should be used with an additional diagnostic tool and it should be more improved to avoid excessive removal of the affected dentin .

conventionally referred to as intersex conditions and affecting an estimated 50,000 americans , disorders of sex development ( dsd ) and best practices of care have remained a controversial topic in the health community . affecting an estimated 1 in 15,000 births , congenital adrenal hyperplasia ( cah ) results from an inherited enzyme deficiency leading to an overproduction of adrenal hormones . in karyotpyic females ( 46 , xx ) this elevated level of androgenic hormones impacts development in utero by virilizing or masculinizing / defeminizing the genitalia . since cah is often first treated by pediatric endocrinologists , surgeons , and urologists , research on outcomes and interventions has primarily centered on issues most clinically relative to these fields , specifically concerns related to sex of rearing , psychosexual outcomes thought to influence sex assignment such as gendered play in childhood , gender identity , and sexual orientation ( see , for a review ) and on ethical considerations related to treatment . thus , while cah 's impact on sexual and psychosexual development is well - established in the literature , its impact on health - related quality of life ( hrql ) , including mental health outcomes , is less clear . in recent years , a multidisciplinary approach for the treatment of dsd has been recommended to also include gynecological , genetic , neonatal , psychological , ethical , and social work specialists working with children , adults , and their families . specific to mental health specialists , psychotherapeutic recommendations have been proposed , yet little is known about what interventions are most efficacious or what specific quality of life and mental health concerns are most salient for women diagnosed with cah . thus the aim of this study was to determine the manner in which having cah impacts hrql ( defined as physical , functional , emotional , and social well - being ) , mental health experience , and counseling needs for women diagnosed with this condition . although the historical focus was primarily on the medical management of cah , within the past thirty years research has focused on understanding how chronic illnesses such as cah impact quality of life ( qol ) through measures specific to health - related quality of life ( hrql ) . hrql typically focuses on those domains of qol that would be directly affected by one 's medical status including physical well - being ( symptoms , impairments ) , functional well - being ( daily living , role performance ) , emotional well - being , ( coping , distress ) , and social well - being ( relationship quality , family ; ) . findings on hrql specific to women with cah have been mixed but have generally revealed implications for identity and relationships . one study found that women with cah had a significantly impaired hrql compared to controls . this study also revealed that women with cah were presently engaged in fewer relationships ( either heterosexual or homosexual ) than controls . although one must be cautious in implying that a lack of relationships inherently denotes impaired hrql , women with cah have often expressed concerns related to romantic partnerships , specifically fears of rejection , impacted body image , and self - esteem [ 10 , 11 ] . though kuhnle et al . , revealed no significant differences on a generic qol measure , women with cah did report trends of feeling less attractive , less pleasant in appearance , less erotic , less romantic , less feminine , and less confident compared to age - matched control women and were more often single , less sexually active and had less children compared to controls . research also finds women with cah have other issues relevant to their condition that may impact their quality of life , including those related to disclosing health - related information , weight and well - being , as well as having a chronic life - threatening condition . additionally , despite improving techniques , female identity may be compromised by imperfect appearing or performing genitalia , even after surgery . indeed , cah participants reported greater amounts of pain during intercourse [ 11 , 13 ] and were only moderately satisfied with the cosmetic appearance of their genitalia , rating their appearance lower than physicians ' ratings . liao reports that women diagnosed with a dsd such as cah may also have difficulty disclosing about their atypical genitalia to potential sexual partners . shame , secrecy and stigma often accompany ambiguous genitalia and the surgeries are , at times , framed as normalizing . an emphasis on normalcy versus a focus on the enjoyment of sex and intimacy may in turn lead to anxiety and/or avoidance of sexual intimacy . this potentially explains the low rates of romantic partnerships and sexual activity reported in the investigations of women diagnosed with cah [ 9 , 11 , 12 , 17 , 18 ] . furthermore , for women who may want to get pregnant , problems with fertility ( see for a review of fertility research ) may also impact willingness to pursue relationships , and some individuals have reported the inability to satisfy a potential mate 's needs for children as a barrier to marriage ( c. migeon , personal communication , january 8 , 2006 ) . cah , a chronic illness that may require repeated surgeries and challenge notions of identity and normalcy , influences those aspects of hrql related to emotional well - being . the sparse research on mental health outcomes related to disorders of sex developments in general has focused primarily on the presence of psychopathology . some evidence suggests that children with a disorder of sex development are at risk for psychological concerns , with roughly 40% of participants presenting with at least one dsm - iv diagnosis and an additional 20% presenting with less severe problems related to learning of their diagnosis and/or surgical interventions . yet , findings are mixed with another study reporting no differences between adult participants diagnosed with a dsd and both healthy and chronic illness control groups on measures of general mental health , posttraumatic stress , and depression . the dsd group , however , did exhibit higher trait anxiety and lower self - esteem than unaffected controls , but were no different than those with a chronic congenital disorder unrelated to sex development . although some participants in one study met diagnostic criteria for dsm diagnoses similar to those reported in the previous study ( i.e. , major depressive disorder , social phobia , panic disorder , generalized anxiety disorder ) , their overall scores on measures of depression , social adjustment , and eating behaviors and attitudes were within normal ranges . in contrast , johannsen et al . , found greater mental distress in this population as well as greater suicidal ideation and attempts when compared to controls . similarly , when compared to participants with other dsd diagnoses , women with cah had more suicide attempts . women with cah also had significantly higher scores on an anxiety measure , whereas participants with different dsds did not differ from controls . this suggests that women with cah , in particular , may be at a greater risk for psychopathology . though research has begun to explore factors associated with poorer mental health ( e.g. , self - concept , personality traits , interpersonal functioning ) for women with cah , no study has directly examined the influence of cah on hrql and mental health . though the above findings are useful in developing recommendations , little is known about what empirically - based psychotherapeutic interventions are most useful for this population . based on its success in reproductive medicine , group work has been recommended for individuals with a dsd . individual therapy focusing on interpersonal relationships has also been suggested , yet stigma exists surrounding seeking psychological help , especially for those diagnosed with a dsd . it is thought that support groups may be beneficial in ending stigma and isolation , and some women with cah do take advantage of them . support for families is an additional recommendation , as some women with a dsd report difficulty talking about condition - related issues with family members . this family intervention may be especially beneficial given findings that psychopathology in individuals with a dsd occurred twice as often among those with children and parents who did not receive counseling at diagnosis . given the inconsistent findings for hrql and mental health concerns as well as limited research regarding psychotherapeutic interventions for women with cah , a qualitative methodology , consensual qualitative research ( cqr ) [ 22 , 23 ] , was deemed a valuable approach to exploring these important treatment issues . given the history of treatment for dsd and ethical concerns regarding treatment decision - making , cqr was also determined to be a culturally sensitive method in that it gives much - needed voices to individuals . similarly , it does not seek to present one universal understanding and instead provides diverse perspectives based on the participants ' experiences ; an important consideration given differences within the population . other authors have also reported that interviews were more successful in helping participants disclose thoughts and feelings regarding their condition than paper and pencil measures . cqr was also chosen specifically for its demonstrated usefulness in gathering a large amount of data from a small sample , as research on this population is often constrained by small numbers given the rarity of this condition . this included characteristics like race , age , and so forth , as well as cah diagnosis , medical , surgical , and psychiatric histories . unpublished data from a longitudinal study as well as consultations with various stakeholders informed the development of appropriate and sensitive questions . hrql questions included inquiries about physical , functional , social , and mental / emotional well - being . given findings on social outcomes and existing recommendations , questions assessing support group use were included . similarly , as gaining psychotherapeutic knowledge was a goal of the investigation , questions assessing therapy / counseling use , experiences , and recommendations were included . open - ended questions were employed to promote full explanations with prompts / probes used for clarification . all identified as caucasian or white and ages ranged from 23 to 51 ( m = 33 , median = 31 ) . seven were diagnosed with the salt losing form of cah , and one with simple virilizing type . six participants identified as heterosexual or straight with the two identifying as lesbian or queer . all had at least an undergraduate degree with the average and median years of education being 18 . all had at least one genital surgery ( clitoroplasty and/or vaginoplasty , range = 1 to 3 surgeries , median = 2 ) , with a mean age of 3.88 years at first surgery ( range = 6 to 18 , median = 1 ) . regarding surgery / treatment , participants were treated at different facilities representing diverse geographic locations in the us . additionally , all but one participant reported a current or past mental health concern , six sought counseling for it and three sought counseling for issues surrounding cah . / cah support groups including the congenital adrenal research education and support foundation ( cares ) and the congenital adrenal hyperplasia support forum , through the nih 's clinicaltrials.gov website and a stand - alone study website . multiple sources were used due to concerns about how unique clinical experiences might steer individual towards or away from various medical , research , or support organizations . upon contacting the researcher , a letter was mailed explaining the study and inclusion criteria along with an irb - approved consent form . the demographic questionnaire was mailed as a follow - up . after returning the questionnaire , participants were screened for inclusion on the basis of self - reported diagnosis ( classical cah ) , biological sex ( female ) , and age ( 2150 ) . geographic location , treating - hospital / s , surgical history , severity ( salt - losing or simple virilizing ) , and referral method ( support group versus other ) were monitored in attempts to avoid over - representation . participants were then contacted by the primary investigator to set up a phone interview , lasting approximately one hour . as recommended by hill et al . , prior to conducting the analysis , the team shared their individual identity labels as well as general biases about the topic . the team consisted of three counseling psychology doctoral students ; one identified as male , aged 30 , and two identified as female , aged 29 and 27 . the auditor was also a counseling doctoral student and identified as female , aged 27 . two members identified as queer , one as queer / bisexual , and another as heterosexual . all were trained in cqr and had previously been engaged in multicultural counseling research that included its use . one had experience working with individuals diagnosed with cah , two noted their pre - existing awareness of intersexuality but hoped to increase their knowledge , whereas one reported only having been exposed to the topic via a television program and interactions with others on the team . common biases included believing that participants ' cah - related identities were different than dichotomous sexuality or sex / gender identities , that individuals may report shame surrounding their diagnoses / treatments , that family considerations would be important , and that this population was , at least theoretically , similar to other historically marginalized populations in regards to mental health . analysis followed methods from hill et al . , and consisted of the following : transcription , domains , core ideas , and cross - analysis . each step was checked either by the participants or an auditor with feedback then incorporated by the team prior to continuing . three participants responded that the transcripts accurately reflected their interview , and five did not respond . all transcripts ( cases ) were then reviewed by the team and general thematic domains were developed . after coding individually , team members then met for consensus on how to split each case into domains . following auditing , each case was summarized / paraphrased ( by domain ) into core ideas , with team members taking turns leading by sharing their cores for an entire case . the final step consisted of a cross - case - analysis during which the team derived categories and subcategories from all participants ' data within single domains . the team then came to consensus about exact placement of core ideas under each category / sub category and sent these results for auditing . lastly , frequencies for each category were calculated based upon the number of cases that had core ideas within a category . six domains described the participants ' experiences and sub jective views of how having cah impacted their hrql , their mental health , and their recommendations for counseling ( table 1 ) . within each domain categories revealed general , typical , and variant themes , which , for some categories were further clarified by sub categories . 's standards were used to determine frequency cutoffs with general representing the majority ( 7 - 8 ) of cases , typical representing more than half ( 6 ) of the cases , and variant for less than half ( 25 ) but more than one case . this domain revealed seven categories encompassing issues related to the impact of illness and treatment on physical / physiological functioning . generally , participants reported feeling like they were functioning normally , a category comprised of two subcategories . the first category , able to function well with no or minimal cah - related restrictions , was typical and included statements like , i function day to day ( and ) go to work like everybody else . the second , a variant subcategory , was normal functioning except for having to take medication daily . participants generally commented on the impact of cah on sexual functioning , ranging from difficulty getting pleasure / orgasm from sex to feeling that they could have sex just like anyone else to stating that penetrative sex was not an issue given the gender of their romantic partners . participants also generally noted an increased risk for and sensitivity to sickness , including both a history and fear of being sick or tired ( e.g. , i did not want to have them take me to the hospital . during ( the ) couple of days that i was sick it was on my mind the whole time ) . under the general category medication , four sub - categories emerged , all of which were typical . first were observations regarding medical regimens , for example , having to take medication daily or wear a medic alert bracelet and information regarding the level of their doses . second were observations about stress dosing with participants sharing their practice or importance of altering their medications based upon stress . third were general side effects from medication , including problems with blood pressure , weakened teeth / bones , and sleep / energy problems . one participant noted that sensitivity to light made it hard for her to do things outdoors . lastly , a subcategory titled weight concerns from medication included reports of weight gain , difficulty losing weight , and a resulting ambivalence about medication . finally , within this domain there were two additional categories that were typical in nature . the first , physical side effects of having cah , mainly included observations about hirsutism and short stature . second were comments related to adrenal crises and hospitalizations , both in childhood and adulthood ( e.g. , i did not show up for work , and when someone called and i did not make sense they sent an ambulance . lastly , in the variant reproductive functioning three participants noted issues with their fertility surrounding cah , while one stated , i will not be able to have kids and i do not think it 's related to cah . this domain encompassed seven unique categories , three of which were general and all of which described various processes related to emotions and the mind . the first general category , processes involved in understanding or becoming aware of cah , described moments of awareness and acceptance and included desires to understand more . for example , upon reading details about her condition , one participant stated , in the letter it talked about having ambiguous genitals , and i was like , just devastated when i read that . other participants reported positives including learning to accept their condition and an ability to relate to others or increased self - awareness , as one participant reported , i think this thing has resulted in me just being more aware or in tune in a good way . the next general category was understanding of identity and included two opposing subcategories , both of which were variant . on one hand , participants talked about feeling normal , for example , some people see cah as an identifier . no , my identity is me , i have a certain job , i have a masters degree . i have always had this sense of not quite fitting in and i wonder now if ( cah ) contributed . another general category was emotions and cognitions related to cah , including two subcategories , negative , which was general , and positive , which was variant . negative included things like general irritability , stress , problems with confidence , and reactions to treatment such as frustration , anger ( e.g. , i believe i was traumatized by the procedure ; it haunts me to this day ) . i never had any sense of shame or anything associated with it ( genital surgery ) . typical for this domain was the category mental health concerns , and this included participant and family mental health and substance abuse histories . according to participants , some concerns were directly related to cah , ( e.g. , ( after having a second surgery ) i actually did get depressed and ( was ) diagnosed with depression the following summer . ( thinking back i think that kinda started it ; i felt like i could not talk to anyone and it was not that i wanted to talk a lot about it ) , while others were unrelated ( e.g. , i do not think my bipolar was caused by the cah , because others in my family are bipolar and schizoaffective ) . sexuality included statements about understanding both attraction ( either to men , women , or both ) as well as identity ( e.g. , i found out that i 'm gay and i 'm very happy with that ) . i just feel like a man sometimes , and growing up i got the message it was ok to be a tomboy but that i needed to know how to be a lady too . lastly , some participants reported concentration and learning issues , such as , growing up i was hyper or i was tired and it was just hard and focusing , sometimes i get hyper focused or i 'd just totally screw around . that still happens now . this domain included eight categories pertaining to interpersonal or relational issues as a function of having cah . generally , disclosure and others ' awareness and knowledge of cah described processes related to telling others about cah as well as their understanding and reactions . now , i do not correct people when they say yes , sir unless it is going to be a continuing relationship ) , and some selected how much they told ( e.g.,if i travel with someone i want them to know i carry a stress dose but that 's about it ) . some participants seemed unsure of who actually knew or how much was known about their cah . for example , one participant shared , my family on my dad 's side i 'm sure they knew or that they know but they do not know the extent of what 's wrong with me and it 's untalked about because they 're weird in the first place and i think its taboo for them . on my mom 's side of the family , they are more open . this category included ; negative feelings and experiences by family members and friends like frustration , guilt , and embarrassment as typified by the following quote : he ( husband ) does not like it because it 's frustrating because i 'll go months and months being sick and there 's no light at the end of the tunnel , as well as no impact on friends and family . the interaction of cah 's genetic component with relationships was also general and included past issues regarding heredity , for example , there are only two times i have seen my parents feel guilty about passing on cah , as well as future considerations such as , i 've thought about it and thought about if i would be able to pass it along to my children . utilization of support groups was a general category and captured participants ' experience in or desire to be involved in various organizations or support groups . some participants spoke about organizations like congenital adrenal hyperplasia research and support foundation ( cares ) or congenitaladrenalhyperplasia.org with statements including , i think the best thing i found was this website where we all share and now that makes me feel less alone . additionally , one participant was uninterested in support groups saying , my endocrinologist has suggested that i go to support groups but ( i ) never really thought about or felt the need . three subcategories were present with parents and other important relationships being general and romantic relationships being typical for participants . supportive parental behaviors included helping with medical management ( e.g. , because my mom was a nurse she was able to ask questions and keep on top of my medications in a way i 'm not sure other people would have ) and normalizing the condition ( e.g. , i really am grateful to my parents for never making me feel odd or sick or anything or ashamed ) . other important relationships included siblings ( e.g. , my brother 's very protective of me ) , friends ( e.g. , i would have to say if i had to be at any friend 's house , that would have been the best place to be just because they are more mature and they would have taken care of me if i needed it ) , religion ( e.g. , going to church again has helped me accept cah ) , and doctors ( e.g. , my doctor , my mom , and two support groups are my only support ) . unsupportive people and behaviors were also typically present in participant interviews . included in this category were parents and siblings , reflected in the following statement : it really hurt me to know my siblings resented me because i did not get abused though i know where they 're coming from ) . also included were coworkers , as one participant stated , i was working with a psychiatrist who asked me point blank , was i trying to have a sex change , you know , was i pre - op ? , with the facial hair and stuff like that , and i had a really good relationship with the guy but it still just pissed me off that he said it , and people in general , for example , i want people to understand where i 'm coming from , but i feel frustrated because people tell me i do not look like i have a problem . also typical was the impact of cah on dating . within this category some participants reported a negative influence ( e.g. , in my 20s and high school it was really hard ; everybody else was dating and saying how fun it was and all of that and i was even afraid to go out with anybody ) , whereas others reported no impact on dating . lastly , and also typical , participants reported having never met anyone else with cah . this domain described women 's experience with health care ( including both physical and mental health ) and was comprised of three categories , all of which were general . comments regarding physicians ' knowledge of cah were general and included statements about family doctors ( e.g. , i was just lucky that my doctor knew the condition so that he could tell that there were issues with me ) , endocrinologists ( e.g. , i do not even feel that most endocrinologists i go to know much about the condition ) , and other physicians ( e.g. , i had to educate the ( athletic ) team 's doctor at school because he just freaked out about the whole idea of ( me ) having adrenal insufficiency and the chance of getting hurt ) . also within this category were negative medical interventions , and participants often described physicians overmedicating patients or not listening and ignoring . this was exemplified by one participant 's statement , at the time my doctor told me ( the weight gain ) was not from the medication , that i was just pretty much lazy and was eating all the time and not exercising and so , when they finally put me on a different medication , which is prednisone , from there i lost all the weight . positive interventions were typical and included comments such as , this doctor i have down here listens and he understands and he actually asked me if he could be my doctor when we sat down and talked when i moved down here . also typical were physicians ' abilities to make appropriate referrals ( e.g. , to other physicians , research studies , or support groups ) . the next general category focused on experiences with mental health clinicians including therapists , psychologists and psychiatrists and revealed four subcategories . first , participants generally described reasons for and goals of mental health treatment and/or therapy . three participants were reportedly forced into treatment after suicide attempts or overdoses , and participants sought treatment voluntarily for issues both related to cah ( e.g. , the third time i got counseling was for eating issues and cah came up in that when we got deeper into them ) and unrelated to cah ( e.g. , i just got overwhelmed with work and school and everything else , so i found a licensed therapist ) . a second general sub category described positive interventions , including those related to cah like accepting and exploring cah and its relation to other issues or simply listening and empathizing ( e.g. , it felt good to talk to someone . you know , all the years i always held it in and did not talk to anybody about my feelings about the health problem i felt more content after i started sharing my feelings ) , as well as those interventions unrelated to cah ( e.g. , relaxation techniques for anxiety ) . negative interventions were variant and primarily included comments about bias and lack of understanding , ( e.g. , she was very opinionated and very judgmental , if i remember correctly . her job was to help me work through whatever issues i 'm dealing with ) . lastly , and also variant , was the sub category clinicians ' knowledge of cah , which spoke to mental health workers ' prior knowledge or lack of knowledge about cah . the last general category , intrapersonal processes in healthcare treatment , encompassed participants ' internal thoughts , feelings , and reactions within the context of treatment . examples included , the only time i really think about it ( cah ) is when i go to the doctor and it feels funny trying to explain to a shrink , yeah , they built me a vagina and they cut off my nerve center . this domain included five categories of recommendations for medical and mental health professionals . the first and only general category was helpful therapeutic traits and was specific to mental health clinicians . this captured helpful traits of therapists ( e.g. , a sickly therapist who could understand that sickness ) , therapeutic approaches and techniques ( e.g. , you still kind of have to fall back to what the individual needs of that person are ) , and foci of therapy ( e.g. , counseling exploring family dynamics and relationships would be helpful ) . the first , the need for more education about cah , emphasized the importance of increasing basic awareness and knowledge about the disease . the second , things to consider when treating individuals with cah , went into greater detail about specific things for professionals to be aware of and contained two subcategories . the second included comments for therapists about the medical considerations related to cah ( e.g. , appropriate ( psychotropic ) medication and when to consult an endocrinologist ) . finally , two variant categories emphasized the need to connect individuals with support groups ( e.g. , it 'd also be good for them to have resources for you to go to . if they were able to find a good ( internet message ) board , like if you did not know about congenitaladrenalhyperplasia.org , they could inform you about it and you could talk to other people , ) and the importance of access to and timing of therapeutic services for both individuals ( e.g. , counseling around the time of surgery would be helpful , ) and families ( e.g. , you have two healthy parents , and then suddenly they have got a sick baby that 's dependent on drug therapy and mean doctors and stuff , but the kid just has it ; the parents have to deal with it ) . the last domain spoke to systemic challenges faced by our participants and contained four categories . the first typical category was generational and cohort differences in women with cah and contained participant observations about differences between individuals with cah of different ages as well as over time , as knowledge about cah increases . for example , some participants noted the lack of interest in , or resources for , adults compared to those for children , while several observed changes in how doctors interact with patients . i do know that there was not as much information given at that time you know , as far as stress dosing . it might be that we did not even know about it back then , that we as the patients did not know about it . i do not know what the medical community was doing then but there was a lot less , you know , interactions between doctors and patients at that time . the next two categories , availability and appropriateness of care and insurance and other financial concerns related to cah , were both typical , and related to concerns about receiving adequate health care . the first included comments about difficulty finding experts , especially for adult patients / clients . the second included statements like , we do not make much money so just thinking about how to financially be able to afford counseling is one thing , and i feel so unsafe out here because i do not have any type of insurance . the last category in this domain was variant and spoke to societal biases and expectations , including statements such as , in our society if you 're a little different people look at you weird . the aim of this study was to determine the manner in which having cah impacts hrql , mental health experiences , and counseling needs for women diagnosed with this condition . though findings suggest that cah does indeed influence aspects of all of the above , the results both confirm and extend previous findings . specifically , despite some common concerns related to their condition , our participants understood themselves as functioning normally . most importantly , they indicated that for women with cah , feelings of normalcy are not solely explained by physical or functional outcomes but that the social and emotional well - being aspects of hrql play an important role in recommendations for treatment . these findings provide critical information for both medical and mental health practitioners working with women diagnosed with cah . our findings support previous results indicating that aspects of hrql are indeed influenced by cah . however , according to the results of the current study , certain aspects of hrql , especially some physical concerns , may actually be more salient of a concern for clinicians than for individuals . for example , although the desire to maximize fertility is something which often plays an important role in determining the sex of a newborn with dsd , and was a concern reported by women with cah in another study , this theme was variant , with just half of our participants bringing up fertility and only half of those mentioning it as a physical problem . similarly , though research has often focused on concerns with sexual / genital functioning ( see for review ) , for many women in this study it was a non issue given their satisfaction with their sexual functioning or lack of interest in penetrative sex . surprisingly , many concerns which were salient to participants did not abnormally impact their functioning as they saw it ( e.g. , having to take medication ) , and women described themselves as functioning normally both in childhood and adulthood . they attributed this to parents , friends , and partners not treating them as sick , to effective medical interventions , and to their own coming to terms with cah . the processes involved in understanding or becoming aware of cah was a blend of both positive and negative experiences . the latter were most often associated with discovering specifics about their history or the extremes of cah ( sometimes unintentionally as one participant did when doing research for a school project ) and were experiences that could have been circumvented through increased education and openness regarding the condition . in relation to social functioning , results from the study provided additional support to previous findings , which suggest that cah does negatively impact participants ' relationships [ 7 , 15 ] . however , supportive relationships were more common than unsupportive ones in the present study , with participants emphasizing the importance of positive , normalizing interactions between parents , family , and partners . similarly , though the impact of cah on dating was noted , most participants reported being in very satisfying and supportive relationships , findings which somewhat contradict those from prior research , which found women less likely to engage in intimate relationships . for both areas , the negative impact of cah was more present in women 's anxiety about disclosing or in fears of others ' reactions than actual negative responses from others . previously documented concerns regarding increased mental illness among individuals with cah were reflected in the results of this study . for those participants who saw them as cah - related , they more often attributed them to physiological changes in mood and to external problems with the way physicians and families handled or disclosed cah rather than from internal processes related to aspects of sexual or gender identity . it is , however , important to note that many participants saw their mental health concerns as typical and unrelated to issues of cah , or were unsure of a link between cah and their emotional or mental health functioning . for example , it was unclear if one participant 's substance use was a coping mechanism for negative emotions surrounding her cah or if it was influenced by a family history of alcoholism or both . similarly , though not all participants had received counseling , all shared a wide range of reasons and goals for counseling , the majority of which were unrelated to cah . unlike their experience with medical professionals , which were more negative than positive , as described both in this study and in one other , the reverse was true for experiences with mental health practitioners . while therapist 's knowledge of cah or willingness to learn about cah was positive , our findings lent support for the greater importance of the working alliance and basic helping skills . since issues of disclosure figured prominently in several domains , therapist empathy , openness , and listening skills can not be emphasized enough . they become all the more important in light of findings that participants had a tendency to overlook or be reticent in telling a mental health clinician about cah . similarly , participants wanted both mental health and medical clinicians to be more attuned to the medical implications and the impact of cah on emotions and mood regulation , which suggests that general knowledge about cah on its own is important , but must be integrated with a tailored understanding of the individual . a related finding , noteworthy for practitioners working with individuals with cah , was that six of the eight participants reported never having met anyone with cah . as consistent with previous findings , this is something that contributed to feelings of isolation for the women in this study . it is unlikely that individuals would know others with cah because the population is small in comparison to the broader population , and disclosure of this information to others is rare . interestingly , few participants recommended that health care professionals connect them with support groups , which would seem to be one solution to deter isolation for individuals with cah . since many of the women in this study strove to achieve normal functioning and identities , the use of a support group may potentially challenge this . the domain systemic considerations included several barriers to finding individual endocrine or therapeutic care providers which may have contributed to women emphasizing this need over the need for support groups and also serves as an important link between individual relationships ( e.g. , between doctors and patients , or individuals and friends ) and societal norms or challenges which may impact them . while attempts were made to examine cah degree - or - type - specific factors related to hrql , almost all of our participants were diagnosed in infancy with salt wasting type . similarly related to sampling , participants self - selected into our study , these individuals may be relatively healthy and interested in therapeutic issues pertaining to cah as compared to individuals who chose not to participate in the study . as such , our sample may not be representative of the broader cah population , including those individuals who hold strong negative feelings towards cah research or who do not identify strongly as a woman with cah . also , much of the research on cah exists within a larger dsd literature base , and this study did not include individuals with any other dsd diagnoses . present in our findings was the importance of considering cohort effects , which participants attributed to the relative newness of cah as a disease , related changes in standards of care , and unique needs of individuals dependent upon age . indeed , future research could expand upon these findings on identity in women with cah by further exploring variables related to identity development processes . that being said , given the rarity of this population , it is unlikely that recommendations from this and other qualitative studies will be meaningfully examined in a large enough sample of individuals with cah using quantitative methods . therefore , it is recommended that mixed methods or qualitative approaches employing samples of practitioners or individual counseling case studies be used in future research . perhaps the most limiting factor for women was the way their cah impacted their functional well - being . their general increased risk for illness impacted not only the activities they could engage in , but also everything from travel plans to choices about where to live . this impacted other domains of hrql as well ( e.g. , socially , not wanting to be a burden on friends ) , yet is something that could be improved through better medical management and compliance . physical concerns regarding medication and its side effects , especially weight gain , were often exacerbated by parents and doctors who minimized them or were hostile towards participants who brought them up . the impact of these concerns may be lessened by increased sensitivity and awareness on the part of physicians , potentially leading to greater medical compliance and , in turn , an improvement in several hrql areas . our results remind clinicians to be mindful of their assumptions and lend support to the primacy of the working alliance . several participants noted that being forced into therapy was counterproductive towards establishing shared tasks and goals . indeed , therapist traits and skills ( e.g. , empathy and appropriate disclosure ) were more frequently mentioned than was therapist knowledge of cah . furthermore , it is important that clinicians not assume that cah is a presenting concern for all individuals . similarly , though some participants brought up gender by mentioning that they did indeed identify as women , only half of participants discussed components of gender identity or role . also , only half discussed sexual orientation . therefore , despite the gendered nature of cah , as well as some evidence suggesting the presence of less heterosexuality in this population , prior recommendations to assist clients with the examination of their gender and sexuality should be tempered with an appreciation of individual clients ' needs . relatedly , clinicians should be sure to consider the diversity within this population , especially as it relates to terminology and identity ( e.g. , intersex , dsd , cah , gender queer ) and to recognize the importance of parallel identities ( i.e. , individual versus group membership identities ) . lastly , given our findings on the role of systemic challenges , we would like to further emphasize prior recommendations that clinicians consider diversity issues and social stigma and serve as advocates for girls and women with cah . from improving access to care by working around insurance limitations to challenging societal bias , there is much that professionals can do in and out of their day - to - day practice . individuals with cah are doing much to advocate for themselves as well as to help increase medicine and psychology 's understanding of cah . when ending our phone interviews , several participants shared their reasons for participating , it 's hard , you know , just growing up with it and i know that from my situation , and i want others to feel more comfortable in the future , and i figure anything that can help with the next generation is you know , i 'm here .

little is known about the long - term health - related quality of life ( hrql ) and mental health outcomes for women diagnosed with congenital adrenal hyperplasia ( cah ) , a disorder of sex development . though recommendations for therapists exist , no research has empirically investigated women 's experiences in therapy or their recommendations for therapy . thus the purpose of the study was to investigate hrql , mental health concerns of women with cah , and patients ' recommendations for therapists . a qualitative methodology , consensual qualitative research , was used to answer these questions . eight women with cah participated in the study . results from their interviews revealed six domains : physical functioning , psychological functioning , interpersonal processes and relationships , healthcare experiences of women with cah , recommendations for health care professionals , and systemic considerations . domains were further broken down into categories with results yielding implications for both medical and mental health professionals and shedding light on issues related to normalcy , concerns both related and unrelated to cah , sex , and gender , and highlighting effective supports and therapeutic interventions .

1. Introduction 2. Health-Related Quality of Life Specific to Disorders of Sex Development and CAH 3. Mental Health Concerns and CAH 4. Research on Psychotherapeutic Interventions 5. Materials and Methods 6. Results 7. Discussion 8. Conclusions

conventionally referred to as intersex conditions and affecting an estimated 50,000 americans , disorders of sex development ( dsd ) and best practices of care have remained a controversial topic in the health community . affecting an estimated 1 in 15,000 births , congenital adrenal hyperplasia ( cah ) results from an inherited enzyme deficiency leading to an overproduction of adrenal hormones . since cah is often first treated by pediatric endocrinologists , surgeons , and urologists , research on outcomes and interventions has primarily centered on issues most clinically relative to these fields , specifically concerns related to sex of rearing , psychosexual outcomes thought to influence sex assignment such as gendered play in childhood , gender identity , and sexual orientation ( see , for a review ) and on ethical considerations related to treatment . thus , while cah 's impact on sexual and psychosexual development is well - established in the literature , its impact on health - related quality of life ( hrql ) , including mental health outcomes , is less clear . in recent years , a multidisciplinary approach for the treatment of dsd has been recommended to also include gynecological , genetic , neonatal , psychological , ethical , and social work specialists working with children , adults , and their families . specific to mental health specialists , psychotherapeutic recommendations have been proposed , yet little is known about what interventions are most efficacious or what specific quality of life and mental health concerns are most salient for women diagnosed with cah . thus the aim of this study was to determine the manner in which having cah impacts hrql ( defined as physical , functional , emotional , and social well - being ) , mental health experience , and counseling needs for women diagnosed with this condition . although the historical focus was primarily on the medical management of cah , within the past thirty years research has focused on understanding how chronic illnesses such as cah impact quality of life ( qol ) through measures specific to health - related quality of life ( hrql ) . hrql typically focuses on those domains of qol that would be directly affected by one 's medical status including physical well - being ( symptoms , impairments ) , functional well - being ( daily living , role performance ) , emotional well - being , ( coping , distress ) , and social well - being ( relationship quality , family ; ) . findings on hrql specific to women with cah have been mixed but have generally revealed implications for identity and relationships . one study found that women with cah had a significantly impaired hrql compared to controls . this study also revealed that women with cah were presently engaged in fewer relationships ( either heterosexual or homosexual ) than controls . although one must be cautious in implying that a lack of relationships inherently denotes impaired hrql , women with cah have often expressed concerns related to romantic partnerships , specifically fears of rejection , impacted body image , and self - esteem [ 10 , 11 ] . , revealed no significant differences on a generic qol measure , women with cah did report trends of feeling less attractive , less pleasant in appearance , less erotic , less romantic , less feminine , and less confident compared to age - matched control women and were more often single , less sexually active and had less children compared to controls . research also finds women with cah have other issues relevant to their condition that may impact their quality of life , including those related to disclosing health - related information , weight and well - being , as well as having a chronic life - threatening condition . liao reports that women diagnosed with a dsd such as cah may also have difficulty disclosing about their atypical genitalia to potential sexual partners . this potentially explains the low rates of romantic partnerships and sexual activity reported in the investigations of women diagnosed with cah [ 9 , 11 , 12 , 17 , 18 ] . furthermore , for women who may want to get pregnant , problems with fertility ( see for a review of fertility research ) may also impact willingness to pursue relationships , and some individuals have reported the inability to satisfy a potential mate 's needs for children as a barrier to marriage ( c. migeon , personal communication , january 8 , 2006 ) . cah , a chronic illness that may require repeated surgeries and challenge notions of identity and normalcy , influences those aspects of hrql related to emotional well - being . the sparse research on mental health outcomes related to disorders of sex developments in general has focused primarily on the presence of psychopathology . some evidence suggests that children with a disorder of sex development are at risk for psychological concerns , with roughly 40% of participants presenting with at least one dsm - iv diagnosis and an additional 20% presenting with less severe problems related to learning of their diagnosis and/or surgical interventions . yet , findings are mixed with another study reporting no differences between adult participants diagnosed with a dsd and both healthy and chronic illness control groups on measures of general mental health , posttraumatic stress , and depression . the dsd group , however , did exhibit higher trait anxiety and lower self - esteem than unaffected controls , but were no different than those with a chronic congenital disorder unrelated to sex development . , major depressive disorder , social phobia , panic disorder , generalized anxiety disorder ) , their overall scores on measures of depression , social adjustment , and eating behaviors and attitudes were within normal ranges . similarly , when compared to participants with other dsd diagnoses , women with cah had more suicide attempts . women with cah also had significantly higher scores on an anxiety measure , whereas participants with different dsds did not differ from controls . this suggests that women with cah , in particular , may be at a greater risk for psychopathology . though research has begun to explore factors associated with poorer mental health ( e.g. , self - concept , personality traits , interpersonal functioning ) for women with cah , no study has directly examined the influence of cah on hrql and mental health . though the above findings are useful in developing recommendations , little is known about what empirically - based psychotherapeutic interventions are most useful for this population . it is thought that support groups may be beneficial in ending stigma and isolation , and some women with cah do take advantage of them . support for families is an additional recommendation , as some women with a dsd report difficulty talking about condition - related issues with family members . given the inconsistent findings for hrql and mental health concerns as well as limited research regarding psychotherapeutic interventions for women with cah , a qualitative methodology , consensual qualitative research ( cqr ) [ 22 , 23 ] , was deemed a valuable approach to exploring these important treatment issues . hrql questions included inquiries about physical , functional , social , and mental / emotional well - being . similarly , as gaining psychotherapeutic knowledge was a goal of the investigation , questions assessing therapy / counseling use , experiences , and recommendations were included . seven were diagnosed with the salt losing form of cah , and one with simple virilizing type . / cah support groups including the congenital adrenal research education and support foundation ( cares ) and the congenital adrenal hyperplasia support forum , through the nih 's clinicaltrials.gov website and a stand - alone study website . upon contacting the researcher , a letter was mailed explaining the study and inclusion criteria along with an irb - approved consent form . after returning the questionnaire , participants were screened for inclusion on the basis of self - reported diagnosis ( classical cah ) , biological sex ( female ) , and age ( 2150 ) . geographic location , treating - hospital / s , surgical history , severity ( salt - losing or simple virilizing ) , and referral method ( support group versus other ) were monitored in attempts to avoid over - representation . one had experience working with individuals diagnosed with cah , two noted their pre - existing awareness of intersexuality but hoped to increase their knowledge , whereas one reported only having been exposed to the topic via a television program and interactions with others on the team . common biases included believing that participants ' cah - related identities were different than dichotomous sexuality or sex / gender identities , that individuals may report shame surrounding their diagnoses / treatments , that family considerations would be important , and that this population was , at least theoretically , similar to other historically marginalized populations in regards to mental health . , and consisted of the following : transcription , domains , core ideas , and cross - analysis . six domains described the participants ' experiences and sub jective views of how having cah impacted their hrql , their mental health , and their recommendations for counseling ( table 1 ) . within each domain categories revealed general , typical , and variant themes , which , for some categories were further clarified by sub categories . 's standards were used to determine frequency cutoffs with general representing the majority ( 7 - 8 ) of cases , typical representing more than half ( 6 ) of the cases , and variant for less than half ( 25 ) but more than one case . this domain revealed seven categories encompassing issues related to the impact of illness and treatment on physical / physiological functioning . the first category , able to function well with no or minimal cah - related restrictions , was typical and included statements like , i function day to day ( and ) go to work like everybody else . the second , a variant subcategory , was normal functioning except for having to take medication daily . lastly , a subcategory titled weight concerns from medication included reports of weight gain , difficulty losing weight , and a resulting ambivalence about medication . lastly , in the variant reproductive functioning three participants noted issues with their fertility surrounding cah , while one stated , i will not be able to have kids and i do not think it 's related to cah . for example , upon reading details about her condition , one participant stated , in the letter it talked about having ambiguous genitals , and i was like , just devastated when i read that . i have always had this sense of not quite fitting in and i wonder now if ( cah ) contributed . another general category was emotions and cognitions related to cah , including two subcategories , negative , which was general , and positive , which was variant . typical for this domain was the category mental health concerns , and this included participant and family mental health and substance abuse histories . according to participants , some concerns were directly related to cah , ( e.g. now , i do not correct people when they say yes , sir unless it is going to be a continuing relationship ) , and some selected how much they told ( e.g.,if i travel with someone i want them to know i carry a stress dose but that 's about it ) . this category included ; negative feelings and experiences by family members and friends like frustration , guilt , and embarrassment as typified by the following quote : he ( husband ) does not like it because it 's frustrating because i 'll go months and months being sick and there 's no light at the end of the tunnel , as well as no impact on friends and family . some participants spoke about organizations like congenital adrenal hyperplasia research and support foundation ( cares ) or congenitaladrenalhyperplasia.org with statements including , i think the best thing i found was this website where we all share and now that makes me feel less alone . , going to church again has helped me accept cah ) , and doctors ( e.g. lastly , and also typical , participants reported having never met anyone else with cah . this domain described women 's experience with health care ( including both physical and mental health ) and was comprised of three categories , all of which were general . , i do not even feel that most endocrinologists i go to know much about the condition ) , and other physicians ( e.g. three participants were reportedly forced into treatment after suicide attempts or overdoses , and participants sought treatment voluntarily for issues both related to cah ( e.g. , the third time i got counseling was for eating issues and cah came up in that when we got deeper into them ) and unrelated to cah ( e.g. a second general sub category described positive interventions , including those related to cah like accepting and exploring cah and its relation to other issues or simply listening and empathizing ( e.g. you know , all the years i always held it in and did not talk to anybody about my feelings about the health problem i felt more content after i started sharing my feelings ) , as well as those interventions unrelated to cah ( e.g. lastly , and also variant , was the sub category clinicians ' knowledge of cah , which spoke to mental health workers ' prior knowledge or lack of knowledge about cah . examples included , the only time i really think about it ( cah ) is when i go to the doctor and it feels funny trying to explain to a shrink , yeah , they built me a vagina and they cut off my nerve center . this domain included five categories of recommendations for medical and mental health professionals . , a sickly therapist who could understand that sickness ) , therapeutic approaches and techniques ( e.g. , you still kind of have to fall back to what the individual needs of that person are ) , and foci of therapy ( e.g. the first , the need for more education about cah , emphasized the importance of increasing basic awareness and knowledge about the disease . the second , things to consider when treating individuals with cah , went into greater detail about specific things for professionals to be aware of and contained two subcategories . the second included comments for therapists about the medical considerations related to cah ( e.g. if they were able to find a good ( internet message ) board , like if you did not know about congenitaladrenalhyperplasia.org , they could inform you about it and you could talk to other people , ) and the importance of access to and timing of therapeutic services for both individuals ( e.g. the first typical category was generational and cohort differences in women with cah and contained participant observations about differences between individuals with cah of different ages as well as over time , as knowledge about cah increases . the next two categories , availability and appropriateness of care and insurance and other financial concerns related to cah , were both typical , and related to concerns about receiving adequate health care . the aim of this study was to determine the manner in which having cah impacts hrql , mental health experiences , and counseling needs for women diagnosed with this condition . most importantly , they indicated that for women with cah , feelings of normalcy are not solely explained by physical or functional outcomes but that the social and emotional well - being aspects of hrql play an important role in recommendations for treatment . these findings provide critical information for both medical and mental health practitioners working with women diagnosed with cah . however , according to the results of the current study , certain aspects of hrql , especially some physical concerns , may actually be more salient of a concern for clinicians than for individuals . for example , although the desire to maximize fertility is something which often plays an important role in determining the sex of a newborn with dsd , and was a concern reported by women with cah in another study , this theme was variant , with just half of our participants bringing up fertility and only half of those mentioning it as a physical problem . similarly , though research has often focused on concerns with sexual / genital functioning ( see for review ) , for many women in this study it was a non issue given their satisfaction with their sexual functioning or lack of interest in penetrative sex . , having to take medication ) , and women described themselves as functioning normally both in childhood and adulthood . they attributed this to parents , friends , and partners not treating them as sick , to effective medical interventions , and to their own coming to terms with cah . in relation to social functioning , results from the study provided additional support to previous findings , which suggest that cah does negatively impact participants ' relationships [ 7 , 15 ] . however , supportive relationships were more common than unsupportive ones in the present study , with participants emphasizing the importance of positive , normalizing interactions between parents , family , and partners . similarly , though the impact of cah on dating was noted , most participants reported being in very satisfying and supportive relationships , findings which somewhat contradict those from prior research , which found women less likely to engage in intimate relationships . for both areas , the negative impact of cah was more present in women 's anxiety about disclosing or in fears of others ' reactions than actual negative responses from others . previously documented concerns regarding increased mental illness among individuals with cah were reflected in the results of this study . for those participants who saw them as cah - related , they more often attributed them to physiological changes in mood and to external problems with the way physicians and families handled or disclosed cah rather than from internal processes related to aspects of sexual or gender identity . it is , however , important to note that many participants saw their mental health concerns as typical and unrelated to issues of cah , or were unsure of a link between cah and their emotional or mental health functioning . similarly , though not all participants had received counseling , all shared a wide range of reasons and goals for counseling , the majority of which were unrelated to cah . unlike their experience with medical professionals , which were more negative than positive , as described both in this study and in one other , the reverse was true for experiences with mental health practitioners . similarly , participants wanted both mental health and medical clinicians to be more attuned to the medical implications and the impact of cah on emotions and mood regulation , which suggests that general knowledge about cah on its own is important , but must be integrated with a tailored understanding of the individual . a related finding , noteworthy for practitioners working with individuals with cah , was that six of the eight participants reported never having met anyone with cah . it is unlikely that individuals would know others with cah because the population is small in comparison to the broader population , and disclosure of this information to others is rare . interestingly , few participants recommended that health care professionals connect them with support groups , which would seem to be one solution to deter isolation for individuals with cah . , between doctors and patients , or individuals and friends ) and societal norms or challenges which may impact them . while attempts were made to examine cah degree - or - type - specific factors related to hrql , almost all of our participants were diagnosed in infancy with salt wasting type . similarly related to sampling , participants self - selected into our study , these individuals may be relatively healthy and interested in therapeutic issues pertaining to cah as compared to individuals who chose not to participate in the study . as such , our sample may not be representative of the broader cah population , including those individuals who hold strong negative feelings towards cah research or who do not identify strongly as a woman with cah . also , much of the research on cah exists within a larger dsd literature base , and this study did not include individuals with any other dsd diagnoses . indeed , future research could expand upon these findings on identity in women with cah by further exploring variables related to identity development processes . , intersex , dsd , cah , gender queer ) and to recognize the importance of parallel identities ( i.e. lastly , given our findings on the role of systemic challenges , we would like to further emphasize prior recommendations that clinicians consider diversity issues and social stigma and serve as advocates for girls and women with cah . when ending our phone interviews , several participants shared their reasons for participating , it 's hard , you know , just growing up with it and i know that from my situation , and i want others to feel more comfortable in the future , and i figure anything that can help with the next generation is you know , i 'm here .

heart failure ( hf ) predominantly affects seniors , many of whom are frail and disabled . according to a recent systematic review , the prevalence of hf in long - term care ( ltc ) homes , which provide 24-hour nursing care to frail persons no longer able to reside in the community , reaches 20% . the one - year mortality of hf in ltc reaches 40% , a rate 50% higher than among residents without hf . hf accounts for approximately 20% of transfers of ltc residents to hospital , and it is considered that many admissions and resulting complications could be prevented with better hf management in ltc . older persons with hf are less likely to be prescribed recommended hf therapies , despite evidence that these can be beneficial even among frail seniors . an important barrier to appropriate prescribing of hf medications to frail seniors is diagnostic uncertainty . the diagnosis , treatment , and prognosis of hf in older adults is often complicated by geriatric syndromes including frailty and psychogeriatric disorders . frail older hf patients , particularly those with difficulty completing activities of daily living , often manifest atypical signs and symptoms , leading to diagnostic delays , inappropriate prescribing , functional decline , and increased health care utilization . furthermore , when an older person is admitted to ltc , the transfer of health information from sending organizations is often inadequate . such poor transitions have been associated with suboptimal care and an increased risk of hospitalization and complications . ensuring the adequacy of diagnostic information upon ltc admission is crucial for optimal hf management . the objective of this paper is to determine the utility of the admission clinical assessment for ltc residents in confirming a prior hf diagnosis . the geriatric outcomes and longitudinal decline in heart failure ( gold - hf ) study took place in south - central ontario from february 2004 to november 2006 , and included hamilton ( 25 ltc homes ) , cambridge ( seven homes ) , and kitchener - waterloo ( nine homes ) . the gold - hf study was a prospective longitudinal study designed to compare over a one - year period the clinical course of newly admitted ltc residents with hf to those without hf . this study complies with the declaration of helsinki , was approved by the research ethics board of mcmaster university , and informed consent was obtained from all subjects or guardians . newly admitted and consecutive ltc residents aged 65 years or over were considered for inclusion . excluded were residents with advanced malignant or non - malignant illness and expected to die within 6 weeks ; those admitted from another ltc home ( unless they had been residing there less than 6 weeks ) ; those admitted to ltc for temporary respite to primary caregivers and expected to return to the community ; and those for whom informed consent could not be obtained . staff at participating homes sought permission from new residents or substitute decision - makers for referral to study nurses , who were then allowed to formally approach potential participants for consent . the period of 6 weeks for inclusion into the study was required by ltc homes to complete routine admission procedures prior to resident recruitment . a trained research nurse assessed all participants and reviewed the ltc home chart . for patients with communication difficulties or cognitive impairment baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . of 546 resident charts reviewed , there were 75 cases in which reviewers disagreed and 12 cases in which both reviewers were initially uncertain ( weighted kappa = 0.73 ) ; all cases were resolved by discussion . the diagnosis of hf was based on accepted diagnostic criteria . the presence of other diagnoses was also verified from review of this information . baseline characteristics were summarized using mean and standard deviation for continuous measures , frequency , and percentage for categorical measures , and compared using t - test and chi - square test , respectively . unconditional estimates of sensitivity , specificity , positive predictive value , negative predictive value , and c - statistic were calculated for an admission history of hf , history of fluid on the lungs , symptoms and signs of hf , other indicators considered were co - morbidities , including hypertension , coronary artery disease ( cad ) , diabetes mellitus , atrial fibrillation , and renal insufficiency ( defined as a calculated creatinine clearance < 60 mls / min ) , and use of hf medications ( loop diuretics , acei , -blockers , and digoxin ) . multiple logistic regression was used to identify the strongest predictors of a prior hf diagnosis . multi - collinearity was considered and determined not to be significant , with estimates of the pearson correlation less than 0.7 and variance inflation factors less than 2.5 . stepwise elimination was used to develop the final model , with remaining variables significant at the 5% level . model fit was assessed using the likelihood ratio test , comparing the full and reduced models , and was found not to be significant . the integrated discriminant improvement ( idi ) index was determined by sequentially adding variables in order of most to least informative c - statistic using the sas rocplus macro . variables were included in the final model if inclusion resulted in significant improvement at the 5% level . all analyses were conducted in sas version 9.1 ( sas institute inc . , cary , nc ) . newly admitted and consecutive ltc residents aged 65 years or over were considered for inclusion . excluded were residents with advanced malignant or non - malignant illness and expected to die within 6 weeks ; those admitted from another ltc home ( unless they had been residing there less than 6 weeks ) ; those admitted to ltc for temporary respite to primary caregivers and expected to return to the community ; and those for whom informed consent could not be obtained . staff at participating homes sought permission from new residents or substitute decision - makers for referral to study nurses , who were then allowed to formally approach potential participants for consent . the period of 6 weeks for inclusion into the study was required by ltc homes to complete routine admission procedures prior to resident recruitment . a trained research nurse assessed all participants and reviewed the ltc home chart . for patients with communication difficulties or cognitive impairment , history was obtained from family caregivers . baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . of 546 resident charts reviewed , there were 75 cases in which reviewers disagreed and 12 cases in which both reviewers were initially uncertain ( weighted kappa = 0.73 ) ; all cases were resolved by discussion . a trained research nurse assessed all participants and reviewed the ltc home chart . for patients with communication difficulties or cognitive impairment , history was obtained from family caregivers . baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . of 546 resident charts reviewed , there were 75 cases in which reviewers disagreed and 12 cases in which both reviewers were initially uncertain ( weighted kappa = 0.73 ) ; all cases were resolved by discussion . baseline characteristics were summarized using mean and standard deviation for continuous measures , frequency , and percentage for categorical measures , and compared using t - test and chi - square test , respectively . unconditional estimates of sensitivity , specificity , positive predictive value , negative predictive value , and c - statistic were calculated for an admission history of hf , history of fluid on the lungs , symptoms and signs of hf , baseline physical findings , and calculated creatinine clearance . other indicators considered were co - morbidities , including hypertension , coronary artery disease ( cad ) , diabetes mellitus , atrial fibrillation , and renal insufficiency ( defined as a calculated creatinine clearance < 60 mls / min ) , and use of hf medications ( loop diuretics , acei , -blockers , and digoxin ) . multiple logistic regression was used to identify the strongest predictors of a prior hf diagnosis . multi - collinearity was considered and determined not to be significant , with estimates of the pearson correlation less than 0.7 and variance inflation factors less than 2.5 . stepwise elimination was used to develop the final model , with remaining variables significant at the 5% level . model fit was assessed using the likelihood ratio test , comparing the full and reduced models , and was found not to be significant . the integrated discriminant improvement ( idi ) index was determined by sequentially adding variables in order of most to least informative c - statistic using the sas rocplus macro . variables were included in the final model if inclusion resulted in significant improvement at the 5% level . all analyses were conducted in sas version 9.1 ( sas institute inc . , cary , nc ) . the study enrolled 546 residents , and analysis will focus on 449 residents for whom creatinine clearance could be estimated . mean age was 84.36.5 years and 66% were women , and a prior hf diagnosis was confirmed in 97 ( 21.6% ) residents . almost half were admitted from hospital , with hf patients more likely to have been so . participants had multiple medical co - morbidities , were prescribed multiple medications , and had significant functional and cognitive deficits . residents with prior hf were older , more likely to have hypertension , cad and atrial fibrillation , and had more acute care visits prior to ltc admission , than those with no prior hf . results of an echocardiogram were available for 69 ( 71% ) of residents with prior hf , 67% of whom had a left ventricular ejection fraction greater than 40% ( preserved ejection fraction ) . baseline characteristics of ltc residents renal insufficiency is define as a calculated creatinine clearance < 60 mls / min , according to the cockcroft - gault equation . all residents on lipid lowering agents were receiving hmg - coa reductase inhibitors , and one resident was also receiving treatment with a fibrate . hf = heart failure ; ltc = long - term care ; ed = emergency department ; cad = coronary artery disease ( history of myocardial infarction , angina / unstable angina , or history of coronary revascularization ) ; pvd = peripheral vascular disease ( history of intermittent claudication , revascularization , or abdominal aortic aneurysm ) ; cvd = cerebrovascular disease ( history of transient ischemic attack , stroke , or revascularization procedure ) ; lvef = left ventricular ejection fraction table 2 presents data from the admission clinical assessment . a history of hf and symptoms and signs of hf were more common in patients with prior hf . however , some were also common in residents without hf , such as peripheral edema , which was reported by almost 60% of residents without hf . among residents without prior hf , there were no statistically significant differences in the prevalence of physical findings of peripheral edema and auscultatory rales between both groups . jugular venous pressures ( jvp ) were generally in the normal range and third heart sounds infrequent , suggesting that most residents are clinically stable following ltc admission . heart failure history , symptoms , and signs elicited at the baseline assessment either from the resident / caregiver interview or from the ltc home chart review not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer , resulting in missing data . table 3 presents diagnostic properties of elements from the admission clinical assessment pertinent to hf . elements pertaining to dyspnea have more modest sensitivities and specificities ; specificity generally rises while sensitivity falls with increasing dyspnea severity . orthopnea and paroxysmal nocturnal dyspnea ( pnd ) were relatively specific , though not very sensitive . in contrast , history of peripheral edema was sensitive , but non - specific . there are notable differences between the properties of elements derived from the ltc chart and those obtained from the resident / caregiver interview . for histories of peripheral edema and varying degrees of dyspnea , the sensitivity of chart - derived information is uniformly lower , and the specificity higher , than that of interview - derived information . however , differences are less marked for elements suggestive of severe hf , such as pnd , orthopnea , or dyspnea at rest or when performing basic activities of daily living . with respect to physical findings , both auscultatory rales and peripheral edema had poor sensitivity and positive predictive value , and modest specificity and negative predictive values . the utility of the jvp and auscultion for a third heart sound was limited in this sample . table 4 presents the utility of cardiovascular co - morbidities and admission medications with respect to a prior hf diagnosis . the absence of cardiovascular morbidities was associated with good to very good negative predictive values , particularly a history of cad . the sensitivity and specificity of individual prescribed medications were modest , other than for digoxin , which appears to be specific for a prior hf diagnosis . properties of individual elements of the admission clinical assessment to predict the diagnosis of hf not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer . hf = heart failure ; pnd = paroxysmal nocturnal dyspnea ; sn = sensitivity ; sp = specificity ; ppv = positive predictive value ; npv = negative predictive value ; adls = activities of daily living ; acei = angiotensin converting enzyme inhibitor ; n / a = not applicable results of the multivariate analysis to predict the diagnosis of hf based on admission clinical characteristics , cardiovascular co - morbidities , and medication profile not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer . aor = adjusted odds ratio ; ci = confidence interval ; hf = heart failure ; pnd = paroxysmal nocturnal dyspnea ; adls = activities of daily living ; idi = integrated discrimination improvement index logistic regression models were derived to determine which combination of elements from the admission assessment was most predictive of a prior hf diagnosis . the final model includes histories of hf , fluid on the lungs , orthopnea , cad , and the use of -blockers and furosemide . the c - statistic was 0.910 ; the idi method arrived at the same final model . this study confirms the high prevalence of hf in ltc and the complexity of residents with this condition . ensuring an accurate hf diagnosis during the transition of residents into ltc is crucial for optimal management of this condition . this study provides important information on the value of the admission assessment of new ltc residents in confirming a prior hf diagnosis . our findings are consistent with other literature showing that features of hf can be non - specific in frail seniors . for example , dyspnea , a cardinal symptom of hf , is only reported for 38.5% to 62.4% residents with prior hf . we observed differences in the sensitivity and specificity of elements of the admission assessment depending on whether information was obtained from the resident chart or from resident / caregiver interview . direct history was more sensitive but less specific than chart information for peripheral edema , orthopnea , and dyspnea , though differences were less marked for more severe symptoms such as resting dyspnea . these suggest discrepancy in the importance ascribed to hf symptoms by residents and by ltc staff recording observations in resident charts . symptoms are experienced subjectively by patients , and thus ltc staff charting may be inherently sensitive to symptom identification than resident reporting . these data are consistent with a recently reported communication gap between ltc staff and residents , either leading to under - reporting of mild symptoms by residents , or to staff erroneously dismissing milder symptoms as normal aging . these data reinforce the critical importance of a thorough history , including collection of collateral information , in order to accurately assess frail seniors with hf . that a history of hf is predictive of a prior hf diagnosis may seem self - evident , but it is an important finding given the uncertainty surrounding the accuracy of health information available to clinicians about new ltc residents . older persons with hf are often hospitalized repeatedly , experience functional decline , and ultimately discharged to ltc . in our study it is therefore likely that because most new ltc residents with hf had a recent hospitalization , available clinical information was more reliable . furthermore , all ltc residents in ontario undergo standardized assessment using the rai ( home care ) instrument prior to admission . the rai family of instruments have been shown to have a high positive predictive value for hf . fluid on the lungs may be useful to explain hf to lay persons ( www.heartfailure.org/eng_site/hf_lungs.asp ) . while the low sensitivity implies that the idiom is not universally used , the high specificity suggests that it is an effective descriptor . specifically , using this idiom when interviewing a ltc resident / caregiver improves the predictive value of the assessment . our data are consistent with other studies in older patients . our findings that orthopnea and pnd are specific but not very sensitive are similar to those from several community - based epidemiologic studies in the u.s . and europe . a systematic review of studies of the utility of signs and symptoms for detecting hf in primary care showed sensitivities of 2947% and 44% , and specificities of 7398% and 89% , for pnd and orthopnea , respectively . in contrast , our study found lower specificities for histories of peripheral edema and dyspnea , likely reflecting the non - specific presentation of hf in ltc residents . physical findings of hf were infrequent in this sample and unhelpful for confirming a prior hf diagnosis , possibly reflecting the relative clinical stability of new ltc residents . these results do not negate the importance of these physical examination maneuvers when assessing acutely unwell residents . cardiovascular co - morbidities were common in the entire sample . not surprisingly , cad and hypertension were relatively sensitive for a prior hf diagnosis , though only cad was included in the final model . admission hf medications had poor to modest sensitivity for prior hf , consistent with their underuse in older patients . the absence of hf medications from the admission drug profile makes a prior hf diagnosis less likely . data were collected prospectively , and we obtained substantial clinical information from multiple sources to facilitate the confirmation of a prior hf diagnosis by two independent reviewers . this information is not readily available in usual practice . though our procedures may have missed a small proportion of residents with mild hf who might never have been hospitalized , it is likely that the majority of those with prior hf were identified . the prevalence of hf in our sample is consistent with that of a recent systematic review . we relied on residents / caregivers to accurately recall their medical history , and ltc staff to identify and accurately document symptoms and signs among residents for whom they cared , limitations that reflect the clinical conditions under which canadian ltc clinicians operate . since the completion of this study , the indications for aldosterone antagonists in the care of hf with reduced ejection fraction have expanded . while it is possible that this has translated into greater usage of this class of medications in the ltc setting , it is not clear whether this would improve upon the diagnostic accuracy of hf in ltc , given the limited utility of other hf medications in this regard . finally , the average accrual rate for this study was lower than expected . recruitment required that potential participants be first contacted by ltc staff within six weeks of admission , a period of turmoil during which clinical and administrative priorities take precedent over research studies . finally , nine ltc homes underwent significant expansion and were unable to participate in our study , but also diverted all new admissions from other participating homes for extended periods . despite these concerns , clinical characteristics of residents enrolled in this study are similar to those from other studies , providing reassurance as to the representativeness of the sample . in summary , hf is prevalent ltc . correctly diagnosing hf is crucial to ensure that affected residents receive optimal management . our data suggest that the most useful indicator of a prior hf diagnosis in new ltc residents include histories of hf , fluid on the lungs , orthopnea , cad , and use of loop diuretics and -blockers . the transfer of health information during the transition to ltc is problematic , and clinicians must rely on limited information upon which to formulate a diagnosis of hf . our findings reinforce the importance of a thorough history , including collateral information from family caregivers , when assessing frail seniors upon admission to ltc .

backgroundheart failure ( hf ) is common in long - term care ( ltc ) . diagnostic uncertainty is important barrier to optimal hf management , stemming from inadequate health information transfer upon ltc admission . we determine the utility of admission clinical information to confirm a hf diagnosis in new ltc residents.methodsthis was a prospective cohort study . from february 2004 to november 2006 , information about new residents from 41 ltc homes in ontario , canada , was collected from residents and caregivers , and all available health records . a prior hf diagnosis was confirmed by consensus review of available data by two independent experts . multivariate modelling was utilized to determine the utility of the admission clinical assessment in confirming a prior hf diagnosis.resultsa total of 449 residents were included for analysis , aged 84.36.5 years , and 21.6% had a prior hf diagnosis . the most useful clinical item for diagnosing hf was a history of hf . the final model included history of hf ( or [ odds ratio ] 13.66 , 95% ci 6.6128.24 ) , fluid on the lungs ( or 2.01 , 95% ci 1.043.89 ) , orthopnea ( or 1.76 , 95% ci 0.933.33 ) , taking -blocker ( or 2.09 , 95% ci 1.103.94 ) , taking loop diuretics ( or 2.11 , 95% ci 1.123.98 ) , and history of coronary artery disease ( or 2.83 , 95% ci 1.425.64).conclusionelements of the clinical assessment for new ltc residents can help confirm a prior hf diagnosis . an admission history of hf is highly predictive .

INTRODUCTION METHODS Participants Data Collection Baseline Assessment Ascertaining a Prior Heart Failure Diagnosis Statistical Considerations RESULTS DISCUSSION CONCLUSION CONFLICT OF INTEREST DISCLOSURES

heart failure ( hf ) predominantly affects seniors , many of whom are frail and disabled . according to a recent systematic review , the prevalence of hf in long - term care ( ltc ) homes , which provide 24-hour nursing care to frail persons no longer able to reside in the community , reaches 20% . the one - year mortality of hf in ltc reaches 40% , a rate 50% higher than among residents without hf . hf accounts for approximately 20% of transfers of ltc residents to hospital , and it is considered that many admissions and resulting complications could be prevented with better hf management in ltc . an important barrier to appropriate prescribing of hf medications to frail seniors is diagnostic uncertainty . the diagnosis , treatment , and prognosis of hf in older adults is often complicated by geriatric syndromes including frailty and psychogeriatric disorders . furthermore , when an older person is admitted to ltc , the transfer of health information from sending organizations is often inadequate . ensuring the adequacy of diagnostic information upon ltc admission is crucial for optimal hf management . the objective of this paper is to determine the utility of the admission clinical assessment for ltc residents in confirming a prior hf diagnosis . the geriatric outcomes and longitudinal decline in heart failure ( gold - hf ) study took place in south - central ontario from february 2004 to november 2006 , and included hamilton ( 25 ltc homes ) , cambridge ( seven homes ) , and kitchener - waterloo ( nine homes ) . the gold - hf study was a prospective longitudinal study designed to compare over a one - year period the clinical course of newly admitted ltc residents with hf to those without hf . this study complies with the declaration of helsinki , was approved by the research ethics board of mcmaster university , and informed consent was obtained from all subjects or guardians . staff at participating homes sought permission from new residents or substitute decision - makers for referral to study nurses , who were then allowed to formally approach potential participants for consent . the period of 6 weeks for inclusion into the study was required by ltc homes to complete routine admission procedures prior to resident recruitment . for patients with communication difficulties or cognitive impairment baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . the diagnosis of hf was based on accepted diagnostic criteria . the presence of other diagnoses was also verified from review of this information . baseline characteristics were summarized using mean and standard deviation for continuous measures , frequency , and percentage for categorical measures , and compared using t - test and chi - square test , respectively . unconditional estimates of sensitivity , specificity , positive predictive value , negative predictive value , and c - statistic were calculated for an admission history of hf , history of fluid on the lungs , symptoms and signs of hf , other indicators considered were co - morbidities , including hypertension , coronary artery disease ( cad ) , diabetes mellitus , atrial fibrillation , and renal insufficiency ( defined as a calculated creatinine clearance < 60 mls / min ) , and use of hf medications ( loop diuretics , acei , -blockers , and digoxin ) . multiple logistic regression was used to identify the strongest predictors of a prior hf diagnosis . multi - collinearity was considered and determined not to be significant , with estimates of the pearson correlation less than 0.7 and variance inflation factors less than 2.5 . stepwise elimination was used to develop the final model , with remaining variables significant at the 5% level . variables were included in the final model if inclusion resulted in significant improvement at the 5% level . newly admitted and consecutive ltc residents aged 65 years or over were considered for inclusion . the period of 6 weeks for inclusion into the study was required by ltc homes to complete routine admission procedures prior to resident recruitment . baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . baseline information collection included demographic data and medical history , hf signs and symptoms , and the most recent diagnostic investigations . medical history information included the following disease diagnoses : pulmonary disease , coronary artery disease , valvular heart disease , hypertension , atrial fibrillation , hyperlipidemia , peripheral vascular disease ( pvd ) , cerebrovascular events , diabetes mellitus , dementia , arthritis , osteoporosis and/or fragility fractures , cancer , renal insufficiency , and mood disorders . specific note was made of baseline use of angiotensin converting enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arb ) , beta - adrenergic receptor blockers ( -blockers ) , spironolactone , digoxin , loop diuretics , anti - platelets , anticoagulants , calcium channel blockers , antidepressants , and major and minor tranquilizers . assessment of functional , cognitive , and neuropsychiatric status , based on a review of the ltc chart and interview with the resident s primary caregiver or nurse , was performed using the barthel index ( bi ) , the minimum data set cognitive scale , the cohen - mansfield agitation inventory , and neuropsychiatric inventory . in order to ascertain a prior hf diagnosis , the research nurse obtained consent from participants ( or substitute decision - makers ) to search for medical records from previous physicians , hospitalizations , and diagnostic procedures , information generally not readily available to admitting ltc homes . all data thus gathered were reviewed independently by two experts ( gah , rsm ) , who categorized the diagnosis of hf as true , possible , nor not present ; disagreements resolved through discussion . baseline characteristics were summarized using mean and standard deviation for continuous measures , frequency , and percentage for categorical measures , and compared using t - test and chi - square test , respectively . unconditional estimates of sensitivity , specificity , positive predictive value , negative predictive value , and c - statistic were calculated for an admission history of hf , history of fluid on the lungs , symptoms and signs of hf , baseline physical findings , and calculated creatinine clearance . other indicators considered were co - morbidities , including hypertension , coronary artery disease ( cad ) , diabetes mellitus , atrial fibrillation , and renal insufficiency ( defined as a calculated creatinine clearance < 60 mls / min ) , and use of hf medications ( loop diuretics , acei , -blockers , and digoxin ) . multiple logistic regression was used to identify the strongest predictors of a prior hf diagnosis . stepwise elimination was used to develop the final model , with remaining variables significant at the 5% level . model fit was assessed using the likelihood ratio test , comparing the full and reduced models , and was found not to be significant . variables were included in the final model if inclusion resulted in significant improvement at the 5% level . the study enrolled 546 residents , and analysis will focus on 449 residents for whom creatinine clearance could be estimated . mean age was 84.36.5 years and 66% were women , and a prior hf diagnosis was confirmed in 97 ( 21.6% ) residents . residents with prior hf were older , more likely to have hypertension , cad and atrial fibrillation , and had more acute care visits prior to ltc admission , than those with no prior hf . results of an echocardiogram were available for 69 ( 71% ) of residents with prior hf , 67% of whom had a left ventricular ejection fraction greater than 40% ( preserved ejection fraction ) . baseline characteristics of ltc residents renal insufficiency is define as a calculated creatinine clearance < 60 mls / min , according to the cockcroft - gault equation . hf = heart failure ; ltc = long - term care ; ed = emergency department ; cad = coronary artery disease ( history of myocardial infarction , angina / unstable angina , or history of coronary revascularization ) ; pvd = peripheral vascular disease ( history of intermittent claudication , revascularization , or abdominal aortic aneurysm ) ; cvd = cerebrovascular disease ( history of transient ischemic attack , stroke , or revascularization procedure ) ; lvef = left ventricular ejection fraction table 2 presents data from the admission clinical assessment . a history of hf and symptoms and signs of hf were more common in patients with prior hf . however , some were also common in residents without hf , such as peripheral edema , which was reported by almost 60% of residents without hf . among residents without prior hf , there were no statistically significant differences in the prevalence of physical findings of peripheral edema and auscultatory rales between both groups . jugular venous pressures ( jvp ) were generally in the normal range and third heart sounds infrequent , suggesting that most residents are clinically stable following ltc admission . heart failure history , symptoms , and signs elicited at the baseline assessment either from the resident / caregiver interview or from the ltc home chart review not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer , resulting in missing data . table 3 presents diagnostic properties of elements from the admission clinical assessment pertinent to hf . in contrast , history of peripheral edema was sensitive , but non - specific . for histories of peripheral edema and varying degrees of dyspnea , the sensitivity of chart - derived information is uniformly lower , and the specificity higher , than that of interview - derived information . however , differences are less marked for elements suggestive of severe hf , such as pnd , orthopnea , or dyspnea at rest or when performing basic activities of daily living . the utility of the jvp and auscultion for a third heart sound was limited in this sample . table 4 presents the utility of cardiovascular co - morbidities and admission medications with respect to a prior hf diagnosis . the absence of cardiovascular morbidities was associated with good to very good negative predictive values , particularly a history of cad . the sensitivity and specificity of individual prescribed medications were modest , other than for digoxin , which appears to be specific for a prior hf diagnosis . properties of individual elements of the admission clinical assessment to predict the diagnosis of hf not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer . hf = heart failure ; pnd = paroxysmal nocturnal dyspnea ; sn = sensitivity ; sp = specificity ; ppv = positive predictive value ; npv = negative predictive value ; adls = activities of daily living ; acei = angiotensin converting enzyme inhibitor ; n / a = not applicable results of the multivariate analysis to predict the diagnosis of hf based on admission clinical characteristics , cardiovascular co - morbidities , and medication profile not all residents underwent a complete physical examination by the research nurses due to refusal to do so , limited cooperation , significantly limited bed mobility or inability to transfer . aor = adjusted odds ratio ; ci = confidence interval ; hf = heart failure ; pnd = paroxysmal nocturnal dyspnea ; adls = activities of daily living ; idi = integrated discrimination improvement index logistic regression models were derived to determine which combination of elements from the admission assessment was most predictive of a prior hf diagnosis . the final model includes histories of hf , fluid on the lungs , orthopnea , cad , and the use of -blockers and furosemide . the c - statistic was 0.910 ; the idi method arrived at the same final model . this study confirms the high prevalence of hf in ltc and the complexity of residents with this condition . ensuring an accurate hf diagnosis during the transition of residents into ltc is crucial for optimal management of this condition . this study provides important information on the value of the admission assessment of new ltc residents in confirming a prior hf diagnosis . our findings are consistent with other literature showing that features of hf can be non - specific in frail seniors . for example , dyspnea , a cardinal symptom of hf , is only reported for 38.5% to 62.4% residents with prior hf . we observed differences in the sensitivity and specificity of elements of the admission assessment depending on whether information was obtained from the resident chart or from resident / caregiver interview . direct history was more sensitive but less specific than chart information for peripheral edema , orthopnea , and dyspnea , though differences were less marked for more severe symptoms such as resting dyspnea . these suggest discrepancy in the importance ascribed to hf symptoms by residents and by ltc staff recording observations in resident charts . symptoms are experienced subjectively by patients , and thus ltc staff charting may be inherently sensitive to symptom identification than resident reporting . that a history of hf is predictive of a prior hf diagnosis may seem self - evident , but it is an important finding given the uncertainty surrounding the accuracy of health information available to clinicians about new ltc residents . in our study it is therefore likely that because most new ltc residents with hf had a recent hospitalization , available clinical information was more reliable . furthermore , all ltc residents in ontario undergo standardized assessment using the rai ( home care ) instrument prior to admission . fluid on the lungs may be useful to explain hf to lay persons ( www.heartfailure.org/eng_site/hf_lungs.asp ) . specifically , using this idiom when interviewing a ltc resident / caregiver improves the predictive value of the assessment . a systematic review of studies of the utility of signs and symptoms for detecting hf in primary care showed sensitivities of 2947% and 44% , and specificities of 7398% and 89% , for pnd and orthopnea , respectively . in contrast , our study found lower specificities for histories of peripheral edema and dyspnea , likely reflecting the non - specific presentation of hf in ltc residents . physical findings of hf were infrequent in this sample and unhelpful for confirming a prior hf diagnosis , possibly reflecting the relative clinical stability of new ltc residents . cardiovascular co - morbidities were common in the entire sample . not surprisingly , cad and hypertension were relatively sensitive for a prior hf diagnosis , though only cad was included in the final model . the absence of hf medications from the admission drug profile makes a prior hf diagnosis less likely . data were collected prospectively , and we obtained substantial clinical information from multiple sources to facilitate the confirmation of a prior hf diagnosis by two independent reviewers . though our procedures may have missed a small proportion of residents with mild hf who might never have been hospitalized , it is likely that the majority of those with prior hf were identified . the prevalence of hf in our sample is consistent with that of a recent systematic review . we relied on residents / caregivers to accurately recall their medical history , and ltc staff to identify and accurately document symptoms and signs among residents for whom they cared , limitations that reflect the clinical conditions under which canadian ltc clinicians operate . since the completion of this study , the indications for aldosterone antagonists in the care of hf with reduced ejection fraction have expanded . while it is possible that this has translated into greater usage of this class of medications in the ltc setting , it is not clear whether this would improve upon the diagnostic accuracy of hf in ltc , given the limited utility of other hf medications in this regard . recruitment required that potential participants be first contacted by ltc staff within six weeks of admission , a period of turmoil during which clinical and administrative priorities take precedent over research studies . finally , nine ltc homes underwent significant expansion and were unable to participate in our study , but also diverted all new admissions from other participating homes for extended periods . despite these concerns , clinical characteristics of residents enrolled in this study are similar to those from other studies , providing reassurance as to the representativeness of the sample . in summary , hf is prevalent ltc . correctly diagnosing hf is crucial to ensure that affected residents receive optimal management . our data suggest that the most useful indicator of a prior hf diagnosis in new ltc residents include histories of hf , fluid on the lungs , orthopnea , cad , and use of loop diuretics and -blockers . the transfer of health information during the transition to ltc is problematic , and clinicians must rely on limited information upon which to formulate a diagnosis of hf . our findings reinforce the importance of a thorough history , including collateral information from family caregivers , when assessing frail seniors upon admission to ltc .

the comparative analysis of dna molecules looks back on a history of over 40 years . increasingly complex models of nucleotide substitution patterns at point mutations have been developed ( sullivan and joyce 2005 ) and are routinely applied on dna sequence markers . another type of mutation , the gain or loss of nucleotides within a defined locus , termed insertion / deletion ( indel ) mutation , has received much less attention ( lunter et al . this lack of attention is not due to a lack of relevance on the contrary , indels constitute a considerable fraction of mutations in coding and noncoding parts of the genome , are responsible for copy number variants , and are the signature of transposable elements ( korbel et al . rather , the lack of attention is due to a lack of profound knowledge on the evolution of indels ( ogden and rosenberg 2007 and references therein , sunday and hart 2013 ) , for which reason they are usually considered uninformative and removed during data preparation , except in the context of gene finding ( kellis et al . the special case of recent indel , henceforth reindel mutations , that is , mutations where both ancestral and derived state are seen in the sample , allows us witnessing indel evolution in real time . knowledge of reindels based on broad sampling should therefore facilitate a better understanding of indel evolution generally . the preconditions for reindel identification are that we know the pedigree in our sample and that our sample is large ( ellegren 2000 ; schltterer 2000 ) ; examples of such situations include studies on extrapair paternity , intraspecific brood parasitism , and eusocial societies , ideally with a single female reproductive ( see fig.1 for an example study system ) . whenever we find previously unknown allelic size variation in genotype data for such systems ( see box 1 for microsatellites as an example ) , reindels can be identified easily . for example , a validated allele detected in a singly mated female 's offspring that is unknown from the parents can be deduced to represent a recent mutation . the reindel can then be described in base - pair length and understood as either insertion or deletion . eusocial insects are among study systems that facilitate generating large population - genetic or population - genomic data sets for individuals with known pedigree and that are thus ideal for studying reindels . here , one of the 1,00010,000 workers ( steiner et al . 2004 ) of a colony of the ant lasius austriacus carries a worker sister at the pupal stage , all these workers stemming from the same mother and father ( steiner et al . microsatellites are noncoding , codominantly inherited dna loci consisting of simple sequence repeats ( ssrs ) , sometimes also termed short tandem repeats ( strs ) or simple sequence length polymorphisms ( sslps ) . due to frequent indel mutations via loss and gain of repeat units , the design of studies with known pedigree allows precise expectations on the number and size of alleles in the data set based on the knowledge of the parental alleles . the vast majority of recent mutations in microsatellites will cause variation in allele size ( estoup et al . 2002 ) , and any deviation from the original expectations thus is a reindel candidate . for correct data interpretation , validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error are necessary . hitherto studies on microsatellite evolution have shed some light on , for example , the factors increasing slippage events and multistep changes ( primmer et al . 1996 ; chakraborty et al . 2012 ) and the existence of differences between male and female germline ( anmarkrud et al . however , many questions remain such as about the influence of base composition of repeat motifs , about mating and/or sex - determination systems , about cross - taxa and cross - loci variation , and about differences between experimental and natural populations ( ellegren 2000 , 2004 ; schltterer 2000 ; leclercq et al . thinking about the increase of accuracy the improvement of nucleotide models of evolution brought about in phylogenetic applications , we can expect comparable advances in microsatellite - based analyses . currently existing microsatellite models such as the stepwise mutation , the generalized stepwise , or the k - allele model ( reviewed in estoup et al . 2002 ) are either rather simplistic or make rather unrealistic assumptions , both of which can lead to poor performance in empirical tests ( see , e.g. , balloux and lugon - moulin 2002 on stepwise - mutation - model - based rst values or , peery et al . 2012 on the reliability of microsatellite - based bottleneck tests for detecting recent population declines ) . however , with profound knowledge about microsatellite evolution , future models might incorporate the full range of relevant aspects for which maximum likelihood or bayesian approaches can make good estimates ( cf . potential implementations include different mutability rates according to motif sequence , motif and allele size , allele - frequency dependence , different probabilities for expansion or contraction events , and different rates for male and female germline . more realistic models will aid multiple research areas in ecology and evolution : kinship , parentage , and behavior analyses will be more accurate facilitating a better understanding of mating systems , dispersal patterns , and social organization ; better estimations of effective population sizes and detection of bottlenecks will aid nature conservation research ; hybridization and backcrossing patterns will be more correctly mirrored , which in turn will increase our understanding of these evolutionary forces ; aberrant modes of reproduction might be elucidated , phylogeography exploring the recent past improved , and signals of selection better recognized . microsatellites are noncoding , codominantly inherited dna loci consisting of simple sequence repeats ( ssrs ) , sometimes also termed short tandem repeats ( strs ) or simple sequence length polymorphisms ( sslps ) . due to frequent indel mutations via loss and gain of repeat units , the design of studies with known pedigree allows precise expectations on the number and size of alleles in the data set based on the knowledge of the parental alleles . the vast majority of recent mutations in microsatellites will cause variation in allele size ( estoup et al . 2002 ) , and any deviation from the original expectations thus is a reindel candidate . for correct data interpretation , validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error are necessary . hitherto studies on microsatellite evolution have shed some light on , for example , the factors increasing slippage events and multistep changes ( primmer et al . 1996 ; chakraborty et al . 2012 ) and the existence of differences between male and female germline ( anmarkrud et al . however , many questions remain such as about the influence of base composition of repeat motifs , about mating and/or sex - determination systems , about cross - taxa and cross - loci variation , and about differences between experimental and natural populations ( ellegren 2000 , 2004 ; schltterer 2000 ; leclercq et al . thinking about the increase of accuracy the improvement of nucleotide models of evolution brought about in phylogenetic applications , we can expect comparable advances in microsatellite - based analyses . currently existing microsatellite models such as the stepwise mutation , the generalized stepwise , or the k - allele model ( reviewed in estoup et al . 2002 ) are either rather simplistic or make rather unrealistic assumptions , both of which can lead to poor performance in empirical tests ( see , e.g. , balloux and lugon - moulin 2002 on stepwise - mutation - model - based rst values or , peery et al . 2012 on the reliability of microsatellite - based bottleneck tests for detecting recent population declines ) . however , with profound knowledge about microsatellite evolution , future models might incorporate the full range of relevant aspects for which maximum likelihood or bayesian approaches can make good estimates ( cf . potential implementations include different mutability rates according to motif sequence , motif and allele size , allele - frequency dependence , different probabilities for expansion or contraction events , and different rates for male and female germline . more realistic models will aid multiple research areas in ecology and evolution : kinship , parentage , and behavior analyses will be more accurate facilitating a better understanding of mating systems , dispersal patterns , and social organization ; better estimations of effective population sizes and detection of bottlenecks will aid nature conservation research ; hybridization and backcrossing patterns will be more correctly mirrored , which in turn will increase our understanding of these evolutionary forces ; aberrant modes of reproduction might be elucidated , phylogeography exploring the recent past improved , and signals of selection better recognized . a better understanding of indel evolution , achievable via the study of reindels , will allow the inclusion of indels as genetic variation in our analyses rather than their discard . our phylogenetic trees and genetic networks will be based on more and more solid information , and our population - genetic and population - genomic inferences will be more accurate ( see box 1 for microsatellite examples ) . this will have far - reaching effects on any research area using molecular data to make ecological and evolutionary inferences , such as speciation research , sociobiology , the study of species interactions , conservation genetics , invasion biology , and climate change biology . from the obviously vast range of potential implications of including indel information in the data analyses , there is broad consensus on the existence of a male mutation bias in vertebrates and some plants caused by the higher number of cell divisions during spermatogenesis than during oogenesis ( kirkpatrick and hall 2004 ) . anyway , the underlying factors shaping the extent of male mutation bias are still poorly understood ( bartosch - harlid et al . 2003 ; goetting - minesky and makova 2006 ) , information from invertebrates is scarce , and recent observations indicate that the mutation rate can be highly different among closely related species ( venn et al . thus , more information on patterns at individual loci and in different organisms is needed ( ellegren 2007 ) . such information will then open up additional research avenues ; for example , such bias may even influence the long - term persistence of populations with skewed sex allocation ( cotton and wedekind 2010 ) . the advent of next - generation sequencing ( ngs ) , the massive parallel sequencing of dna , has opened up a previously unthinkable array of opportunities ( andrew et al . possibly the most important consequence is that ngs makes available genomics for the study of nonmodel organisms ( hudson 2008 ; tautz et al . compared with population genetics , the promises of population genomics include improved identification of adaptive molecular variation as well as improved inferences about population demography and evolutionary history ( luikart et al . 2003 ; stapley et al . mutation rates in microsatellites tend to vary considerably within taxa , from 10 to 10 per site and generation , relatively homogenously across the tree of life ( bhargava and fuentes 2010 ) . in contrast , estimations of genome - wide rates of nonmicrosatellite indel mutations per site and generation are in the range of 2.1 10 ( sacharomyces cerevisiae ; lang and murray 2008 ) to 1.2 10 ( caenorhabditis elegans ; denver et al . 2004 ) , that is , two to eight orders of magnitude lower than the estimated rates of mutations in microsatellites . anyway , the sheer amount of data in ngs projects compensates for the lower mutation rate . 2008 ) , one of the more established ngs methods for population genomics looking at just fractions of the genome in the range of typically 1% , targets thousands or tens of thousands of loci in a single analysis , compared with some dozens or , rarely , hundreds in the most comprehensive microsatellite studies . some ngs data sets have been found to contain considerable amounts of indels indeed ( e.g. , baldwin et al . 2012 ) . currently , it is common to filter out indels in an early stage of the bioinformatics pipeline ( e.g. , toonen et al . 2013 ) , but there is growing awareness that indels in ngs data are not just a nuisance in the alignment process but also a valuable source of information ( e.g. , pacurar et al . the unparalleled promises of ngs have caused many researchers in ecology and evolution to switch from traditional , locus - based to whole - genome - based approaches or to plan doing so . however , to complete the transition , multiple challenges coming with the new technologies need be overcome ( sboner et al . 2011 ; dewoody et al . 2013 ; poisot et al . 2013 ; andrews and luikart 2014 ; mesak et al 2014 ) . the transition is slower in the study of nonmodel organisms than in that of model organisms ( mccormack et al . 2013 ) , given that for nonmodel organisms , resources tend to be more limited both in terms of relevant genomic background information ( nevado et al . 2014 ) and money . in any case , not the technology per se but the relevance of the question addressed and the stringency in testing the hypotheses raised make the quality of research . for various research questions , using ngs technology might be like using a sledgehammer to crack a nut ( cf . 2014 ) viewed matter - of - factly , there are both advantages and disadvantages to microsatellites compared with ngs techniques , and microsatellites are still being used massively ( box 2 ) . in short , some believe in the fast replacement of microsatellites by ngs approaches ( andrew et al . 2013 ) , others in the persistence of microsatellites in the study of populations also in the future ( e.g. , zalapa et al . , researchers addressing population - level questions can decide among a range of classical population genetic and next - generation sequencing ( ngs ) approaches . the decision is not easy , and the criteria to be considered range from scientific to resource related . in compiling a list of characteristics ( table b1 ) , we considered just microsatellites among classical approaches due to their common use but two different approaches among ngs techniques , representing the two extremes in effort and information . 2008 ) , the most frequently used among approaches analyzing just a fraction of the genome , and whole - genome resequencing ( huang et al . we used recent protocols and manuals and our own experience ; we aimed at covering a wide range of characteristics and at objectivity but take responsibility for any failure in doing so . characteristics ( as of october 2014 ) of microsatellites versus two selected next - generation sequencing ( ngs ) approaches in studying populations , radseq ( baird et al . 2008 ) and whole - genome resequencing ( huang et al . 2009 ) . all sequencing information is based on the assumption that illumina ( http://www.illumina.com/ ) technology is used , except the sequencing for developing microsatellite loci , for which the use of roche 454 ( http://www.454.com/ ) technology is assumed . where feasible , we classified using a five - step scale of very low , low , intermediate , high , and very high , for each characteristic calibrated relatively across the three techniques . secondary bioinformatics : sequence alignment and variant calling ; tertiary bioinformatics : further downstream steps of sequence annotation and interpretation ( wright et al . 2011 ; primary bioinformatics , that is , base calling , usually is performed by the software of the sequencing machine ) we also performed a web - of - science - based analysis of the numbers of annually published original articles on microsatellites and population genomics , from 1995 , when the first population - genomic paper came out , to 2013 ( fig.b1 ; appendix 1 ) and made two inferences . first , microsatellites still represent the major research approach with about 4500 contributions in 2013 and thus the 47-fold of population - genomic papers . second , population genomics should indeed have a bright future with a growth rate better explained by an exponential than a linear function . in contrast , microsatellites ' growth rate is , over the observed period , better explained by a linear than an exponential function , and , in fact , the number of contributions on microsatellites seems to level off in recent years . the annual numbers of publications on microsatellites and population genomics published 19952013 included in web of science . the results of regression analyses comparing the r of linear ( lin ) and exponential ( exp ) functions are added . see appendix 1 for the database query protocols used and for the regression analysis results . years are given as relative years as used in the regression analyses : 2 = 1995 , 20 = 2013 . today , researchers addressing population - level questions can decide among a range of classical population genetic and next - generation sequencing ( ngs ) approaches . the decision is not easy , and the criteria to be considered range from scientific to resource related . in compiling a list of characteristics ( table b1 ) , we considered just microsatellites among classical approaches due to their common use but two different approaches among ngs techniques , representing the two extremes in effort and information . 2008 ) , the most frequently used among approaches analyzing just a fraction of the genome , and whole - genome resequencing ( huang et al . 2009 ) , the approach using the maximum of information that can be used . we used recent protocols and manuals and our own experience ; we aimed at covering a wide range of characteristics and at objectivity but take responsibility for any failure in doing so . characteristics ( as of october 2014 ) of microsatellites versus two selected next - generation sequencing ( ngs ) approaches in studying populations , radseq ( baird et al . all sequencing information is based on the assumption that illumina ( http://www.illumina.com/ ) technology is used , except the sequencing for developing microsatellite loci , for which the use of roche 454 ( http://www.454.com/ ) technology is assumed . where feasible , we classified using a five - step scale of very low , low , intermediate , high , and very high , for each characteristic calibrated relatively across the three techniques . secondary bioinformatics : sequence alignment and variant calling ; tertiary bioinformatics : further downstream steps of sequence annotation and interpretation ( wright et al . 2011 ; primary bioinformatics , that is , base calling , usually is performed by the software of the sequencing machine ) we also performed a web - of - science - based analysis of the numbers of annually published original articles on microsatellites and population genomics , from 1995 , when the first population - genomic paper came out , to 2013 ( fig.b1 ; appendix 1 ) and made two inferences . first , microsatellites still represent the major research approach with about 4500 contributions in 2013 and thus the 47-fold of population - genomic papers . second , population genomics should indeed have a bright future with a growth rate better explained by an exponential than a linear function . in contrast , microsatellites ' growth rate is , over the observed period , better explained by a linear than an exponential function , and , in fact , the number of contributions on microsatellites seems to level off in recent years . the annual numbers of publications on microsatellites and population genomics published 19952013 included in web of science . the results of regression analyses comparing the r of linear ( lin ) and exponential ( exp ) functions are added . see appendix 1 for the database query protocols used and for the regression analysis results . years are given as relative years as used in the regression analyses : 2 = 1995 , 20 = 2013 . can we simply use the published literature to study reindels ? we did a standardized analysis of microsatellite literature ( see appendix 2 for details ) and found unexpectedly few reindel reports . in detail , in 16 animal microsatellite studies from 2013 in which sufficient information on pedigree was available , between 264 and 93,140 microsatellite alleles were seen per study . one study reported the absence of reindels explicitly ( liu et al . 2013 ) . one study ( 32,788 alleles seen ) reported 17 putative reindels , but did not report the validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error ( mayer and pasinelli 2013 ) . in that study , the position of the loci on autosomes was indicated as was the sex that introduced the mutations , but no information was provided on the identity of the locus or loci , mutation type(s ) , and allele size(s ) . we also calculated the binomial proportion confidence interval to identify mutation rates in line with the number of microsatellite reindels reported in the 16 papers surveyed using a confidence level of 0.95 , as implemented in a custom fortran program : 17 reindels of 32,788 alleles are in line with mutation rates of 3 10 to 8 10 which is well in the middle range of the reported per - locus - per - generation mutation rates of microsatellites of 10 to 10 ( bhargava and fuentes 2010 ) . on the other hand , zero reindels of the combined 184,660 alleles seen in the 15 studies without reindel reports ( eight animal classes ) are compatible with , at most , a mutation rate of 2 10 . there are two explanations thinkable for a mutation rate at the lower end of the rate range known : ( 1 ) the mutation rate could be very low indeed , or ( 2 ) researchers may be insufficiently reindel aware . we are not able to decide definitively in favor of one of these explanations but for two reasons hypothesize that ( 2 ) applies . firstly , microsatellite loci used in the 15 studies lacking reindel detection were all chosen for maximum polymorphism by the authors , rendering ( 1 ) rather implausible . secondly , 14 of the 15 studies lacking successful reindel detection lack any statement about the absence of or scanning for reindels . apparently , an increase of reindel awareness is needed , irrespective of the future prime methods in ecology and evolution . to prevent further loss of reindel information , we appeal to researchers to scan their data for reindels and report them together with a few easy - to - convey and crucial details on the nature of locus and mutation . we recommend the standard reindel report to contain the following information : report of absence , or , alternatively , ( 1 ) identity of locus affected including genbank accession number , ( 2 ) ( putative ) ancestral allele and derived allele , ( 3 ) sex of ( putative ) ancestral - allele donor , as well as ( 4 ) pipeline of reindel validation . importantly , we suggest that in both instances , absence and presence of reindels , the result of the reindel search should be reported : only when reindel absence is exported explicitly , will it be possible to use absence data in mutability calculations , in contrast to our inability of doing so with the results of our literature analysis . by managing to efficiently tap all sources for reindel knowledge from now on , we will rapidly create a comprehensive set of information including on insertion / deletion lengths , flanking regions , and chromosomal locations , suitable for the development of indel mutation models . no matter whether the study of reindels proposed here will be based on microsatellite or ngs data , it seems that the time for advanced indel modeling is near .

today , the comparative analysis of dna molecules mainly uses information inferred from nucleotide substitutions . insertion / deletion ( indel ) mutations , in contrast , are largely considered uninformative and discarded , due to our lacking knowledge on their evolution . however , including rather than discarding indels would be relevant to any research area in ecology and evolution that uses molecular data . as a practical approach to better understanding indel evolution in general , we propose the study of recent indel ( reindel ) mutations mutations where both ancestral and derived state are seen in the sample . the precondition for reindel identification is knowledge about the pedigree of the individuals sampled . sound reindel knowledge will allow the improved modeling needed for including indels in the downstream analysis of molecular data . both microsatellites , currently still the predominant marker system in the analysis of populations , and sequences generated by next - generation sequencing , a promising and rapidly developing range of technologies , offer the opportunity for reindel identification . however , a 2013 sample of animal microsatellite studies contained unexpectedly few reindels identified . as most likely explanation , we hypothesize that reindels are underreported rather than absent and that this underreporting stems from common reindel unawareness . if our hypothesis applies , increased reindel awareness should allow gathering data rapidly . we recommend the routine reporting of either the absence or presence of reindels together with standardized key information on the nature of mutations when they are detected and the use of the keyword reindel to increase visibility in both instances of successful and unsuccessful search .

Introducing reINDELs: Recent Insertion/Deletion Mutations Box 1. Microsatellites as a fast road to understanding reINDELs The Relevance of (recent) INDEL Mutations to the Study of Ecology and Evolution Studying reINDELs in the NGS Era Box 2. Microsatellites versus next-generation sequencing in the study of populations Currently Few reINDEL Reports we Hypothesize Little reINDEL Awareness Recommendation Conflict of Interest

the comparative analysis of dna molecules looks back on a history of over 40 years . another type of mutation , the gain or loss of nucleotides within a defined locus , termed insertion / deletion ( indel ) mutation , has received much less attention ( lunter et al . this lack of attention is not due to a lack of relevance on the contrary , indels constitute a considerable fraction of mutations in coding and noncoding parts of the genome , are responsible for copy number variants , and are the signature of transposable elements ( korbel et al . rather , the lack of attention is due to a lack of profound knowledge on the evolution of indels ( ogden and rosenberg 2007 and references therein , sunday and hart 2013 ) , for which reason they are usually considered uninformative and removed during data preparation , except in the context of gene finding ( kellis et al . the special case of recent indel , henceforth reindel mutations , that is , mutations where both ancestral and derived state are seen in the sample , allows us witnessing indel evolution in real time . knowledge of reindels based on broad sampling should therefore facilitate a better understanding of indel evolution generally . the preconditions for reindel identification are that we know the pedigree in our sample and that our sample is large ( ellegren 2000 ; schltterer 2000 ) ; examples of such situations include studies on extrapair paternity , intraspecific brood parasitism , and eusocial societies , ideally with a single female reproductive ( see fig.1 for an example study system ) . for example , a validated allele detected in a singly mated female 's offspring that is unknown from the parents can be deduced to represent a recent mutation . eusocial insects are among study systems that facilitate generating large population - genetic or population - genomic data sets for individuals with known pedigree and that are thus ideal for studying reindels . here , one of the 1,00010,000 workers ( steiner et al . 2004 ) of a colony of the ant lasius austriacus carries a worker sister at the pupal stage , all these workers stemming from the same mother and father ( steiner et al . due to frequent indel mutations via loss and gain of repeat units , the design of studies with known pedigree allows precise expectations on the number and size of alleles in the data set based on the knowledge of the parental alleles . the vast majority of recent mutations in microsatellites will cause variation in allele size ( estoup et al . 2002 ) , and any deviation from the original expectations thus is a reindel candidate . for correct data interpretation , validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error are necessary . hitherto studies on microsatellite evolution have shed some light on , for example , the factors increasing slippage events and multistep changes ( primmer et al . 2012 ) and the existence of differences between male and female germline ( anmarkrud et al . however , many questions remain such as about the influence of base composition of repeat motifs , about mating and/or sex - determination systems , about cross - taxa and cross - loci variation , and about differences between experimental and natural populations ( ellegren 2000 , 2004 ; schltterer 2000 ; leclercq et al . thinking about the increase of accuracy the improvement of nucleotide models of evolution brought about in phylogenetic applications , we can expect comparable advances in microsatellite - based analyses . currently existing microsatellite models such as the stepwise mutation , the generalized stepwise , or the k - allele model ( reviewed in estoup et al . 2012 on the reliability of microsatellite - based bottleneck tests for detecting recent population declines ) . however , with profound knowledge about microsatellite evolution , future models might incorporate the full range of relevant aspects for which maximum likelihood or bayesian approaches can make good estimates ( cf . potential implementations include different mutability rates according to motif sequence , motif and allele size , allele - frequency dependence , different probabilities for expansion or contraction events , and different rates for male and female germline . more realistic models will aid multiple research areas in ecology and evolution : kinship , parentage , and behavior analyses will be more accurate facilitating a better understanding of mating systems , dispersal patterns , and social organization ; better estimations of effective population sizes and detection of bottlenecks will aid nature conservation research ; hybridization and backcrossing patterns will be more correctly mirrored , which in turn will increase our understanding of these evolutionary forces ; aberrant modes of reproduction might be elucidated , phylogeography exploring the recent past improved , and signals of selection better recognized . due to frequent indel mutations via loss and gain of repeat units , the design of studies with known pedigree allows precise expectations on the number and size of alleles in the data set based on the knowledge of the parental alleles . the vast majority of recent mutations in microsatellites will cause variation in allele size ( estoup et al . 2002 ) , and any deviation from the original expectations thus is a reindel candidate . for correct data interpretation , validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error are necessary . hitherto studies on microsatellite evolution have shed some light on , for example , the factors increasing slippage events and multistep changes ( primmer et al . 2012 ) and the existence of differences between male and female germline ( anmarkrud et al . however , many questions remain such as about the influence of base composition of repeat motifs , about mating and/or sex - determination systems , about cross - taxa and cross - loci variation , and about differences between experimental and natural populations ( ellegren 2000 , 2004 ; schltterer 2000 ; leclercq et al . thinking about the increase of accuracy the improvement of nucleotide models of evolution brought about in phylogenetic applications , we can expect comparable advances in microsatellite - based analyses . currently existing microsatellite models such as the stepwise mutation , the generalized stepwise , or the k - allele model ( reviewed in estoup et al . 2012 on the reliability of microsatellite - based bottleneck tests for detecting recent population declines ) . however , with profound knowledge about microsatellite evolution , future models might incorporate the full range of relevant aspects for which maximum likelihood or bayesian approaches can make good estimates ( cf . potential implementations include different mutability rates according to motif sequence , motif and allele size , allele - frequency dependence , different probabilities for expansion or contraction events , and different rates for male and female germline . more realistic models will aid multiple research areas in ecology and evolution : kinship , parentage , and behavior analyses will be more accurate facilitating a better understanding of mating systems , dispersal patterns , and social organization ; better estimations of effective population sizes and detection of bottlenecks will aid nature conservation research ; hybridization and backcrossing patterns will be more correctly mirrored , which in turn will increase our understanding of these evolutionary forces ; aberrant modes of reproduction might be elucidated , phylogeography exploring the recent past improved , and signals of selection better recognized . a better understanding of indel evolution , achievable via the study of reindels , will allow the inclusion of indels as genetic variation in our analyses rather than their discard . this will have far - reaching effects on any research area using molecular data to make ecological and evolutionary inferences , such as speciation research , sociobiology , the study of species interactions , conservation genetics , invasion biology , and climate change biology . from the obviously vast range of potential implications of including indel information in the data analyses , there is broad consensus on the existence of a male mutation bias in vertebrates and some plants caused by the higher number of cell divisions during spermatogenesis than during oogenesis ( kirkpatrick and hall 2004 ) . anyway , the underlying factors shaping the extent of male mutation bias are still poorly understood ( bartosch - harlid et al . 2003 ; goetting - minesky and makova 2006 ) , information from invertebrates is scarce , and recent observations indicate that the mutation rate can be highly different among closely related species ( venn et al . thus , more information on patterns at individual loci and in different organisms is needed ( ellegren 2007 ) . such information will then open up additional research avenues ; for example , such bias may even influence the long - term persistence of populations with skewed sex allocation ( cotton and wedekind 2010 ) . the advent of next - generation sequencing ( ngs ) , the massive parallel sequencing of dna , has opened up a previously unthinkable array of opportunities ( andrew et al . possibly the most important consequence is that ngs makes available genomics for the study of nonmodel organisms ( hudson 2008 ; tautz et al . compared with population genetics , the promises of population genomics include improved identification of adaptive molecular variation as well as improved inferences about population demography and evolutionary history ( luikart et al . in contrast , estimations of genome - wide rates of nonmicrosatellite indel mutations per site and generation are in the range of 2.1 10 ( sacharomyces cerevisiae ; lang and murray 2008 ) to 1.2 10 ( caenorhabditis elegans ; denver et al . 2004 ) , that is , two to eight orders of magnitude lower than the estimated rates of mutations in microsatellites . anyway , the sheer amount of data in ngs projects compensates for the lower mutation rate . 2008 ) , one of the more established ngs methods for population genomics looking at just fractions of the genome in the range of typically 1% , targets thousands or tens of thousands of loci in a single analysis , compared with some dozens or , rarely , hundreds in the most comprehensive microsatellite studies . currently , it is common to filter out indels in an early stage of the bioinformatics pipeline ( e.g. 2013 ) , but there is growing awareness that indels in ngs data are not just a nuisance in the alignment process but also a valuable source of information ( e.g. the unparalleled promises of ngs have caused many researchers in ecology and evolution to switch from traditional , locus - based to whole - genome - based approaches or to plan doing so . however , to complete the transition , multiple challenges coming with the new technologies need be overcome ( sboner et al . the transition is slower in the study of nonmodel organisms than in that of model organisms ( mccormack et al . in any case , not the technology per se but the relevance of the question addressed and the stringency in testing the hypotheses raised make the quality of research . 2014 ) viewed matter - of - factly , there are both advantages and disadvantages to microsatellites compared with ngs techniques , and microsatellites are still being used massively ( box 2 ) . in short , some believe in the fast replacement of microsatellites by ngs approaches ( andrew et al . 2013 ) , others in the persistence of microsatellites in the study of populations also in the future ( e.g. , researchers addressing population - level questions can decide among a range of classical population genetic and next - generation sequencing ( ngs ) approaches . the decision is not easy , and the criteria to be considered range from scientific to resource related . in compiling a list of characteristics ( table b1 ) , we considered just microsatellites among classical approaches due to their common use but two different approaches among ngs techniques , representing the two extremes in effort and information . 2008 ) , the most frequently used among approaches analyzing just a fraction of the genome , and whole - genome resequencing ( huang et al . we used recent protocols and manuals and our own experience ; we aimed at covering a wide range of characteristics and at objectivity but take responsibility for any failure in doing so . characteristics ( as of october 2014 ) of microsatellites versus two selected next - generation sequencing ( ngs ) approaches in studying populations , radseq ( baird et al . all sequencing information is based on the assumption that illumina ( http://www.illumina.com/ ) technology is used , except the sequencing for developing microsatellite loci , for which the use of roche 454 ( http://www.454.com/ ) technology is assumed . where feasible , we classified using a five - step scale of very low , low , intermediate , high , and very high , for each characteristic calibrated relatively across the three techniques . 2011 ; primary bioinformatics , that is , base calling , usually is performed by the software of the sequencing machine ) we also performed a web - of - science - based analysis of the numbers of annually published original articles on microsatellites and population genomics , from 1995 , when the first population - genomic paper came out , to 2013 ( fig.b1 ; appendix 1 ) and made two inferences . in contrast , microsatellites ' growth rate is , over the observed period , better explained by a linear than an exponential function , and , in fact , the number of contributions on microsatellites seems to level off in recent years . years are given as relative years as used in the regression analyses : 2 = 1995 , 20 = 2013 . today , researchers addressing population - level questions can decide among a range of classical population genetic and next - generation sequencing ( ngs ) approaches . the decision is not easy , and the criteria to be considered range from scientific to resource related . in compiling a list of characteristics ( table b1 ) , we considered just microsatellites among classical approaches due to their common use but two different approaches among ngs techniques , representing the two extremes in effort and information . 2008 ) , the most frequently used among approaches analyzing just a fraction of the genome , and whole - genome resequencing ( huang et al . 2009 ) , the approach using the maximum of information that can be used . we used recent protocols and manuals and our own experience ; we aimed at covering a wide range of characteristics and at objectivity but take responsibility for any failure in doing so . characteristics ( as of october 2014 ) of microsatellites versus two selected next - generation sequencing ( ngs ) approaches in studying populations , radseq ( baird et al . all sequencing information is based on the assumption that illumina ( http://www.illumina.com/ ) technology is used , except the sequencing for developing microsatellite loci , for which the use of roche 454 ( http://www.454.com/ ) technology is assumed . where feasible , we classified using a five - step scale of very low , low , intermediate , high , and very high , for each characteristic calibrated relatively across the three techniques . 2011 ; primary bioinformatics , that is , base calling , usually is performed by the software of the sequencing machine ) we also performed a web - of - science - based analysis of the numbers of annually published original articles on microsatellites and population genomics , from 1995 , when the first population - genomic paper came out , to 2013 ( fig.b1 ; appendix 1 ) and made two inferences . in contrast , microsatellites ' growth rate is , over the observed period , better explained by a linear than an exponential function , and , in fact , the number of contributions on microsatellites seems to level off in recent years . years are given as relative years as used in the regression analyses : 2 = 1995 , 20 = 2013 . we did a standardized analysis of microsatellite literature ( see appendix 2 for details ) and found unexpectedly few reindel reports . in detail , in 16 animal microsatellite studies from 2013 in which sufficient information on pedigree was available , between 264 and 93,140 microsatellite alleles were seen per study . one study reported the absence of reindels explicitly ( liu et al . one study ( 32,788 alleles seen ) reported 17 putative reindels , but did not report the validation of allele calling to exclude a scoring error and re - genotyping of the respective individual to exclude a pcr error ( mayer and pasinelli 2013 ) . in that study , the position of the loci on autosomes was indicated as was the sex that introduced the mutations , but no information was provided on the identity of the locus or loci , mutation type(s ) , and allele size(s ) . we also calculated the binomial proportion confidence interval to identify mutation rates in line with the number of microsatellite reindels reported in the 16 papers surveyed using a confidence level of 0.95 , as implemented in a custom fortran program : 17 reindels of 32,788 alleles are in line with mutation rates of 3 10 to 8 10 which is well in the middle range of the reported per - locus - per - generation mutation rates of microsatellites of 10 to 10 ( bhargava and fuentes 2010 ) . on the other hand , zero reindels of the combined 184,660 alleles seen in the 15 studies without reindel reports ( eight animal classes ) are compatible with , at most , a mutation rate of 2 10 . there are two explanations thinkable for a mutation rate at the lower end of the rate range known : ( 1 ) the mutation rate could be very low indeed , or ( 2 ) researchers may be insufficiently reindel aware . we are not able to decide definitively in favor of one of these explanations but for two reasons hypothesize that ( 2 ) applies . firstly , microsatellite loci used in the 15 studies lacking reindel detection were all chosen for maximum polymorphism by the authors , rendering ( 1 ) rather implausible . secondly , 14 of the 15 studies lacking successful reindel detection lack any statement about the absence of or scanning for reindels . apparently , an increase of reindel awareness is needed , irrespective of the future prime methods in ecology and evolution . to prevent further loss of reindel information , we appeal to researchers to scan their data for reindels and report them together with a few easy - to - convey and crucial details on the nature of locus and mutation . we recommend the standard reindel report to contain the following information : report of absence , or , alternatively , ( 1 ) identity of locus affected including genbank accession number , ( 2 ) ( putative ) ancestral allele and derived allele , ( 3 ) sex of ( putative ) ancestral - allele donor , as well as ( 4 ) pipeline of reindel validation . importantly , we suggest that in both instances , absence and presence of reindels , the result of the reindel search should be reported : only when reindel absence is exported explicitly , will it be possible to use absence data in mutability calculations , in contrast to our inability of doing so with the results of our literature analysis . by managing to efficiently tap all sources for reindel knowledge from now on , we will rapidly create a comprehensive set of information including on insertion / deletion lengths , flanking regions , and chromosomal locations , suitable for the development of indel mutation models . no matter whether the study of reindels proposed here will be based on microsatellite or ngs data , it seems that the time for advanced indel modeling is near .

services of curative care are defined as those where the principal medical intent of care is to relieve symptoms or reduce severity or protect against exacerbation or complication of illness or injury . in addition to in - patient and day care , hc1 includes out - patient care , defined as basic medical and diagnostic services , out - patient dental care and other specialist health care provided to outpatients by physicians or paramedics , including services provided at home . primary legislation addresses the establishment and promotion of a comprehensive health service , requiring the secretary of state for health to provide or secure the effective provision of services. however , discretion is at the heart of these duties and politicians are entitled to take into account the resources available to them . under the 1977 national health service act ( chap . 49 ) , the secretary of state has a duty to provide to such extent as he considers necessary to meet all reasonable requirements : ( a ) hospital accommodation [ s. 3 ( 1(a ) ) ] , including high security psychiatric services ( s. 4 ) ; ( b ) other accommodation necessary for the purpose of any other services required by the act [ s. 3 ( 1(b ) ) ] ; ( c ) medical , dental and nursing services [ s. 3 ( 1(c ) ) ] ( n.b . : surgical ) ; and ( d ) such other services as are required for the treatment of illness [ s. 3 ( 1(f ) ) ] ( n.b . : illness includes a mental disorder and any injury or disability requiring medical or dental treatment or nursing ) . the secretary of state for health also has powers to provide as he considers appropriate facilities for the care of persons suffering from illness [ s. 3 ( 1(e ) ) ] . the responsibility for providing these services has passed to local health authorities , which in the current organisational structure are known as primary care trusts ( pcts ) . the 1977 nhs act also places a duty on pcts to provide general medical services ( s. 29 ) , general dental services ( s. 35 ) , pharmaceutical services ( s. 41 ) and general ophthalmic services ( s. 38 ) . in the law courts r v nw lancashire health authority , ex p a , d and g examined the case of three applicants suffering from gender identity dysphoria . the health authority had identified this illness as amongst the bottom 10% in terms of need ( together with cosmetic surgery , reversal of sterilisation , correction of myopia and most alternative medicines ) and therefore transsexual surgery would be provided only in cases of overriding clinical need. the court acknowledged the need for priority setting in which issues of effectiveness , the seriousness of the condition and cost were taken into account . however , the court found that the health authority had in practice adopted a blanket ban and recommended the authorities introduced a fair and consistent policy for decision making that adequately assessed exceptional cases by considering each request for treatment on its individual merits . the case therefore made it illegal for health authorities to refuse to provide specific services , with the possible exception of a treatment where the clinical evidence of its inefficacy is overwhelming . the case implies that costs and benefits should be evaluated on an individual patient basis , rather basing entitlement on the typical or average case . primary legislation and case law have therefore not prescribed which services are to be included in or excluded from the english health basket . however , secondary legislation on professional contracts goes some way towards defining entitlement to specific services . for example , the 2004 national health service ( general medical services contracts ) regulations provide details of the terms of service for general practitioners ( gps ) and state that essential services must be provided , covering emergency treatment and treatment for patients with chronic , terminal and self - limiting disease [ national health service , general medical services contracts , regulation si 2004/291 , reg . 15 ( 3:5,6:8 ) , 2004 ; see table 2].table 2 eligibility for primary care essential services under the 2004 general medical services contract ( from ) type of essential serviceeligibility for treatmentimmediate and necessary emergency treatmentany person to whom the contractor has been requested to provide treatment ... at any place in its practice area [ reg.15 ( 6)]management of terminal illnessregistered patients and temporary residentstreatment of conditions from which recovery is generally expectedregistered patients and temporary residentstreatment of chronic diseaseregistered patients and temporary residentsadvice in connection with the patient s health , including relevant health promotion adviceregistered patients and temporary residentsreferral of the patient for other services under the 1977 actregistered patients and temporary residentshome visitswhere contractor considers it inappropriate , because of a patient s medical condition , for the patient to attend the practice premisesannual health checkspatients aged over 75 yearspatients not seen within 3 years ; newly registered patients eligibility for primary care essential services under the 2004 general medical services contract ( from ) for dental practitioners the national nhs ( general dental services ) regulations 1992 make provision for the secretary of state for health to determine dentists remuneration , including a scale of fees for providing particular services [ reg . , the statement of dental remuneration describes over 400 services covered by the fees , including clinical examinations and treatment planning , diagnostic procedures , such as radiographic examinations , preventative , periodontal , conservation and surgical treatments and the supply of prostheses ; patients pay 80% of the charge up to a stated maximum , although some exemptions apply ( department of health , statement of dental remuneration , amendment no . the doctors and dentists review body reviews existing fee scales and makes recommendations regarding uplift . the department of health then considers these recommendations and the chief dental officer for england notifies strategic health authorities , pcts , nhs trusts ( groups of nhs hospitals , each operating as a single legal entity ) and all general dental practitioners of the updated fee scales ( ibid . ) . similarly , the 1986 nhs ( general ophthalmic services ) regulations make provision for opticians and ophthalmic medical practitioners to charge for sight tests and for optical appliances ( national health service , general ophthalmic services , regulation si 1986/975 , 1986 ) . charges are determined by the secretary of state in consultation with professional bodies , updated at least annually and amended by secondary legislation . for certain patients vouchers eligibility for general ophthalmic services , outlined in section 13 of the health and medicines act 1988 , is determined by age , disease ( or risk of disease ) or income . , nhs provision is shaped by a considerable amount of quasi - law. quasi - law is defined as rules which are not usually legally binding , although they may have some legal force , but which will in practice determine the way in which people act . amongst the regulation helping to define patient entitlement to services are nsfs , nice guidance , waiting time guarantees , fee schedules , and incentive schemes . the healthcare commission is the key regulator , assessing the performance of nhs organisations against national standards in its annual health check , a monitoring process that assesses both existing performance ( core standards ) and capacity to improve ( developmental standards ) . the programme of nsfs , launched by the department of health in april 1998 , usually produces one new framework a year . nsfs set national standards , identify key interventions for a defined service or care group that should be available , establish strategies to support implementation and outline ways to ensure progress within an agreed time scale . each nsf is developed with the assistance of an external reference group ( erg ) , which seeks to engage a range of views from health professionals , service users and carers , health service managers , partner agencies and other advocates . it is unclear how nsfs are to be updated to reflect changes in the evidence base that underpins them.table 3 services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative carecategoryserviceschildrenstandard 7 : guidance on hospital - based services for childrenstandard 3 : guidance on community - based carecoronary heart diseasestandard 7 : nhs trusts to provide appropriate investigations and treatments for patients with suspected or confirmed coronary heart diseasediabetesstandard 7 : nhs to provide rapid and effective treatment for diabetic emergenciesmental healthin - patient hospital beds for persons needing a short period of intensive intervention and observationelderlystandard 4 : need for appropriate specialist carestandard 7 : effective diagnosis , treatment and support for those with mental health problemsrenal diseasequality requirement 2 : timely , appropriate and effective investigation , treatment and follow - up for those with chronic kidney diseasestandard 5 : all likely to benefit from a kidney transplant to receive a high quality service which supports them in managing their transplant(from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative care ( from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) there is no statutory obligation on health care organisations to implement nsf standards . however , the health and social care ( community health and standards ) act 2003 [ health and social care ( community health and standards ) act , chap . 43 , 2003 ) gave the secretary of state powers to publish standards for health care [ s. 46 ( 1 ) ] that nhs bodies are bound to take into account [ s. 46 ( 4 ) ] ; nsfs could inform these standards . furthermore , the healthcare commission reviews health care organisations implementation of nsfs as part of its annual health check , reinforcing the quasi - legislative nature of nsf guidance . nice , the organisation responsible for assessing whether new or existing technologies should be available on the nhs , produces three types of guidance which help define the availability of nhs services of curative care . firstly , technology appraisals give guidance on the use of new and existing treatments within the nhs . of the 91 technology appraisals published to date ( july 2005 ) some relate to in - patient care , such as appraisal no . 11 ( the use of implantable cardioverter defibrillators for arrhythmias ) ; to day care , such as appraisal no . 48 ( home compared with hospital haemodialysis for patients with end - stage renal failure ) ; and to out - patient care , such as appraisal no . 24 ( debriding agents for difficult to heal surgical wounds ) . secondly , nice clinical guidelines offer guidance on the appropriate treatment and care of people with specific diseases and conditions within the nhs . clinical guidelines are based on the best available evidence and are intended to help health care professionals in their work but not to replace their knowledge and skills . for example , the dyspepsia guideline ( clinical guideline , cg , 18 ) recommends life - style advice and appropriate medication as secondary preventative measures and the guideline on head injury ( cg4 ) specifies treatment pathways covering emergency , out - patient and in - patient care . thirdly , nice produces guidance on whether interventional procedures used for diagnosis or treatment are sufficiently safe and effective for routine use in the nhs . of the 127 currently published topics most relate to hospital - based care , such as radiotherapy for age - related macular degeneration ( interventional procedure guidance , ipg , 048 ) and auditory brainstem implants ( ipg108 ) . guidance on technology appraisals is formulated by the nice appraisal committee , an independent advisory body with individuals from a range of professional backgrounds . preliminary guidance is based on evidence from an academic assessment group and from company submissions . following consultation , the final appraisal determination is developed , approved by the nice guidance executive and put out again for consultation . all guidance is reviewed at regular intervals . to develop a guideline , nice commissions one of the national collaborating centres to establish a guideline development group . the group appraises clinical and cost - effectiveness evidence from systematic reviews of the research evidence . views of clinicians , consumers and stakeholders also inform the guideline . to produce guidance on interventional procedures , the interventional procedures advisory committee , an independent body of 24 members with a range of expertise , considers the safety and efficacy of procedures , but does not examine clinical or cost effectiveness . the committee produces a consultation document and published guidance reflects comments received over the 4-week consultation period . nice guidance generally acts as quasi - law , but one aspect of guidance on technology appraisals is supported by statute . if nice guidance is that a new technology should be made available to certain nhs patients , the funding bodies ( pcts ) are obliged by law to ensure there are adequate resources to facilitate the implementation of nice guidance ( secretary of state for health national health service act 1977 : directions to health authorities , primary care trusts and nhs trusts in england , 2001 ) . however , the guidance is not binding on individual clinicians , who must assess whether the technology is appropriate for the patients they treat . published annually , these specify national goals within the public sector , including waiting time targets ( or guarantees ) for the nhs : ( a ) by the end of 2005 , patients will wait a maximum of 6 months for in - patient admission and no more than 13 weeks for an out - patient appointment . ( b ) by the end of 2008 the maximum wait from gp referral to hospital treatment will be 18 weeks . targets that should already be achieved , and henceforth maintained , include the 4-hour maximum wait for emergency care and the 24/48 target for accessing primary care . ( the 24/48 target refers to patients being able to see a general practitioner within 2 working days or another primary care professional within 1 working day , whilst the 4-hour wait target is that patients should spend no more than 4 hours in an accident and emergency hospital department from arrival to admission , transfer or discharge . ) where fee schedules exist for nhs care , patient for example , the new national tariff system of payments for hospital services , whilst in no way guaranteeing provision , suggests services that should be accessible on the nhs . the national price schedule for patient services is classified by health care resource group ( hrg ) codes . a range of clinical procedures , treatments and diagnoses is included in the current list of 550 hrg tariffs for elective in - patient care . the quality and outcomes framework , part of the gp contract , is a voluntary mechanism for encouraging primary care provision of some services of curative care , such as antiplatelets for patients with coronary heart disease . with total payments amounting to 1520% of available total practice remuneration , there is a clear incentive for gps to make these services available to nhs patients . primary legislation addresses the establishment and promotion of a comprehensive health service , requiring the secretary of state for health to provide or secure the effective provision of services. however , discretion is at the heart of these duties and politicians are entitled to take into account the resources available to them . under the 1977 national health service act ( chap . 49 ) , the secretary of state has a duty to provide to such extent as he considers necessary to meet all reasonable requirements : ( a ) hospital accommodation [ s. 3 ( 1(a ) ) ] , including high security psychiatric services ( s. 4 ) ; ( b ) other accommodation necessary for the purpose of any other services required by the act [ s. 3 ( 1(b ) ) ] ; ( c ) medical , dental and nursing services [ s. 3 ( 1(c ) ) ] ( n.b . : surgical ) ; and ( d ) such other services as are required for the treatment of illness [ s. 3 ( 1(f ) ) ] ( n.b . : illness includes a mental disorder and any injury or disability requiring medical or dental treatment or nursing ) . the secretary of state for health also has powers to provide as he considers appropriate facilities for the care of persons suffering from illness [ s. 3 ( 1(e ) ) ] . the responsibility for providing these services has passed to local health authorities , which in the current organisational structure are known as primary care trusts ( pcts ) . the 1977 nhs act also places a duty on pcts to provide general medical services ( s. 29 ) , general dental services ( s. 35 ) , pharmaceutical services ( s. 41 ) and general ophthalmic services ( s. 38 ) . in the law courts r v nw lancashire health authority , ex p a , d and g examined the case of three applicants suffering from gender identity dysphoria . the health authority had identified this illness as amongst the bottom 10% in terms of need ( together with cosmetic surgery , reversal of sterilisation , correction of myopia and most alternative medicines ) and therefore transsexual surgery would be provided only in cases of overriding clinical need. the court acknowledged the need for priority setting in which issues of effectiveness , the seriousness of the condition and cost were taken into account . blanket ban and recommended the authorities introduced a fair and consistent policy for decision making that adequately assessed exceptional cases by considering each request for treatment on its individual merits . the case therefore made it illegal for health authorities to refuse to provide specific services , with the possible exception of a treatment where the clinical evidence of its inefficacy is overwhelming . the case implies that costs and benefits should be evaluated on an individual patient basis , rather basing entitlement on the typical or average case . primary legislation and case law have therefore not prescribed which services are to be included in or excluded from the english health basket . however , secondary legislation on professional contracts goes some way towards defining entitlement to specific services . for example , the 2004 national health service ( general medical services contracts ) regulations provide details of the terms of service for general practitioners ( gps ) and state that essential services must be provided , covering emergency treatment and treatment for patients with chronic , terminal and self - limiting disease [ national health service , general medical services contracts , regulation si 2004/291 , reg . 15 ( 3:5,6:8 ) , 2004 ; see table 2].table 2 eligibility for primary care essential services under the 2004 general medical services contract ( from ) type of essential serviceeligibility for treatmentimmediate and necessary emergency treatmentany person to whom the contractor has been requested to provide treatment ... at any place in its practice area [ reg.15 ( 6)]management of terminal illnessregistered patients and temporary residentstreatment of conditions from which recovery is generally expectedregistered patients and temporary residentstreatment of chronic diseaseregistered patients and temporary residentsadvice in connection with the patient s health , including relevant health promotion adviceregistered patients and temporary residentsreferral of the patient for other services under the 1977 actregistered patients and temporary residentshome visitswhere contractor considers it inappropriate , because of a patient s medical condition , for the patient to attend the practice premisesannual health checkspatients aged over 75 yearspatients not seen within 3 years ; newly registered patients eligibility for primary care essential services under the 2004 general medical services contract ( from ) for dental practitioners the national nhs ( general dental services ) regulations 1992 make provision for the secretary of state for health to determine dentists remuneration , including a scale of fees for providing particular services [ reg . 19 ( 1 ) ] . updated at least annually , the statement of dental remuneration describes over 400 services covered by the fees , including clinical examinations and treatment planning , diagnostic procedures , such as radiographic examinations , preventative , periodontal , conservation and surgical treatments and the supply of prostheses ; patients pay 80% of the charge up to a stated maximum , although some exemptions apply ( department of health , statement of dental remuneration , amendment no . the doctors and dentists review body reviews existing fee scales and makes recommendations regarding uplift . the department of health then considers these recommendations and the chief dental officer for england notifies strategic health authorities , pcts , nhs trusts ( groups of nhs hospitals , each operating as a single legal entity ) and all general dental practitioners of the updated fee scales ( ibid . ) . similarly , the 1986 nhs ( general ophthalmic services ) regulations make provision for opticians and ophthalmic medical practitioners to charge for sight tests and for optical appliances ( national health service , general ophthalmic services , regulation si 1986/975 , 1986 ) . charges are determined by the secretary of state in consultation with professional bodies , updated at least annually and amended by secondary legislation . for certain patients vouchers eligibility for general ophthalmic services , outlined in section 13 of the health and medicines act 1988 , is determined by age , disease ( or risk of disease ) or income . in addition to legislation , nhs provision is shaped by a considerable amount of quasi - law. quasi - law is defined as rules which are not usually legally binding , although they may have some legal force , but which will in practice determine the way in which people act . amongst the regulation helping to define patient entitlement to services are nsfs , nice guidance , waiting time guarantees , fee schedules , and incentive schemes . the healthcare commission is the key regulator , assessing the performance of nhs organisations against national standards in its annual health check , a monitoring process that assesses both existing performance ( core standards ) and capacity to improve ( developmental standards ) . the programme of nsfs , launched by the department of health in april 1998 , usually produces one new framework a year . nsfs set national standards , identify key interventions for a defined service or care group that should be available , establish strategies to support implementation and outline ways to ensure progress within an agreed time scale . each nsf is developed with the assistance of an external reference group ( erg ) , which seeks to engage a range of views from health professionals , service users and carers , health service managers , partner agencies and other advocates . it is unclear how nsfs are to be updated to reflect changes in the evidence base that underpins them.table 3 services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative carecategoryserviceschildrenstandard 7 : guidance on hospital - based services for childrenstandard 3 : guidance on community - based carecoronary heart diseasestandard 7 : nhs trusts to provide appropriate investigations and treatments for patients with suspected or confirmed coronary heart diseasediabetesstandard 7 : nhs to provide rapid and effective treatment for diabetic emergenciesmental healthin - patient hospital beds for persons needing a short period of intensive intervention and observationelderlystandard 4 : need for appropriate specialist carestandard 7 : effective diagnosis , treatment and support for those with mental health problemsrenal diseasequality requirement 2 : timely , appropriate and effective investigation , treatment and follow - up for those with chronic kidney diseasestandard 5 : all likely to benefit from a kidney transplant to receive a high quality service which supports them in managing their transplant(from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative care ( from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) there is no statutory obligation on health care organisations to implement nsf standards . however , the health and social care ( community health and standards ) act 2003 [ health and social care ( community health and standards ) act , chap . 43 , 2003 ) gave the secretary of state powers to publish standards for health care [ s. 46 ( 1 ) ] that nhs bodies are bound to take into account [ s. 46 ( 4 ) ] ; nsfs could inform these standards . furthermore , the healthcare commission reviews health care organisations implementation of nsfs as part of its annual health check , reinforcing the quasi - legislative nature of nsf guidance . nice , the organisation responsible for assessing whether new or existing technologies should be available on the nhs , produces three types of guidance which help define the availability of nhs services of curative care . firstly , technology appraisals give guidance on the use of new and existing treatments within the nhs . of the 91 technology appraisals published to date ( july 2005 ) some relate to in - patient care , such as appraisal no . 11 ( the use of implantable cardioverter defibrillators for arrhythmias ) ; to day care , such as appraisal no . 48 ( home compared with hospital haemodialysis for patients with end - stage renal failure ) ; and to out - patient care , such as appraisal no secondly , nice clinical guidelines offer guidance on the appropriate treatment and care of people with specific diseases and conditions within the nhs . clinical guidelines are based on the best available evidence and are intended to help health care professionals in their work but not to replace their knowledge and skills . for example , the dyspepsia guideline ( clinical guideline , cg , 18 ) recommends life - style advice and appropriate medication as secondary preventative measures and the guideline on head injury ( cg4 ) specifies treatment pathways covering emergency , out - patient and in - patient care . thirdly , nice produces guidance on whether interventional procedures used for diagnosis or treatment are sufficiently safe and effective for routine use in the nhs . of the 127 currently published topics most relate to hospital - based care , such as radiotherapy for age - related macular degeneration ( interventional procedure guidance , ipg , 048 ) and auditory brainstem implants ( ipg108 ) . guidance on technology appraisals is formulated by the nice appraisal committee , an independent advisory body with individuals from a range of professional backgrounds . preliminary guidance is based on evidence from an academic assessment group and from company submissions . following consultation , the final appraisal determination is developed , approved by the nice guidance executive and put out again for consultation . all guidance is reviewed at regular intervals . to develop a guideline , nice commissions one of the national collaborating centres to establish a guideline development group . the group appraises clinical and cost - effectiveness evidence from systematic reviews of the research evidence . views of clinicians , consumers and stakeholders also inform the guideline . to produce guidance on interventional procedures , the interventional procedures advisory committee , an independent body of 24 members with a range of expertise , considers the safety and efficacy of procedures , but does not examine clinical or cost effectiveness . the committee produces a consultation document and published guidance reflects comments received over the 4-week consultation period . nice guidance generally acts as quasi - law , but one aspect of guidance on technology appraisals is supported by statute . if nice guidance is that a new technology should be made available to certain nhs patients , the funding bodies ( pcts ) are obliged by law to ensure there are adequate resources to facilitate the implementation of nice guidance ( secretary of state for health national health service act 1977 : directions to health authorities , primary care trusts and nhs trusts in england , 2001 ) . however , the guidance is not binding on individual clinicians , who must assess whether the technology is appropriate for the patients they treat . . published annually , these specify national goals within the public sector , including waiting time targets ( or guarantees ) for the nhs : ( a ) by the end of 2005 , patients will wait a maximum of 6 months for in - patient admission and no more than 13 weeks for an out - patient appointment . ( b ) by the end of 2008 the maximum wait from gp referral to hospital treatment will be 18 weeks . targets that should already be achieved , and henceforth maintained , include the 4-hour maximum wait for emergency care and the 24/48 target for accessing primary care . ( the 24/48 target refers to patients being able to see a general practitioner within 2 working days or another primary care professional within 1 working day , whilst the 4-hour wait target is that patients should spend no more than 4 hours in an accident and emergency hospital department from arrival to admission , transfer or discharge . ) where fee schedules exist for nhs care , patient for example , the new national tariff system of payments for hospital services , whilst in no way guaranteeing provision , the national price schedule for patient services is classified by health care resource group ( hrg ) codes . a range of clinical procedures , treatments and diagnoses is included in the current list of 550 hrg tariffs for elective in - patient care . the quality and outcomes framework , part of the gp contract , is a voluntary mechanism for encouraging primary care provision of some services of curative care , such as antiplatelets for patients with coronary heart disease . with total payments amounting to 1520% of available total practice remuneration , there is a clear incentive for gps to make these services available to nhs patients . the nhs is a complex and heavily regulated health care system in which the roles of actors are in general clearly defined . as the primary emphasis has historically been upon local cost control rather than quality or access issues , geographical variations in quality and quantity of provision have emerged . the lack of explicitness in the definition of the health basket has led to a great deal of uncertainty about entitlement . even if there is a statutory duty governing provision for example , the 1977 nhs act obliges pcts to provide , or to arrange for the provision of , general dental services ( national health service act , chap . 49 , 1977 ) . however , a large proportion of the population is unable to access nhs dental services because of a shortage of dentists willing to provide these services for the nhs . the rapid growth of private practice since 1992 was apparently precipitated by a 7% cut in nhs fees , which was designed to redress perceived excessive income [ 15 , 16 ] . access problems to nhs dentistry have triggered an urgent review of the regulations governing the dentists terms of service with a recent cash injection of over 350 ( 504 ) million , aimed at increasing the number of dentists working for the nhs . the british dental association in is negotiations with the department of health to develop a new contract which is expected in april 2006 . this example illustrates the general principle that private practice flourishes where there are access or quality problems within the nhs . the use of regulation to address quality and access issues has had mixed results . a national evaluation of compliance by nhs organisations with nice guidance found variable implementation . looking at rates of prescribing and use of procedures and medical devices , the time - series analysis found significantly increased prescribing of some taxanes for cancer and orlistat for obesity in line with guidance . nice guidance specifies entitlement in terms of patient groups and pcts are obliged to provide funding only for these patients . whether this practice is equivalent to the pct operating a blanket ban for patients whose condition lies outside the specified guidance is debatable and has not been tested in the courts . over the past decade the growth in quasi - law suggests that the english system is heading towards a more formal statement of benefits and entitlements . positive list of technologies that the nhs should fund ; nsfs describe interventions that should be implemented to achieve standards of care . the payment by results system will be expanded to cover non - elective care and the healthcare commission will increasingly encourage nice and nsf implementation , reinforcing standards through the annual health check . these factors together will take the nhs forward in defining a more explicit health basket for england . however , variations in local capacity to comply with national standards may result in a more explicit rationing of healthcare services at the local level .

a health benefit basket is a range of publicly entitled health - related goods and services . primary legislation ensures the provision of broad categories of healthcare , but this provision is subject to political discretion . case law has established that healthcare organisations may not operate a blanket ban for particular services . this means that the english health basket currently has very few specific services explicitly included or excluded . regulation may , however , be important in determining citizens rights . with reference to services of curative care , this paper explores whether the nhs is moving towards a more explicit definition of a health basket .

Englands Health Benefit Basket: the case of services of curative care The legal framework Quasi-law Discussion

services of curative care are defined as those where the principal medical intent of care is to relieve symptoms or reduce severity or protect against exacerbation or complication of illness or injury . in addition to in - patient and day care , hc1 includes out - patient care , defined as basic medical and diagnostic services , out - patient dental care and other specialist health care provided to outpatients by physicians or paramedics , including services provided at home . primary legislation addresses the establishment and promotion of a comprehensive health service , requiring the secretary of state for health to provide or secure the effective provision of services. however , discretion is at the heart of these duties and politicians are entitled to take into account the resources available to them . however , the court found that the health authority had in practice adopted a blanket ban and recommended the authorities introduced a fair and consistent policy for decision making that adequately assessed exceptional cases by considering each request for treatment on its individual merits . the case therefore made it illegal for health authorities to refuse to provide specific services , with the possible exception of a treatment where the clinical evidence of its inefficacy is overwhelming . primary legislation and case law have therefore not prescribed which services are to be included in or excluded from the english health basket . however , secondary legislation on professional contracts goes some way towards defining entitlement to specific services . 15 ( 3:5,6:8 ) , 2004 ; see table 2].table 2 eligibility for primary care essential services under the 2004 general medical services contract ( from ) type of essential serviceeligibility for treatmentimmediate and necessary emergency treatmentany person to whom the contractor has been requested to provide treatment ... at any place in its practice area [ reg.15 ( 6)]management of terminal illnessregistered patients and temporary residentstreatment of conditions from which recovery is generally expectedregistered patients and temporary residentstreatment of chronic diseaseregistered patients and temporary residentsadvice in connection with the patient s health , including relevant health promotion adviceregistered patients and temporary residentsreferral of the patient for other services under the 1977 actregistered patients and temporary residentshome visitswhere contractor considers it inappropriate , because of a patient s medical condition , for the patient to attend the practice premisesannual health checkspatients aged over 75 yearspatients not seen within 3 years ; newly registered patients eligibility for primary care essential services under the 2004 general medical services contract ( from ) for dental practitioners the national nhs ( general dental services ) regulations 1992 make provision for the secretary of state for health to determine dentists remuneration , including a scale of fees for providing particular services [ reg . the department of health then considers these recommendations and the chief dental officer for england notifies strategic health authorities , pcts , nhs trusts ( groups of nhs hospitals , each operating as a single legal entity ) and all general dental practitioners of the updated fee scales ( ibid . ) . , nhs provision is shaped by a considerable amount of quasi - law. quasi - law is defined as rules which are not usually legally binding , although they may have some legal force , but which will in practice determine the way in which people act . amongst the regulation helping to define patient entitlement to services are nsfs , nice guidance , waiting time guarantees , fee schedules , and incentive schemes . the healthcare commission is the key regulator , assessing the performance of nhs organisations against national standards in its annual health check , a monitoring process that assesses both existing performance ( core standards ) and capacity to improve ( developmental standards ) . the programme of nsfs , launched by the department of health in april 1998 , usually produces one new framework a year . each nsf is developed with the assistance of an external reference group ( erg ) , which seeks to engage a range of views from health professionals , service users and carers , health service managers , partner agencies and other advocates . it is unclear how nsfs are to be updated to reflect changes in the evidence base that underpins them.table 3 services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative carecategoryserviceschildrenstandard 7 : guidance on hospital - based services for childrenstandard 3 : guidance on community - based carecoronary heart diseasestandard 7 : nhs trusts to provide appropriate investigations and treatments for patients with suspected or confirmed coronary heart diseasediabetesstandard 7 : nhs to provide rapid and effective treatment for diabetic emergenciesmental healthin - patient hospital beds for persons needing a short period of intensive intervention and observationelderlystandard 4 : need for appropriate specialist carestandard 7 : effective diagnosis , treatment and support for those with mental health problemsrenal diseasequality requirement 2 : timely , appropriate and effective investigation , treatment and follow - up for those with chronic kidney diseasestandard 5 : all likely to benefit from a kidney transplant to receive a high quality service which supports them in managing their transplant(from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative care ( from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) there is no statutory obligation on health care organisations to implement nsf standards . however , the health and social care ( community health and standards ) act 2003 [ health and social care ( community health and standards ) act , chap . nice , the organisation responsible for assessing whether new or existing technologies should be available on the nhs , produces three types of guidance which help define the availability of nhs services of curative care . firstly , technology appraisals give guidance on the use of new and existing treatments within the nhs . of the 91 technology appraisals published to date ( july 2005 ) some relate to in - patient care , such as appraisal no . 11 ( the use of implantable cardioverter defibrillators for arrhythmias ) ; to day care , such as appraisal no . 48 ( home compared with hospital haemodialysis for patients with end - stage renal failure ) ; and to out - patient care , such as appraisal no . secondly , nice clinical guidelines offer guidance on the appropriate treatment and care of people with specific diseases and conditions within the nhs . thirdly , nice produces guidance on whether interventional procedures used for diagnosis or treatment are sufficiently safe and effective for routine use in the nhs . of the 127 currently published topics most relate to hospital - based care , such as radiotherapy for age - related macular degeneration ( interventional procedure guidance , ipg , 048 ) and auditory brainstem implants ( ipg108 ) . guidance on technology appraisals is formulated by the nice appraisal committee , an independent advisory body with individuals from a range of professional backgrounds . to develop a guideline , nice commissions one of the national collaborating centres to establish a guideline development group . the group appraises clinical and cost - effectiveness evidence from systematic reviews of the research evidence . to produce guidance on interventional procedures , the interventional procedures advisory committee , an independent body of 24 members with a range of expertise , considers the safety and efficacy of procedures , but does not examine clinical or cost effectiveness . nice guidance generally acts as quasi - law , but one aspect of guidance on technology appraisals is supported by statute . however , the guidance is not binding on individual clinicians , who must assess whether the technology is appropriate for the patients they treat . published annually , these specify national goals within the public sector , including waiting time targets ( or guarantees ) for the nhs : ( a ) by the end of 2005 , patients will wait a maximum of 6 months for in - patient admission and no more than 13 weeks for an out - patient appointment . where fee schedules exist for nhs care , patient for example , the new national tariff system of payments for hospital services , whilst in no way guaranteeing provision , suggests services that should be accessible on the nhs . a range of clinical procedures , treatments and diagnoses is included in the current list of 550 hrg tariffs for elective in - patient care . the quality and outcomes framework , part of the gp contract , is a voluntary mechanism for encouraging primary care provision of some services of curative care , such as antiplatelets for patients with coronary heart disease . with total payments amounting to 1520% of available total practice remuneration , there is a clear incentive for gps to make these services available to nhs patients . primary legislation addresses the establishment and promotion of a comprehensive health service , requiring the secretary of state for health to provide or secure the effective provision of services. however , discretion is at the heart of these duties and politicians are entitled to take into account the resources available to them . the responsibility for providing these services has passed to local health authorities , which in the current organisational structure are known as primary care trusts ( pcts ) . blanket ban and recommended the authorities introduced a fair and consistent policy for decision making that adequately assessed exceptional cases by considering each request for treatment on its individual merits . the case therefore made it illegal for health authorities to refuse to provide specific services , with the possible exception of a treatment where the clinical evidence of its inefficacy is overwhelming . primary legislation and case law have therefore not prescribed which services are to be included in or excluded from the english health basket . however , secondary legislation on professional contracts goes some way towards defining entitlement to specific services . 15 ( 3:5,6:8 ) , 2004 ; see table 2].table 2 eligibility for primary care essential services under the 2004 general medical services contract ( from ) type of essential serviceeligibility for treatmentimmediate and necessary emergency treatmentany person to whom the contractor has been requested to provide treatment ... at any place in its practice area [ reg.15 ( 6)]management of terminal illnessregistered patients and temporary residentstreatment of conditions from which recovery is generally expectedregistered patients and temporary residentstreatment of chronic diseaseregistered patients and temporary residentsadvice in connection with the patient s health , including relevant health promotion adviceregistered patients and temporary residentsreferral of the patient for other services under the 1977 actregistered patients and temporary residentshome visitswhere contractor considers it inappropriate , because of a patient s medical condition , for the patient to attend the practice premisesannual health checkspatients aged over 75 yearspatients not seen within 3 years ; newly registered patients eligibility for primary care essential services under the 2004 general medical services contract ( from ) for dental practitioners the national nhs ( general dental services ) regulations 1992 make provision for the secretary of state for health to determine dentists remuneration , including a scale of fees for providing particular services [ reg . similarly , the 1986 nhs ( general ophthalmic services ) regulations make provision for opticians and ophthalmic medical practitioners to charge for sight tests and for optical appliances ( national health service , general ophthalmic services , regulation si 1986/975 , 1986 ) . in addition to legislation , nhs provision is shaped by a considerable amount of quasi - law. quasi - law is defined as rules which are not usually legally binding , although they may have some legal force , but which will in practice determine the way in which people act . amongst the regulation helping to define patient entitlement to services are nsfs , nice guidance , waiting time guarantees , fee schedules , and incentive schemes . the programme of nsfs , launched by the department of health in april 1998 , usually produces one new framework a year . each nsf is developed with the assistance of an external reference group ( erg ) , which seeks to engage a range of views from health professionals , service users and carers , health service managers , partner agencies and other advocates . it is unclear how nsfs are to be updated to reflect changes in the evidence base that underpins them.table 3 services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative carecategoryserviceschildrenstandard 7 : guidance on hospital - based services for childrenstandard 3 : guidance on community - based carecoronary heart diseasestandard 7 : nhs trusts to provide appropriate investigations and treatments for patients with suspected or confirmed coronary heart diseasediabetesstandard 7 : nhs to provide rapid and effective treatment for diabetic emergenciesmental healthin - patient hospital beds for persons needing a short period of intensive intervention and observationelderlystandard 4 : need for appropriate specialist carestandard 7 : effective diagnosis , treatment and support for those with mental health problemsrenal diseasequality requirement 2 : timely , appropriate and effective investigation , treatment and follow - up for those with chronic kidney diseasestandard 5 : all likely to benefit from a kidney transplant to receive a high quality service which supports them in managing their transplant(from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) services of curative care covered by selected national service frameworks ; icha category hc.1 : services of curative care ( from : dh website , http://www.dh.gov.uk/policyandguidance/healthandsocialcaretopics/healthandsocialcarearticle/fs/en?content_id=4070951&chk=w3ar/w , accessed 12 july 2005 ) there is no statutory obligation on health care organisations to implement nsf standards . however , the health and social care ( community health and standards ) act 2003 [ health and social care ( community health and standards ) act , chap . 43 , 2003 ) gave the secretary of state powers to publish standards for health care [ s. 46 ( 1 ) ] that nhs bodies are bound to take into account [ s. 46 ( 4 ) ] ; nsfs could inform these standards . nice , the organisation responsible for assessing whether new or existing technologies should be available on the nhs , produces three types of guidance which help define the availability of nhs services of curative care . firstly , technology appraisals give guidance on the use of new and existing treatments within the nhs . of the 91 technology appraisals published to date ( july 2005 ) some relate to in - patient care , such as appraisal no . 11 ( the use of implantable cardioverter defibrillators for arrhythmias ) ; to day care , such as appraisal no . 48 ( home compared with hospital haemodialysis for patients with end - stage renal failure ) ; and to out - patient care , such as appraisal no secondly , nice clinical guidelines offer guidance on the appropriate treatment and care of people with specific diseases and conditions within the nhs . for example , the dyspepsia guideline ( clinical guideline , cg , 18 ) recommends life - style advice and appropriate medication as secondary preventative measures and the guideline on head injury ( cg4 ) specifies treatment pathways covering emergency , out - patient and in - patient care . thirdly , nice produces guidance on whether interventional procedures used for diagnosis or treatment are sufficiently safe and effective for routine use in the nhs . of the 127 currently published topics most relate to hospital - based care , such as radiotherapy for age - related macular degeneration ( interventional procedure guidance , ipg , 048 ) and auditory brainstem implants ( ipg108 ) . guidance on technology appraisals is formulated by the nice appraisal committee , an independent advisory body with individuals from a range of professional backgrounds . following consultation , the final appraisal determination is developed , approved by the nice guidance executive and put out again for consultation . to produce guidance on interventional procedures , the interventional procedures advisory committee , an independent body of 24 members with a range of expertise , considers the safety and efficacy of procedures , but does not examine clinical or cost effectiveness . nice guidance generally acts as quasi - law , but one aspect of guidance on technology appraisals is supported by statute . however , the guidance is not binding on individual clinicians , who must assess whether the technology is appropriate for the patients they treat . published annually , these specify national goals within the public sector , including waiting time targets ( or guarantees ) for the nhs : ( a ) by the end of 2005 , patients will wait a maximum of 6 months for in - patient admission and no more than 13 weeks for an out - patient appointment . ( the 24/48 target refers to patients being able to see a general practitioner within 2 working days or another primary care professional within 1 working day , whilst the 4-hour wait target is that patients should spend no more than 4 hours in an accident and emergency hospital department from arrival to admission , transfer or discharge . ) where fee schedules exist for nhs care , patient for example , the new national tariff system of payments for hospital services , whilst in no way guaranteeing provision , the national price schedule for patient services is classified by health care resource group ( hrg ) codes . a range of clinical procedures , treatments and diagnoses is included in the current list of 550 hrg tariffs for elective in - patient care . the quality and outcomes framework , part of the gp contract , is a voluntary mechanism for encouraging primary care provision of some services of curative care , such as antiplatelets for patients with coronary heart disease . with total payments amounting to 1520% of available total practice remuneration , there is a clear incentive for gps to make these services available to nhs patients . the nhs is a complex and heavily regulated health care system in which the roles of actors are in general clearly defined . the lack of explicitness in the definition of the health basket has led to a great deal of uncertainty about entitlement . even if there is a statutory duty governing provision for example , the 1977 nhs act obliges pcts to provide , or to arrange for the provision of , general dental services ( national health service act , chap . however , a large proportion of the population is unable to access nhs dental services because of a shortage of dentists willing to provide these services for the nhs . access problems to nhs dentistry have triggered an urgent review of the regulations governing the dentists terms of service with a recent cash injection of over 350 ( 504 ) million , aimed at increasing the number of dentists working for the nhs . this example illustrates the general principle that private practice flourishes where there are access or quality problems within the nhs . nice guidance specifies entitlement in terms of patient groups and pcts are obliged to provide funding only for these patients . whether this practice is equivalent to the pct operating a blanket ban for patients whose condition lies outside the specified guidance is debatable and has not been tested in the courts . over the past decade the growth in quasi - law suggests that the english system is heading towards a more formal statement of benefits and entitlements . positive list of technologies that the nhs should fund ; nsfs describe interventions that should be implemented to achieve standards of care . the payment by results system will be expanded to cover non - elective care and the healthcare commission will increasingly encourage nice and nsf implementation , reinforcing standards through the annual health check . these factors together will take the nhs forward in defining a more explicit health basket for england . however , variations in local capacity to comply with national standards may result in a more explicit rationing of healthcare services at the local level .

if africa is to achieve the millennium development goal ( mdg ) of reducing childhood mortality by two thirds by 2015 , new approaches to healthcare delivery must be developed . despite various national and international efforts , malaria still remains a major cause of childhood mortality accounting for about 800,000 deaths annually [ 2 , 3 ] . as over 80% of these deaths occur in african children under the age of five , it will be difficult to achieve the mdg goals without substantially reducing the burden of malaria . the roll back malaria ( rbm ) partnership was established to coordinate a global approach to combat malaria , and at an rbm meeting in 2000 , african heads of the state pledged to ensure that 60% of people suffering from malaria have access to affordable and appropriate treatment within 24 hours of the onset of illness and 60% of children and pregnant women sleep under insecticide - treated nets . treatment starting within 24 hours of the onset of symptoms prevents progression to severe malaria or death . however strategies to achieve this goal must take into account populations that do not patronize public health facilities or are inadequately served by them . often children do not reach public facilities untill three or more days after the onset of symptoms because they are treated at home or by private health providers in the community [ 59 ] . yet until recently private health providers were not the focus of intervention programs but now the roll back malaria initiative is encouraging participation of all private health providers within overall government strategies for malaria control . patent medicine sellers ( pms ) are a large group of informal private health providers found in many countries who operate as shop keepers , traders in periodic markets , itinerant drug peddlers , and wholesale drug merchants [ 10 , 11 ] . those who operate from shops are patronized by people from all segments of the community especially the poor because they are perceived to be more accessible , caring , and affordable than public health facilities [ 7 , 8 , 1012 ] . they are a source of drugs , advice , and treatment for illnesses in many communities [ 1016 ] . they have no formal pharmacy training but are licensed to sell proprietary and over - the - counter drugs . however many of their practices have been reported to be substandard [ 10 , 14 , 15 ] . policy makers have generally ignored them , attempted to redirect customers away from them , or tried ( usually unsuccessfully ) to enforce regulations preventing them from acting as health providers [ 10 , 17 ] . few reported interventions have tried to work with them to improve management of malaria , and there is little or no exchange of information or collaboration between government bodies and pms . the result is that there is a huge , largely unregulated resource which is not being utilized to contribute to national child survival initiatives [ 10 , 17 ] . but because 15%82% of caregivers in sub - saharan africa first consult them for treatment of their sick children , it is increasingly being recognized that they must be included in malaria control programmes [ 10 , 17 ] . various strategies like information , incentives , persuasion , social marketing , franchise , or accreditation have been suggested to improve their practices [ 10 , 17 , 18 ] . these all need research into which method or combination of methods is the best , but whichever method is used , pms would need to acquire some basic knowledge and probably training . in order to determine how best pms can be used in malaria control in children , it is important to determine exactly what they do for sick children . such information however has been based mainly on interviews , use of mystery clients and anecdotes [ 1922 ] . few studies have documented what actually happens when caregivers visit pms shops . partly because of the attitude of some health professionals and partly for fear of legal repercussions , pms are often reluctant to divulge information about their activities . even where they do , there is sometimes a discrepancy between what they say they do and what they actually do . ideally , studies about pms should involve a combination of methods , for example , direct observations of their activities and interviews to allow for validation and increase of the relevance and generalization of results . for this reason a combination of methods was used in this study whose aim was to investigate how pms manage presumptive malaria in children under 5 years in kaduna , nigeria , specifically to determine the characteristics of pms and the services they offer , and for those who manage children , to document how they identify and treat presumptive malaria . it is hoped that the findings would provide program managers and policy makers substantial grounds to integrate pms into malaria control programs and assist in the development of interventions for them . nigeria , the most populous country in africa , has one of the highest childhood mortality rates in the world with malaria accounting for 30% of mortality [ 25 , 26 ] . it was the capital of the former northern region and is an important city in the north of nigeria . it is situated in the northwestern health zone which has the highest under - five mortality rate of the six health zones in the country . in the early 1900s , various parts of the country people from the major ethnic groups ( hausa , yoruba , and ibo ) and numerous minor ethnic groups have settled in kaduna . the main local language spoken by most residents is hausa , but a lot of people are multilingual . sometimes ethnic and religious tensions erupt into riots ( the last occurred in 2000 ) . many people work in the textile , car assembly , and oil refining industries , the civil service , and the small - scale businesses . the city is made up of 2 administrative areas : kaduna north and south local government areas ( lgas ) . each lga is a mixture of high- , medium- , and low - density areas supporting various commercial and industrial activities with people of different sociocultural , ethnic , and religious backgrounds . the city had an estimated population of a million at the time of the study . knowledge about pms management of sick children in kaduna is scanty as most studies about pms were carried out in southern nigeria where the population , culture , and perceptions differ from those of the north . studies from the south were carried out in urban and rural areas but involved populations which were homogenous in ethnicity and religion [ 7 , 20 , 29 ] . in contrast , kaduna is an urban area with a heterogeneous population in terms of religion , ethnicity and culture . though there are no published data , it appears that the proportion of people patronizing pms in kaduna is increasing in spite of the presence of many orthodox health facilities including a university teaching hospital , a paediatric hospital , 3 general hospitals , 10 primary healthcare centres , and 93 registered fee - paying private hospitals . pms are expected to be over 21 years of age with at least primary school education before they can be registered by the state ministry of health ( smoh ) . registered pms are issued licenses which they are supposed to renew every year and display in their shops . the task force of the smoh carries out periodic inspections to make sure that pms comply with regulations . there are also an unknown number of pms who , because they are not registered by the smoh , are not supposed to sell drugs but who nevertheless do so . both registered and unregistered pms belong to the national association of patent and proprietary medicine dealers ( nappmed ) , membership of which is voluntary the study carried out in 2003 was a cross - sectional , descriptive survey of all pms in kaduna south lga . only pms operating in shops ( registered and unregistered ) were studied because these appear to be the ones most patronized by caregivers of sick children [ 10 , 11 , 17 ] . traders in periodic markets , itinerant drug peddlers , and wholesale drug merchants were excluded . two research instruments were used a questionnaire used to interview pms and a detailed checklist used during direct observation of customer - pms interactions . a pretest was carried out in an area similar to but far away from the study area to validate both instruments . the questionnaire was translated from english to hausa ( the commonest local language in the study area ) and backtranslated into english by an independent translator to ensure correct translation . the translation was done so that it could be used for pms who were not fluent in english . the questionnaire contained questions about the characteristics of pms , the shop , the services they offered both adults and children , and the methods they used to identify and manage or refer children with presumptive malaria . an inventory of drugs sold for malaria in the week preceding the interview was taken . questions were also asked about the type , the methods of administration , and the doses of medicines ( if any ) given to treat children they thought had malaria . the questionnaire was administered by trained research assistants , and interviews were carried out with the pms who attended to customers . the detailed checklist was used to record the number , sex , estimated ages , and actions of all visits by customers whether these visits were of a medical nature ; it also recorded the response of pms ( including whether the customer was asked questions about illness , sale of drugs , giving of advice , physical examination , and any treatment given ) . during the visits , trained research assistants recorded activities in shops and observations this was done at different times of the day for one hour using the detailed checklist . a maximum of 20 observations per shop were recorded in the one - hour period . data recorded were based on consecutive arrival of clients at shops . in order to minimize interference with customer - pms interactions , the research assistants did not interfere with shop activities or ask customers any questions and no exit interviews of customers were done . studies which conducted observations of customer - health worker interactions at government health facilities recorded different numbers of observations ranging from two observations per condition to six observations per facility . none of these studies provided a rationale for the number of observations made or the time spent . shop observations studies from southern nigeria indicated that 10 observations per shop were adequate to obtain significant information and avoid the cluster effect of the same individual repeating his behaviour . further the study pretest indicated that approximately 50% of customers ' visits were for nonmedical purposes and about 20% of sales concern children less than 5 years . thus the number of total observations needed for the shops was calculated as 220 10 = 2200 . a 5% level of precision was used taking into account the resources available for the study . at expected sales for children of 20% ( 5% ) , with = 0.05 and = 0.20 , the minimum number of visits by customers that would give an adequate number of customer - pms observations was calculated to be 471 . the visits that represent under-5 children equal 2200 thus the number of visits that will represent total children visits equals 5 220 = 1100 . with an estimated 10 customer - pms observations per shop , the total number of shops to be studied equals 1100 10 = 110 . since this represented the approximate number of shops in one lga of the city , all the pms in the lga were to be surveyed . as approximately 50% of customers ' visits were for non - medical purposes , the number of observations was increased to 20 . this was the maximum number of customer - pms observations recorded in the one - hour period the by the trained researcher . approval was obtained from the scientific and ethical committee of ahmadu bello university teaching hospital , zaria , nigeria and the institutional review board of boston university , north america . informed consent was obtained from the state chairman of the national association of patent and propriety medicine dealers ( nappmed ) , each local head of nappmed , the owner of each shop , and individuals enrolled in the survey . variables analyzed included characteristics of the pms , the shop , customers , and child for whom help was sought . drugs pms recommended for malaria were classified into pharmacological groups and doses were classified as correct , underdose , or overdose in accordance with national treatment guidelines , for example , chloroquine dose was deemed appropriate when given orally in a dose of 25 mg / kg base over 3 days . possible factors that could influence the behaviour of different groups of pms ( e.g. , gender , age , educational background , and location of shop ) were compared in terms of proportions and tests of significance using chi - squared tested and student t - test to detect statistically significant differences ( p < .05 ) . all pms shops in the kaduna south lga were to be included in the study . the expected number of shops was 110 , but on the field , a total of 544 shops were identified . however 53 ( 9.7% ) owners were unavailable or refused to participate in the study mainly because of suspicion that researchers were members of the state monitoring task force . most 440 ( 89.6% ) of the remaining 491 shops claimed to be registered but only 56% displayed licenses . the majority ( 87.8% ) of shops was situated near busy places ( main roads or markets ) , 8.1% near health facilities , and the rest near hotels , places of worship , or residential areas . most of them ( 94.7% ) were open 6 or 7 days a week from early morning untill late at night , and 48.3% also sold general store items . services available for children less than 5 included giving medical advice ( 97.8% ) , consultation for illness ( 94.7% ) , administration of injections ( 40.7% ) , and intravenous fluids ( 18.5% ) . some shops ( 37% ) had the services of a medical doctor or nurse ( mostly on a part - time basis ) to see patients . pms consisted of 370 owners ( 75% ) , 63 apprentices ( 13.8% ) , and 58 employees or relatives ( 11.8% ) . while some , 117 ( 23.8% ) , had received training in healthcare , most had not . 104 pms ( 21.2% ) had other jobs , with 63 ( 14.7% ) working in hospitals . most 1584 ( 61.9% ) of 2557 visits were made for medical reasons ( the others were for purchase of non - medical items or social visits ) . of these medical visits , 1266(79.9% ) were of customers who came to request for drugs without prescription and 117 of these requests were obviously for children . just 19 ( 1.2% ) customers presented prescriptions for drugs for children . in most instances only 29.5% gave customers instructions about doses , 2.6% about side effects , and 0.8% about precautions . of 291 people who complained about illness , 86 ( 29.6% ) complained about children 's illnesses , but 49 ( 57% ) did not bring them . three customers were asked to go back and bring the children but 19 pms sold caregivers drugs without asking any questions about the illness . of 37 ( 43% ) children brought for treatment , 5 were asked to return later and 3 were sent into an inner room where the observer could not determine what happened . pms were observed treating 29 children for fever , diarrhea , injuries , and skin lesions . twenty - seven ( 93% ) pms asked for clarification , 21 ( 75% ) examined the children , 7 ( 18.9% ) dressed or stitched wounds , and one gave a child an intravenous infusion . thus at least 222 visits ( 117 drug requests , 19 prescriptions , and 86 consultations ) involved children representing 14% of shop visits . pms were specifically asked about how they identified and managed children they thought had malaria . only 18 ( 3.7% ) of the 491 pms studied stated that they did not treat children for malaria because they did not see children or they referred them . 226 ( 47.8% ) used past experience , 189 ( 39.6% ) , physical examination , and 21(4.4% ) used laboratory tests . the main symptoms used to identify malaria were fever 318 ( 66.1% ) , inability to eat 98 ( 20.7% ) , weakness 70 ( 14.2% ) , eye discoloration 29 ( 6.1% ) and vomiting 29 ( 6.1% ) . there was no difference between types and doses of drugs used by pms who had received professional health training and those who had not ( p > .5 ) . 429 pms who gave antimalarials ( 90.7% ) used chloroquine , 36 ( 7.6% ) used sulfadoxine - pyrimethamine ( at the client 's request ) , 6 ( 1.3% ) used halofantrine , and 2 ( 0.4% ) used quinine . no pms mentioned artemisinin based - products . only 116 ( 19.4% ) pms recommended that caregivers should use methods to reduce the child 's temperature ( exposure , tepid sponging , and cold baths ) . 23 pms ( 4.9% ) recommended practices ( hot baths , covering the child ) which could lead to increase in temperature . just 60 ( 12.7% ) pms mentioned use of mosquito nets ( mainly more often by pms who were health professionals ; p = .03 ) . forty ( 57% ) of 70 pms who had recently referred children had treated them for three or more days . reasons for referral were inability to handle the illness , 27 ( 38.6% ) , lack of improvement , 25(35.7% ) , severe illness , 6 ( 8.6% ) , or laboratory tests , 2 ( 2.9% ) . in the week preceding the survey , pms estimated that they had sold 8,430 doses of paracetamol , 5,413 doses of chloroquine ( mainly syrups , also tablets , and injections ) , 102 doses of sulfadoxine - pyrimethamine , and 3 doses of quinine . chloroquine and paracetamol were popular because they were effective , well known to customers , and cheap ( less than two us dollars ) . improving access to prompt , effective , and appropriate treatment is a key element of the global malaria programme strategy [ 2 , 3 ] . the study shows that pms have the potential to improve access , but they need skills and knowledge to manage malaria . they acted mainly as facilitators for home care ( as the great number of requests by their customers for drugs without prescription indicates a community that was actively involved in self - care ) . some did not ask questions about illness for which they were consulted , and many used injections to treat children . other studies reveal that pms frequently give no or inappropriate advice about drugs [ 10 , 14 , 15 , 17 ] . this highlights one weakness of home management of malaria ( hmm ) which aims to ensure prompt and effective care of sick children by educating mothers and distributors of antimalarials in the community [ 33 , 34 ] . many mothers in developing countries get drugs from pms or drug hawkers usually without prescription [ 10 , 17 , 19 ] . unfortunately , because the person selling the drug or the one administering it does not know the correct dose , children are given inappropriate doses of antimalarials leading to development of severe disease and death [ 10 , 17 , 35 , 36 ] . the study shows that interventions involving pms should focus on training them to determine the correct dose of antimalarials and encourage them to give customers advice about administration . children 's doses vary and are usually calculated using weight , surface area , or age . as most children were not taken to the shops , use of weight or surface area is clearly impractical but using age is feasible . in some countries antimalarials are prepackaged or colour coded according to age , and these have been used with great success [ 37 , 38 ] . in many areas , though , such drugs are not available or affordable , and so other simple methods of determining doses are needed . age could be used to calculate dose as a percentage of the normal adult dose but pms would need training on how to do this . pictorial guides in the shop and inserts in drugs packets could help but inserts increase cost of drugs . it is striking that none of them mentioned artemisinin - combination therapies ( acts ) . this might have been because , at the time of the study , first - line treatment consisted of chloroquine which was cheap and effective . due to increasing drug resistance , the government changed its malaria treatment policy and adopted acts as first - line drugs in 2005 , but many pms were unaware of this [ 39 , 40 ] . at present , though , acts can not legally be sold by pms because they are classified as prescription drugs . changing their status to over - the - counter requires legislation , a process that is currently underway as the federal ministry of health proposes to use act at community level to improve hmm . use of acts might also help reduce the high rate of injections given to children treated in shops and so reduce risk of infection , nerve paralysis , and danger to the community caused by improper disposal of needles . it is of note that some pms dispensed drugs without asking questions ( in essence relying on the caregivers ' diagnosis ) . the importance of pms asking questions about illness , for which they are consulted needs to be stressed because it helps identify those children who need urgent referral to health facilities for example , those with danger signs like vomiting , refusal , or inability to feed . the study was limited in that some pms were cautious about letting researchers see how they treated children because the government task force was threatening to close down shops for illegal activities . thus it is likely that many more children were treated than was recorded . also some pms may have adopted different behaviours to their normal ones because they were being observed but the most probable result of this would be that more , not less , pms than normal would give customers advice about drugs . determining the effect medical personnel who were not pms had on patient management was outside the scope of the study , so was determining the accuracy of pms diagnosis of illnesses , the doses they actually gave , or the extent their advice or lack of it had on the health of their customers . the study shows that pms need training in order to improve their role in malaria control in the community . however generally governments of developing countries have been reluctant to train informal health providers and instead have concentrated on training health workers in the public sector [ 10 , 17 ] . training pms on a range of common conditions in adults led to an improvement in their knowledge though the impact on their practice was not evaluated . in kenya , training resulted in an increase in the proportion of antimalarial drug users who received an adequate dose . the hmm strategy advocates training community - based service providers to dispense antimalarials and educate mothers about the correct dose to give . however surprisingly few programs have considered using pms as the community - based service providers . . carried out a malaria intervention program in southwest nigeria using pms to distribute drugs to mothers , and they reported that most caregivers perceived pms as accessible and efficient at drug distribution . they were quick to add , though that , for good distribution to be maintained , pms would need to receive incentives in the form of commission for the antimalarials they sold or distributed . training however arouses fears that this would be interpreted by the public as official recognition of pms as health providers . definitely continued supervision is needed to sustain changes and ensure a minimum standard of care . further improvement in availability , and quality of public services by the government is necessary as pms can not take the place of public facilities . however as people are likely to continue to patronize pms for a long time , it is recommended that training and retraining should be made a prerequisite for registering and reregistering shops . the effect training has on doses and drugs dispensed in communities needs to be monitored at regular intervals . further , more research is needed to determine what other sorts of interventions involving pms could assist in prevention and management of childhood malaria . the feasibility or otherwise of officially recognizing pms roles in healthcare needs further consideration and discussion . pms have the potential to improve malaria control in the community through improving access of caregivers to effective and appropriate treatment . interventions involving them should focus on certain key issues training them on how to determine the correct dose of appropriate antimalarials , give advice about drug administration , and use insecticide - treated nets . they should also be encouraged to use oral medications instead of injections and ask questions about illness for which they are consulted . if we ignore pms , we do so at our peril ( or more realisticallyat that of our children ) .

roll back malaria initiative encourages participation of private health providers in malaria control because mothers seek care for sick children from them . this study investigated patent medicine sellers ( pms ) management of presumptive malaria in children in order to identify how they can assist malaria control . a cross - sectional survey of 491 pms in kaduna , nigeria , was done using interviews and observation of shop activities . most ( 80% ) customers bought drugs without prescriptions . only 29.5% were given instructions about doses . between 40100% doses of recommended antimalarials were incorrect . some ( 22% ) pms did not ask questions about illness for which they were consulted . most children treated in shops received injections . pms facilitate homecare but have deficiencies in knowledge and practice . interventions must focus on training them to accurately determine doses , give advice about drug administration , use oral medication , and ask about illness . training should be made a prerequisite for registering and reregistering shops .

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

despite various national and international efforts , malaria still remains a major cause of childhood mortality accounting for about 800,000 deaths annually [ 2 , 3 ] . the roll back malaria ( rbm ) partnership was established to coordinate a global approach to combat malaria , and at an rbm meeting in 2000 , african heads of the state pledged to ensure that 60% of people suffering from malaria have access to affordable and appropriate treatment within 24 hours of the onset of illness and 60% of children and pregnant women sleep under insecticide - treated nets . often children do not reach public facilities untill three or more days after the onset of symptoms because they are treated at home or by private health providers in the community [ 59 ] . yet until recently private health providers were not the focus of intervention programs but now the roll back malaria initiative is encouraging participation of all private health providers within overall government strategies for malaria control . patent medicine sellers ( pms ) are a large group of informal private health providers found in many countries who operate as shop keepers , traders in periodic markets , itinerant drug peddlers , and wholesale drug merchants [ 10 , 11 ] . those who operate from shops are patronized by people from all segments of the community especially the poor because they are perceived to be more accessible , caring , and affordable than public health facilities [ 7 , 8 , 1012 ] . they are a source of drugs , advice , and treatment for illnesses in many communities [ 1016 ] . however many of their practices have been reported to be substandard [ 10 , 14 , 15 ] . policy makers have generally ignored them , attempted to redirect customers away from them , or tried ( usually unsuccessfully ) to enforce regulations preventing them from acting as health providers [ 10 , 17 ] . few reported interventions have tried to work with them to improve management of malaria , and there is little or no exchange of information or collaboration between government bodies and pms . but because 15%82% of caregivers in sub - saharan africa first consult them for treatment of their sick children , it is increasingly being recognized that they must be included in malaria control programmes [ 10 , 17 ] . these all need research into which method or combination of methods is the best , but whichever method is used , pms would need to acquire some basic knowledge and probably training . in order to determine how best pms can be used in malaria control in children , it is important to determine exactly what they do for sick children . such information however has been based mainly on interviews , use of mystery clients and anecdotes [ 1922 ] . few studies have documented what actually happens when caregivers visit pms shops . partly because of the attitude of some health professionals and partly for fear of legal repercussions , pms are often reluctant to divulge information about their activities . ideally , studies about pms should involve a combination of methods , for example , direct observations of their activities and interviews to allow for validation and increase of the relevance and generalization of results . for this reason a combination of methods was used in this study whose aim was to investigate how pms manage presumptive malaria in children under 5 years in kaduna , nigeria , specifically to determine the characteristics of pms and the services they offer , and for those who manage children , to document how they identify and treat presumptive malaria . it is hoped that the findings would provide program managers and policy makers substantial grounds to integrate pms into malaria control programs and assist in the development of interventions for them . nigeria , the most populous country in africa , has one of the highest childhood mortality rates in the world with malaria accounting for 30% of mortality [ 25 , 26 ] . it is situated in the northwestern health zone which has the highest under - five mortality rate of the six health zones in the country . in the early 1900s , various parts of the country people from the major ethnic groups ( hausa , yoruba , and ibo ) and numerous minor ethnic groups have settled in kaduna . many people work in the textile , car assembly , and oil refining industries , the civil service , and the small - scale businesses . each lga is a mixture of high- , medium- , and low - density areas supporting various commercial and industrial activities with people of different sociocultural , ethnic , and religious backgrounds . knowledge about pms management of sick children in kaduna is scanty as most studies about pms were carried out in southern nigeria where the population , culture , and perceptions differ from those of the north . studies from the south were carried out in urban and rural areas but involved populations which were homogenous in ethnicity and religion [ 7 , 20 , 29 ] . though there are no published data , it appears that the proportion of people patronizing pms in kaduna is increasing in spite of the presence of many orthodox health facilities including a university teaching hospital , a paediatric hospital , 3 general hospitals , 10 primary healthcare centres , and 93 registered fee - paying private hospitals . pms are expected to be over 21 years of age with at least primary school education before they can be registered by the state ministry of health ( smoh ) . registered pms are issued licenses which they are supposed to renew every year and display in their shops . there are also an unknown number of pms who , because they are not registered by the smoh , are not supposed to sell drugs but who nevertheless do so . both registered and unregistered pms belong to the national association of patent and proprietary medicine dealers ( nappmed ) , membership of which is voluntary the study carried out in 2003 was a cross - sectional , descriptive survey of all pms in kaduna south lga . only pms operating in shops ( registered and unregistered ) were studied because these appear to be the ones most patronized by caregivers of sick children [ 10 , 11 , 17 ] . traders in periodic markets , itinerant drug peddlers , and wholesale drug merchants were excluded . two research instruments were used a questionnaire used to interview pms and a detailed checklist used during direct observation of customer - pms interactions . the questionnaire was translated from english to hausa ( the commonest local language in the study area ) and backtranslated into english by an independent translator to ensure correct translation . the translation was done so that it could be used for pms who were not fluent in english . the questionnaire contained questions about the characteristics of pms , the shop , the services they offered both adults and children , and the methods they used to identify and manage or refer children with presumptive malaria . an inventory of drugs sold for malaria in the week preceding the interview was taken . questions were also asked about the type , the methods of administration , and the doses of medicines ( if any ) given to treat children they thought had malaria . the questionnaire was administered by trained research assistants , and interviews were carried out with the pms who attended to customers . the detailed checklist was used to record the number , sex , estimated ages , and actions of all visits by customers whether these visits were of a medical nature ; it also recorded the response of pms ( including whether the customer was asked questions about illness , sale of drugs , giving of advice , physical examination , and any treatment given ) . during the visits , trained research assistants recorded activities in shops and observations this was done at different times of the day for one hour using the detailed checklist . in order to minimize interference with customer - pms interactions , the research assistants did not interfere with shop activities or ask customers any questions and no exit interviews of customers were done . studies which conducted observations of customer - health worker interactions at government health facilities recorded different numbers of observations ranging from two observations per condition to six observations per facility . none of these studies provided a rationale for the number of observations made or the time spent . shop observations studies from southern nigeria indicated that 10 observations per shop were adequate to obtain significant information and avoid the cluster effect of the same individual repeating his behaviour . further the study pretest indicated that approximately 50% of customers ' visits were for nonmedical purposes and about 20% of sales concern children less than 5 years . thus the number of total observations needed for the shops was calculated as 220 10 = 2200 . a 5% level of precision was used taking into account the resources available for the study . at expected sales for children of 20% ( 5% ) , with = 0.05 and = 0.20 , the minimum number of visits by customers that would give an adequate number of customer - pms observations was calculated to be 471 . the visits that represent under-5 children equal 2200 thus the number of visits that will represent total children visits equals 5 220 = 1100 . since this represented the approximate number of shops in one lga of the city , all the pms in the lga were to be surveyed . as approximately 50% of customers ' visits were for non - medical purposes , the number of observations was increased to 20 . approval was obtained from the scientific and ethical committee of ahmadu bello university teaching hospital , zaria , nigeria and the institutional review board of boston university , north america . informed consent was obtained from the state chairman of the national association of patent and propriety medicine dealers ( nappmed ) , each local head of nappmed , the owner of each shop , and individuals enrolled in the survey . variables analyzed included characteristics of the pms , the shop , customers , and child for whom help was sought . possible factors that could influence the behaviour of different groups of pms ( e.g. , gender , age , educational background , and location of shop ) were compared in terms of proportions and tests of significance using chi - squared tested and student t - test to detect statistically significant differences ( p < .05 ) . the majority ( 87.8% ) of shops was situated near busy places ( main roads or markets ) , 8.1% near health facilities , and the rest near hotels , places of worship , or residential areas . most of them ( 94.7% ) were open 6 or 7 days a week from early morning untill late at night , and 48.3% also sold general store items . services available for children less than 5 included giving medical advice ( 97.8% ) , consultation for illness ( 94.7% ) , administration of injections ( 40.7% ) , and intravenous fluids ( 18.5% ) . some shops ( 37% ) had the services of a medical doctor or nurse ( mostly on a part - time basis ) to see patients . pms consisted of 370 owners ( 75% ) , 63 apprentices ( 13.8% ) , and 58 employees or relatives ( 11.8% ) . 104 pms ( 21.2% ) had other jobs , with 63 ( 14.7% ) working in hospitals . most 1584 ( 61.9% ) of 2557 visits were made for medical reasons ( the others were for purchase of non - medical items or social visits ) . of these medical visits , 1266(79.9% ) were of customers who came to request for drugs without prescription and 117 of these requests were obviously for children . just 19 ( 1.2% ) customers presented prescriptions for drugs for children . in most instances only 29.5% gave customers instructions about doses , 2.6% about side effects , and 0.8% about precautions . of 291 people who complained about illness , 86 ( 29.6% ) complained about children 's illnesses , but 49 ( 57% ) did not bring them . three customers were asked to go back and bring the children but 19 pms sold caregivers drugs without asking any questions about the illness . of 37 ( 43% ) children brought for treatment , 5 were asked to return later and 3 were sent into an inner room where the observer could not determine what happened . pms were observed treating 29 children for fever , diarrhea , injuries , and skin lesions . twenty - seven ( 93% ) pms asked for clarification , 21 ( 75% ) examined the children , 7 ( 18.9% ) dressed or stitched wounds , and one gave a child an intravenous infusion . thus at least 222 visits ( 117 drug requests , 19 prescriptions , and 86 consultations ) involved children representing 14% of shop visits . pms were specifically asked about how they identified and managed children they thought had malaria . only 18 ( 3.7% ) of the 491 pms studied stated that they did not treat children for malaria because they did not see children or they referred them . 226 ( 47.8% ) used past experience , 189 ( 39.6% ) , physical examination , and 21(4.4% ) used laboratory tests . the main symptoms used to identify malaria were fever 318 ( 66.1% ) , inability to eat 98 ( 20.7% ) , weakness 70 ( 14.2% ) , eye discoloration 29 ( 6.1% ) and vomiting 29 ( 6.1% ) . there was no difference between types and doses of drugs used by pms who had received professional health training and those who had not ( p > .5 ) . 429 pms who gave antimalarials ( 90.7% ) used chloroquine , 36 ( 7.6% ) used sulfadoxine - pyrimethamine ( at the client 's request ) , 6 ( 1.3% ) used halofantrine , and 2 ( 0.4% ) used quinine . no pms mentioned artemisinin based - products . only 116 ( 19.4% ) pms recommended that caregivers should use methods to reduce the child 's temperature ( exposure , tepid sponging , and cold baths ) . just 60 ( 12.7% ) pms mentioned use of mosquito nets ( mainly more often by pms who were health professionals ; p = .03 ) . forty ( 57% ) of 70 pms who had recently referred children had treated them for three or more days . reasons for referral were inability to handle the illness , 27 ( 38.6% ) , lack of improvement , 25(35.7% ) , severe illness , 6 ( 8.6% ) , or laboratory tests , 2 ( 2.9% ) . in the week preceding the survey , pms estimated that they had sold 8,430 doses of paracetamol , 5,413 doses of chloroquine ( mainly syrups , also tablets , and injections ) , 102 doses of sulfadoxine - pyrimethamine , and 3 doses of quinine . chloroquine and paracetamol were popular because they were effective , well known to customers , and cheap ( less than two us dollars ) . improving access to prompt , effective , and appropriate treatment is a key element of the global malaria programme strategy [ 2 , 3 ] . they acted mainly as facilitators for home care ( as the great number of requests by their customers for drugs without prescription indicates a community that was actively involved in self - care ) . some did not ask questions about illness for which they were consulted , and many used injections to treat children . other studies reveal that pms frequently give no or inappropriate advice about drugs [ 10 , 14 , 15 , 17 ] . this highlights one weakness of home management of malaria ( hmm ) which aims to ensure prompt and effective care of sick children by educating mothers and distributors of antimalarials in the community [ 33 , 34 ] . many mothers in developing countries get drugs from pms or drug hawkers usually without prescription [ 10 , 17 , 19 ] . unfortunately , because the person selling the drug or the one administering it does not know the correct dose , children are given inappropriate doses of antimalarials leading to development of severe disease and death [ 10 , 17 , 35 , 36 ] . the study shows that interventions involving pms should focus on training them to determine the correct dose of antimalarials and encourage them to give customers advice about administration . children 's doses vary and are usually calculated using weight , surface area , or age . as most children were not taken to the shops , use of weight or surface area is clearly impractical but using age is feasible . in some countries antimalarials are prepackaged or colour coded according to age , and these have been used with great success [ 37 , 38 ] . in many areas , though , such drugs are not available or affordable , and so other simple methods of determining doses are needed . age could be used to calculate dose as a percentage of the normal adult dose but pms would need training on how to do this . it is striking that none of them mentioned artemisinin - combination therapies ( acts ) . this might have been because , at the time of the study , first - line treatment consisted of chloroquine which was cheap and effective . at present , though , acts can not legally be sold by pms because they are classified as prescription drugs . changing their status to over - the - counter requires legislation , a process that is currently underway as the federal ministry of health proposes to use act at community level to improve hmm . use of acts might also help reduce the high rate of injections given to children treated in shops and so reduce risk of infection , nerve paralysis , and danger to the community caused by improper disposal of needles . it is of note that some pms dispensed drugs without asking questions ( in essence relying on the caregivers ' diagnosis ) . the importance of pms asking questions about illness , for which they are consulted needs to be stressed because it helps identify those children who need urgent referral to health facilities for example , those with danger signs like vomiting , refusal , or inability to feed . the study was limited in that some pms were cautious about letting researchers see how they treated children because the government task force was threatening to close down shops for illegal activities . also some pms may have adopted different behaviours to their normal ones because they were being observed but the most probable result of this would be that more , not less , pms than normal would give customers advice about drugs . the study shows that pms need training in order to improve their role in malaria control in the community . however generally governments of developing countries have been reluctant to train informal health providers and instead have concentrated on training health workers in the public sector [ 10 , 17 ] . carried out a malaria intervention program in southwest nigeria using pms to distribute drugs to mothers , and they reported that most caregivers perceived pms as accessible and efficient at drug distribution . they were quick to add , though that , for good distribution to be maintained , pms would need to receive incentives in the form of commission for the antimalarials they sold or distributed . training however arouses fears that this would be interpreted by the public as official recognition of pms as health providers . definitely continued supervision is needed to sustain changes and ensure a minimum standard of care . further improvement in availability , and quality of public services by the government is necessary as pms can not take the place of public facilities . however as people are likely to continue to patronize pms for a long time , it is recommended that training and retraining should be made a prerequisite for registering and reregistering shops . the effect training has on doses and drugs dispensed in communities needs to be monitored at regular intervals . further , more research is needed to determine what other sorts of interventions involving pms could assist in prevention and management of childhood malaria . pms have the potential to improve malaria control in the community through improving access of caregivers to effective and appropriate treatment . interventions involving them should focus on certain key issues training them on how to determine the correct dose of appropriate antimalarials , give advice about drug administration , and use insecticide - treated nets . they should also be encouraged to use oral medications instead of injections and ask questions about illness for which they are consulted . if we ignore pms , we do so at our peril ( or more realisticallyat that of our children ) .

reagents and media : unless otherwise specified , all chemicals were purchased from sigma chemical co. ( st . louis , mo , u.s.a . ) , and media were from gibco ( invitrogen , burlington , on , canada ) . isolation and culture : all experiments were authorized by the animal center for biomedical experimentation at gyeongsang national university . tissues of three female and three male pigs from the same litter were recovered under standard surgical procedures at both 1 week and 8 months after birth . porcine mscs from subcutaneous adipose tissue ( a - mscs ) were isolated following a previously described method by digestion with 0.075% collagenase type i ( millipore , temecula , ca , u.s.a . ) and subsequent separation by filtration with 100- and 40-m cell strainers . porcine dermal skin mscs ( ds - mscs ) were isolated after removal of the epidermis of the ear skin as previously described . as a control , mscs were isolated from porcine bone marrow ( bm - mscs ) as described previously . all cells were isolated from each sample in triplicates and cultured in advanced dulbecco s modified eagle medium ( admem ) supplemented with 10% fetal calf serum ( fcs ) and 1% penicillin - streptomycin ( 10,000 iu and 10,000 g / ml ) at 38.5c in a humidified atmosphere of 5% co2 in air . after cells reaching 70% confluence , they were sub - cultured / passaged for further analysis using 0.25% trypsin - edta solution . alkaline phosphatase ( ap ) activity : to detect ap activity , porcine mscs grown on 35-mm dishes for 2 weeks were stained with an ap chromogen kit ( bcip / nbt ) ( abcam inc . , cambridge , ma , u.s.a . ) after fixing with 4% formaldehyde . cd marker analysis : the expression of cell surface marker cd90 in pmscs was analyzed by flow cytometer as previously described . in brief , 1 10 cells at passage 3 were suspended in 100 l of dulbecco s phosphate - buffered saline ( dpbs ) and labeled with fluorescein isothiocyanate ( fitc ) conjugated mouse anti - human cd90 ( thy-1 , bd biosciences , san jose , ca , u.s.a . ) or isotype - matched control antibodies ( mouse igg1 , bd biosciences ) . a total of 10,000 cells were analyzed by flow cytometer using cellquest software ( becton dickinson , frankin lakes , nj , u.s.a . ) . in vitro differentiation potential : cells at ~80% confluence were induced toward adipogenic or osteogenic differentiation by culturing in admem supplemented with lineage specific constituents for 3 weeks in triplicates . adipogenic differentiation medium consisted of 10 m insulin , 200 m indomethacin , 500 m isobutylmethylxanthine and 1 m dexamethasone . osteogenic differentiation medium consisted of 1 m dexamethasone , 100 m ascorbic acid and 10 mm -glycerophosphate . senescence - associated -galactosidase ( sa--gal ) staining : to evaluate cellular senescence , an sa--gal assay was performed according to manufacturer s protocol using sa--gal staining kit ( cell signaling technology , cambridge , ma , u.s.a . ) . briefly , cells at 60% confluence grown in 35-mm dishes in triplicates were fixed with 3.7% formalin for 10 min at room temperature . after washing twice with dpbs , 1 ml of sa--gal staining solution was added to each dish and incubated at 37c overnight in a dry incubator ( no co2 ) . the cells were observed under a microscope for the development of blue color , while the sa--gal staining solution was still on the dishes . analysis of telomere length : genomic dna from each sample was extracted using the gene alltm tissue sv plus mini kit ( general biosystem , seoul , korea ) . the dna was subjected to telomere length analysis using the telo taggg telomere length assay kit ( roche , mannheim , germany ) following manufacturer s instructions . briefly , genomic dna was digested with rsa1 and hinf1 for 2 hr at 37c and separated by 0.8% agarose gel electrophoresis at 30 v / cm for 5 hr . after gel depurination and denaturation , dna fragments were transferred overnight by southern blotting in 20 ssc buffer to a positively charged nylon membrane . dna was fixed to the membrane by uv cross - linking , and the membrane was probed using a dig - labeled telomere probe , washed and blocked . bands were detected using ap anti - dig antibody and cdp - star chemiluminescent substrate . the telomere length was calculated by exposing the x - ray film for 15 min and then imaging it . the signals were scanned and evaluated using the unok-8000 gel manager system gel viewer 1.5 system ( biotechnology , seoul , korea ) . the mean telomere length was calculated using the formula : mean trf (odi)/ (odi / li ) , where odi is the optical density at a position i on the lane and li is the corresponding terminal restriction fragment ( trf ) length at position i on the lane . this formula takes into account the higher signal intensity generated by larger trf fragments . female bm - mscs derived from the same litter both at 1 week and 8 months after birth were used for an internal control , whereas cells derived from the testis tissue of 1-year - old pig were used for a positive control . cell cycle analysis : for cell cycle analysis by flow cytometry , the cells were fixed with 70% ethanol , washed twice with dpbs and stained with 10 g / ml propidium iodide . the cell cycle analysis was performed in triplicate using 10,000 cells per sample each time . western blot analysis : the expression of proliferation , senescence and apoptosis associated proteins was analyzed using western blot . briefly , protein lysate was prepared from respective mscs at passage 3 using ripa buffer ( all samples were isolated from three female and three male pigs from the same litter ) ( thermo scientific , rockford , il , u.s.a . ) and then centrifuged at 14,000 g for 5 min at 4c . the supernatant was used for determining protein concentration using bicinchoninic acid ( bca ) protein assay reagent kit ( pierce biotechnology , rockford , il , u.s.a . ) . a total of 25 g each protein sample was separated on 12% sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page , mini protean , biorad , hercules , ca , u.s.a . ) and electroblotted onto polyvinylidene difluoride ( pvdf , millipore ) membranes . female bm - mscs derived from 1 week old pig were used as an internal control for mscs . hela and f9 whole cell lysates ( santa cruz , ca ) were used as positive controls for telomerase reverse transcriptase ( tert ; hela ) , p21 ( hela ) and oct 3/4 ( f9 ) , whereas lysates from human fibroblast ( mcf 5 ) and cancer cells ( mda - mb-231 ) were used as negative controls for oct 3/4 protein expression . the membranes were blocked and incubated with anti - tert ( 1:200 dilution ; santa cruz biotechnology , ca , u.s.a . ) , anti - oct 3/4 ( 1:100 dilution ; santa cruz biotechnology ) , anti - il6 ( 1:500 dilution ; abcam , cambridge , uk ) , anti - p53 ( 1:100 dilution ; santa cruz biotechnology ) , anti - c - myc ( 1:500 dilution ; abcam ) , anti - tgf ( 1;500 dilution ; abcam ) , anti - p21 ( 1:200 dilution ; santa cruz biotechnology ) , anti - bak ( 1:1,000 dilution ; abcam ) , anti - bcl2 ( 1:200 dilution ; abcam ) and anti--actin ( 1:1,000 dilution ; cell signaling technology , cambridge , ma , u.s.a . ) antibodies overnight at 4c . the membranes were then incubated with horseradish peroxidase ( hrp)-conjugated secondary antibodies , such as goat anti - mouse ( 1:1,000 dilution ; p53 and bcl2 ) , goat anti - rabbit ( 1:5,000 dilution ; p21 , il6 , c - myc , tgf , bax and -actin ) and donkey anti - goat ( 1:5,000 dilution ; oct3/4 and tert ) for 1 hr at room temperature . protein bands were detected by enhanced chemiluminescence ( ecl ) ( amersham biosciences corp . , piscataway , nj , u.s.a . ) . statistical analysis : data were analyzed by one - way analysis of variance ( anova ) using spss 12.0 ( spss inc . , general observations for mscs : all mscs have shown fibroblast - like morphology in monolayer culture and formed colonies regardless of donor sex and age . as shown in fig . 1.analysis of alkaline phosphatase ( ap ) activity and cd90 expression of mscs harvested at 1 week and 8 months after birth . ( a ) ap activity . ba , open and filled blue histograms display isotype - matched controls and positive reaction , respectively . female - derived mscs obtained at 1 week after birth showed higher ap activity than male - derived mscs obtained at the same age . however , the ap activity was decreased in mscs harvested at 8 months after birth regardless of gender compared to that in mscs harvested at 1 week after birth . there was no notable difference in ap activity in mscs of both ages with respect to the source of tissue . all mscs were strongly positive for cd90 with staining in 87% of cells , except for adult female a - mscs , which showed cd90 expression in 80% of cells ( fig . cd90 expression was maintained in cells isolated at 8 months after birth without any significant differences ( p<0.05 ) , indicating the mesodermal lineage of the cells ( fig . analysis of alkaline phosphatase ( ap ) activity and cd90 expression of mscs harvested at 1 week and 8 months after birth . ba , open and filled blue histograms display isotype - matched controls and positive reaction , respectively . analysis of msc differentiation potential : the stemness of mscs harvested at 1 week and 8 months after birth was assessed by in vitro differentiation into osteocytes and adipocytes . all mscs differentiated into osteocytes and adipocytes in vitro regardless of donor age , sex and tissue source with varying differentiation capacity ( fig . mscs harvested at 8 months after birth showed enhanced osteogenic differentiation , where male - derived cells have shown higher propensity toward osteogenesis than female - derived cells . furthermore , mscs from adipose tissue displayed a higher differentiation capacity than those from dermal skin . in contrast , all mscs harvested at 8 months after birth showed markedly diminished adipogenic differentiation , except for male - derived a - mscs which maintained their differentiation capacity with the advancement in age . on the other hand , a - mscs harvested at 1 week after birth regardless of their sex have shown higher adipogenic differentiation ability when compared to both male and female derived ds - mscs harvested at the same age . analysis of sa--galactosidase activity : the results of sa--gal staining for the detection of cell aging are displayed in fig . 3.fig . 3.senescence-associated ( sa)--galactosidase activity of mscs obtained at 1 week and 8 months after birth . all mscs harvested at 8 months after birth were more positive for sa--gal staining , except for female a - mscs , when compared to those harvested at 1 week after birth ( macrographs of fig . , ds - mscs showed stronger sa--gal staining than a - mscs harvested at 8 months after birth . however , there was no difference found in sa--gal staining among mscs harvested at 1 week age regardless of their sex and tissue source . senescence - associated ( sa)--galactosidase activity of mscs obtained at 1 week and 8 months after birth . analysis of msc life - span and proliferation : msc life - span was analyzed according to donor sex , age and tissue source based on telomere length ( southern blotting ) and telomerase activity ( tert ; western blotting ) ( fig . 4.analysis of telomere length , telomere activity - related tert protein expression and oct 3/4 protein expression in mscs harvested at 1 week and 8 months after birth . ( a ) telomere restriction fragment lengths determined by southern blotting using a telomere length assay . ( b ) tert and oct3/4 protein expression determined by western blot analysis . ) . the telomere length of female - derived bm - mscs was 18.5 1.3 and 19.0 0.0 kb at 1 week and 8 months of age , respectively . the telomere length ranged from 17.0 1.0 to 19.3 1.0 kb in all mscs with no significant differences among donor sex , age or tissue source . the telomere length in the testis tissue of 1-year - old pig was 22.3 2.5 kb , which was significantly higher than that in all other mscs under current investigation . analysis of telomere length , telomere activity - related tert protein expression and oct 3/4 protein expression in mscs harvested at 1 week and 8 months after birth . ( a ) telomere restriction fragment lengths determined by southern blotting using a telomere length assay . expression of oct 3/4 protein , an early stem cell transcription factor and a potent indicator of proliferation capacity , was observed to be diminished with the advancement in age . female - derived mscs showed slightly stronger oct 3/4 protein expression than male - derived mscs at both the ages . further , its expression was slightly higher in a - mscs than in ds - mscs . cell cycle analysis : the cell cycle distribution of mscs is presented in table 1table 1.analysis of cell cycle status in mscs harvested at 1 week and 8 months after birthgroups*mean s.d . ( % ) g0/g1sg2/m1 week after birthfa - mscs68.8 4.8 27.1 3.3 4.1 2.0 fds - mscs67.8 2.9 16.8 5.6 15.4 3.5 ma - mscs62.7 10.1 25.6 5.2 11.7 5.0 mds - mscs61.6 4.3 25.0 7.0 13.4 2.6 8 months after birthfa - mscs78.5 0.8 16.8 5.6 4.7 4.9 fds - mscs77.9 2.8 7.3 4.7 14.8 5.1 ma - mscs71.6 4.0 21.0 5.0 7.4 4.4 mds - mscs70.5 1.5 27.3 3.7 2.2 2.2 this experiment was performed in triplicate , and significant differences are shown within columns . each sample was analyzed with 10,000 cells at a time by flow cytometry . * fa - msc , msc derived from female adipose tissue ; fds - msc , msc derived from female dermal skin tissue ; ma - msc , msc derived from male adipose tissue ; mds - msc , msc derived from male dermal skin tissue . and fig . 5.cell cycle analysis of mscs harvested at 1 week ( a ) and 8 months after birth ( b ) . the first and second black filled peaks indicate the g0/g1 and g2/m phases , respectively , and the black oblique line - filled peak in the middle indicates the s phase .. a higher proportion of mscs harvested at 8 months after birth was in the g0/g1 phase compared to that of mscs harvested at 1 week after birth . each sample was analyzed with 10,000 cells at a time by flow cytometry . * fa - msc , msc derived from female adipose tissue ; fds - msc , msc derived from female dermal skin tissue ; ma - msc , msc derived from male adipose tissue ; mds - msc , msc derived from male dermal skin tissue . cell cycle analysis of mscs harvested at 1 week ( a ) and 8 months after birth ( b ) . the first and second black filled peaks indicate the g0/g1 and g2/m phases , respectively , and the black oblique line - filled peak in the middle indicates the s phase . analysis of cell - aging - related proteins : the expression of proteins related to cell proliferation , differentiation and apoptosis was analyzed by western blotting ( fig . 6afig . ( a ) il6 as an immunomodulator , c - myc as a cell proliferation factor and oncoprotein , tgf as a differentiation and cell proliferation factor , p53 as an external stress and antitumor factor , and p21 as a cell cycle arrest protein were assayed . ( b ) bak as a proapoptotic protein and bcl2 as an antiapoptotic protein were analyzed . ) . the pro - inflammatory cytokine il6 was expressed in all types of mscs without change in its expression with the advancement in age . however , il6 expression was higher in male - derived mscs than in female - derived mscs , wherein adipose - derived mscs have shown slightly higher expression than dermal skin - derived mscs at both the ages . c - myc , a cell proliferation factor and oncogene , was expressed at higher levels in female ds - mscs derived at one week after birth than that in other types of mscs . expression of tgf , a cell proliferation and differentiation factor , was found to be elevated in male ds - mscs derived at one week after birth compared to other types of mscs . p53 , an antitumor molecule and stress factor , was expressed in all types of mscs and did not vary according to donor age , sex and tissue source , except in male ds - mscs harvested at 8 months after birth . further , p53 expression was higher in a - mscs compared to that in ds - mscs regardless of donor age and sex . the expression of p21 was increased in mscs harvested at 8 months after birth and was similar to that of female - derived bm - mscs . senescence - related proteins expression in mscs harvested at 1 week and 8 months after birth . ( a ) il6 as an immunomodulator , c - myc as a cell proliferation factor and oncoprotein , tgf as a differentiation and cell proliferation factor , p53 as an external stress and antitumor factor , and p21 as a cell cycle arrest protein were assayed . ( b ) bak as a proapoptotic protein and bcl2 as an antiapoptotic protein were analyzed . analysis of apoptosis - related proteins : expression of bak and bcl2 is known to be directly correlated with apoptosis , and the expression patterns of these proteins in mscs are shown in fig . bak , a pro - apoptotic protein , was expressed in all msc types , although with varying expression levels , female - derived mscs harvested at 1 week after birth showed higher expression . in contrast , bcl2 protein was expressed at extremely low levels in all mscs , except ds - mscs harvested at 1 week after birth . bcl2 expression in female - derived ds - mscs and male - derived ds - mscs harvested 1 week after birth was similar to that of female bm - mscs . here , we report that the donor age , gender and tissue source are linked to the change in physiological , functional and molecular characteristics of porcine mscs . both human and rat mscs are previously reported to be associated with age - dependent change in biological properties [ 45 , 50 ] , and especially , the proliferative nature of mscs may vary upon in vitro cultivation depending on the tissue source as well as the donor age [ 25 , 40 ] . the present study also demonstrated the characteristics of porcine mscs , such as stemness , proliferation , senescence and apoptosis , were affected by donor age , gender and tissue source . stemness was evaluated by ap activity , cd marker expression and differentiation ability of mscs . in porcine , ap expression has been reported as a marker for assessing pluripotency of stem cells including porcine umbilical cord ( pucs ) and skin - derived cells , and its expression was found to be affected by donor age . our study showed the decreased ap activity with aging regardless of donor sex and tissue source , indicating the progressive loss of differentiation capacity of mscs with the advancement in age of porcine . cd90 is a representative msc - specific marker to confirm mesodermal origin of porcine mscs . donor aging has been reported to be associated with the decline in number of cd90-positive cells [ 12 , 45 ] . however , in the present study , its expression was not altered by either donor age , sex or tissue source . this may be due to the species specific variability . as cd90 also participates in immunosuppression by reducing the levels of hla - g or il10 , our results indicate that porcine mscs are thought to maintain their immunosuppressive capacity at least for up to 8 months after birth . to address whether donor age , sex and tissue source will affect the multipotency of porcine mscs , we have evaluated in vitro osteogenesis and adipogenesis . previously , it has been reported that donor aging will affect multipotency of mscs , and especially , osteogenic propensity will decline with the advancement in age [ 48 , 51 , 52 ] . however , in this study , donor aging had only affected adipogenic ability while maintaining osteogenic ability of porcine mscs . the reason for conflicting results obtained in this study may possibly be due to the early procurement of mscs at 8 months after birth when compared to previous studies where mscs were derived from the bone marrow of elderly humans ( > 50 years old ) and rats ( 26 months old ) [ 48 , 52 ] . further , adipose tissue derived mscs showed higher multipotency than those derived from dermal skin , indicating mscs present in adipose tissue may be more primitive than those in dermal skin . nevertheless , higher adipogenic differentiation of a - mscs obtained at 1 week after birth regardless of their sex suggests that the epigenetic memory could also play an important role in the differentiation ability of mscs . embryonic stem cells possess long telomeres and high telomerase activity and expression of the early stem cell transcription factor oct 3/4 [ 19 , 22 ] . these factors are involved in cell proliferation and maintenance of cellular aging in stem cells . however , in adult stem cells , such as mscs , the expression of telomerase and oct 3/4 is still unclear as some studies have reported very low telomerase activity and the absence or extremely low levels of oct 3/4 expression [ 19 , 24 , 34 ] . in this study , telomere length was unaffected by the effect of donor age , sex and tissue source . however , telomerase activity was absent or extremely low in all types of mscs . the proliferative capacity of porcine mscs is found to be declined with the advancement of donor age as indicated by the higher expression of p21 protein with higher proportion of cells in g0/g1 phase of cell cycle in mscs harvested at 8 months after birth compared to that in mscs harvested at 1 week after birth . apart from donor age , it is also evident that sex and tissue source can affect proliferative capacity of mscs as revealed by the expression of oct 3/4 protein . female- , adipose - derived mscs are more likely to maintain their proliferative capacity than male- , dermal skin - derived mscs based on their expression of oct 3/4 protein . further , lower c - myc expression in a - mscs compared to ds - mscs indicates that the porcine adipose tissue may be the best source of mscs in terms of safety for in vivo transplantation . as short - term activation of c - myc promotes cell cycle progression , its prolonged activation may result in the initial hyper - proliferation of cells to subsequently undergo cellular senescence . sa--galactosidase is known to specifically indicate the senescence of cells , whereas il6 acts as both pro - inflammatory and anti - inflammatory cytokine . adult mouse bm - mscs were found to secrete significantly higher levels of il6 than neonatal mouse bm - mscs , and this elevated il6 levels have been linked to various pathologies [ 15 , 32 , 47 ] . these findings indicate an increased secretion of il6 might directly be associated with stem cell aging . although mscs were obtained at a relatively younger age of 8 months , positive staining was observed in the present study . mscs isolated at 8 months of age displayed reduced proliferation with higher sa--galactosidase activity indicating donor age may directly affect the cellular characteristics . female- , adipose - derived mscs with reduced expression of sa--galactosidase and il6 protein even at 8 months of age suggest that these cells might be more resistant to senescence in vitro compared to male- , ds - derived mscs in porcine . this increased resistance to senescence may possibly due to their higher expression of oct3/4 protein , which might contribute to prolonged proliferative capacity under in vitro condition . in the present study , apoptotic behavior of mscs bcl2 is an anti - apoptotic protein that extends the cell life - span and facilitates tumorigenesis [ 6 , 42 ] , and therefore , our study suggests that the ds - mscs may possess tumorigenic properties due to their higher bcl2 protein expression observed at 1 week after birth . in conclusion , biological characteristics of porcine mscs were affected by both donor age , gender and tissue source . higher differentiation potential was noted in adipose - derived mscs compared to dermal skin - derived mscs . female adipose - derived mscs displayed prolonged proliferation potential . increased c - myc and bcl2 levels in ds - mscs at 1 week after birth suggested their tumorigenic potential . although we did not use very old porcine donors , our results suggest that mscs display a cellular and molecular variability depending on the donor age , sex and tissue source even under similar genetic and environmental conditions . based on these findings , it is concluded that adipose - tissue - derived mscs from young female individuals tend to have higher resistance to senescence under in vitro culture conditions .

the biological properties of mesenchymal stem cells ( mscs ) are influenced by donor age , gender and/or tissue sources . the present study investigated the cellular and molecular properties of porcine mesenchymal stromal / stem cells ( mscs ) isolated from different tissues ( adipose & dermal skin ) and sex at different ages ( 1 week & 8 months after birth ) with similar genetic and environmental backgrounds . mscs were analyzed for alkaline phosphatase ( ap ) activity , cd90 and oct3/4 expression , in vitro differentiation ability , senescence - associated -galactosidase ( sa--gal ) activity , telomeric properties , cell cycle status and expression of senescence ( il6 , c - myc , tgf , p53 and p21)- and apoptosis ( bak and bcl2)-related proteins . an age - dependent decline in ap activity and adipogenesis was observed in all mscs , except for male a - mscs . cd90 expression did not change , but sa--gal activity increased with advancement in age , except in a - mscs . telomeric properties were similar in all mscs , whereas expression levels of oct3/4 protein declined with the advancement in age . p21 expression was increased with increase in donor age . male derived cells have shown higher il6 expression . the expression of p53 was slightly lower in mscs of dermal tissue than in adipose tissue . bak was expressed in all mscs regardless of age , but up regulation of bcl2 was observed in ds - mscs derived at 1 week after birth . in conclusion , adipose tissue - derived mscs from young female individuals were found to be more resistant to senescence under in vitro culture conditions .

MATERIALS AND METHODS RESULTS DISCUSSION

tissues of three female and three male pigs from the same litter were recovered under standard surgical procedures at both 1 week and 8 months after birth . porcine mscs from subcutaneous adipose tissue ( a - mscs ) were isolated following a previously described method by digestion with 0.075% collagenase type i ( millipore , temecula , ca , u.s.a . ) porcine dermal skin mscs ( ds - mscs ) were isolated after removal of the epidermis of the ear skin as previously described . as a control , mscs were isolated from porcine bone marrow ( bm - mscs ) as described previously . all cells were isolated from each sample in triplicates and cultured in advanced dulbecco s modified eagle medium ( admem ) supplemented with 10% fetal calf serum ( fcs ) and 1% penicillin - streptomycin ( 10,000 iu and 10,000 g / ml ) at 38.5c in a humidified atmosphere of 5% co2 in air . alkaline phosphatase ( ap ) activity : to detect ap activity , porcine mscs grown on 35-mm dishes for 2 weeks were stained with an ap chromogen kit ( bcip / nbt ) ( abcam inc . senescence - associated -galactosidase ( sa--gal ) staining : to evaluate cellular senescence , an sa--gal assay was performed according to manufacturer s protocol using sa--gal staining kit ( cell signaling technology , cambridge , ma , u.s.a . ) female bm - mscs derived from the same litter both at 1 week and 8 months after birth were used for an internal control , whereas cells derived from the testis tissue of 1-year - old pig were used for a positive control . western blot analysis : the expression of proliferation , senescence and apoptosis associated proteins was analyzed using western blot . female bm - mscs derived from 1 week old pig were used as an internal control for mscs . hela and f9 whole cell lysates ( santa cruz , ca ) were used as positive controls for telomerase reverse transcriptase ( tert ; hela ) , p21 ( hela ) and oct 3/4 ( f9 ) , whereas lysates from human fibroblast ( mcf 5 ) and cancer cells ( mda - mb-231 ) were used as negative controls for oct 3/4 protein expression . , anti - oct 3/4 ( 1:100 dilution ; santa cruz biotechnology ) , anti - il6 ( 1:500 dilution ; abcam , cambridge , uk ) , anti - p53 ( 1:100 dilution ; santa cruz biotechnology ) , anti - c - myc ( 1:500 dilution ; abcam ) , anti - tgf ( 1;500 dilution ; abcam ) , anti - p21 ( 1:200 dilution ; santa cruz biotechnology ) , anti - bak ( 1:1,000 dilution ; abcam ) , anti - bcl2 ( 1:200 dilution ; abcam ) and anti--actin ( 1:1,000 dilution ; cell signaling technology , cambridge , ma , u.s.a . ) the membranes were then incubated with horseradish peroxidase ( hrp)-conjugated secondary antibodies , such as goat anti - mouse ( 1:1,000 dilution ; p53 and bcl2 ) , goat anti - rabbit ( 1:5,000 dilution ; p21 , il6 , c - myc , tgf , bax and -actin ) and donkey anti - goat ( 1:5,000 dilution ; oct3/4 and tert ) for 1 hr at room temperature . , general observations for mscs : all mscs have shown fibroblast - like morphology in monolayer culture and formed colonies regardless of donor sex and age . 1.analysis of alkaline phosphatase ( ap ) activity and cd90 expression of mscs harvested at 1 week and 8 months after birth . female - derived mscs obtained at 1 week after birth showed higher ap activity than male - derived mscs obtained at the same age . however , the ap activity was decreased in mscs harvested at 8 months after birth regardless of gender compared to that in mscs harvested at 1 week after birth . there was no notable difference in ap activity in mscs of both ages with respect to the source of tissue . all mscs were strongly positive for cd90 with staining in 87% of cells , except for adult female a - mscs , which showed cd90 expression in 80% of cells ( fig . cd90 expression was maintained in cells isolated at 8 months after birth without any significant differences ( p<0.05 ) , indicating the mesodermal lineage of the cells ( fig . analysis of alkaline phosphatase ( ap ) activity and cd90 expression of mscs harvested at 1 week and 8 months after birth . analysis of msc differentiation potential : the stemness of mscs harvested at 1 week and 8 months after birth was assessed by in vitro differentiation into osteocytes and adipocytes . all mscs differentiated into osteocytes and adipocytes in vitro regardless of donor age , sex and tissue source with varying differentiation capacity ( fig . mscs harvested at 8 months after birth showed enhanced osteogenic differentiation , where male - derived cells have shown higher propensity toward osteogenesis than female - derived cells . furthermore , mscs from adipose tissue displayed a higher differentiation capacity than those from dermal skin . in contrast , all mscs harvested at 8 months after birth showed markedly diminished adipogenic differentiation , except for male - derived a - mscs which maintained their differentiation capacity with the advancement in age . on the other hand , a - mscs harvested at 1 week after birth regardless of their sex have shown higher adipogenic differentiation ability when compared to both male and female derived ds - mscs harvested at the same age . 3.senescence-associated ( sa)--galactosidase activity of mscs obtained at 1 week and 8 months after birth . all mscs harvested at 8 months after birth were more positive for sa--gal staining , except for female a - mscs , when compared to those harvested at 1 week after birth ( macrographs of fig . , ds - mscs showed stronger sa--gal staining than a - mscs harvested at 8 months after birth . however , there was no difference found in sa--gal staining among mscs harvested at 1 week age regardless of their sex and tissue source . senescence - associated ( sa)--galactosidase activity of mscs obtained at 1 week and 8 months after birth . 4.analysis of telomere length , telomere activity - related tert protein expression and oct 3/4 protein expression in mscs harvested at 1 week and 8 months after birth . the telomere length of female - derived bm - mscs was 18.5 1.3 and 19.0 0.0 kb at 1 week and 8 months of age , respectively . analysis of telomere length , telomere activity - related tert protein expression and oct 3/4 protein expression in mscs harvested at 1 week and 8 months after birth . expression of oct 3/4 protein , an early stem cell transcription factor and a potent indicator of proliferation capacity , was observed to be diminished with the advancement in age . further , its expression was slightly higher in a - mscs than in ds - mscs . cell cycle analysis : the cell cycle distribution of mscs is presented in table 1table 1.analysis of cell cycle status in mscs harvested at 1 week and 8 months after birthgroups*mean s.d . ( % ) g0/g1sg2/m1 week after birthfa - mscs68.8 4.8 27.1 3.3 4.1 2.0 fds - mscs67.8 2.9 16.8 5.6 15.4 3.5 ma - mscs62.7 10.1 25.6 5.2 11.7 5.0 mds - mscs61.6 4.3 25.0 7.0 13.4 2.6 8 months after birthfa - mscs78.5 0.8 16.8 5.6 4.7 4.9 fds - mscs77.9 2.8 7.3 4.7 14.8 5.1 ma - mscs71.6 4.0 21.0 5.0 7.4 4.4 mds - mscs70.5 1.5 27.3 3.7 2.2 2.2 this experiment was performed in triplicate , and significant differences are shown within columns . 5.cell cycle analysis of mscs harvested at 1 week ( a ) and 8 months after birth ( b ) . the first and second black filled peaks indicate the g0/g1 and g2/m phases , respectively , and the black oblique line - filled peak in the middle indicates the s phase .. a higher proportion of mscs harvested at 8 months after birth was in the g0/g1 phase compared to that of mscs harvested at 1 week after birth . cell cycle analysis of mscs harvested at 1 week ( a ) and 8 months after birth ( b ) . ( a ) il6 as an immunomodulator , c - myc as a cell proliferation factor and oncoprotein , tgf as a differentiation and cell proliferation factor , p53 as an external stress and antitumor factor , and p21 as a cell cycle arrest protein were assayed . the pro - inflammatory cytokine il6 was expressed in all types of mscs without change in its expression with the advancement in age . however , il6 expression was higher in male - derived mscs than in female - derived mscs , wherein adipose - derived mscs have shown slightly higher expression than dermal skin - derived mscs at both the ages . c - myc , a cell proliferation factor and oncogene , was expressed at higher levels in female ds - mscs derived at one week after birth than that in other types of mscs . expression of tgf , a cell proliferation and differentiation factor , was found to be elevated in male ds - mscs derived at one week after birth compared to other types of mscs . p53 , an antitumor molecule and stress factor , was expressed in all types of mscs and did not vary according to donor age , sex and tissue source , except in male ds - mscs harvested at 8 months after birth . further , p53 expression was higher in a - mscs compared to that in ds - mscs regardless of donor age and sex . the expression of p21 was increased in mscs harvested at 8 months after birth and was similar to that of female - derived bm - mscs . senescence - related proteins expression in mscs harvested at 1 week and 8 months after birth . ( a ) il6 as an immunomodulator , c - myc as a cell proliferation factor and oncoprotein , tgf as a differentiation and cell proliferation factor , p53 as an external stress and antitumor factor , and p21 as a cell cycle arrest protein were assayed . analysis of apoptosis - related proteins : expression of bak and bcl2 is known to be directly correlated with apoptosis , and the expression patterns of these proteins in mscs are shown in fig . bak , a pro - apoptotic protein , was expressed in all msc types , although with varying expression levels , female - derived mscs harvested at 1 week after birth showed higher expression . in contrast , bcl2 protein was expressed at extremely low levels in all mscs , except ds - mscs harvested at 1 week after birth . bcl2 expression in female - derived ds - mscs and male - derived ds - mscs harvested 1 week after birth was similar to that of female bm - mscs . here , we report that the donor age , gender and tissue source are linked to the change in physiological , functional and molecular characteristics of porcine mscs . both human and rat mscs are previously reported to be associated with age - dependent change in biological properties [ 45 , 50 ] , and especially , the proliferative nature of mscs may vary upon in vitro cultivation depending on the tissue source as well as the donor age [ 25 , 40 ] . the present study also demonstrated the characteristics of porcine mscs , such as stemness , proliferation , senescence and apoptosis , were affected by donor age , gender and tissue source . stemness was evaluated by ap activity , cd marker expression and differentiation ability of mscs . in porcine , ap expression has been reported as a marker for assessing pluripotency of stem cells including porcine umbilical cord ( pucs ) and skin - derived cells , and its expression was found to be affected by donor age . our study showed the decreased ap activity with aging regardless of donor sex and tissue source , indicating the progressive loss of differentiation capacity of mscs with the advancement in age of porcine . donor aging has been reported to be associated with the decline in number of cd90-positive cells [ 12 , 45 ] . however , in the present study , its expression was not altered by either donor age , sex or tissue source . as cd90 also participates in immunosuppression by reducing the levels of hla - g or il10 , our results indicate that porcine mscs are thought to maintain their immunosuppressive capacity at least for up to 8 months after birth . to address whether donor age , sex and tissue source will affect the multipotency of porcine mscs , we have evaluated in vitro osteogenesis and adipogenesis . previously , it has been reported that donor aging will affect multipotency of mscs , and especially , osteogenic propensity will decline with the advancement in age [ 48 , 51 , 52 ] . the reason for conflicting results obtained in this study may possibly be due to the early procurement of mscs at 8 months after birth when compared to previous studies where mscs were derived from the bone marrow of elderly humans ( > 50 years old ) and rats ( 26 months old ) [ 48 , 52 ] . further , adipose tissue derived mscs showed higher multipotency than those derived from dermal skin , indicating mscs present in adipose tissue may be more primitive than those in dermal skin . nevertheless , higher adipogenic differentiation of a - mscs obtained at 1 week after birth regardless of their sex suggests that the epigenetic memory could also play an important role in the differentiation ability of mscs . embryonic stem cells possess long telomeres and high telomerase activity and expression of the early stem cell transcription factor oct 3/4 [ 19 , 22 ] . however , in adult stem cells , such as mscs , the expression of telomerase and oct 3/4 is still unclear as some studies have reported very low telomerase activity and the absence or extremely low levels of oct 3/4 expression [ 19 , 24 , 34 ] . the proliferative capacity of porcine mscs is found to be declined with the advancement of donor age as indicated by the higher expression of p21 protein with higher proportion of cells in g0/g1 phase of cell cycle in mscs harvested at 8 months after birth compared to that in mscs harvested at 1 week after birth . apart from donor age , it is also evident that sex and tissue source can affect proliferative capacity of mscs as revealed by the expression of oct 3/4 protein . female- , adipose - derived mscs are more likely to maintain their proliferative capacity than male- , dermal skin - derived mscs based on their expression of oct 3/4 protein . further , lower c - myc expression in a - mscs compared to ds - mscs indicates that the porcine adipose tissue may be the best source of mscs in terms of safety for in vivo transplantation . as short - term activation of c - myc promotes cell cycle progression , its prolonged activation may result in the initial hyper - proliferation of cells to subsequently undergo cellular senescence . adult mouse bm - mscs were found to secrete significantly higher levels of il6 than neonatal mouse bm - mscs , and this elevated il6 levels have been linked to various pathologies [ 15 , 32 , 47 ] . although mscs were obtained at a relatively younger age of 8 months , positive staining was observed in the present study . mscs isolated at 8 months of age displayed reduced proliferation with higher sa--galactosidase activity indicating donor age may directly affect the cellular characteristics . female- , adipose - derived mscs with reduced expression of sa--galactosidase and il6 protein even at 8 months of age suggest that these cells might be more resistant to senescence in vitro compared to male- , ds - derived mscs in porcine . this increased resistance to senescence may possibly due to their higher expression of oct3/4 protein , which might contribute to prolonged proliferative capacity under in vitro condition . in the present study , apoptotic behavior of mscs bcl2 is an anti - apoptotic protein that extends the cell life - span and facilitates tumorigenesis [ 6 , 42 ] , and therefore , our study suggests that the ds - mscs may possess tumorigenic properties due to their higher bcl2 protein expression observed at 1 week after birth . in conclusion , biological characteristics of porcine mscs were affected by both donor age , gender and tissue source . higher differentiation potential was noted in adipose - derived mscs compared to dermal skin - derived mscs . increased c - myc and bcl2 levels in ds - mscs at 1 week after birth suggested their tumorigenic potential . although we did not use very old porcine donors , our results suggest that mscs display a cellular and molecular variability depending on the donor age , sex and tissue source even under similar genetic and environmental conditions . based on these findings , it is concluded that adipose - tissue - derived mscs from young female individuals tend to have higher resistance to senescence under in vitro culture conditions .

the benefit of colonoscopy on cancer prevention is dependent on effective polyp detection and removal . three important aspects of our approach to managing colon polyps are in evolution : the first aspect is related to the management of diminutive polyps ( polyps up to 5 mm in size ) . because diminutive polyps are very common and almost never contain cancer , new management strategies to improve cost - effectiveness have been proposed . the resect - and - discard strategy that uses real - time polyp diagnosis of diminutive polyps has been recently endorsed by endoscopy societies . hot electrocautery snare resection has been the standard - of - care for several decades , recent studies suggest that cold snare resection without electrocautery may be as safe and effective as hot snare resection . finally , several studies have focused on the management of large 20 mm polyps , particularly with respect to lowering the risk of bleeding complications . prophylactic clip closure of the mucosal defect has become a common practice ; however , whether clipping truly decreases bleeding risk remains unclear . in this article , we discuss recent research developments and controversies with regards to the management of diminutive polyps , cold snare resection of mid - size polyps , and bleeding prophylaxis after resection of large polyps . the discussed observations call for refocusing our cancer prevention efforts in practice and research from removing diminutive polyps to the detection and safe resection of higher - risk polyps . current colonoscopy practice guidelines recommend to remove , whenever possible , all polypoid lesions for histopathology assessment irrespective of the size or appearance . of all detected polyps , 7080% cancer is exceedingly rare , and previous studies have described cancer prevalence between 0 and 0.08% for diminutive polyps and cancer prevalence between 0 and 1.5% in polyps up to 10 mm . a recent and largest cross - sectional study to date included > 42,000 polypectomies of up to 9 mm polyps did not find any cancer in any of these diminutive or small polyps . resection of diminutive polyps increases patient risk and cost , yet the benefit on cancer prevention by removing diminutive polyps is questionable . however , histopathology evaluation of diminutive polyps remains important because presence of adenoma may determine low- or high - risk status of the patient and affect the colonoscopy surveillance interval . one avenue to reduce colonoscopy related cost would be to replace histopathology assessment by using endoscopic image , enhancing modalities to distinguish neoplastic from non - neoplastic polyps ( figure 1 ) . novel image modalities have shown to predict neoplastic polyps with high accuracy and thus allow to determine the interval for the subsequent surveillance colonoscopy . this new ability has inspired the concept of the resect - and - discard strategy . according to this strategy , diminutive polyps are diagnosed real time as adenomas or non - adenomatous polyps by using digital chromoendoscopy , like narrow band imaging ( nbi ) . polyps that are diagnosed with high confidence are resected and discarded , while others are sent for pathology evaluation . calculated cost savings of this approach have been estimated to be 33,000,000 $ per year in the united states . a > 90% agreement between the optical and the pathology - based surveillance recommendations has been set as the required quality benchmark in order to adopt the resect - and - discard strategy . in recent years , multiple studies have shown that this benchmark can be accomplished , but only if optical diagnosis is done with high confidence by experienced endoscopists in an academic setting . on the basis of these results , the resect - and - discard strategy has been endorsed by the european and american societies for gastrointestinal endoscopy ( esge and asge ) . both societies are well aware of challenges with successful implementation of resect - and - discard . further , the resect - and - discard approach requires additional efforts by the endoscopist during the examination , added photo and text documentation , and the need to combine optical with pathology based diagnoses in a large proportion of patients . alternative concepts to simplify the resect - and - discard strategy and minimize or eliminate the need for optical and histopathology assessment have recently been proposed . in a post hoc analysis , a non - optical resect - and - discard strategy was examined , in which all rectosigmoid diminutive polyps were considered as hyperplastic and all polyps proximal to the rectosigmoid as neoplastic . the non - optical strategy agreed with the pathology - based surveillance recommendations in 89% of patients , just shy of the 90% benchmark , but not significantly different from the optical strategy . the non - optical strategy also reduced the number of required pathology examinations and provided more patients with surveillance recommendations immediately following the colonoscopy compared to the optical resect - and - discard strategy . while resect - and - discard is a promising idea to reduce colonoscopy associated cost , further research on how to make the concept feasible for community practice the low risk of diminutive polyps to develop cancer might support an even more radical approach to polyp management , namely to defer resection of diminutive polyp , and to only remove those that have grown to higher - risk polyps during the surveillance interval . according to ct colonography guidelines , polyps 5 mm are not reported and exams repeated every 5 years . available studies on natural history of polyps , albeit few support this approach . the risk of transition to cancer increases with size , and cost - effectiveness studies have only considered that polyps larger than 5 mm would transition to cancer . in two follow - up ct colonography studies , the majority of 6 - 9 mm polyps ( 6578% ) did not grow within 23 years . only a small proportion grew and none progressed to cancer among a total of 401 polyps . when considering even smaller 5 mm polyps , it is plausible that their resection does not sufficiently contribute to crc prevention to justify their removal , and its associated risk and cost . instead although leaving diminutive polyps in place would constitute a paradigm shift , in a recent survey 72% of gastroenterologists would be agreeable to leave diminutive polyps in place if such an approach was endorsed by governing societies . also , in daily practice , gastroenterologists may not resect diminutive polyps when their appearance suggests non - adenomatous tissue . it has been estimated that deferring resection of diminutive polyps would result in a 64% reduction of therapeutic interventions during colonoscopies . prospective studies will have to show the safety and efficacy of this approach and whether it truly does not affect overall effectiveness of screening . in addition , patients ' expectations and fears with regard to perceived cancer risk of deferring polyp resection would have to be evaluated . polyps are removed either by forceps or by snare . while both are comparable for 3 mm polyps , larger polyps are insufficiently removed with a forceps and should be resected with a snare . in current practice , the most common approach to removing medium and large sized polyps 5 mm is to use electrocautery or hot snare resection . added cautery ablates marginal tissue and may therefore improve completeness of resection ( figure 2 ) . further , it might lower the risk of immediate bleeding . however , there is little proof that these assertions are true . hot snare resection is often incomplete . in the complete adenoma resection ( care ) , study 10% of 520 incomplete resection increased with size and varied broadly across endoscopists . a recent smaller study from australia presented in abstract form reported an only 5% incomplete resection of up to 20 mm polyps when using a cold snare suggesting that resection may be at least be similarly complete when using a cold snare than a hot snare . with respect to bleeding risk a randomized trial among patients on anticoagulation with up to 10 mm polyps suggests that cold snare resection may actually lower the risk of bleeding . cold snare resection resulted in a lower - immediate ( 6 vs. 23% ) and delayed bleeding risk ( 0 vs. 14% ) when compared to hot snare resection . two other randomized trials compared cold to hot snare resection for up to 8 mm polyps . in one study , the other study reported immediate bleeding in 9% with cold snare resection ; however , all resolved spontaneously , and none required an intervention . these studies are small and bleeding is not well defined , however , the results question the assumed benefit of cautery on bleeding risk . aside from randomized trials , an increasing number of uncontrolled cohort studies suggest that cold snare polypectomy is safe and effective for up to 10 mm polyps . current commonly used snares have been designed to be used with electrocautery and may not easily cut through the polyp base without cautery . specialized fine wire snares to facilitate cold resection have been introduced . a first randomized trial compared the use of a dedicated cold snare with a standard snare for cold resection of up to 10 mm polyps and found a lower incomplete resection rate with using a dedicated cold snare ( 9 vs. 21% ) . while there are increasing number of studies on the safety and efficacy of cold snare resection for polyps up to 10 mm , the data on cold snare resection for larger polyps are limited to feasibility . case series have reported on piecemeal resection of up to > 20 mm colorectal polyps . among those three studies , immediate bleeding requiring intervention only occurred in one patient who was on anticoagulation treatment . finally , it should be noted , that cold snare polypectomy has no risk of cautery damage to the colonic wall or the resected polyp . this absence of electrocautery may decrease complications ( perforation , post - polypectomy syndrome ) and allow for better histopathology evaluation of the polyp and examination of the resection margins after polyp removal . remnant polyp tissue may be more readily visible , which may improve completeness of resection . although the data on cold snare resection of larger polyps is still emerging , cold snare resection appears to be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . future studies should systematically examine efficacy and safety of cold snare resection ; particularly determine the upper limit for en - bloc and piecemeal resection , the need for submucosal injection , and associated bleeding risks . the prevalence of cancer in polyps equal or larger than 10 mm has been reported to be between 2.4 and 10.2% . therefore , it is of vital importance to assure complete resection particularly of large lesions . the standard - of - care for large polyps used to be surgical resection . a growing number of studies have demonstrated that resection can effectively and safely remove 8590% of these polyps and has therefore become the preferred treatment . for non - pedunculated polyps , endoscopic mucosal resection ( emr ) is the current standard - of - care in western countries . en bloc resection is the goal because the risk of recurrence is lower when compared to piecemeal resection . however , in the majority of these polyps en bloc resection can not be achieved and are therefore removed piece - meal . emr typically includes submucosal injection with a contrasting agent ( methylene blue or indigo carmine ) to provide a submucosal safety cushion and to better delineate the submucosal layer . with the application of electrocautery to a larger area of the colonic wall during resection and the resultant large mucosal defect , the risk for perforation , post - polypectomy syndrome , and bleeding increases . bleeding typically occurs within 710 days after emr and may require hospital admission , endoscopic intervention , and blood transfusion . proximal polyp location , size , and bleeding during the resection have been identified as risk factors for delayed bleeding . age , comorbidities , use of anticoagulation , and electrocautery setting have been reported risks in some but not other studies . recent publications suggest that closing the mucosal defect with clips may reduce the bleeding risk ( figure 3 ) . a large retrospective single endoscopist study found that complete clip closure after removal of 20 mm polyps among 225 patients was associated with a 2% bleeding risk , significantly lower than the 10% observed in 247 historical controls , who did not undergo prophylactic clipping . similarly , clip closure was associated with lower bleeding risk in a recent prospective multicenter cohort study from spain . although both studies support the use of clips , a major concern is an uncontrolled study design with the possibility of patient selection bias and unmeasured factors that may affect bleeding risk . although the study from spain was large ( 1214 patients ) , the lack of a standardized resection protocol , a clear definition of outcomes and assurance of complete outcome assessment are some of the limitations . for instance , clip closure may have been more frequently applied to lower - risk lesions because they may have been easier to clip ( preferential clipping ) , which may have confounded risk assessment . prophylactic clip closure was compared to no clip closure after endoscopic resection of 10 mm sessile polyps among 348 patients . polyps were removed either by emr , endoscopic submucosal dissection ( esd ) or hybrid esd ( combination of emr and esd ) . delayed bleeding occurred less frequently after clip closure compared to no clipping ( 1 vs. 7% ) . however , the study included smaller polyps , allowed different endoscopic resection techniques , observed a higher than expected rate of complications in the control group , and used an unclear definition of bleeding events . therefore , the results are not sufficient to inform current emr practice . despite the lack of good evidence additional studies are under way and will hopefully provide a more definitive answer in the near future . at present stage , however , there is no adequate evidence to support prophylactic use of clips after emr . current colonoscopy practice guidelines recommend to remove , whenever possible , all polypoid lesions for histopathology assessment irrespective of the size or appearance . of all detected polyps , 7080% cancer is exceedingly rare , and previous studies have described cancer prevalence between 0 and 0.08% for diminutive polyps and cancer prevalence between 0 and 1.5% in polyps up to 10 mm . a recent and largest cross - sectional study to date included > 42,000 polypectomies of up to 9 mm polyps did not find any cancer in any of these diminutive or small polyps . resection of diminutive polyps increases patient risk and cost , yet the benefit on cancer prevention by removing diminutive polyps is questionable . however , histopathology evaluation of diminutive polyps remains important because presence of adenoma may determine low- or high - risk status of the patient and affect the colonoscopy surveillance interval . one avenue to reduce colonoscopy related cost would be to replace histopathology assessment by using endoscopic image , enhancing modalities to distinguish neoplastic from non - neoplastic polyps ( figure 1 ) . novel image modalities have shown to predict neoplastic polyps with high accuracy and thus allow to determine the interval for the subsequent surveillance colonoscopy . this new ability has inspired the concept of the resect - and - discard strategy . according to this strategy , diminutive polyps are diagnosed real time as adenomas or non - adenomatous polyps by using digital chromoendoscopy , like narrow band imaging ( nbi ) . polyps that are diagnosed with high confidence are resected and discarded , while others are sent for pathology evaluation . calculated cost savings of this approach have been estimated to be 33,000,000 $ per year in the united states . a > 90% agreement between the optical and the pathology - based surveillance recommendations has been set as the required quality benchmark in order to adopt the resect - and - discard strategy . in recent years , multiple studies have shown that this benchmark can be accomplished , but only if optical diagnosis is done with high confidence by experienced endoscopists in an academic setting . on the basis of these results , the resect - and - discard strategy has been endorsed by the european and american societies for gastrointestinal endoscopy ( esge and asge ) . both societies are well aware of challenges with successful implementation of resect - and - discard . further , the resect - and - discard approach requires additional efforts by the endoscopist during the examination , added photo and text documentation , and the need to combine optical with pathology based diagnoses in a large proportion of patients . alternative concepts to simplify the resect - and - discard strategy and minimize or eliminate the need for optical and histopathology assessment have recently been proposed . in a post hoc analysis , a non - optical resect - and - discard strategy was examined , in which all rectosigmoid diminutive polyps were considered as hyperplastic and all polyps proximal to the rectosigmoid as neoplastic . the non - optical strategy agreed with the pathology - based surveillance recommendations in 89% of patients , just shy of the 90% benchmark , but not significantly different from the optical strategy . the non - optical strategy also reduced the number of required pathology examinations and provided more patients with surveillance recommendations immediately following the colonoscopy compared to the optical resect - and - discard strategy . while resect - and - discard is a promising idea to reduce colonoscopy associated cost , further research on how to make the concept feasible for community practice the low risk of diminutive polyps to develop cancer might support an even more radical approach to polyp management , namely to defer resection of diminutive polyp , and to only remove those that have grown to higher - risk polyps during the surveillance interval . according to ct colonography guidelines , polyps 5 mm are not reported and exams repeated every 5 years . available studies on natural history of polyps , albeit few support this approach . the risk of transition to cancer increases with size , and cost - effectiveness studies have only considered that polyps larger than 5 mm would transition to cancer . in two follow - up ct colonography studies , the majority of 6 - 9 mm polyps ( 6578% ) did not grow within 23 years . only a small proportion grew and none progressed to cancer among a total of 401 polyps . when considering even smaller 5 mm polyps , it is plausible that their resection does not sufficiently contribute to crc prevention to justify their removal , and its associated risk and cost . instead although leaving diminutive polyps in place would constitute a paradigm shift , in a recent survey 72% of gastroenterologists would be agreeable to leave diminutive polyps in place if such an approach was endorsed by governing societies . also , in daily practice , gastroenterologists may not resect diminutive polyps when their appearance suggests non - adenomatous tissue . it has been estimated that deferring resection of diminutive polyps would result in a 64% reduction of therapeutic interventions during colonoscopies . prospective studies will have to show the safety and efficacy of this approach and whether it truly does not affect overall effectiveness of screening . in addition , patients ' expectations and fears with regard to perceived cancer risk of deferring polyp resection would have to be evaluated . polyps are removed either by forceps or by snare . while both are comparable for 3 mm polyps , larger polyps are insufficiently removed with a forceps and should be resected with a snare . in current practice , the most common approach to removing medium and large sized polyps 5 added cautery ablates marginal tissue and may therefore improve completeness of resection ( figure 2 ) . further , it might lower the risk of immediate bleeding . however , there is little proof that these assertions are true . hot snare resection is often incomplete . in the complete adenoma resection ( care ) , study 10% of 520 mm neoplastic polyps were not completely removed . incomplete resection increased with size and varied broadly across endoscopists . a recent smaller study from australia presented in abstract form reported an only 5% incomplete resection of up to 20 mm polyps when using a cold snare suggesting that resection may be at least be similarly complete when using a cold snare than a hot snare . with respect to bleeding risk a randomized trial among patients on anticoagulation with up to 10 mm polyps suggests that cold snare resection may actually lower the risk of bleeding . cold snare resection resulted in a lower - immediate ( 6 vs. 23% ) and delayed bleeding risk ( 0 vs. 14% ) when compared to hot snare resection . two other randomized trials compared cold to hot snare resection for up to 8 mm polyps . in one study , the other study reported immediate bleeding in 9% with cold snare resection ; however , all resolved spontaneously , and none required an intervention . these studies are small and bleeding is not well defined , however , the results question the assumed benefit of cautery on bleeding risk . aside from randomized trials , an increasing number of uncontrolled cohort studies suggest that cold snare polypectomy is safe and effective for up to 10 mm polyps . current commonly used snares have been designed to be used with electrocautery and may not easily cut through the polyp base without cautery . specialized fine wire snares to facilitate cold resection have been introduced . a first randomized trial compared the use of a dedicated cold snare with a standard snare for cold resection of up to 10 mm polyps and found a lower incomplete resection rate with using a dedicated cold snare ( 9 vs. 21% ) . while there are increasing number of studies on the safety and efficacy of cold snare resection for polyps up to 10 mm , the data on cold snare resection for larger polyps are limited to feasibility . case series have reported on piecemeal resection of up to > 20 mm colorectal polyps . among those three studies , immediate bleeding requiring intervention only occurred in one patient who was on anticoagulation treatment . finally , it should be noted , that cold snare polypectomy has no risk of cautery damage to the colonic wall or the resected polyp . this absence of electrocautery may decrease complications ( perforation , post - polypectomy syndrome ) and allow for better histopathology evaluation of the polyp and examination of the resection margins after polyp removal . remnant polyp tissue may be more readily visible , which may improve completeness of resection . although the data on cold snare resection of larger polyps is still emerging , cold snare resection appears to be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . future studies should systematically examine efficacy and safety of cold snare resection ; particularly determine the upper limit for en - bloc and piecemeal resection , the need for submucosal injection , and associated bleeding risks . the prevalence of cancer in polyps equal or larger than 10 mm has been reported to be between 2.4 and 10.2% . therefore , it is of vital importance to assure complete resection particularly of large lesions . the standard - of - care for large polyps used to be surgical resection . a growing number of studies have demonstrated that resection can effectively and safely remove 8590% of these polyps and has therefore become the preferred treatment . for non - pedunculated polyps , endoscopic mucosal resection ( emr ) is the current standard - of - care in western countries . en bloc resection is the goal because the risk of recurrence is lower when compared to piecemeal resection . however , in the majority of these polyps en bloc resection can not be achieved and are therefore removed piece - meal . emr typically includes submucosal injection with a contrasting agent ( methylene blue or indigo carmine ) to provide a submucosal safety cushion and to better delineate the submucosal layer . with the application of electrocautery to a larger area of the colonic wall during resection and the resultant large mucosal defect , the risk for perforation , post - polypectomy syndrome , and bleeding increases . bleeding typically occurs within 710 days after emr and may require hospital admission , endoscopic intervention , and blood transfusion . proximal polyp location , size , and bleeding during the resection have been identified as risk factors for delayed bleeding . age , comorbidities , use of anticoagulation , and electrocautery setting have been reported risks in some but not other studies . recent publications suggest that closing the mucosal defect with clips may reduce the bleeding risk ( figure 3 ) . a large retrospective single endoscopist study found that complete clip closure after removal of 20 mm polyps among 225 patients was associated with a 2% bleeding risk , significantly lower than the 10% observed in 247 historical controls , who did not undergo prophylactic clipping . similarly , clip closure was associated with lower bleeding risk in a recent prospective multicenter cohort study from spain . although both studies support the use of clips , a major concern is an uncontrolled study design with the possibility of patient selection bias and unmeasured factors that may affect bleeding risk . although the study from spain was large ( 1214 patients ) , the lack of a standardized resection protocol , a clear definition of outcomes and assurance of complete outcome assessment are some of the limitations . for instance , clip closure may have been more frequently applied to lower - risk lesions because they may have been easier to clip ( preferential clipping ) , which may have confounded risk assessment . a randomized trial from china prophylactic clip closure was compared to no clip closure after endoscopic resection of 10 mm sessile polyps among 348 patients . polyps were removed either by emr , endoscopic submucosal dissection ( esd ) or hybrid esd ( combination of emr and esd ) . delayed bleeding occurred less frequently after clip closure compared to no clipping ( 1 vs. 7% ) . however , the study included smaller polyps , allowed different endoscopic resection techniques , observed a higher than expected rate of complications in the control group , and used an unclear definition of bleeding events . therefore , the results are not sufficient to inform current emr practice . despite the lack of good evidence , several observational studies indicated that clip closure is increasingly applied . additional studies are under way and will hopefully provide a more definitive answer in the near future . at present stage , however , there is no adequate evidence to support prophylactic use of clips after emr . while we spent most of our time and resources on the detection and removal of diminutive polyps , it is unclear that this effort is worthwhile . the proposed resect - and - discard strategy is an approach to shift this balance . however , training , monitoring , auditing requirements , challenges in implementation , and the added complexity may further hinder adoption into clinical practice . alternative strategies include a simplified resect - and - discard strategy or deferring removal of diminutive polyps until they grow to higher - risk polyps . new strategies need to be studied , particularly with a focus on trade - offs of safety compared to cost - savings . as we may de - emphasize the importance of removing diminutive polyps , our effort should have a renewed focus on the detection and complete resection of higher - risk polyps . cold snare resection may be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . the larger the polyp the higher the risk for complications . although clipping of the mucosal defect after resection seems to be increasingly performed , there is currently insufficient evidence to support this practice for all non - pedunculated 20 mm polyps . new developments like the resect - anddiscard strategy , deferring removal of diminutive polyps and considerations how polypectomy techniques can be improved are discussed in this article .

detection and complete removal of precancerous neoplastic polyps are central to effective colorectal cancer screening . the prevalence of neoplastic polyps in the screening population in the united states is likely > 50% . however , most persons with neoplastic polyps are never destined to develop cancer , and do not benefit for finding and removing polyps , and may only be harmed by the procedure . further 7080% of polyps are diminutive ( 5 mm ) and such polyps almost never contain cancer . given the questionable benefit , the high - cost and the potential risk changing our approach to the management of diminutive polyps is currently debated . deemphasizing diminutive polyps and shifting our efforts to detection and complete removal of larger and higher - risk polyps deserves discussion and study . this article explores three controversies , and emerging concepts related to endoscopic polyp resection . first , we discuss challenges of optical resect - and - discard strategy and possible alternatives . second , we review recent studies that support the use of cold snare resection for 5 mm polyps . thirdly , we examine current evidence for prophylactic clipping after resection of large polyps .

INTRODUCTION Should we adopt a resect-and-discard strategy for diminutive polyps? Should we use cold snare resection for all polyps? Should we use prophylactic clipping after large polyp resection? SUMMARY Study Highlights

the benefit of colonoscopy on cancer prevention is dependent on effective polyp detection and removal . three important aspects of our approach to managing colon polyps are in evolution : the first aspect is related to the management of diminutive polyps ( polyps up to 5 mm in size ) . because diminutive polyps are very common and almost never contain cancer , new management strategies to improve cost - effectiveness have been proposed . the resect - and - discard strategy that uses real - time polyp diagnosis of diminutive polyps has been recently endorsed by endoscopy societies . hot electrocautery snare resection has been the standard - of - care for several decades , recent studies suggest that cold snare resection without electrocautery may be as safe and effective as hot snare resection . finally , several studies have focused on the management of large 20 mm polyps , particularly with respect to lowering the risk of bleeding complications . in this article , we discuss recent research developments and controversies with regards to the management of diminutive polyps , cold snare resection of mid - size polyps , and bleeding prophylaxis after resection of large polyps . the discussed observations call for refocusing our cancer prevention efforts in practice and research from removing diminutive polyps to the detection and safe resection of higher - risk polyps . of all detected polyps , 7080% cancer is exceedingly rare , and previous studies have described cancer prevalence between 0 and 0.08% for diminutive polyps and cancer prevalence between 0 and 1.5% in polyps up to 10 mm . resection of diminutive polyps increases patient risk and cost , yet the benefit on cancer prevention by removing diminutive polyps is questionable . however , histopathology evaluation of diminutive polyps remains important because presence of adenoma may determine low- or high - risk status of the patient and affect the colonoscopy surveillance interval . one avenue to reduce colonoscopy related cost would be to replace histopathology assessment by using endoscopic image , enhancing modalities to distinguish neoplastic from non - neoplastic polyps ( figure 1 ) . this new ability has inspired the concept of the resect - and - discard strategy . according to this strategy , diminutive polyps are diagnosed real time as adenomas or non - adenomatous polyps by using digital chromoendoscopy , like narrow band imaging ( nbi ) . calculated cost savings of this approach have been estimated to be 33,000,000 $ per year in the united states . a > 90% agreement between the optical and the pathology - based surveillance recommendations has been set as the required quality benchmark in order to adopt the resect - and - discard strategy . on the basis of these results , the resect - and - discard strategy has been endorsed by the european and american societies for gastrointestinal endoscopy ( esge and asge ) . both societies are well aware of challenges with successful implementation of resect - and - discard . further , the resect - and - discard approach requires additional efforts by the endoscopist during the examination , added photo and text documentation , and the need to combine optical with pathology based diagnoses in a large proportion of patients . alternative concepts to simplify the resect - and - discard strategy and minimize or eliminate the need for optical and histopathology assessment have recently been proposed . in a post hoc analysis , a non - optical resect - and - discard strategy was examined , in which all rectosigmoid diminutive polyps were considered as hyperplastic and all polyps proximal to the rectosigmoid as neoplastic . the non - optical strategy also reduced the number of required pathology examinations and provided more patients with surveillance recommendations immediately following the colonoscopy compared to the optical resect - and - discard strategy . while resect - and - discard is a promising idea to reduce colonoscopy associated cost , further research on how to make the concept feasible for community practice the low risk of diminutive polyps to develop cancer might support an even more radical approach to polyp management , namely to defer resection of diminutive polyp , and to only remove those that have grown to higher - risk polyps during the surveillance interval . available studies on natural history of polyps , albeit few support this approach . the risk of transition to cancer increases with size , and cost - effectiveness studies have only considered that polyps larger than 5 mm would transition to cancer . in two follow - up ct colonography studies , the majority of 6 - 9 mm polyps ( 6578% ) did not grow within 23 years . when considering even smaller 5 mm polyps , it is plausible that their resection does not sufficiently contribute to crc prevention to justify their removal , and its associated risk and cost . instead although leaving diminutive polyps in place would constitute a paradigm shift , in a recent survey 72% of gastroenterologists would be agreeable to leave diminutive polyps in place if such an approach was endorsed by governing societies . it has been estimated that deferring resection of diminutive polyps would result in a 64% reduction of therapeutic interventions during colonoscopies . while both are comparable for 3 mm polyps , larger polyps are insufficiently removed with a forceps and should be resected with a snare . in current practice , the most common approach to removing medium and large sized polyps 5 mm is to use electrocautery or hot snare resection . a recent smaller study from australia presented in abstract form reported an only 5% incomplete resection of up to 20 mm polyps when using a cold snare suggesting that resection may be at least be similarly complete when using a cold snare than a hot snare . with respect to bleeding risk a randomized trial among patients on anticoagulation with up to 10 mm polyps suggests that cold snare resection may actually lower the risk of bleeding . cold snare resection resulted in a lower - immediate ( 6 vs. 23% ) and delayed bleeding risk ( 0 vs. 14% ) when compared to hot snare resection . two other randomized trials compared cold to hot snare resection for up to 8 mm polyps . in one study , the other study reported immediate bleeding in 9% with cold snare resection ; however , all resolved spontaneously , and none required an intervention . these studies are small and bleeding is not well defined , however , the results question the assumed benefit of cautery on bleeding risk . aside from randomized trials , an increasing number of uncontrolled cohort studies suggest that cold snare polypectomy is safe and effective for up to 10 mm polyps . a first randomized trial compared the use of a dedicated cold snare with a standard snare for cold resection of up to 10 mm polyps and found a lower incomplete resection rate with using a dedicated cold snare ( 9 vs. 21% ) . while there are increasing number of studies on the safety and efficacy of cold snare resection for polyps up to 10 mm , the data on cold snare resection for larger polyps are limited to feasibility . case series have reported on piecemeal resection of up to > 20 mm colorectal polyps . finally , it should be noted , that cold snare polypectomy has no risk of cautery damage to the colonic wall or the resected polyp . this absence of electrocautery may decrease complications ( perforation , post - polypectomy syndrome ) and allow for better histopathology evaluation of the polyp and examination of the resection margins after polyp removal . although the data on cold snare resection of larger polyps is still emerging , cold snare resection appears to be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . future studies should systematically examine efficacy and safety of cold snare resection ; particularly determine the upper limit for en - bloc and piecemeal resection , the need for submucosal injection , and associated bleeding risks . the prevalence of cancer in polyps equal or larger than 10 mm has been reported to be between 2.4 and 10.2% . however , in the majority of these polyps en bloc resection can not be achieved and are therefore removed piece - meal . with the application of electrocautery to a larger area of the colonic wall during resection and the resultant large mucosal defect , the risk for perforation , post - polypectomy syndrome , and bleeding increases . bleeding typically occurs within 710 days after emr and may require hospital admission , endoscopic intervention , and blood transfusion . age , comorbidities , use of anticoagulation , and electrocautery setting have been reported risks in some but not other studies . a large retrospective single endoscopist study found that complete clip closure after removal of 20 mm polyps among 225 patients was associated with a 2% bleeding risk , significantly lower than the 10% observed in 247 historical controls , who did not undergo prophylactic clipping . although both studies support the use of clips , a major concern is an uncontrolled study design with the possibility of patient selection bias and unmeasured factors that may affect bleeding risk . prophylactic clip closure was compared to no clip closure after endoscopic resection of 10 mm sessile polyps among 348 patients . however , the study included smaller polyps , allowed different endoscopic resection techniques , observed a higher than expected rate of complications in the control group , and used an unclear definition of bleeding events . at present stage , however , there is no adequate evidence to support prophylactic use of clips after emr . of all detected polyps , 7080% cancer is exceedingly rare , and previous studies have described cancer prevalence between 0 and 0.08% for diminutive polyps and cancer prevalence between 0 and 1.5% in polyps up to 10 mm . resection of diminutive polyps increases patient risk and cost , yet the benefit on cancer prevention by removing diminutive polyps is questionable . however , histopathology evaluation of diminutive polyps remains important because presence of adenoma may determine low- or high - risk status of the patient and affect the colonoscopy surveillance interval . one avenue to reduce colonoscopy related cost would be to replace histopathology assessment by using endoscopic image , enhancing modalities to distinguish neoplastic from non - neoplastic polyps ( figure 1 ) . novel image modalities have shown to predict neoplastic polyps with high accuracy and thus allow to determine the interval for the subsequent surveillance colonoscopy . this new ability has inspired the concept of the resect - and - discard strategy . according to this strategy , diminutive polyps are diagnosed real time as adenomas or non - adenomatous polyps by using digital chromoendoscopy , like narrow band imaging ( nbi ) . calculated cost savings of this approach have been estimated to be 33,000,000 $ per year in the united states . a > 90% agreement between the optical and the pathology - based surveillance recommendations has been set as the required quality benchmark in order to adopt the resect - and - discard strategy . on the basis of these results , the resect - and - discard strategy has been endorsed by the european and american societies for gastrointestinal endoscopy ( esge and asge ) . both societies are well aware of challenges with successful implementation of resect - and - discard . further , the resect - and - discard approach requires additional efforts by the endoscopist during the examination , added photo and text documentation , and the need to combine optical with pathology based diagnoses in a large proportion of patients . alternative concepts to simplify the resect - and - discard strategy and minimize or eliminate the need for optical and histopathology assessment have recently been proposed . in a post hoc analysis , a non - optical resect - and - discard strategy was examined , in which all rectosigmoid diminutive polyps were considered as hyperplastic and all polyps proximal to the rectosigmoid as neoplastic . the non - optical strategy also reduced the number of required pathology examinations and provided more patients with surveillance recommendations immediately following the colonoscopy compared to the optical resect - and - discard strategy . while resect - and - discard is a promising idea to reduce colonoscopy associated cost , further research on how to make the concept feasible for community practice the low risk of diminutive polyps to develop cancer might support an even more radical approach to polyp management , namely to defer resection of diminutive polyp , and to only remove those that have grown to higher - risk polyps during the surveillance interval . available studies on natural history of polyps , albeit few support this approach . the risk of transition to cancer increases with size , and cost - effectiveness studies have only considered that polyps larger than 5 mm would transition to cancer . in two follow - up ct colonography studies , the majority of 6 - 9 mm polyps ( 6578% ) did not grow within 23 years . when considering even smaller 5 mm polyps , it is plausible that their resection does not sufficiently contribute to crc prevention to justify their removal , and its associated risk and cost . instead although leaving diminutive polyps in place would constitute a paradigm shift , in a recent survey 72% of gastroenterologists would be agreeable to leave diminutive polyps in place if such an approach was endorsed by governing societies . it has been estimated that deferring resection of diminutive polyps would result in a 64% reduction of therapeutic interventions during colonoscopies . while both are comparable for 3 mm polyps , larger polyps are insufficiently removed with a forceps and should be resected with a snare . in current practice , the most common approach to removing medium and large sized polyps 5 added cautery ablates marginal tissue and may therefore improve completeness of resection ( figure 2 ) . in the complete adenoma resection ( care ) , study 10% of 520 mm neoplastic polyps were not completely removed . a recent smaller study from australia presented in abstract form reported an only 5% incomplete resection of up to 20 mm polyps when using a cold snare suggesting that resection may be at least be similarly complete when using a cold snare than a hot snare . with respect to bleeding risk a randomized trial among patients on anticoagulation with up to 10 mm polyps suggests that cold snare resection may actually lower the risk of bleeding . cold snare resection resulted in a lower - immediate ( 6 vs. 23% ) and delayed bleeding risk ( 0 vs. 14% ) when compared to hot snare resection . two other randomized trials compared cold to hot snare resection for up to 8 mm polyps . in one study , the other study reported immediate bleeding in 9% with cold snare resection ; however , all resolved spontaneously , and none required an intervention . these studies are small and bleeding is not well defined , however , the results question the assumed benefit of cautery on bleeding risk . aside from randomized trials , an increasing number of uncontrolled cohort studies suggest that cold snare polypectomy is safe and effective for up to 10 mm polyps . a first randomized trial compared the use of a dedicated cold snare with a standard snare for cold resection of up to 10 mm polyps and found a lower incomplete resection rate with using a dedicated cold snare ( 9 vs. 21% ) . while there are increasing number of studies on the safety and efficacy of cold snare resection for polyps up to 10 mm , the data on cold snare resection for larger polyps are limited to feasibility . finally , it should be noted , that cold snare polypectomy has no risk of cautery damage to the colonic wall or the resected polyp . this absence of electrocautery may decrease complications ( perforation , post - polypectomy syndrome ) and allow for better histopathology evaluation of the polyp and examination of the resection margins after polyp removal . although the data on cold snare resection of larger polyps is still emerging , cold snare resection appears to be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . future studies should systematically examine efficacy and safety of cold snare resection ; particularly determine the upper limit for en - bloc and piecemeal resection , the need for submucosal injection , and associated bleeding risks . the prevalence of cancer in polyps equal or larger than 10 mm has been reported to be between 2.4 and 10.2% . a growing number of studies have demonstrated that resection can effectively and safely remove 8590% of these polyps and has therefore become the preferred treatment . however , in the majority of these polyps en bloc resection can not be achieved and are therefore removed piece - meal . with the application of electrocautery to a larger area of the colonic wall during resection and the resultant large mucosal defect , the risk for perforation , post - polypectomy syndrome , and bleeding increases . bleeding typically occurs within 710 days after emr and may require hospital admission , endoscopic intervention , and blood transfusion . age , comorbidities , use of anticoagulation , and electrocautery setting have been reported risks in some but not other studies . a large retrospective single endoscopist study found that complete clip closure after removal of 20 mm polyps among 225 patients was associated with a 2% bleeding risk , significantly lower than the 10% observed in 247 historical controls , who did not undergo prophylactic clipping . although both studies support the use of clips , a major concern is an uncontrolled study design with the possibility of patient selection bias and unmeasured factors that may affect bleeding risk . although the study from spain was large ( 1214 patients ) , the lack of a standardized resection protocol , a clear definition of outcomes and assurance of complete outcome assessment are some of the limitations . however , the study included smaller polyps , allowed different endoscopic resection techniques , observed a higher than expected rate of complications in the control group , and used an unclear definition of bleeding events . therefore , the results are not sufficient to inform current emr practice . at present stage , however , there is no adequate evidence to support prophylactic use of clips after emr . while we spent most of our time and resources on the detection and removal of diminutive polyps , it is unclear that this effort is worthwhile . the proposed resect - and - discard strategy is an approach to shift this balance . however , training , monitoring , auditing requirements , challenges in implementation , and the added complexity may further hinder adoption into clinical practice . alternative strategies include a simplified resect - and - discard strategy or deferring removal of diminutive polyps until they grow to higher - risk polyps . as we may de - emphasize the importance of removing diminutive polyps , our effort should have a renewed focus on the detection and complete resection of higher - risk polyps . cold snare resection may be at least as safe and effective as hot snare resection for polyps up to 10 mm in size . although clipping of the mucosal defect after resection seems to be increasingly performed , there is currently insufficient evidence to support this practice for all non - pedunculated 20 mm polyps . new developments like the resect - anddiscard strategy , deferring removal of diminutive polyps and considerations how polypectomy techniques can be improved are discussed in this article .

heart failure ( hf ) is a prevalent chronic disease characterized by high physical and psychosocial burdens that dramatically impair patients ' quality of life and performance status . specialist palliative care is a multidisciplinary intervention that focuses on optimizing quality of life for patients and families affected by serious illness , independent of prognosis . it is related to , but distinct from hospice , which in the united states , is restricted to patients with a maximum expected prognosis of 6 months . nonhospice palliative care may be initiated at any point in the disease trajectory and in conjunction with curative or lifeprolonging treatments . in this study , we considered palliative care as the service delivered by care teams with specialty expertise . specialist palliative care includes : expert symptom identification and management ; psychological , spiritual , and logistical support ; assistance with treatment decisionmaking and setting care goals ; and complex care coordination . palliative care has been shown to improve survival , quality of life , symptom burden , and caregiver outcomes , as well as reduce healthcare expenditures , and hospitalizations for patients with serious illness ; to date , the bulk of this research has focused on patients with cancer . nevertheless , the role of palliative care for patients with nonmalignant illnesses , including hf , remains somewhat undefined . growing evidence supports the role of palliative care in reducing distress among patients with hf . for example , a recent casecontrol study of outpatient palliative care consultations found reduced symptom burden and improved quality of life , while another study suggests improved satisfaction with care among stage d hf patients receiving palliative care consultations . additionally , palliative care may hold particular promise in hf management as a mechanism of advance care planning and goalsofcare identification ; this is especially salient recognizing that 80% of medicare beneficiaries with hf are hospitalized within the last 6 months of life . the 2013 hf management guidelines of the american college of cardiology and american heart association recommend considering palliative care across the hf trajectory , with particular focus paid to patients with advanced symptomatic hf and during inpatient discharge planning . in parallel , others have similarly called for palliative care integration within standard hf management . however , despite similar symptom burden and prognosis as advanced cancer patients , hf patients use palliative services less often than cancer patients . provider behavior may contribute to underuse : in one study , more than half of cardiologists surveyed would not discuss palliative care with elderly advanced hf patients . several additional factors appear to be related to the low rates of palliative care enrollment by hf patients , including : the unpredictable disease trajectory of hf ; the view of hf as a chronic , manageable disease ; and patient and caregiver confusion regarding prognosis . prior qualitative studies exploring palliative care referral barriers for patients with hf in the united kingdom and australia suggest organizational , professional , and cognitive factors potentially at play . recognizing differences in healthcare systems and culture , we conducted an exploratory qualitative study with primary care , cardiology , and palliative care providers to uncover potential barriers to palliative care referral for hf patients in the united states . we used stratified purposeful sampling to recruit primary care , cardiology , and palliative care physicians and nonphysician providers from diverse practice settings ( ie , academic / nonacademic , urban / rural ) . chain referral was used to allow participants to suggest colleagues who might provide valuable insights based on clinical experience and expertise ; study coauthors also suggested potential participants . eligibility criteria were ( a ) physician , nurse practitioner , or physician assistant ; ( b ) practicing in north carolina ; and ( c ) cared for 3 hf patients in the preceding 6 months . one author ( dk ) conducted all interviews , in person or by telephone , between december 2011 and may 2012 . based upon a literature review regarding hf care transitions and discussions with 3 experts , we developed an interview guide ( table 1 ) containing 10 questions in 4 domains : needs of hf patients ; knowledge and perceptions of palliative care ; indications for , and optimal timing of , palliative care referral in hf ; and barriers to palliative care referral in hf . to increase validity , we developed probes for iterative questioning to further explore providers ' responses . semistructured interview guide : domains of interest and sample questions prior to the interview , participants reviewed a vignette of a patient with advanced hf , typical comorbidities , and unaddressed palliative needs ( table 2 ) . participants completed a survey collecting demographics as well as selfreported information about their hf patient caseload . hypothetical heart failure patient vignette used to frame interviews bid indicates twice daily ; bmi , body mass index ; hf , heart failure ; hr , heart rate ; jvp , jugular venous pressure ; ntprobnp , nterminal prohormone of brain natriuretic protein ; qd , daily ; sbp , systolic blood pressure . we used template analysis , a qualitative approach that combines content analysis and grounded theory . template analysis flexibly integrates a priori assumptions and hypotheses for a hybrid inductive / deductive analytic process . data analysis was performed iteratively , with the initial codebook informed by an extensive literature review . two authors ( dk , emm ) independently coded a random 50% sample of transcripts in 3 rounds . after each round , consensus meetings were held to discuss and arbitrate discrepancies . dk coded all remaining transcripts , which emm reviewed and verified . using the constant comparative technique , text units were compared with previously coded data to ensure stability and relevance of themes . we used nvivo9 ( version 9 , qsr international ) to code and query transcripts . to further explore our data , we performed matrix and compound queries . we applied 9 techniques ( table 3 ) to improve the trustworthiness of our findings across 4 domains : credibility ; transferability ; dependability ; and confirmability . regarding saturation , we followed the guidance of strauss and corbin , who suggest ceasing data collection when additional interviews do not significantly expand upon prior findings . as a method of quality assurance , we compared every additional interview to prior interviews , scrutizing the degree of theme overlap versus marginal gain . we used stratified purposeful sampling to recruit primary care , cardiology , and palliative care physicians and nonphysician providers from diverse practice settings ( ie , academic / nonacademic , urban / rural ) . chain referral was used to allow participants to suggest colleagues who might provide valuable insights based on clinical experience and expertise ; study coauthors also suggested potential participants . eligibility criteria were ( a ) physician , nurse practitioner , or physician assistant ; ( b ) practicing in north carolina ; and ( c ) cared for 3 hf patients in the preceding 6 months . one author ( dk ) conducted all interviews , in person or by telephone , between december 2011 and may 2012 . based upon a literature review regarding hf care transitions and discussions with 3 experts , we developed an interview guide ( table 1 ) containing 10 questions in 4 domains : needs of hf patients ; knowledge and perceptions of palliative care ; indications for , and optimal timing of , palliative care referral in hf ; and barriers to palliative care referral in hf . to increase validity , we developed probes for iterative questioning to further explore providers ' responses . semistructured interview guide : domains of interest and sample questions prior to the interview , participants reviewed a vignette of a patient with advanced hf , typical comorbidities , and unaddressed palliative needs ( table 2 ) . participants completed a survey collecting demographics as well as selfreported information about their hf patient caseload . hypothetical heart failure patient vignette used to frame interviews bid indicates twice daily ; bmi , body mass index ; hf , heart failure ; hr , heart rate ; jvp , jugular venous pressure ; ntprobnp , nterminal prohormone of brain natriuretic protein ; qd , daily ; sbp , systolic blood pressure . we used template analysis , a qualitative approach that combines content analysis and grounded theory . template analysis flexibly integrates a priori assumptions and hypotheses for a hybrid inductive / deductive analytic process . data analysis was performed iteratively , with the initial codebook informed by an extensive literature review . two authors ( dk , emm ) independently coded a random 50% sample of transcripts in 3 rounds . after each round , consensus meetings were held to discuss and arbitrate discrepancies . dk coded all remaining transcripts , which emm reviewed and verified . using the constant comparative technique , text units were compared with previously coded data to ensure stability and relevance of themes . we used nvivo9 ( version 9 , qsr international ) to code and query transcripts . to further explore our data , we performed matrix and compound queries . we applied 9 techniques ( table 3 ) to improve the trustworthiness of our findings across 4 domains : credibility ; transferability ; dependability ; and confirmability . regarding saturation , we followed the guidance of strauss and corbin , who suggest ceasing data collection when additional interviews do not significantly expand upon prior findings . as a method of quality assurance , we compared every additional interview to prior interviews , scrutizing the degree of theme overlap versus marginal gain . of 22 individuals contacted , 19 agreed to participate ; we interviewed 18 . within each specialty all but one participant had both inpatient and outpatient palliative care services within their geographic area ( data not shown ) . participants represented 2 academic medical centers , the veterans affairs medical center , and communitybased nonacademic practices . interviews lasted a median of 37 minutes ( range : 25 to 51 ) . during analysis , we allowed themes to naturally emerge from interview data ; coincidentally , we discovered a latent what , when , why , who , where , and how structure to the themes ( figure 1 ) . this organizational framework was applied once all themes had been identified , and not a priori . common referrals made by participants for their patients with hf are displayed in figure 2 . characteristics of study participants columns may not total 100% due to rounding and missing data . qualitative themes identified related to palliative care referral for patients with heart failure ( hf ) . although all providers reported that they could define palliative care , probing revealed variation across cardiology and primary care providers . specifically , nearly all primary care and cardiology providers lacked clarity that palliative care is not prognosis dependent and may be administered concurrently with lifeprolonging therapy . many cardiology and primary care providers failed to recognize specialist palliative care as a tangible clinical service . for example , a primary care physician said : palliative care is a philosophy . hospice is a treatment approach complete with billables , and payers , and all that crap , within that treatment philosophy . when asked to describe eligibility and appropriateness criteria for palliative care ( for which there are none , aside from patient need ) , cardiology and primary care providers used the terms hospice and palliative care interviewer : so in your opinion , what makes a hf patient eligible for palliative care?cardiologist : i mean , that 's a legislative decision.interviewer:and , so in your mind , is there a distinction between palliative care and hospice care?cardiologist : no . not in my mind . so in your mind , is there a distinction between palliative care and hospice care ? incorrectly believing that palliative care mandates suspension of lifeprolonging therapies , cardiology and primary care providers reported that patient goals determine eligibility : if the patient wants to focus on symptoms and is willing to accept the sort of basics then the patient 's eligible for palliative care . ( primary care physician ) if the patient wants to focus on symptoms and is willing to accept the sort of basics ( primary care physician ) some cardiology and primary care providers recognized differences between palliative care and hospice . i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . i prefer to view palliative care as clear management of symptoms and emphasis of the patient 's quality of life in decisionmaking . i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . i prefer to view palliative care as clear management of symptoms and emphasis of the patient 's quality of life in decisionmaking . ( primary care physician ) most nonpalliative care providers lacked experience with specialist palliative care providers / teams . they frequently acknowledged not knowing how to access palliative care ; a primary care physician from a large academic medical center said : i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . most providers based referral on triggers , including physiological findings ( eg , symptom presence ) , disease status ( eg , functional decline ) , or events ( eg , device implantation ) . among primary and palliative care providers , repeated hospitalizations over a short interval ( eg , 3 in 6 months ) suggested that palliative care might be appropriate . however , cardiology providers frequently discussed the point at which you are unable to do more as another trigger : i think that the trigger to get [ the palliative care service ] involved was knowing that my patient was dying and that i did n't have other medical options for them . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) i think that the trigger to get [ the palliative care service ] involved was knowing that my patient was dying and that i did n't have other medical options for them . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) the unpredictable trajectory of hf was a frequently cited barrier , especially when providers believed that palliative care eligibility or reimbursement were dependent upon prognosis ( as with hospice ) . moreover , some providers spoke of the difficulty of recognizing and acting upon triggers: i think the main challenge is both for the cardiologist and for the patient , recognizing those prognostic signs that say this is an individual who is moving into the last phase of their disease . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , i think the main challenge is both for the cardiologist and for the patient , recognizing those prognostic signs that say this is an individual who is moving into the last phase of their disease . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , ( palliative care physician ) providers commonly mentioned the insistence on lifeprolonging treatments as a barrier to palliative care referral . one palliative care physician thought, that [ cardiologists ] are not only procedure driven but intervention driven they have a hard time seeing an endpoint in the same way that oncologists have a difficult time really seeing when their treatments just are n't working anymore . that [ cardiologists ] are not only procedure driven but intervention driven they have a hard time seeing an endpoint in the same way that oncologists have a difficult time really seeing when their treatments just are n't working anymore . all providers endorsed palliative care in hf and believed that palliative care providers are experts regarding : symptom management ; care coordination ; and facilitating difficult discussions about prognosis , and advance care planning . many appreciated its focus on quality of life : [ t]o have a voice from the palliative care team where we do n't forget to focus on quality of life as much as survival would be team where we do n't forget to focus on quality of life as much as survival would be ( cardiologist ) providers also discussed how sociocultural attitudes regarding death influenced provider referral , but often alluded to hospice when discussing palliative care . one primary care physician described , a cultural bias against adopting palliative care unless you know unlike the cureoriented goal of oncology , providers believed that hf , as a progressive , incurable disease , poses challenges to integrating palliative care into standard hf management : [ a]ll medical therapy for hf is really to relieve their symptoms . and so , in a sense , to me , it all feels like palliative care . ( primary care physician ) to improve familiarity and acceptance , providers thought that palliative care must demonstrate and market its benefit to patients and providers . trust and rapport were identified as key facilitators to palliative care referral , especially because palliative care knowledge is limited . palliative care providers discussed how networking and peer education have resulted in greater and earlier referrals , by winning over previously skeptical colleagues: i do a whole lot more than start morphine drips , thank god. so i actually , in a goodnatured way , really try to do a little education with folks , and i think they really appreciate it . i think once he realized that we 're not the grim reaper service and that we 're really about what does the patient want, they sort of lay down their baggage . i do a whole lot more than start morphine drips , thank god. so i actually , in a goodnatured way , really try to do a little education with folks , and i think they really appreciate it . i think once he realized that we 're not the grim reaper service and that we 're really about what does the patient want, they sort of lay down their baggage . ( palliative care physician ) nearly all providers felt that primary care or cardiology providers should initiate conversations about palliative care due to their preexisting relationships with patients . however , palliative care providers feared that such conversations are inhibited by providers ' discomfort with discussing palliation.i think it 's perfectly fine for the primary treating cardiologist to begin that conversation to say , we need to start talking about your hf being in its latest stages . we need to think about what our options are for how to give you the best quality of life under these circumstances. but it 's also perfectly fine for them to duck that conversation and say , i think it 's perfectly fine for the primary treating cardiologist to begin that conversation to say , we need to start talking about your hf being in its latest stages . we need to think about what our options are for how to give you the best quality of life under these circumstances. but it 's also perfectly fine for them to duck that conversation and say , ( palliative care physician ) a major barrier to palliative care referral was lack of knowledge within the healthcare community ; overcoming this barrier requires intervention directed at providers during graduate or postgraduate training: [e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . i do n't think we get a good job of learning about that during our medical school or residency or fellowship training and if you do n't train us at that point , you ca n't expect us to understand or know how to use them at this point . [ m]any of us physicians struggle with even bringing up the palliative care concept with patients because we 're just not skilled necessarily at doing it . [ e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . i do n't think we get a good job of learning about that during our medical school or residency or fellowship training and if you do n't train us at that point , you ca n't expect us to understand or know how to use them at this point . [ m]any of us physicians struggle with even bringing up the palliative care concept with patients because we 're just not skilled necessarily at doing it . ( cardiologist ) all providers expressed the need to develop palliative care basics ( eg , symptom identification / management in advanced illness , communication skills regarding goals of care ) , albeit for different reasons . some cardiology providers desired to gain confidence in difficult communication , whereas palliative care providers often spoke of workforce constraints: there are still only 3,000 boardcertified palliative medicine physicians in the u.s . one is the dominant model in the next several years would be to promote early inpatient consultation maybe through identifying mutually agreedupon triggers for referrals with a cardiology service . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) one is the dominant model in the next several years would be to promote early inpatient consultation maybe through identifying mutually agreedupon triggers for referrals with a cardiology service . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) finally , providers discussed various practical strategies to encourage palliative care referral . for example: [palliative care referral ] relies on me asking for it when it probably should be more automated . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) [ palliative care referral ] relies on me asking for it when it probably should be more automated . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) although all providers reported that they could define palliative care , probing revealed variation across cardiology and primary care providers . specifically , nearly all primary care and cardiology providers lacked clarity that palliative care is not prognosis dependent and may be administered concurrently with lifeprolonging therapy . many cardiology and primary care providers failed to recognize specialist palliative care as a tangible clinical service . for example , a primary care physician said : palliative care is a philosophy . hospice is a treatment approach complete with billables , and payers , and all that crap , within that treatment philosophy . when asked to describe eligibility and appropriateness criteria for palliative care ( for which there are none , aside from patient need ) , cardiology and primary care providers used the terms hospice and palliative care interviewer : so in your opinion , what makes a hf patient eligible for palliative care?cardiologist : i mean , that 's a legislative decision.interviewer:and , so in your mind , is there a distinction between palliative care and hospice care?cardiologist : no . not in my mind . so in your mind , is there a distinction between palliative care and hospice care ? is there ? incorrectly believing that palliative care mandates suspension of lifeprolonging therapies , cardiology and primary care providers reported that patient goals determine eligibility : if the patient wants to focus on symptoms and is willing to accept the sort of basics ( primary care physician ) if the patient wants to focus on symptoms and is willing to accept the sort of basics then the patient 's eligible for palliative care . ( primary care physician ) some cardiology and primary care providers recognized differences between palliative care and hospice . for example : i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . i prefer to view palliative care as clear management of symptoms and emphasis of the patient 's quality of life in decisionmaking . i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . i prefer to view palliative care as clear management of symptoms and emphasis of the patient 's quality of life in decisionmaking . ( primary care physician ) most nonpalliative care providers lacked experience with specialist palliative care providers / teams . they frequently acknowledged not knowing how to access palliative care ; a primary care physician from a large academic medical center said : i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . most providers based referral on triggers , including physiological findings ( eg , symptom presence ) , disease status ( eg , functional decline ) , or events ( eg , device implantation ) . among primary and palliative care providers , repeated hospitalizations over a short interval ( eg , 3 in 6 months ) suggested that palliative care might be appropriate . however , cardiology providers frequently discussed the point at which you are unable to do more as another trigger : i think that the trigger to get [ the palliative care service ] involved was knowing that my patient was dying and that i did n't have other medical options for them . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) i think that the trigger to get [ the palliative care service ] involved was knowing that my patient was dying and that i did n't have other medical options for them . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) the unpredictable trajectory of hf was a frequently cited barrier , especially when providers believed that palliative care eligibility or reimbursement were dependent upon prognosis ( as with hospice ) . moreover , some providers spoke of the difficulty of recognizing and acting upon triggers: i think the main challenge is both for the cardiologist and for the patient , recognizing those prognostic signs that say this is an individual who is moving into the last phase of their disease . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , i think the main challenge is both for the cardiologist and for the patient , recognizing those prognostic signs that say this is an individual who is moving into the last phase of their disease . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , ( palliative care physician ) providers commonly mentioned the insistence on lifeprolonging treatments as a barrier to palliative care referral . one palliative care physician thought, that [ cardiologists ] are not only procedure driven but intervention driven they have a hard time seeing an endpoint in the same way that oncologists have a difficult time really seeing when their treatments just are n't working anymore . that [ cardiologists ] are not only procedure driven but intervention driven they have a hard time seeing an endpoint in the same way that oncologists have a difficult time really seeing when their treatments just are n't working anymore . all providers endorsed palliative care in hf and believed that palliative care providers are experts regarding : symptom management ; care coordination ; and facilitating difficult discussions about prognosis , and advance care planning . many appreciated its focus on quality of life : [ t]o have a voice from the palliative care team where we do n't forget to focus on quality of life as much as survival would be team where we do n't forget to focus on quality of life as much as survival would be ( cardiologist ) providers also discussed how sociocultural attitudes regarding death influenced provider referral , but often alluded to hospice when discussing palliative care . one primary care physician described , a cultural bias against adopting palliative care unless you know unlike the cureoriented goal of oncology , providers believed that hf , as a progressive , incurable disease , poses challenges to integrating palliative care into standard hf management : [ a]ll medical therapy for hf is really to relieve their symptoms . and so , in a sense , to me , it all feels like palliative care . to improve familiarity and acceptance , providers thought that palliative care must demonstrate and market its benefit to patients and providers . trust and rapport were identified as key facilitators to palliative care referral , especially because palliative care knowledge is limited . palliative care providers discussed how networking and peer education have resulted in greater and earlier referrals , by winning over previously skeptical colleagues: i do a whole lot more than start morphine drips , thank god. so i actually , in a goodnatured way , really try to do a little education with folks , and i think they really appreciate it . that same cardiologist has sent me several more consults since then i think once he realized that we 're not the grim reaper service and that we 're really about what does the patient want, they sort of lay down their baggage . i do a whole lot more than start morphine drips , thank god. so i actually , in a goodnatured way , really try to do a little education with folks , and i think they really appreciate it . i think once he realized that we 're not the grim reaper service and that we 're really about what does the patient want, they sort of lay down their baggage . nearly all providers felt that primary care or cardiology providers should initiate conversations about palliative care due to their preexisting relationships with patients . however , palliative care providers feared that such conversations are inhibited by providers ' discomfort with discussing palliation.i think it 's perfectly fine for the primary treating cardiologist to begin that conversation to say , we need to start talking about your hf being in its latest stages . we need to think about what our options are for how to give you the best quality of life under these circumstances. but it 's also perfectly fine for them to duck that conversation and say , i think it 's perfectly fine for the primary treating cardiologist to begin that conversation to say , we need to start talking about your hf being in its latest stages . we need to think about what our options are for how to give you the best quality of life under these circumstances. but it 's also perfectly fine for them to duck that conversation and say , a major barrier to palliative care referral was lack of knowledge within the healthcare community ; overcoming this barrier requires intervention directed at providers during graduate or postgraduate training: [e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . i do n't think we get a good job of learning about that during our medical school or residency or fellowship training and if you do n't train us at that point , you ca n't expect us to understand or know how to use them at this point . [ m]any of us physicians struggle with even bringing up the palliative care concept with patients because we 're just not skilled necessarily at doing it . [ e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . i do n't think we get a good job of learning about that during our medical school or residency or fellowship training and if you do n't train us at that point , you ca n't expect us to understand or know how to use them at this point . [ m]any of us physicians struggle with even bringing up the palliative care concept with patients because we 're just not skilled necessarily at doing it . ( cardiologist ) all providers expressed the need to develop palliative care basics ( eg , symptom identification / management in advanced illness , communication skills regarding goals of care ) , albeit for different reasons . some cardiology providers desired to gain confidence in difficult communication , whereas palliative care providers often spoke of workforce constraints: there are still only 3,000 boardcertified palliative medicine physicians in the u.s . one is the dominant model in the next several years would be to promote early inpatient consultation maybe through identifying mutually agreedupon triggers for referrals with a cardiology service . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) one is the dominant model in the next several years would be to promote early inpatient consultation maybe through identifying mutually agreedupon triggers for referrals with a cardiology service . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) finally , providers discussed various practical strategies to encourage palliative care referral . for example: [palliative care referral ] relies on me asking for it when it probably should be more automated . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) [ palliative care referral ] relies on me asking for it when it probably should be more automated . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) this is the first us study to explore barriers to palliative care referral for advanced hf patients among providers frequently caring for these patients . despite the majority of cardiology and primary care providers practicing near specialist palliative care services , we found limited knowledge related to : what nonhospice palliative care is ( especially differences from hospice ) ; what it offers patients , families , and providers ; when it is indicated ; and , how to access it . one barrier is confusion about the term palliative care itself . all cardiology and primary care providers reported familiarity with nonhospice palliative care ; however , phrases such as comfort care or just the basics implied their equating nonhospice and hospice palliative care . consistent with previous studies , nonpalliative care providers often reported criteria associated with the medicare hospice benefit ( ie , 6 months expected survival , suspending lifeprolonging treatments ) as those for nonhospice palliative care . notably , because providers almost unanimously used triggers to initiate palliative care , using inappropriate triggers ( eg , active dying ) may result in late referrals ( if any ) . prior work suggests that oncologists are more comfortable and are more likely to refer patients to a service called supportive care as opposed to palliative care . similarly , in a 22 trial comparing the 2 names , patients with cancer had more favorable impressions of the term supportive care . the implications of the name palliative care have yet to be elucidated in the cardiogy population . nevertheless , we must not lose sight of the fact that perception is paramount , particularly in sensitive contexts , such as serious illness . providers from all specialties interviewed frequently reported the unpredictability of hf as a barrier to palliative care referral , as seen elsewhere . unlike the current hospice framework , nonhospice palliative care may present a more flexible alternative for addressing the needs of patients with diseases with volatile trajectories . crisp demarcations between curative and palliative modalities reflect an unnecessary dichotomy that defers focusing on quality of life improvement until disease futility has been established ( ie , hospice ) . confusion regarding nonhospice palliative care and hospice was evident from both explicit statements and implicit comments . for example , the prognostic difficulty of hf and the insistence on lifeprolonging treatments were frequently discussed as barriers ; however , they are actually only barriers to hospice referral , not to nonhospice palliative care . all nonpalliative care providers interviewed explicitly expressed interest in exploring how to collaborate with palliative care . prior qualitative research with physicians and nurses , in the united kingdom and australia suggests a mixture of professional , organizational , and cognitive factors impeding palliative care referral for hf patients , including concerns that patients might be stolen by palliative care providers . nevertheless , nonpalliative care providers reported being unsure of how and when to integrate specialist palliative care into standard hf management , the overarching reason cited for low referral . indeed , our research provides supporting evidence that collaboration between palliative and cardiology providers is suboptimal , despite mutual agreement of the likely benefit to patients that could result from palliative care . as such such training should strive to : correct misconceptions about palliative care and hospice ( ie , that hospice is a specific subset of palliative care , largely defined by policy ) ; teach providers how to identify palliative needs in their patients ; how to provide primary palliative care themselves when appropriate ; how to determine when specialist palliative care referral or consultation is indicated ; and , how to effectively work with palliative care specialists . consistent with previous work , nonpalliative care providers in our study were interested in learning more about palliative care from palliative care experts themselves . in the same vein , a palliative care physician said , we must reach out and give [ cardiology and primary care providers ] the language [ of palliative care ] . additionally , identifying and supporting internal champions within primary care or cardiology to serve as interdisciplinary liaisons with palliative care may be another mechanism by which to enhance meaningful collaboration between specialties . nevertheless , palliative care specialists may serve as valuable resources to correct misconceptions regarding palliative care and hospice among medical professionals , patients , and caregivers . our findings support that the role of specialist palliative care has yet to be articulated for hf patients . several steps pave the way to a clearer understanding of how specialist palliative care can be integrated into hf care . first , it is critical to determine whether palliative care improves hf patient outcomes in a randomized , controlled trial ( as has been demonstrated in lung cancer ) ; these studies are underway , although preliminary studies offer signals that palliative care indeed holds promise in reducing hfrelated suffering . second , research should define the optimal model for palliative care to maximize benefit while being flexible enough to accomodate patients with unpredictable trajectories ( eg , hf ) . it is unlikely that the contemporary model of palliative care in oncology ( ie , referral at the point of diagnosis ) will seamlessly translate to hf . needsbased , as opposed to diagnosisbased , referral may be a better approach to leveraging palliative care resources . if so , future research will need to identify clinically relevant , patientcentered referral triggers specific to hf ( eg , transition to stage c hf ) . the increasing prevalence of patients with lifelimiting illnesses warrants exploring strategies to expand palliative care coverage , such as outpatient and communitybased palliative care programs . first , generalizability may be limited because providers come from a single state and many practice in large , academic medical centers with palliative care programs . second , although we used sound methodological techniques that maximize analytic rigor and trustworthiness , others may have ultimately identified different themes . third , whereas some may find our sample of 18 providers too small to provide generalizations to all cardiology , primary care , and palliative care providers , we caution that such conclusions are inappropriate given our qualitative study design . furthermore , qualitative theory fundamentally discourages insistence upon sample size requirements and broadbased generalizable inference . nevertheless , guest and colleagues suggest that thematic saturation generally occurs within the first 12 interviews , with basic themes emerging after 6 interviews ; we conducted 18 . our work naturally sets the stage for survey research to confirm our findings on a national level . first , generalizability may be limited because providers come from a single state and many practice in large , academic medical centers with palliative care programs . second , although we used sound methodological techniques that maximize analytic rigor and trustworthiness , others may have ultimately identified different themes . third , whereas some may find our sample of 18 providers too small to provide generalizations to all cardiology , primary care , and palliative care providers , we caution that such conclusions are inappropriate given our qualitative study design . furthermore , qualitative theory fundamentally discourages insistence upon sample size requirements and broadbased generalizable inference . nevertheless , guest and colleagues suggest that thematic saturation generally occurs within the first 12 interviews , with basic themes emerging after 6 interviews ; we conducted 18 . our work naturally sets the stage for survey research to confirm our findings on a national level . our research suggests that deficits in providers ' knowledge and comfort in discussing palliative care for a difficulttopredict disease present major barriers to referring patients with advanced hf for palliative care . this issue is of high relevance due to the recent acc / aha guidelines which emphasize the potential benefit of integrating palliative care in hf management ; our work provides a glimpse at impediments that pave the road to eventual operationalization of the guidelines . indeed , our research highlights the need for increasing awareness of palliative care among healthcare providers , correcting misconceptions that it is only appropriate for the terminally ill . future work should seek to develop provider and patientcentered interventions to reduce actionable barriers to palliative care uptake in hf . additionally , we thank sarah birken , phd , for her thoughtful and constructive comments during the planning and interpretation of this study .

backgroundalthough similar to cancer patients regarding symptom burden and prognosis , patients with heart failure ( hf ) tend to receive palliative care far less frequently . we sought to explore factors perceived by cardiology , primary care , and palliative care providers to impede palliative care referral for hf patients.methods and resultswe conducted semistructured interviews regarding ( 1 ) perceived needs of patients with advanced hf ; ( 2 ) knowledge , attitudes , and experiences with specialist palliative care ; ( 3 ) perceived indications for and optimal timing of palliative care referral in hf ; and ( 4 ) perceived barriers to palliative care referral . two investigators analyzed data using template analysis , a qualitative technique . we interviewed 18 physician , nurse practitioner , and physician assistant providers from 3 specialties : cardiology , primary care , and palliative care . providers had limited knowledge regarding what palliative care is , and how it can complement traditional hf therapy to decrease hfrelated suffering . interviews identified several potential barriers : the unpredictable course of hf ; lack of clear referral triggers across the hf trajectory ; and ambiguity regarding what differentiates standard hf therapy from palliative care . nevertheless , providers expressed interest for integrating palliative care into traditional hf care , but were unsure of how to initiate collaboration.conclusionspalliative care referral for hf patients may be suboptimal due to limited provider knowledge and misperceptions of palliative care as a service reserved for those near death . these factors represent potentially modifiable targets for provider education , which may help to improve palliative care referral for hf patients with unresolved diseaserelated burden .

Introduction Methods Design Sample and Recruitment Data Collection Analysis Results What: Lack of Functional Knowledge Regarding Palliative Care When: Appropriate Timing for Palliative Care Referral Why: Perceptions of Palliative Care in HF Who: Interprovider Relationships and Responsibilities Where: Origin of Referral How: Strategies to Increase Palliative Care Referral Discussion Limitations Conclusions Acknowledgments

heart failure ( hf ) is a prevalent chronic disease characterized by high physical and psychosocial burdens that dramatically impair patients ' quality of life and performance status . specialist palliative care is a multidisciplinary intervention that focuses on optimizing quality of life for patients and families affected by serious illness , independent of prognosis . it is related to , but distinct from hospice , which in the united states , is restricted to patients with a maximum expected prognosis of 6 months . specialist palliative care includes : expert symptom identification and management ; psychological , spiritual , and logistical support ; assistance with treatment decisionmaking and setting care goals ; and complex care coordination . palliative care has been shown to improve survival , quality of life , symptom burden , and caregiver outcomes , as well as reduce healthcare expenditures , and hospitalizations for patients with serious illness ; to date , the bulk of this research has focused on patients with cancer . nevertheless , the role of palliative care for patients with nonmalignant illnesses , including hf , remains somewhat undefined . growing evidence supports the role of palliative care in reducing distress among patients with hf . for example , a recent casecontrol study of outpatient palliative care consultations found reduced symptom burden and improved quality of life , while another study suggests improved satisfaction with care among stage d hf patients receiving palliative care consultations . additionally , palliative care may hold particular promise in hf management as a mechanism of advance care planning and goalsofcare identification ; this is especially salient recognizing that 80% of medicare beneficiaries with hf are hospitalized within the last 6 months of life . the 2013 hf management guidelines of the american college of cardiology and american heart association recommend considering palliative care across the hf trajectory , with particular focus paid to patients with advanced symptomatic hf and during inpatient discharge planning . however , despite similar symptom burden and prognosis as advanced cancer patients , hf patients use palliative services less often than cancer patients . several additional factors appear to be related to the low rates of palliative care enrollment by hf patients , including : the unpredictable disease trajectory of hf ; the view of hf as a chronic , manageable disease ; and patient and caregiver confusion regarding prognosis . prior qualitative studies exploring palliative care referral barriers for patients with hf in the united kingdom and australia suggest organizational , professional , and cognitive factors potentially at play . recognizing differences in healthcare systems and culture , we conducted an exploratory qualitative study with primary care , cardiology , and palliative care providers to uncover potential barriers to palliative care referral for hf patients in the united states . we used stratified purposeful sampling to recruit primary care , cardiology , and palliative care physicians and nonphysician providers from diverse practice settings ( ie , academic / nonacademic , urban / rural ) . eligibility criteria were ( a ) physician , nurse practitioner , or physician assistant ; ( b ) practicing in north carolina ; and ( c ) cared for 3 hf patients in the preceding 6 months . based upon a literature review regarding hf care transitions and discussions with 3 experts , we developed an interview guide ( table 1 ) containing 10 questions in 4 domains : needs of hf patients ; knowledge and perceptions of palliative care ; indications for , and optimal timing of , palliative care referral in hf ; and barriers to palliative care referral in hf . semistructured interview guide : domains of interest and sample questions prior to the interview , participants reviewed a vignette of a patient with advanced hf , typical comorbidities , and unaddressed palliative needs ( table 2 ) . we used template analysis , a qualitative approach that combines content analysis and grounded theory . we applied 9 techniques ( table 3 ) to improve the trustworthiness of our findings across 4 domains : credibility ; transferability ; dependability ; and confirmability . we used stratified purposeful sampling to recruit primary care , cardiology , and palliative care physicians and nonphysician providers from diverse practice settings ( ie , academic / nonacademic , urban / rural ) . eligibility criteria were ( a ) physician , nurse practitioner , or physician assistant ; ( b ) practicing in north carolina ; and ( c ) cared for 3 hf patients in the preceding 6 months . based upon a literature review regarding hf care transitions and discussions with 3 experts , we developed an interview guide ( table 1 ) containing 10 questions in 4 domains : needs of hf patients ; knowledge and perceptions of palliative care ; indications for , and optimal timing of , palliative care referral in hf ; and barriers to palliative care referral in hf . semistructured interview guide : domains of interest and sample questions prior to the interview , participants reviewed a vignette of a patient with advanced hf , typical comorbidities , and unaddressed palliative needs ( table 2 ) . we used template analysis , a qualitative approach that combines content analysis and grounded theory . during analysis , we allowed themes to naturally emerge from interview data ; coincidentally , we discovered a latent what , when , why , who , where , and how structure to the themes ( figure 1 ) . qualitative themes identified related to palliative care referral for patients with heart failure ( hf ) . although all providers reported that they could define palliative care , probing revealed variation across cardiology and primary care providers . specifically , nearly all primary care and cardiology providers lacked clarity that palliative care is not prognosis dependent and may be administered concurrently with lifeprolonging therapy . many cardiology and primary care providers failed to recognize specialist palliative care as a tangible clinical service . for example , a primary care physician said : palliative care is a philosophy . when asked to describe eligibility and appropriateness criteria for palliative care ( for which there are none , aside from patient need ) , cardiology and primary care providers used the terms hospice and palliative care interviewer : so in your opinion , what makes a hf patient eligible for palliative care?cardiologist : i mean , that 's a legislative decision.interviewer:and , so in your mind , is there a distinction between palliative care and hospice care?cardiologist : no . ( primary care physician ) if the patient wants to focus on symptoms and is willing to accept the sort of basics ( primary care physician ) some cardiology and primary care providers recognized differences between palliative care and hospice . i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . ( primary care physician ) most nonpalliative care providers lacked experience with specialist palliative care providers / teams . they frequently acknowledged not knowing how to access palliative care ; a primary care physician from a large academic medical center said : i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . among primary and palliative care providers , repeated hospitalizations over a short interval ( eg , 3 in 6 months ) suggested that palliative care might be appropriate . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) the unpredictable trajectory of hf was a frequently cited barrier , especially when providers believed that palliative care eligibility or reimbursement were dependent upon prognosis ( as with hospice ) . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , ( palliative care physician ) providers commonly mentioned the insistence on lifeprolonging treatments as a barrier to palliative care referral . all providers endorsed palliative care in hf and believed that palliative care providers are experts regarding : symptom management ; care coordination ; and facilitating difficult discussions about prognosis , and advance care planning . one primary care physician described , a cultural bias against adopting palliative care unless you know unlike the cureoriented goal of oncology , providers believed that hf , as a progressive , incurable disease , poses challenges to integrating palliative care into standard hf management : [ a]ll medical therapy for hf is really to relieve their symptoms . ( primary care physician ) to improve familiarity and acceptance , providers thought that palliative care must demonstrate and market its benefit to patients and providers . trust and rapport were identified as key facilitators to palliative care referral , especially because palliative care knowledge is limited . but it 's also perfectly fine for them to duck that conversation and say , ( palliative care physician ) a major barrier to palliative care referral was lack of knowledge within the healthcare community ; overcoming this barrier requires intervention directed at providers during graduate or postgraduate training: [e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) finally , providers discussed various practical strategies to encourage palliative care referral . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) although all providers reported that they could define palliative care , probing revealed variation across cardiology and primary care providers . specifically , nearly all primary care and cardiology providers lacked clarity that palliative care is not prognosis dependent and may be administered concurrently with lifeprolonging therapy . many cardiology and primary care providers failed to recognize specialist palliative care as a tangible clinical service . for example , a primary care physician said : palliative care is a philosophy . when asked to describe eligibility and appropriateness criteria for palliative care ( for which there are none , aside from patient need ) , cardiology and primary care providers used the terms hospice and palliative care interviewer : so in your opinion , what makes a hf patient eligible for palliative care?cardiologist : i mean , that 's a legislative decision.interviewer:and , so in your mind , is there a distinction between palliative care and hospice care?cardiologist : no . incorrectly believing that palliative care mandates suspension of lifeprolonging therapies , cardiology and primary care providers reported that patient goals determine eligibility : if the patient wants to focus on symptoms and is willing to accept the sort of basics ( primary care physician ) if the patient wants to focus on symptoms and is willing to accept the sort of basics then the patient 's eligible for palliative care . for example : i think i 'm reasonably familiar [ with palliative care ] , but i also think that to me , it 's become somewhat of a confusing term i worry that sometimes we confuse palliative care , meaning it has to equal endoflife care . ( primary care physician ) most nonpalliative care providers lacked experience with specialist palliative care providers / teams . they frequently acknowledged not knowing how to access palliative care ; a primary care physician from a large academic medical center said : i do n't even know where they are or where they exist , or even really what they do i would n't even know where to start to try and get in touch with someone . among primary and palliative care providers , repeated hospitalizations over a short interval ( eg , 3 in 6 months ) suggested that palliative care might be appropriate . meaning that they were n't candidates for advanced therapies and that there was nothing else i could do to alter the natural history of the disease ( cardiologist ) the unpredictable trajectory of hf was a frequently cited barrier , especially when providers believed that palliative care eligibility or reimbursement were dependent upon prognosis ( as with hospice ) . there 's so much experience with successful management of exacerbations that it 's hard for both the patient and the doctor to say , ( palliative care physician ) providers commonly mentioned the insistence on lifeprolonging treatments as a barrier to palliative care referral . all providers endorsed palliative care in hf and believed that palliative care providers are experts regarding : symptom management ; care coordination ; and facilitating difficult discussions about prognosis , and advance care planning . one primary care physician described , a cultural bias against adopting palliative care unless you know unlike the cureoriented goal of oncology , providers believed that hf , as a progressive , incurable disease , poses challenges to integrating palliative care into standard hf management : [ a]ll medical therapy for hf is really to relieve their symptoms . to improve familiarity and acceptance , providers thought that palliative care must demonstrate and market its benefit to patients and providers . trust and rapport were identified as key facilitators to palliative care referral , especially because palliative care knowledge is limited . nearly all providers felt that primary care or cardiology providers should initiate conversations about palliative care due to their preexisting relationships with patients . but it 's also perfectly fine for them to duck that conversation and say , a major barrier to palliative care referral was lack of knowledge within the healthcare community ; overcoming this barrier requires intervention directed at providers during graduate or postgraduate training: [e]ducating hf physicians on the value and availability and the utilization of palliative care services is key . and then i think the second element is to ramp up the level of palliative care expertise that cardiologists have to exercise in their own practice so that it 's not purely dependent on consultative services ( palliative care physician ) finally , providers discussed various practical strategies to encourage palliative care referral . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) [ palliative care referral ] relies on me asking for it when it probably should be more automated . it 's like if someone has a big tumor on a ct scan , it 's pretty quick how they get into an oncologist or get in for a biopsy but if it 's got a really clear indicator for worsening trajectory , they do n't automatically get [ palliative care ] ( primary care nurse ) this is the first us study to explore barriers to palliative care referral for advanced hf patients among providers frequently caring for these patients . despite the majority of cardiology and primary care providers practicing near specialist palliative care services , we found limited knowledge related to : what nonhospice palliative care is ( especially differences from hospice ) ; what it offers patients , families , and providers ; when it is indicated ; and , how to access it . all cardiology and primary care providers reported familiarity with nonhospice palliative care ; however , phrases such as comfort care or just the basics implied their equating nonhospice and hospice palliative care . notably , because providers almost unanimously used triggers to initiate palliative care , using inappropriate triggers ( eg , active dying ) may result in late referrals ( if any ) . prior work suggests that oncologists are more comfortable and are more likely to refer patients to a service called supportive care as opposed to palliative care . providers from all specialties interviewed frequently reported the unpredictability of hf as a barrier to palliative care referral , as seen elsewhere . unlike the current hospice framework , nonhospice palliative care may present a more flexible alternative for addressing the needs of patients with diseases with volatile trajectories . for example , the prognostic difficulty of hf and the insistence on lifeprolonging treatments were frequently discussed as barriers ; however , they are actually only barriers to hospice referral , not to nonhospice palliative care . all nonpalliative care providers interviewed explicitly expressed interest in exploring how to collaborate with palliative care . prior qualitative research with physicians and nurses , in the united kingdom and australia suggests a mixture of professional , organizational , and cognitive factors impeding palliative care referral for hf patients , including concerns that patients might be stolen by palliative care providers . nevertheless , nonpalliative care providers reported being unsure of how and when to integrate specialist palliative care into standard hf management , the overarching reason cited for low referral . as such such training should strive to : correct misconceptions about palliative care and hospice ( ie , that hospice is a specific subset of palliative care , largely defined by policy ) ; teach providers how to identify palliative needs in their patients ; how to provide primary palliative care themselves when appropriate ; how to determine when specialist palliative care referral or consultation is indicated ; and , how to effectively work with palliative care specialists . in the same vein , a palliative care physician said , we must reach out and give [ cardiology and primary care providers ] the language [ of palliative care ] . additionally , identifying and supporting internal champions within primary care or cardiology to serve as interdisciplinary liaisons with palliative care may be another mechanism by which to enhance meaningful collaboration between specialties . nevertheless , palliative care specialists may serve as valuable resources to correct misconceptions regarding palliative care and hospice among medical professionals , patients , and caregivers . our findings support that the role of specialist palliative care has yet to be articulated for hf patients . several steps pave the way to a clearer understanding of how specialist palliative care can be integrated into hf care . third , whereas some may find our sample of 18 providers too small to provide generalizations to all cardiology , primary care , and palliative care providers , we caution that such conclusions are inappropriate given our qualitative study design . third , whereas some may find our sample of 18 providers too small to provide generalizations to all cardiology , primary care , and palliative care providers , we caution that such conclusions are inappropriate given our qualitative study design . our research suggests that deficits in providers ' knowledge and comfort in discussing palliative care for a difficulttopredict disease present major barriers to referring patients with advanced hf for palliative care . this issue is of high relevance due to the recent acc / aha guidelines which emphasize the potential benefit of integrating palliative care in hf management ; our work provides a glimpse at impediments that pave the road to eventual operationalization of the guidelines . future work should seek to develop provider and patientcentered interventions to reduce actionable barriers to palliative care uptake in hf .

sixty years after dobzhansky ( 1950 ) first described the polytene chromosomes of drosophila willistoni , the large number of chromosomal inversions found in populations of this species still attracts the attention of geneticists . when dobzhansky first published his work , the focus was on evolution from a population genetics viewpoint ( dobzhansky and powell , 1975 ; ehrman and powell , 1982 ; krimbas and powell , 1992 ) . now , d. willistoni is part of a bigger scenario of evolutionary studies on drosophila that began with the publication of the comparative genome sequences of 12 closely related fruit fly species ( drosophila 12 genomes consortium , 2007 ) . ten species were sequenced for the first time ( d. sechellia , d. simulans , d. yakuba , d. erecta , d. ananassae , d. persimilis , d. mojavensis , d. virilis , d. grimshawi and d. willistoni ) and two were genomes of drosophila melanogaster , which had previously been sequenced in 2000 ( adam et al . , 2000 ) , and d. pseudoobscura , published in 2005 ( richards et al . , 2005 ) . with these results , a rich source of genomic data is now available and can be used in comparisons to other important organisms , such as humans , as well as for identifying novel genes and other functional elements in insect genomes . the results of the drosophila 12 genomes consortium ( 2007 ) revealed that , despite the overall similarities , several unexpected differences occur among the species studied , especially in aspects related to ecology and behavior , reflecting adjustments to the vastly different environments in which they evolved . among the sequenced species , drosophila willistoni was the only neotropical one , and proved to be exceptional in several aspects of its genome : its large genome size , unusually skewed codon usage , high content of transposable elements ( 15% , only lower than that of d. ananassae : 25% ) and an apparent lack of selenoproteins ( vicario et al . , 2007 ) . this major genome sequencing and analysis effort has brought about the need to reevaluate the available physical maps , in part to aid the mapping of assembled scaffolds ( schaeffer et al . , 2008 ) . in schaeffer et al . ( 2008 ) we presented a definitive and individual photomap of the polytene chromosomes of drosophila willistoni used to anchor the assembled scaffolds of the drosophila 12 genomes consortium ( 2007 ) . the main results of this study are in agreement with muller s idea that the chromosomes of different drosophila species are syntenic with respect to their genic content , although several rearrangements occurred over time , including inversions and fusions ( muller , 1940 ) . so , as a rule , even when an extreme rearrangement of the chromosome elements has occurred , synteny relationships usually remain relatively conserved ( bhutkar et al . , 2008 ) . drosophila willistoni has a wide distribution , extending from mexico and florida to southern brazil , argentina and uruguay , and from the atlantic to the pacific ocean ( spassky et al . , 1971 ; the chromosomes of natural populations of d. willistoni are characterized by multiple chromosome inversions ( sperlich and pfriem , 1986 ; krimbas and powell , 1992 ; powell , 1997 ) and in more than three decades working with this organism , we have never collected stable monomorphic populations in the field . this is the case for the southern borders of its geographical distribution , such as uruguay , where the natural populations of d. willistoni still have a high level of polymorphism ( valente et al . , 2001 , 2003 ) . this said , the vast chromosomal polymorphism for inversions is one of the paramount characteristics of this species ( burla et al . , 1949 ; da cunha et al . , 1950 , 1959 ; townsend , 1952 ; da cunha and dobzhansky , 1954 ; valente and arajo , 1985 , 1986 ; valente et al . , 1993 ; 2003 ; rohde et al . , 2005 ) , and it appears that being chromosomally polymorphic is the rule for d. willistoni . faced with this , it was a special challenge to choose one recent collected isofemale line of drosophila willistoni to represent the species in the genome sequencing project ( drosophila 12 genomes consortium , 2007 ) . gd - h4 - 1 was finally chosen for this purpose due to its lack of inversion heterozygosity . the strain is the result of various laboratory generations of single brother - sister mating descending from an isofemale line collected in the caribbean island of guadeloupe . in common with several other species that belong to the sophophora subgenus of drosophila it is hard to explain why there are more inversions in the subgenus sophophora than in drosophila as a whole , but according to sperlich and pfriem ( 1986 ) this may be due to several factors , including genetics ( more fragile chromosomes ) , ecology ( these flies inhabit diversified environments ) and history ( phylogenetically old species may accumulate more inversions ) . literature reports are unclear regarding the real number of variants segregating in natural populations of d. willistoni . the criteria available for descriptions of polytene chromosome inversions are somewhat confusing , not only due to the more fragile structural nature of the chromosomes , but also due to imprecise descriptions of inversion breakpoints . the camera lucida drawings made by dobzhansky ( 1950 ) , da cunha and dobzhansky ( 1954 ) and da cunha et al . ( 1950 , 1959 ) show large numbers of inversions present in the same chromosome , making the identification of each particular inversion and respective breakpoints very difficult . this difficulty in accurately identifying inversions is also a consequence of the small size of several d. willistoni inversions , which hinders the recognition of several of these in homozygosis . furthermore , the chromosomes of d. willistoni also present several ectopic pairings that make accurate analyses difficult . as a consequence , the estimates available for this high polymorphism in the literature consider only the arrangements in heterozygosis ( burla et al . , 1949 ; da cunha et al . , 1950 ; townsend , 1952 ; da cunha and dobzhansky , 1954 ; dobzhansky , 1957 ; valente and arajo , 1985 ; 1986 ; valente et al . , 1993 ; 2003 ) , resulting in an underestimation of the true number of variants segregating in the natural populations of d. willistoni . over the last three decades our group has worked towards producing a good photomap of d. willistoni polytene chromosomes . at first , valente and morales ( 1985 ) redrew the reference map of dobzhansky ( 1950 ) , improving the identification of landmark bands that are useful to understand complex inversion configurations . ( 1996 ) presented the first photomap of d. willistoni polytene chromosomes , considering the arrangements present in natural populations of southern brazil as a base to map insertion sites of the p transposable element . the value of this photomap , however , has proven to be restricted to populations inhabiting the southern reaches of the american continent . dissertation , federal university of rio grande do sul , porto alegre ) , taking into consideration 22 american populations from florida to uruguay , concluded that different standard arrangements of the x chromosome occur along the geographical distribution of the species . herein we revised all these descriptions of inversions found in the salivary gland chromosomes , presenting new results in an attempt to systematize the knowledge of the d. willistoni chromosomal polymorphism based on 30 neotropical populations . this approach led to the detection of 50 different arrangements in all five chromosomal arms and the description of different geographical patterns of chromosome evolution in autosomes and the sex chromosome ( x ) . we studied 30 drosophila willistoni populations collected at different times and from several areas ( table 1 ) of the neotropical distribution range of this species ( spassky et al . , 1971 ) . the flies , maintained in the laboratory by mass crosses or as isofemale lines , were reared on corn flour culture medium ( marques et al . , 1966 ) and kept at 17 1 c and 60% relative humidity . three populations ( flo , wip and pne ) were old stocks from ufrgs department of genetics . besides flo , two others ( mon and gua ) were also obtained from the stock center at tucson / arizona and represent inbred lines originally collected from central america islands . all five stocks had been maintained for many years in the laboratory and thus a substantial reduction in genetic variability was to be expected . the other 25 populations were recently collected by us or kindly sent in by collaborators ( table 1 ) . squash mounts of polytene chromosomes present in salivary gland cells of third instar larvae were made using a modification of the technique described by ashburner ( 1967 ) , fixed with acetic acid ( 45% ) , transferred to a drop of acetic acid , lactic acid and water ( 3:2:1 ) and stained with acetic orcein ( 2% ) . for each larvae , inversion polymorphism characterization was made by photomicrographic analysis of the five long chromosomal arms ( xl , xr , iil , iir and iii ) of the chromosome set using a zeiss photo - microscope and phase contrast at 1000x magnification . the photomicrographs were edited using adobe photoshop 5.0 and coreldraw x3 . the usual procedure adopted to study chromosomal inversions in drosophila ( analysis of the chromosomes of larvae produced by crossing males of unknown karyotype with homokaryotypic females of known karyotype ) was not employed due to the virtual absence of homokaryotypic viable stocks of d. willistoni . chromosomal polymorphism of d. willistoni was studied according to the reference photomap published in schaeffer et al . this photomap is being presented here in only one standard picture , with all chromosomal arms together ( supplementary material , figure s1 ) . both heterozygous and homozygous for the calculation of inversion frequencies we considered each inversion in the heterozygous state once , and each inversion in the homozygous state ( representing each chromosome homologue ) twice . the similarity index was calculated based on the number and frequency of paracentric inversions among 30 geographical populations . the resulting dendrogram was generated with past version 2.04 ( hammer et al . , 2001 ) and the morisita index ( morisita , 1959 ) . figures 1 to 5 present the configuration of different arrangements of xl , xr , iil , iir and iii chromosome of d. willistoni , with their own inversions . inversions iil - d and iil - e are difficult to tell apart , and for this reason , are grouped for frequency analysis in table 2 . all the photomaps and photomicrographs ( figures 1 to 5 ) are oriented the same way , with the proximal ( centromeric ) regions of the chromosomes to the left and the distal ( free ) regions to the right . the distal regions of the chromosomes are usually well polytenized and allow the different chromosomes to be recognized . since there are different fixed configurations for the xl and xr chromosomal arms , we use capital letters to distinguish between them , like xl - a or xr - a , both presented in the standard photo - map of the species ( figure s1 ) . in total , we observed at least 10 different chromosomal arrangements segregating on the xl arm and eight on the xr arm . two xl arrangements , xl - c and xl - h , were found only in the homozygous state ( data not shown ) and two inversions ( xl - j and xl - i , figure 1 ) were only found by regner and valente ( unpublished data ) for other southern brazilian populations . ( 2005 ) reported the unique pericentric inversion found until now in d. willistoni ( xl / xr - p ) , both in homo and heterozygous states . in the autosomes , we were able to detect 10 different arrangements on the iil arm , eight on the iir arm and 13 on the iii chromosome . as shown in figures 1 to 5 , the precise identification of each breakpoint of the 50 different arrangements was possible , or facilitated , because many inversions were detected in the homozygous state . although heterozygous inversions are easily detected in cytological preparations the breakpoints are very often concealed above the loop , especially in very small inversions that are common in d. willistoni . this is probably one of the reasons why there has been , until now , a virtual absence in the literature of descriptions of homozygous inversions in d. willistoni . figures 1 and 2 also present an estimated evolutionary relationship between the arrangements of xl and those of xr . for example , the inversions ( in ) ( xl)f1+f2 occurred over the fixed inversion ( xl)d , which in turn occurred over the xl - a standard . one possible evolutionary relationship ( that will be presented in detail in another paper , with descriptions and comparisons of their homozygous forms ) is that different arrangements of xl and also of xr can have occurred and were selected along the wide geographic distribution of d. willistoni . we propose that xl - a is the ancestral configuration , and xl - d , xl - e and xl - b are descendant forms with their own inversions , respectively : in(xl)f1+f2 , in(xl)b , g , and in(xl)j , k , i . as shown in table 2 , the inversion frequency of all arrangements of xl , and also xr segregate in different frequencies with a clear latitudinal cline , from north to south america . compared to the xr chromosomal arm , the xl one presents a more fixed arrangement ( 4 to 3 ) among populations . on the other hand , all the autosomes ( iil , iir and iii ) have a basic arrangement , within which may occur different inversions , but with a clear latitudinal cline of the respect frequencies , similar to what was seen for the x chromosome . by combining all inversion frequencies , a dendrogram was generated ( figure 6 ) showing that geographically proximate populations are much more closely related . in the present study , we tried to catalog the chromosomal inversions occurring in 30 drosophila willistoni populations representative of almost its entire geographical distribution and to draw a general picture of the chromosomal inversion polymorphisms for this organism . we were able to analyze the chromosomes of 615 d. willistoni individuals , most of them being isofemale lines from natural populations . we found at least 50 different gene arrangements for various parts of the distribution range of the species , with evidence suggesting that northern and southern groups emerged gradually . with respect to the chromosomal arms xl and xl , our data are comparable with those reported by levitan ( 1992 ) , reviewing data of d. robusta and including his findings and those of carson and stalker ( 1947 ) . the existence of inversion clines , with a tendency to lose heterokaryotypes toward the periphery of the species distribution ( carson , 1959 ) was partially found in drosophila willistoni , especially with respect to the autosomes ( valente et al . , 2003 ) . our data on d. willistoni inversions from uruguayan populations , however , are not indicative of less polymorphism in the marginal populations in the extreme south of the distribution of this species ( valente et al . , 2003 ) . chromosomal variation found in d. willistoni was mainly due to inversions and , for the first time , homozygous inversions were identified and included in the chromosomal polymorphism analysis . this was due to careful maintenance of isofemale lines , which allowed us to obtain chromosomal preparations of high resolution quality . the precise analysis of each inversion was done through the use of several different photomicrographs representing all possible configurations . this was also done for homozygous chromosomal arms . due to the large number of inversions and different types of combination of arrangements , a definitive discussion on their macro - geographic distribution none of the sampled natural population was totally chromosomally monomorphic for all chromosomal arms . with respect to the x chromosome , the 18 central - south brazilian and uruguayan populations ( bra to pir ) mainly presented the xl - b or xl - e , and xr - b or xr - e arrangements . three northern brazilian populations ( zoo , cat and wip ) presented both standard arrangements as fixed : xl - a and xr - a . the other north brazilian populations ( man , tra , par ) and ecu , mex and flo mainly presented the xl - d or xl - f1+f2 , and xr - f or xr - d . finally , two caribbean island populations ( mon and gua ) presented the arrangements xl - a and xr - c as fixed . the xl - a arrangement , considered as the standard arrangement in d. willistoni ( schaeffer et al . , 2008 ) , was found in samples as far away as mex ( close to the north limit of the species range ) and ita ( close to south limit of the species range ) , with frequencies varying between 0.009 and 0.179 , being exclusive to northern populations of brazil ( table 2 ) . with respect to xr - a , found in samples from flo ( in the north limit of the species range ) to rib ( central brazil ) their frequencies ranged from 0.150 to 0.548 , being fixed in six different populations , as presented in table 2 . it is interesting to note in table 2 that in each population analyzed just one or two possible arrangement of the xl or xr chromosomal arms were observed at frequencies above 0.1 ( or 10% ) . wallace ( 1953 a , b ) hypothesized that the simultaneous presence in a population of three successively overlapping inversions creates difficulties in maintaining a co - adaptive system ( i.e. , co - adaptation for the three possible pairs of inversions ) , since crossovers create a linkage of alleles between the two remote members of the triad . our data regarding the arrangements of the x chromosome found that the populations of d. willistoni studied ( except population bra ) are in accordance with wallace s predictions , but krimbas and loukas ( 1980 ) found that there were 46 exceptions ( 11.7% ) to wallace s rule among of a total of 394 cases of polymorphism in one of the chromosomes of a d. subobscura population . the authors suggested that in a chromosome triad , a frequency of 0.0005 ( or 0.05% ) was the minimum possible for any of the three coexisting members . if we accept that this is also true for the x - chromosomes of our d. willistoni populations , then some populations for the xl arm ( par , cip2 , bra , rib , tab , rat , tur and ita2 ) and some for the xr arm ( man , bra , cip2 , rat , isc and tur ) would also be exceptions to wallace s rule . table 2 shows that not just three , but four arrangements exist ( in populations bra , rib and tur ) , produced by successive overlapping inversions , if the frequency limit of the less frequent inversion is raised to 0.001 ( or 0.10% ) . in the autosomes of d. willistoni , we observed regional characteristics regarding inversion frequencies , with some arrangements appearing to be fixed in some populations ( iil - i and iil - f in the wip population and iir - e in the par population , for example ) while other arrangements occurred at very high frequencies ( iil - i in the tra population , iil - f in mex and ecu populations , iir - e in the tra population , iii - w in the tra population and iii - j in tra and pir populations ) . however , it is possible that at least the inversions referred to by da cunha et al . ( 1950 ) as the most common ones ( iil - f , iir - e , iii - j , , one can see that chromosome iii , the most polymorphic among the autosomes , has many inversions that are separated from each other by only a few sections . so it is possible that we may have made mistakes when trying to correlate photomicrographs of this chromosome with the drawings made by previous authors , although we tried to minimize such errors by naming the inversions as they were found and comparing them with the map of valente and morales ( 1985 ) , preserving the original descriptions . from the dendrogram presented in figure 6 , the conclusion is that the analysis of heterozygous and homozygous frequency inversions in d. willistoni chromosomes is a good method for grouping populations . it can be also seen that geographically isolated populations , such as ecu and bra , form two unique clusters , with the bra population equally distant from the two biggest clusters , the southern and northern ones . our results also support the theory that x chromosomes and autosomes are under different selective pressures and shed light on the particular chromosomal phylogeny of the x chromosome of d. willistoni , which will be published in more detail elsewhere . due to the presence of fixed rearrangements in the x and other chromosomes , we do not recommend that only heterozygous inversions be used in inversion polymorphism analyses of this species . unfortunately , it was very difficult to compare our results with other previous publications , since few of them included photomicrographs of the inversions in the heterozygous state or their breakpoints in a photomap . this is the case with two recent interesting chromosomal studies on d. willistoni populations from mexico ( salceda , 2006 , 2010 ) . the author found two frequent arrangements segregating in the xl chromosomal arm ( xl a and d ) plus the standard , and two arrangements in xr ( xl d and e ) plus the standard considering the frequencies of xl and xr presented by the author , it could be suggested that xl st is our xl - f , being an arrangement resulting from two homozygous inversions occurring at the same time , in(xl)f1+f2 ( as presented in figure 1 ) . when both inversions ( f1 and f2 ) occur together in both chromosomal arms xl of the same female ( so , in homozygosis ) they originate the arrangement xl - f , present in our mexican populations at a frequency of 0.875 ( or 87.5% ) ( data not shown ) . st , it s not possible to suggest correlations with our data because we found two xr homozygous arrangements at similar frequencies : xl - a ( 0.357 ) and xl - d ( 0.464 ) . besides xl st , salceda ( 2010 ) found seven more xl inversions , and besides xr due to the presence of paracentric inversions , the rate of genome rearrangement in drosophila is one of the highest among all eukaryotes ( ranz et al . ( 2001 ) estimated that between the drosophila and sophophora subgenera , for example , the evolution rate was 0.91.4 chromosomal inversions fixed per million years , based on the divergence time between the subgenus ( spicer , 1988 ; russo et al . , 1995 ) . ( 2005 ) , selection may favor inversions because they maintain epi - static combinations within the inverted segment ( dobzhansky 1949 ; charlesworth and charlesworth 1973 ; wu and beckenbach , 1983 ; otto and barton , 2001 ) , or selection may favor rearrangements that reorganize genes into clusters of coordinately expressed genes ( roy et al . , 2002 ; once established in populations , chromosomal inversions may play a role in the formation of new species ( noor et al . these aspects made it possible to use the recognition of inversion to reconstruct phylogenies between closely related species , as done by rohde et al . for all these results to be interpreted correctly , one source of error must be accounted for , that is the low resolution of the cytological analysis of populations or species studied . for example , if two subsequent inversions with similar or identical breakpoints are overlooked , then they will produce a chromosome arrangement very similar to the ancestral one and a phylogenetic link could be missed ( ruiz et al . conversely , two different inversions may be mistakenly identified as the same inversion , creating a false phylogenetic link between two unrelated species . all these observations mean that chromosomal polymorphism deserves further studies that could include the detailed description of inversions , like photomicrographs and breakpoint descriptions over a unique photomap , allowing findings to be compared with each other , thus adding to the knowledge about the chromosomal polymorphism of d. willistoni and all related neotropical species . the following online material is available for this article : - figure s1 - reference photomap of drosophila willistoni .

drosophila willistoni ( insecta , diptera ) is considered a paradigm for evolutionary studies . their chromosomes are characterized by multiple paracentric inversions that make it hard to identify and describe chromosomal polymorphisms . in the present report we attempted to systematize the description of all the 50 inversions found in the last three decades , since we have been studying the chromosomes of several individuals of 30 different populations , including the one used in the genome sequencing project ( gd - h4 - 1 ) . we present the photographic register of 11 arrangements in the left arm of the x chromosome ( xl ) , eight in the right arm ( xr ) , 10 in the left arm of chromosome ii ( iil ) , eight in its right arm ( iir ) and 13 in chromosome iii . this information also includes their breakpoints on the reference photomap . a clear geographic difference was detected in xl and xr , with different fixed arrangements depending on the origin of the population studied . through the comparison of all x arrangements it was possible to infer the putative ancestral arrangements , i.e. , those related to all the remaining arrangements through the small number of inversions that occurred in the past , which we will call xl - a and xr - a . in the autosomes ( iil / iir and iii ) , fixed inversions were detected , but most are segregating in different frequencies along the geographical distribution of the d. willistoni populations .

Introduction Materials and Methods Results Discussion Supplementary Material

sixty years after dobzhansky ( 1950 ) first described the polytene chromosomes of drosophila willistoni , the large number of chromosomal inversions found in populations of this species still attracts the attention of geneticists . now , d. willistoni is part of a bigger scenario of evolutionary studies on drosophila that began with the publication of the comparative genome sequences of 12 closely related fruit fly species ( drosophila 12 genomes consortium , 2007 ) . ten species were sequenced for the first time ( d. sechellia , d. simulans , d. yakuba , d. erecta , d. ananassae , d. persimilis , d. mojavensis , d. virilis , d. grimshawi and d. willistoni ) and two were genomes of drosophila melanogaster , which had previously been sequenced in 2000 ( adam et al . the results of the drosophila 12 genomes consortium ( 2007 ) revealed that , despite the overall similarities , several unexpected differences occur among the species studied , especially in aspects related to ecology and behavior , reflecting adjustments to the vastly different environments in which they evolved . among the sequenced species , drosophila willistoni was the only neotropical one , and proved to be exceptional in several aspects of its genome : its large genome size , unusually skewed codon usage , high content of transposable elements ( 15% , only lower than that of d. ananassae : 25% ) and an apparent lack of selenoproteins ( vicario et al . ( 2008 ) we presented a definitive and individual photomap of the polytene chromosomes of drosophila willistoni used to anchor the assembled scaffolds of the drosophila 12 genomes consortium ( 2007 ) . the main results of this study are in agreement with muller s idea that the chromosomes of different drosophila species are syntenic with respect to their genic content , although several rearrangements occurred over time , including inversions and fusions ( muller , 1940 ) . , 1971 ; the chromosomes of natural populations of d. willistoni are characterized by multiple chromosome inversions ( sperlich and pfriem , 1986 ; krimbas and powell , 1992 ; powell , 1997 ) and in more than three decades working with this organism , we have never collected stable monomorphic populations in the field . this is the case for the southern borders of its geographical distribution , such as uruguay , where the natural populations of d. willistoni still have a high level of polymorphism ( valente et al . , 2005 ) , and it appears that being chromosomally polymorphic is the rule for d. willistoni . faced with this , it was a special challenge to choose one recent collected isofemale line of drosophila willistoni to represent the species in the genome sequencing project ( drosophila 12 genomes consortium , 2007 ) . gd - h4 - 1 was finally chosen for this purpose due to its lack of inversion heterozygosity . in common with several other species that belong to the sophophora subgenus of drosophila it is hard to explain why there are more inversions in the subgenus sophophora than in drosophila as a whole , but according to sperlich and pfriem ( 1986 ) this may be due to several factors , including genetics ( more fragile chromosomes ) , ecology ( these flies inhabit diversified environments ) and history ( phylogenetically old species may accumulate more inversions ) . literature reports are unclear regarding the real number of variants segregating in natural populations of d. willistoni . the criteria available for descriptions of polytene chromosome inversions are somewhat confusing , not only due to the more fragile structural nature of the chromosomes , but also due to imprecise descriptions of inversion breakpoints . the camera lucida drawings made by dobzhansky ( 1950 ) , da cunha and dobzhansky ( 1954 ) and da cunha et al . ( 1950 , 1959 ) show large numbers of inversions present in the same chromosome , making the identification of each particular inversion and respective breakpoints very difficult . this difficulty in accurately identifying inversions is also a consequence of the small size of several d. willistoni inversions , which hinders the recognition of several of these in homozygosis . furthermore , the chromosomes of d. willistoni also present several ectopic pairings that make accurate analyses difficult . as a consequence , the estimates available for this high polymorphism in the literature consider only the arrangements in heterozygosis ( burla et al . , 1993 ; 2003 ) , resulting in an underestimation of the true number of variants segregating in the natural populations of d. willistoni . over the last three decades our group has worked towards producing a good photomap of d. willistoni polytene chromosomes . at first , valente and morales ( 1985 ) redrew the reference map of dobzhansky ( 1950 ) , improving the identification of landmark bands that are useful to understand complex inversion configurations . ( 1996 ) presented the first photomap of d. willistoni polytene chromosomes , considering the arrangements present in natural populations of southern brazil as a base to map insertion sites of the p transposable element . dissertation , federal university of rio grande do sul , porto alegre ) , taking into consideration 22 american populations from florida to uruguay , concluded that different standard arrangements of the x chromosome occur along the geographical distribution of the species . herein we revised all these descriptions of inversions found in the salivary gland chromosomes , presenting new results in an attempt to systematize the knowledge of the d. willistoni chromosomal polymorphism based on 30 neotropical populations . this approach led to the detection of 50 different arrangements in all five chromosomal arms and the description of different geographical patterns of chromosome evolution in autosomes and the sex chromosome ( x ) . we studied 30 drosophila willistoni populations collected at different times and from several areas ( table 1 ) of the neotropical distribution range of this species ( spassky et al . the flies , maintained in the laboratory by mass crosses or as isofemale lines , were reared on corn flour culture medium ( marques et al . squash mounts of polytene chromosomes present in salivary gland cells of third instar larvae were made using a modification of the technique described by ashburner ( 1967 ) , fixed with acetic acid ( 45% ) , transferred to a drop of acetic acid , lactic acid and water ( 3:2:1 ) and stained with acetic orcein ( 2% ) . for each larvae , inversion polymorphism characterization was made by photomicrographic analysis of the five long chromosomal arms ( xl , xr , iil , iir and iii ) of the chromosome set using a zeiss photo - microscope and phase contrast at 1000x magnification . the usual procedure adopted to study chromosomal inversions in drosophila ( analysis of the chromosomes of larvae produced by crossing males of unknown karyotype with homokaryotypic females of known karyotype ) was not employed due to the virtual absence of homokaryotypic viable stocks of d. willistoni . chromosomal polymorphism of d. willistoni was studied according to the reference photomap published in schaeffer et al . the similarity index was calculated based on the number and frequency of paracentric inversions among 30 geographical populations . , 2001 ) and the morisita index ( morisita , 1959 ) . figures 1 to 5 present the configuration of different arrangements of xl , xr , iil , iir and iii chromosome of d. willistoni , with their own inversions . all the photomaps and photomicrographs ( figures 1 to 5 ) are oriented the same way , with the proximal ( centromeric ) regions of the chromosomes to the left and the distal ( free ) regions to the right . the distal regions of the chromosomes are usually well polytenized and allow the different chromosomes to be recognized . since there are different fixed configurations for the xl and xr chromosomal arms , we use capital letters to distinguish between them , like xl - a or xr - a , both presented in the standard photo - map of the species ( figure s1 ) . two xl arrangements , xl - c and xl - h , were found only in the homozygous state ( data not shown ) and two inversions ( xl - j and xl - i , figure 1 ) were only found by regner and valente ( unpublished data ) for other southern brazilian populations . ( 2005 ) reported the unique pericentric inversion found until now in d. willistoni ( xl / xr - p ) , both in homo and heterozygous states . in the autosomes , we were able to detect 10 different arrangements on the iil arm , eight on the iir arm and 13 on the iii chromosome . as shown in figures 1 to 5 , the precise identification of each breakpoint of the 50 different arrangements was possible , or facilitated , because many inversions were detected in the homozygous state . although heterozygous inversions are easily detected in cytological preparations the breakpoints are very often concealed above the loop , especially in very small inversions that are common in d. willistoni . this is probably one of the reasons why there has been , until now , a virtual absence in the literature of descriptions of homozygous inversions in d. willistoni . for example , the inversions ( in ) ( xl)f1+f2 occurred over the fixed inversion ( xl)d , which in turn occurred over the xl - a standard . one possible evolutionary relationship ( that will be presented in detail in another paper , with descriptions and comparisons of their homozygous forms ) is that different arrangements of xl and also of xr can have occurred and were selected along the wide geographic distribution of d. willistoni . we propose that xl - a is the ancestral configuration , and xl - d , xl - e and xl - b are descendant forms with their own inversions , respectively : in(xl)f1+f2 , in(xl)b , g , and in(xl)j , k , i . as shown in table 2 , the inversion frequency of all arrangements of xl , and also xr segregate in different frequencies with a clear latitudinal cline , from north to south america . on the other hand , all the autosomes ( iil , iir and iii ) have a basic arrangement , within which may occur different inversions , but with a clear latitudinal cline of the respect frequencies , similar to what was seen for the x chromosome . in the present study , we tried to catalog the chromosomal inversions occurring in 30 drosophila willistoni populations representative of almost its entire geographical distribution and to draw a general picture of the chromosomal inversion polymorphisms for this organism . we were able to analyze the chromosomes of 615 d. willistoni individuals , most of them being isofemale lines from natural populations . we found at least 50 different gene arrangements for various parts of the distribution range of the species , with evidence suggesting that northern and southern groups emerged gradually . with respect to the chromosomal arms xl and xl , our data are comparable with those reported by levitan ( 1992 ) , reviewing data of d. robusta and including his findings and those of carson and stalker ( 1947 ) . the existence of inversion clines , with a tendency to lose heterokaryotypes toward the periphery of the species distribution ( carson , 1959 ) was partially found in drosophila willistoni , especially with respect to the autosomes ( valente et al . our data on d. willistoni inversions from uruguayan populations , however , are not indicative of less polymorphism in the marginal populations in the extreme south of the distribution of this species ( valente et al . chromosomal variation found in d. willistoni was mainly due to inversions and , for the first time , homozygous inversions were identified and included in the chromosomal polymorphism analysis . the precise analysis of each inversion was done through the use of several different photomicrographs representing all possible configurations . due to the large number of inversions and different types of combination of arrangements , a definitive discussion on their macro - geographic distribution none of the sampled natural population was totally chromosomally monomorphic for all chromosomal arms . with respect to the x chromosome , the 18 central - south brazilian and uruguayan populations ( bra to pir ) mainly presented the xl - b or xl - e , and xr - b or xr - e arrangements . three northern brazilian populations ( zoo , cat and wip ) presented both standard arrangements as fixed : xl - a and xr - a . the other north brazilian populations ( man , tra , par ) and ecu , mex and flo mainly presented the xl - d or xl - f1+f2 , and xr - f or xr - d . finally , two caribbean island populations ( mon and gua ) presented the arrangements xl - a and xr - c as fixed . the xl - a arrangement , considered as the standard arrangement in d. willistoni ( schaeffer et al . , 2008 ) , was found in samples as far away as mex ( close to the north limit of the species range ) and ita ( close to south limit of the species range ) , with frequencies varying between 0.009 and 0.179 , being exclusive to northern populations of brazil ( table 2 ) . with respect to xr - a , found in samples from flo ( in the north limit of the species range ) to rib ( central brazil ) their frequencies ranged from 0.150 to 0.548 , being fixed in six different populations , as presented in table 2 . wallace ( 1953 a , b ) hypothesized that the simultaneous presence in a population of three successively overlapping inversions creates difficulties in maintaining a co - adaptive system ( i.e. , co - adaptation for the three possible pairs of inversions ) , since crossovers create a linkage of alleles between the two remote members of the triad . our data regarding the arrangements of the x chromosome found that the populations of d. willistoni studied ( except population bra ) are in accordance with wallace s predictions , but krimbas and loukas ( 1980 ) found that there were 46 exceptions ( 11.7% ) to wallace s rule among of a total of 394 cases of polymorphism in one of the chromosomes of a d. subobscura population . if we accept that this is also true for the x - chromosomes of our d. willistoni populations , then some populations for the xl arm ( par , cip2 , bra , rib , tab , rat , tur and ita2 ) and some for the xr arm ( man , bra , cip2 , rat , isc and tur ) would also be exceptions to wallace s rule . table 2 shows that not just three , but four arrangements exist ( in populations bra , rib and tur ) , produced by successive overlapping inversions , if the frequency limit of the less frequent inversion is raised to 0.001 ( or 0.10% ) . in the autosomes of d. willistoni , we observed regional characteristics regarding inversion frequencies , with some arrangements appearing to be fixed in some populations ( iil - i and iil - f in the wip population and iir - e in the par population , for example ) while other arrangements occurred at very high frequencies ( iil - i in the tra population , iil - f in mex and ecu populations , iir - e in the tra population , iii - w in the tra population and iii - j in tra and pir populations ) . ( 1950 ) as the most common ones ( iil - f , iir - e , iii - j , , one can see that chromosome iii , the most polymorphic among the autosomes , has many inversions that are separated from each other by only a few sections . it can be also seen that geographically isolated populations , such as ecu and bra , form two unique clusters , with the bra population equally distant from the two biggest clusters , the southern and northern ones . our results also support the theory that x chromosomes and autosomes are under different selective pressures and shed light on the particular chromosomal phylogeny of the x chromosome of d. willistoni , which will be published in more detail elsewhere . due to the presence of fixed rearrangements in the x and other chromosomes , we do not recommend that only heterozygous inversions be used in inversion polymorphism analyses of this species . unfortunately , it was very difficult to compare our results with other previous publications , since few of them included photomicrographs of the inversions in the heterozygous state or their breakpoints in a photomap . this is the case with two recent interesting chromosomal studies on d. willistoni populations from mexico ( salceda , 2006 , 2010 ) . the author found two frequent arrangements segregating in the xl chromosomal arm ( xl a and d ) plus the standard , and two arrangements in xr ( xl d and e ) plus the standard considering the frequencies of xl and xr presented by the author , it could be suggested that xl st is our xl - f , being an arrangement resulting from two homozygous inversions occurring at the same time , in(xl)f1+f2 ( as presented in figure 1 ) . when both inversions ( f1 and f2 ) occur together in both chromosomal arms xl of the same female ( so , in homozygosis ) they originate the arrangement xl - f , present in our mexican populations at a frequency of 0.875 ( or 87.5% ) ( data not shown ) . st , it s not possible to suggest correlations with our data because we found two xr homozygous arrangements at similar frequencies : xl - a ( 0.357 ) and xl - d ( 0.464 ) . besides xl st , salceda ( 2010 ) found seven more xl inversions , and besides xr due to the presence of paracentric inversions , the rate of genome rearrangement in drosophila is one of the highest among all eukaryotes ( ranz et al . , 2002 ; once established in populations , chromosomal inversions may play a role in the formation of new species ( noor et al . these aspects made it possible to use the recognition of inversion to reconstruct phylogenies between closely related species , as done by rohde et al . all these observations mean that chromosomal polymorphism deserves further studies that could include the detailed description of inversions , like photomicrographs and breakpoint descriptions over a unique photomap , allowing findings to be compared with each other , thus adding to the knowledge about the chromosomal polymorphism of d. willistoni and all related neotropical species . the following online material is available for this article : - figure s1 - reference photomap of drosophila willistoni .

autophagy is a catabolic process that targets aged or malfunctioning organelles and damaged or misfolded proteins for lysosomal degradation . there are several autophagy - related proteins whose coordinated function is required for conjugation of the autophagy - specific ubiquitin - like proteins ( ubls ) , lc3a , lc3b , and lc3c , and gabarap , gabarap - l1 , and gate-16 to the lipid phosphatidylethanolamine ( pe ) . together with adapter proteins , including p62 and nbr1 , these lipidated ubls help to encapsulate substrates into double - membrane vesicles called autophagosomes which ultimately fuse with lysosomes . at basal levels , this process plays an important role in developing and maintaining cellular homeostasis and genomic integrity . autophagy may also contribute to tumorigenesis by promoting cell survival in response to metabolic or oncogenic stress and to resistance to chemotherapy [ 24 ] . recent studies have shown that the pharmacological and/or genetic inhibition of autophagy can sensitize cancer cells to the lethal effects of various cancer therapies , including chemotherapy , radiotherapy , and targeted therapies [ 5 , 6 ] . these results suggest that inhibition of autophagy may provide a valuable sensitizing strategy for cancer treatments . it is well documented that nutrient starvation results in mtor inhibition and induction of autophagy . as a central regulator of cell growth , mtor plays a key role at the interface of the pathway that regulates the balance between cell growth and autophagy in response to nutritional states , growth factors , and stress signals . hypoxia is also known to induce autophagy in highly proliferating tumor cells and the adverse effects of hypoxia to chemotherapy treatment have been well documented [ 3 , 9 , 10 ] . the recent finding that hypoxia results in mtor pathway inhibition and autophagy upregulation may contribute to this self - protective mechanism in certain cancers . lipid conjugation of the autophagy ubls is essential for autophagosome formation and is mediated by the e1 activating enzyme atg7 along with the e2 conjugating enzyme atg3 and an e3 ligase complex consisting of atg5atg12/atg16 . atg7 knockdown reduces autophagy ubl lipidation and slows down basal constitutive autophagy process resulting in accumulation of the autophagy adapter proteins p62 and nbr1 . these proteins , which function to deliver polyubiquitinated , misfolded , or aggregated proteins and dysfunctional organelles to autophagosomes , are also themselves autophagy substrates [ 1315 ] . thus , monitoring the cellular levels and localization of the autophagy ubls and adapter proteins can be used as a measure for autophagy activation or inhibition . methods to study autophagy regulation in cells in vitro have often employed detection of gfp - labeled autophagy proteins such as lc3b or electron microscopy to observe double membrane - bound autophagosomal structures . however , these approaches are not ideal for monitoring autophagy regulation in an in vivo drug discovery setting . an alternative approach such as ihc would be valuable for assessing autophagy regulation in tissues in clinically relevant settings . unfortunately , significant technical challenges exist , as conventional polymer based ihc methods are largely ineffective for detecting autophagy - specific markers due to the low abundance and transient nature of autophagosomes [ 1620 ] . to address these issues , tyramide signal amplification ( tsa ) technology was applied to the autophagy markers lc3b and nbr1 , which resulted in a more robust and measurable signal as compared to the conventional polymer ihc platform . tsa technology is based on the catalyzed reporter deposition ( card ) principle . using the peroxidase activity of an hrp - conjugated secondary antibody , the label could be a chromophore , a hapten , or an enzyme such as hrp , and the signals are then detected accordingly . in the scheme used in the present study , hq ( a hapten ) was attached to tyramide and later detected using an hrp - conjugated anti - hq antibody . using this methodology , autophagy - related markers were monitored under conditions of autophagy induction and inhibition in calu-6 and hct116 xenograft tumor models . mln0128 is a potent mtor kinase inhibitor that induces autophagy by inhibiting the mtor pathway . in contrast , xenograft tumors in which atg7 was knocked down using shrna have reduced levels of ubl lipidation and impaired autophagy . using these methods to manipulate autophagy , robust changes were observed in both lc3b and nbr1 levels in a time - dependent manner using the sensitive tsa - mediated detection method . this enabled the measurement of dynamic changes in autophagy regulated proteins without the use of engineered artificial protein labeling . using this method , the correlation of different markers in a multiplex format was examined and the relationship between basal levels of hypoxia and autophagy in tumor development was monitored . the methodology used herein should provide a valuable approach to studying autophagy regulation in various disease settings . atg7 shrna , trcn0000007587 ( sigma - aldrich ) , was cloned into the doxycycline - inducible ptripz lentiviral vector ( open biosystem rhs4750 ) . lentivirus was packaged in 293 t cells using the mission lentiviral packaging mix ( sigma - aldrich shp001 ) and viral supernatants were collected at 48 and 72 hours after transfection and used to transduce hct116 cells in the presence of 8 g / ml polybrene ( sigma - aldrich 107689 ) for 72 hours . the cells were then selected for 8 days in the presence of 1 g / ml puromycin ( invitrogen a1113803 ) . doxycycline ( sigma - aldrich d9891 ) induced atg7 knockdown in the atg7 shrna3.3 clone was confirmed by western blot using anti - atg7 antibody ( epitomics , 2054 - 1 ) . scramble control line f6 was generated using the nonsilencing control shrna in ptripz vector ( open biosystem rhs4743 ) alongside with the same process . hct116 cell lines with doxycycline - inducible atg7-shrna ( clone 3.3 ) were propagated in the cell culture medium , mccoy 's 5a , with 10% fetal bovine serum ( fbs ) . cells were grown and expanded for the inoculation of 2 10 cells in 0.1 ml per mouse . using a 26 3/8 gauge needle , 0.1 ml cell suspension in media - free pbs was subcutaneously inoculated into the right dorsal flank area of the female ncr nude mice . when the tumors reached approximately 200 mm , by caliper measurements ( typically around 10 days after inoculation ) , the mice were randomized to receive food with or without doxycycline ( 0.0625 mg / kg ) for 7 , 14 , or 21 days and then sacrificed . calu-6 human lung cancer cells were propagated in cell culture medium mem with the addition of 10% fbs . cells were grown and expanded for inoculation of 5 10 cells in 0.1 ml per mouse . using a 26 3/8 gauge needle , 0.1 ml cell suspension in media - free pbs solution was subcutaneously inoculated into the right dorsal flank area of female nude mice . approximately 3 weeks after inoculation , when the tumor size reached 500800 mm , animals were randomized into treatment groups . all animals received a 0.2 ml single oral dose of mln0128 ( 5.0 mg / kg ) . at the specific time point ( 30 minutes , 1 , 2 , 4 , 8 , and 24 hours ) after dose , the animals were sacrificed . tumors were collected and fixed in 10% neutral buffered formalin ( nbf ) before processing for histology studies . tumor tissues were processed in a covaris e200 tissue homogenizer in accordance with the manufacturer 's instructions . ripa lysis buffer ( millipore ) with 1x protease inhibitor cocktail ( calbiochem ) , 2 mmol / l sodium orthovanadate ( sigma - aldrich ) , 25 mmol / l sodium fluoride , and 25 mmol / l -glycerophosphate was used . cold lysis buffer ( 300 to 800 l ) was added to each tumor sample followed by sonication . the supernatants were transferred to new tubes and protein concentrations determined by spectramax plus 384 ( molecular devices ) . for western blot analysis , tumor lysates ( 20 g protein ) were loaded onto 4% to 12% gradient bis - tris gels ( invitrogen ) and subjected to electrophoresis . separated proteins were transferred to pvdf - fl membranes ( millipore ) using a semidry transfer apparatus . the membranes were blocked with li - cor blocking buffer ( li - cor biosciences , 927 - 40000 ) and then incubated with primary antibody in li - cor blocking buffer plus 0.1% tween-20 ( sigma - aldrich , p 1397 ) overnight at 4c . after three washes with tbst , the membranes were incubated with alexa fluor 680-labeled goat anti - rabbit igg ( life technology ) in li - cor blocking buffer plus 0.1% tween-20 for 1 hour . while protected from light , the membranes were washed five times with tbst , once with tbs , and then dried . the membranes were scanned with the odyssey infrared imaging system ( li - cor biosciences ) and the band intensities were quantified with odyssey software . the following primary antibodies were used : monoclonal rabbit anti - lc3b ( cell signaling technology , 3868 , 412 g / ml , 1 : 1000 ) ; polyclonal rabbit anti - sqstm1/p62 antibody ( abcam , 91526 , 1 g / ml , 1 : 1000 ) ; and polyclonal rabbit anti - atg7 ( epitomics , 2054 - 1 , 1 : 1000 ) . tumor tissues were processed in a covaris e200 tissue homogenizer in accordance with the manufacturer 's instructions . heavy membrane lysis buffer ( 250 mm mannitol , 70 mm sucrose , 0.5 mm egta , and 5 mm hepes - naoh , ph 7.2 ) with 1x protease inhibitor cocktail ( calbiochem ) , 2 mmol / l sodium orthovanadate ( sigma - aldrich ) , 25 mmol / l sodium fluoride , and 25 mmol / l -glycerophosphate ( sigma - aldrich ) was used . cold heavy membrane lysis buffer ( 1000 l ) was added to each tumor sample followed by sonication . lysates were transferred to eppendorf tubes and centrifuged at 3,000 rpm in eppendorf centrifuge 5417r at 4 for 10 minutes , and supernatants were transferred to new eppendorf tubes . after taking 250 l of supernatant and saving as total lysate l ( tl1 ) , the remaining supernatants were centrifuged at 14,000 rpm at 4 for 15 minutes . the hmf pellets were lysed in 150 l of ripa lysis buffer with the previously mentioned inhibitors and kept on ice for 20 minutes . all xenograft tumor samples were fixed in 10% nbf for a minimum of 16 hours before being dehydrated and embedded in paraffin ( ffpe ) . for ihc evaluations , 5 m sections of ffpe tissue were mounted onto glass slides , dried overnight at room temperature , and baked at 60c for 1 hour prior to use . tumor sections were processed by automated staining using either the discovery xt ( ventana systems ) or the bond rx ( leica biosystems ) autostainers , following preset protocols . in brief , the sections were subjected to ethylenediaminetetraacetic acid ( edta ) based antigen retrieval for 20 minutes and protein block ( dako , ds9390 ) for 12 minutes before incubation with primary antibody for 1 hour at room temperature . the following primary antibodies were used : polyclonal rabbit anti - carbonic anhydrase ix ( caix ) antibody ( novus biologicals , nb100 - 417 , 1 : 2500 ) ; polyclonal rabbit anti - sqstm1/p62 antibody ( abcam , 91526 , 1 : 5000 ) ; polyclonal rabbit anti - atg7 ( epitomics , 2054 - 1 , 1 : 800 ) ; monoclonal rabbit anti - ps6 ( cell signaling technology , 5364 , 1 : 2500 ) ; monoclonal rabbit anti - lc3b ( cell signaling technology , 3868 , 1 : 50 ) ; and monoclonal rabbit anti - nbr1 ( inhouse antibody , 1 : 50 ; validation of ml85 - 61 - 3 data not shown ) . 3,3-diaminobenzidine dab chromogenic detection kits , omnimap hrp multimer kit ( ventana systems , 760 - 159 ) or bond polymer refine detection kit ( leica biosystems , 9800 dab and ds9390 red ) , were used following the manufacturer 's instructions . colocalization studies of caix and ps6 used sequential standard ihc detection protocols on bond rx ( leica biosystems ) with both bond polymer refine detection dab and red kits . for detecting low abundance antigens lc3b and nbr1 , discovery amp hq kit ( ventana systems , 760 - 052 ) , a tyramide signal amplification detection kit , was used . ffpe sections were dried overnight at room temperature and then baked at 60c for 1 hour prior to loading onto discovery xt autostainer ( ventana systems ) . the sections were subjected to edta - based cc1 mild antigen retrieval for 20 minutes and protein block ( dako , ds9390 ) for 12 minutes before incubation with primary antibody for 1 hour at room temperature . the lc3b primary antibody ( cell signaling technology , 3868 , 412 g / ml , 1 : 400 ) and nbr1 ( takeda pharmaceuticals inc . , rabbit monoclonal antibody , 1.7 mg / ml , 1 : 2000 ) were used for this study . following primary antibody incubation , the amp hq kit in conjunction with ( 1 ) anti - hq multimer , ( 2 ) omnimap hrp multimer , and ( 3 ) chromogenic detection kit was used following the manufacturer 's instructions ( ventana systems ) . the ihc - stained slides were scanned by aperio scanscopext ( leica biosystems ) using the 20x objective , and the digital images were stored in eslide manager ( leica biosystems ) database . aperio genie ( leica biosystems ) , a pattern recognition tool , was used to select viable areas within the tumor . for caix - lc3b analysis , caix - positive and caix - negative regions were manually annotated and transferred to the lc3b image for subsequent image analysis . whole slide image analysis was performed using positive pixel count algorithm where the area of positive signal was measured and normalized to the corresponding total viable tumor area . one - way anova was used for statistical analysis with a significance level set at p < 0.05 . the use of standard ihc methods for detecting and quantifying autophagy - specific biomarkers has been limited due to the low abundance and transient nature of autophagosomes . with standard ihc methods , signal is typically amplified with antispecies multimer complexes containing multiple enzyme molecules . ffpe calu-6 xenograft tumors stained for lc3b and nbr1 with standard ihc methods showed diffuse brown staining in the cytoplasm with very fine , light staining puncta structures ( figures 1(a ) and 1(c ) ) . in contrast , amp hq uses a tyramide substrate - based amplification where in the presence of hydrogen peroxide , and immobilized hrp converts tyramide substrate into a reactive intermediate that covalently binds to electron rich regions of proteins immediately adjacent to the activating hrp enzyme . subsequent detection of the hq substrate yields large amplification of signal . using the amp hq method on calu-6 xenograft sections , both lc3b and nbr1 were clearly detected as dark - staining puncta structures in the cytoplasm of viable tumor ( figures 1(b ) and 1(d ) ) . to verify the amp hq assays were capable of measuring specific changes to lc3b and nbr1 levels under conditions of autophagy induction and inhibition , the autophagy pathway was manipulated in vivo using the investigational mtor inhibitor , mln0128 , to activate autophagy and using doxycycline - inducible atg7shrna knockdown to downregulate autophagy . calu-6 xenograft tumors were harvested from female nude mice following a single oral 5 mg / kg dose of mln0128 and examined for levels of the mtor pathway marker , ps6 , using standard ihc and the autophagy marker lc3b using amp hq ihc . within one hour of dosing , the level of ps6 was dramatically decreased ( figures 2(a ) and 2(b ) ) indicating mtor pathway inhibition . quantification of ps6 with positive pixel analysis revealed a greater than 4-fold decrease ( p = 0.038 ) in the mln0128-treated tumor compared to the vehicle only control ( figure 2(b ) ) . also , a rapid increase in lc3b puncta in the cytoplasm of tumor cells from treated animals was observed(figures 2(a ) and 2(c ) ) , indicating autophagy pathway induction . quantification of lc3b puncta by positive pixel analysis revealed early and transient changes in signal levels at different time points ( figure 2(c ) ) . the difference of the mean values of lc3b level between the animals treated with mln0128 for 30 minutes and the vehicle group was statistically significant ( p = 0.029 ) . consistent with the lc3b amp hq result , western blot analysis of the hmf from the same tumor tissue also showed the lipidated lc3b increased after mln0128 administration ( figure 2(d ) ) . a central core of necrosis and areas of hypoxia are commonly observed in highly proliferating xenograft tumors . to investigate the relationship of hypoxia conditions with autophagy and mtor pathway activity , calu-6 tumors were analyzed for caix expression to annotate tumor hypoxic and normoxic regions and for levels of ps6 and lc3b to measure mtor and lc3b pathway activities , respectively , using a dual ihc stain on a single slide . a high level of the hypoxia - induced protein caix expression was observed adjacent to the tumor necrotic areas indicating that necrosis in xenograft tumors was likely induced by hypoxia ( figure 3 ) . interestingly , with caix and ps6 double labeling , a dramatic decrease in ps6 signal in the strong caix staining hypoxic region was observed ( figure 3(b ) ) , suggesting reduced mtor signaling due to hypoxia . to assess autophagy activity in the context of hypoxia , sections of calu-6 xenograft tumor were stained for caix and manually annotated as caix positive ( hypoxic ) and caix negative ( normoxic ) . annotated regions were subsequently transposed onto serial slides stained for lc3b ( figure 3(c ) ) . the lc3b puncta in the caix - positive and caix - negative regions were measured using positive pixel image analysis . the areas of viable tumor with caix - positive staining had higher levels of lc3b puncta than caix - negative regions . the data showed a significant difference between the hypoxic region and the normoxic region ( figure 3(d ) ) , when the two values were expressed as the mean sd ( p = 0.012 ) . atg7 knockdown has been shown to impair formation of autophagosomes , the double - membrane vesicles which are responsible for delivering cytoplasm material to lysosomes . a standard ihc with anti - atg7 antibody staining was performed on the xenograft tumor collected at days 3 , 7 , and 14 after treatment with or without doxycycline . ihc images showed a dramatically reduced atg7 protein expression in day 7 doxycycline - treated samples ( figure 4(a ) ) . positive pixel image analysis demonstrated a significant decreased expression of atg7 in the doxycycline - treated tumors ( p < 0.001 ) ( figure 4(b ) ) . further evaluation by western blot analysis also showed decreased atg7 protein levels with the doxycycline - treated group ( figure 4(f ) ) . the same set of samples was analyzed using the amp hq method to detect lc3b puncta . as expected , doxycycline - induced atg7 knockdown led to reduced lc3b expression ( figure 4(a ) ) . positive pixel algorithm analysis showed a greatly decreased lc3b expression in doxycycline - treated groups at 7 and 14 days ( p = 0.030 ) ( figure 4(c ) ) . as a result of atg7 knockdown and impaired formation of autophagosomes , the accumulation of the adapter proteins p62 and positive pixel algorithm analysis demonstrates a greatly increased p62 and nbr1 signal in doxycycline - treated groups on days 7 and 14 ( figures 4(d ) and 4(e ) ) . the western blot analysis is consistent with the ihc results and also showed an increasing intensity of p62 on days 7 and 14 ( figure 4(f ) ) . a panel of xenograft tumors was analyzed for lc3b and nbr1 using the amp hq method , as a way to assess the variability of basal autophagy activity across various tumor types . the data showed a wide range of basal lc3b and nbr1 signals across the various tumor models . u87 mg ( brain ) and skov-3 ( ovary ) xenograft tumor models showed a high level signal of lc3b and low level of nbr1 . ht-29 ( colon ) , mda - md-361 ( breast ) , mm1.s ( myeloma ) , and hct116 ( colon ) xenograft tumor models showed a high level signal of nbr1 and low level of lc3b . oci - ly10 ( lymphoma ) and mda - mb-231 ( breast ) xenograft tumor models showed a good correlation between the lc3b and the nbr1 signals . xenograft tumor models , hl60 ( leukemia ) , a549 ( lung ) , and thp-1 ( leukemia ) , showed a very low signal for both markers ( figures 5(a ) and 5(b ) ) . autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized . during starvation , the nutrient - responsive kinase mtor is inhibited and a cell survival mechanism is upregulated that includes activating autophagy to reuse internal resources [ 7 , 23 , 24 ] . using mln0128 to inhibit mtor activity and mimic starvation - induced autophagy , an increase was observed in lc3b puncta levels using tyramide - based signal amplification technology . the increased signal of the tsa method enabled the quantitative analysis of lc3b expression by an automated image analysis method . this was not possible with the traditional ihc method because of low specific signal and high background staining with the standard ihc . a downstream marker of the mtor pathway , ps6 , was used to monitor inhibition of mtor following mln0128 treatment . these data show a considerable decrease in ps6 levels starting as early as 0.5 hours after treatment . in response to mtor inhibition , autophagy was activated and soluble lc3 ( lc3b - i ) was conjugated to form lc3-pe ( lc3b - ii ) which participates in the formation of autophagosomes . detecting lc3b by immunoblotting or immunofluorescence in addition to direct mtor inhibition , hypoxic conditions in the tumor microenvironment are also capable of rapidly inducing autophagy via induction of the hypoxia - inducible factor-1 ( hif-1 ) leading to mtor inhibition [ 9 , 10 ] . it is common to observe a central core of necrosis in a rapidly growing xenograft tumor where overexpression of carbonic anhydrase ix ( caix ) has been detected in hypoxic regions of tumors and the caix ihc assay serves as a biomarker of hypoxia in many solid tumors . to understand the relationship between hypoxia and mtor and autophagy pathway regulation , a colocalization analysis of caix with ps6 and lc3b was performed . in this study , caix - positive staining was observed in viable tumor adjacent to the tumor necrotic regions . a colocalization study with ps6 showed low ps6 levels in the same area that was positive for caix , demonstrating an inverse correlation between hypoxia and mtor activity . further investigation using caix costaining with lc3b showed that there was higher level of lc3b in the caix - positive region , indicating hypoxia - induced autophagy in the hypoxic regions of the xenograft tumor . a doxycycline - inducible atg7shrna model was used to study the dynamic range of the amp hq lc3b assay . autophagy ubl conjugation is a key feature of autophagosome formation and is mediated by the e1 enzyme atg7 . in this study , the intensity of atg7 expression was drastically diminished in response to atg7shrna expression . as a result of atg7 knockdown , a significant decrease in the number and size of the lc3b puncta this was accompanied by an increase in the level of the adaptor proteins nbr1 and p62 . p62 is a multifunction protein and , in the autophagy pathway , plays a role as a cargo receptor or adapter protein for degradation of ubiquitinated substrates . a direct interaction between the adapter proteins p62 and nbr1 and the autophagosomal protein lc3 results in aggregates which can be cleared through autophagy [ 14 , 15 ] . knocking down atg7 caused impaired autophagy progression and stabilization of p62-positive bodies containing polyubiquitinated proteins . using standard ihc and tsa methods , the accumulation of p62 and nbr1 , respectively , was clearly detected . this data also showed a large dynamic range in lc3b and nbr1 levels in a panel of xenograft tumors , suggesting that the two markers were useful tools to support the intertumor assessment of autophagy pathway status . in conclusion , with this tsa technology , autophagy markers can be monitored in xenograft tumors which will be helpful in understanding the mechanism of autophagy regulation in tumor development .

objective . use of tyramide signal amplification ( tsa ) to detect autophagy biomarkers in formalin fixed and paraffin embedded ( ffpe ) xenograft tissue . materials and methods . autophagy marker regulation was studied in xenograft tissues using amp hq ihc and standard ihc methods . results . the data demonstrate the feasibility of using high sensitivity tsa ihc assays to measure low abundant autophagy markers in ffpe xenograft tissue .

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

autophagy is a catabolic process that targets aged or malfunctioning organelles and damaged or misfolded proteins for lysosomal degradation . together with adapter proteins , including p62 and nbr1 , these lipidated ubls help to encapsulate substrates into double - membrane vesicles called autophagosomes which ultimately fuse with lysosomes . it is well documented that nutrient starvation results in mtor inhibition and induction of autophagy . hypoxia is also known to induce autophagy in highly proliferating tumor cells and the adverse effects of hypoxia to chemotherapy treatment have been well documented [ 3 , 9 , 10 ] . thus , monitoring the cellular levels and localization of the autophagy ubls and adapter proteins can be used as a measure for autophagy activation or inhibition . methods to study autophagy regulation in cells in vitro have often employed detection of gfp - labeled autophagy proteins such as lc3b or electron microscopy to observe double membrane - bound autophagosomal structures . however , these approaches are not ideal for monitoring autophagy regulation in an in vivo drug discovery setting . unfortunately , significant technical challenges exist , as conventional polymer based ihc methods are largely ineffective for detecting autophagy - specific markers due to the low abundance and transient nature of autophagosomes [ 1620 ] . to address these issues , tyramide signal amplification ( tsa ) technology was applied to the autophagy markers lc3b and nbr1 , which resulted in a more robust and measurable signal as compared to the conventional polymer ihc platform . using the peroxidase activity of an hrp - conjugated secondary antibody , the label could be a chromophore , a hapten , or an enzyme such as hrp , and the signals are then detected accordingly . in the scheme used in the present study , hq ( a hapten ) was attached to tyramide and later detected using an hrp - conjugated anti - hq antibody . using these methods to manipulate autophagy , robust changes were observed in both lc3b and nbr1 levels in a time - dependent manner using the sensitive tsa - mediated detection method . this enabled the measurement of dynamic changes in autophagy regulated proteins without the use of engineered artificial protein labeling . using this method , the correlation of different markers in a multiplex format was examined and the relationship between basal levels of hypoxia and autophagy in tumor development was monitored . lentivirus was packaged in 293 t cells using the mission lentiviral packaging mix ( sigma - aldrich shp001 ) and viral supernatants were collected at 48 and 72 hours after transfection and used to transduce hct116 cells in the presence of 8 g / ml polybrene ( sigma - aldrich 107689 ) for 72 hours . scramble control line f6 was generated using the nonsilencing control shrna in ptripz vector ( open biosystem rhs4743 ) alongside with the same process . cells were grown and expanded for the inoculation of 2 10 cells in 0.1 ml per mouse . using a 26 3/8 gauge needle , 0.1 ml cell suspension in media - free pbs was subcutaneously inoculated into the right dorsal flank area of the female ncr nude mice . calu-6 human lung cancer cells were propagated in cell culture medium mem with the addition of 10% fbs . using a 26 3/8 gauge needle , 0.1 ml cell suspension in media - free pbs solution was subcutaneously inoculated into the right dorsal flank area of female nude mice . approximately 3 weeks after inoculation , when the tumor size reached 500800 mm , animals were randomized into treatment groups . all animals received a 0.2 ml single oral dose of mln0128 ( 5.0 mg / kg ) . tumors were collected and fixed in 10% neutral buffered formalin ( nbf ) before processing for histology studies . tumor tissues were processed in a covaris e200 tissue homogenizer in accordance with the manufacturer 's instructions . cold lysis buffer ( 300 to 800 l ) was added to each tumor sample followed by sonication . for western blot analysis , tumor lysates ( 20 g protein ) were loaded onto 4% to 12% gradient bis - tris gels ( invitrogen ) and subjected to electrophoresis . separated proteins were transferred to pvdf - fl membranes ( millipore ) using a semidry transfer apparatus . the membranes were blocked with li - cor blocking buffer ( li - cor biosciences , 927 - 40000 ) and then incubated with primary antibody in li - cor blocking buffer plus 0.1% tween-20 ( sigma - aldrich , p 1397 ) overnight at 4c . after three washes with tbst , the membranes were incubated with alexa fluor 680-labeled goat anti - rabbit igg ( life technology ) in li - cor blocking buffer plus 0.1% tween-20 for 1 hour . while protected from light , the membranes were washed five times with tbst , once with tbs , and then dried . the following primary antibodies were used : monoclonal rabbit anti - lc3b ( cell signaling technology , 3868 , 412 g / ml , 1 : 1000 ) ; polyclonal rabbit anti - sqstm1/p62 antibody ( abcam , 91526 , 1 g / ml , 1 : 1000 ) ; and polyclonal rabbit anti - atg7 ( epitomics , 2054 - 1 , 1 : 1000 ) . heavy membrane lysis buffer ( 250 mm mannitol , 70 mm sucrose , 0.5 mm egta , and 5 mm hepes - naoh , ph 7.2 ) with 1x protease inhibitor cocktail ( calbiochem ) , 2 mmol / l sodium orthovanadate ( sigma - aldrich ) , 25 mmol / l sodium fluoride , and 25 mmol / l -glycerophosphate ( sigma - aldrich ) was used . cold heavy membrane lysis buffer ( 1000 l ) was added to each tumor sample followed by sonication . lysates were transferred to eppendorf tubes and centrifuged at 3,000 rpm in eppendorf centrifuge 5417r at 4 for 10 minutes , and supernatants were transferred to new eppendorf tubes . all xenograft tumor samples were fixed in 10% nbf for a minimum of 16 hours before being dehydrated and embedded in paraffin ( ffpe ) . for ihc evaluations , 5 m sections of ffpe tissue were mounted onto glass slides , dried overnight at room temperature , and baked at 60c for 1 hour prior to use . tumor sections were processed by automated staining using either the discovery xt ( ventana systems ) or the bond rx ( leica biosystems ) autostainers , following preset protocols . in brief , the sections were subjected to ethylenediaminetetraacetic acid ( edta ) based antigen retrieval for 20 minutes and protein block ( dako , ds9390 ) for 12 minutes before incubation with primary antibody for 1 hour at room temperature . the following primary antibodies were used : polyclonal rabbit anti - carbonic anhydrase ix ( caix ) antibody ( novus biologicals , nb100 - 417 , 1 : 2500 ) ; polyclonal rabbit anti - sqstm1/p62 antibody ( abcam , 91526 , 1 : 5000 ) ; polyclonal rabbit anti - atg7 ( epitomics , 2054 - 1 , 1 : 800 ) ; monoclonal rabbit anti - ps6 ( cell signaling technology , 5364 , 1 : 2500 ) ; monoclonal rabbit anti - lc3b ( cell signaling technology , 3868 , 1 : 50 ) ; and monoclonal rabbit anti - nbr1 ( inhouse antibody , 1 : 50 ; validation of ml85 - 61 - 3 data not shown ) . colocalization studies of caix and ps6 used sequential standard ihc detection protocols on bond rx ( leica biosystems ) with both bond polymer refine detection dab and red kits . for detecting low abundance antigens lc3b and nbr1 , discovery amp hq kit ( ventana systems , 760 - 052 ) , a tyramide signal amplification detection kit , was used . ffpe sections were dried overnight at room temperature and then baked at 60c for 1 hour prior to loading onto discovery xt autostainer ( ventana systems ) . the sections were subjected to edta - based cc1 mild antigen retrieval for 20 minutes and protein block ( dako , ds9390 ) for 12 minutes before incubation with primary antibody for 1 hour at room temperature . , rabbit monoclonal antibody , 1.7 mg / ml , 1 : 2000 ) were used for this study . following primary antibody incubation , the amp hq kit in conjunction with ( 1 ) anti - hq multimer , ( 2 ) omnimap hrp multimer , and ( 3 ) chromogenic detection kit was used following the manufacturer 's instructions ( ventana systems ) . the ihc - stained slides were scanned by aperio scanscopext ( leica biosystems ) using the 20x objective , and the digital images were stored in eslide manager ( leica biosystems ) database . for caix - lc3b analysis , caix - positive and caix - negative regions were manually annotated and transferred to the lc3b image for subsequent image analysis . one - way anova was used for statistical analysis with a significance level set at p < 0.05 . the use of standard ihc methods for detecting and quantifying autophagy - specific biomarkers has been limited due to the low abundance and transient nature of autophagosomes . with standard ihc methods , signal is typically amplified with antispecies multimer complexes containing multiple enzyme molecules . ffpe calu-6 xenograft tumors stained for lc3b and nbr1 with standard ihc methods showed diffuse brown staining in the cytoplasm with very fine , light staining puncta structures ( figures 1(a ) and 1(c ) ) . in contrast , amp hq uses a tyramide substrate - based amplification where in the presence of hydrogen peroxide , and immobilized hrp converts tyramide substrate into a reactive intermediate that covalently binds to electron rich regions of proteins immediately adjacent to the activating hrp enzyme . using the amp hq method on calu-6 xenograft sections , both lc3b and nbr1 were clearly detected as dark - staining puncta structures in the cytoplasm of viable tumor ( figures 1(b ) and 1(d ) ) . to verify the amp hq assays were capable of measuring specific changes to lc3b and nbr1 levels under conditions of autophagy induction and inhibition , the autophagy pathway was manipulated in vivo using the investigational mtor inhibitor , mln0128 , to activate autophagy and using doxycycline - inducible atg7shrna knockdown to downregulate autophagy . calu-6 xenograft tumors were harvested from female nude mice following a single oral 5 mg / kg dose of mln0128 and examined for levels of the mtor pathway marker , ps6 , using standard ihc and the autophagy marker lc3b using amp hq ihc . quantification of ps6 with positive pixel analysis revealed a greater than 4-fold decrease ( p = 0.038 ) in the mln0128-treated tumor compared to the vehicle only control ( figure 2(b ) ) . the difference of the mean values of lc3b level between the animals treated with mln0128 for 30 minutes and the vehicle group was statistically significant ( p = 0.029 ) . consistent with the lc3b amp hq result , western blot analysis of the hmf from the same tumor tissue also showed the lipidated lc3b increased after mln0128 administration ( figure 2(d ) ) . a central core of necrosis and areas of hypoxia are commonly observed in highly proliferating xenograft tumors . to investigate the relationship of hypoxia conditions with autophagy and mtor pathway activity , calu-6 tumors were analyzed for caix expression to annotate tumor hypoxic and normoxic regions and for levels of ps6 and lc3b to measure mtor and lc3b pathway activities , respectively , using a dual ihc stain on a single slide . a high level of the hypoxia - induced protein caix expression was observed adjacent to the tumor necrotic areas indicating that necrosis in xenograft tumors was likely induced by hypoxia ( figure 3 ) . to assess autophagy activity in the context of hypoxia , sections of calu-6 xenograft tumor were stained for caix and manually annotated as caix positive ( hypoxic ) and caix negative ( normoxic ) . the lc3b puncta in the caix - positive and caix - negative regions were measured using positive pixel image analysis . the areas of viable tumor with caix - positive staining had higher levels of lc3b puncta than caix - negative regions . the data showed a significant difference between the hypoxic region and the normoxic region ( figure 3(d ) ) , when the two values were expressed as the mean sd ( p = 0.012 ) . atg7 knockdown has been shown to impair formation of autophagosomes , the double - membrane vesicles which are responsible for delivering cytoplasm material to lysosomes . a standard ihc with anti - atg7 antibody staining was performed on the xenograft tumor collected at days 3 , 7 , and 14 after treatment with or without doxycycline . ihc images showed a dramatically reduced atg7 protein expression in day 7 doxycycline - treated samples ( figure 4(a ) ) . positive pixel image analysis demonstrated a significant decreased expression of atg7 in the doxycycline - treated tumors ( p < 0.001 ) ( figure 4(b ) ) . further evaluation by western blot analysis also showed decreased atg7 protein levels with the doxycycline - treated group ( figure 4(f ) ) . the same set of samples was analyzed using the amp hq method to detect lc3b puncta . positive pixel algorithm analysis showed a greatly decreased lc3b expression in doxycycline - treated groups at 7 and 14 days ( p = 0.030 ) ( figure 4(c ) ) . a panel of xenograft tumors was analyzed for lc3b and nbr1 using the amp hq method , as a way to assess the variability of basal autophagy activity across various tumor types . the data showed a wide range of basal lc3b and nbr1 signals across the various tumor models . u87 mg ( brain ) and skov-3 ( ovary ) xenograft tumor models showed a high level signal of lc3b and low level of nbr1 . ht-29 ( colon ) , mda - md-361 ( breast ) , mm1.s ( myeloma ) , and hct116 ( colon ) xenograft tumor models showed a high level signal of nbr1 and low level of lc3b . oci - ly10 ( lymphoma ) and mda - mb-231 ( breast ) xenograft tumor models showed a good correlation between the lc3b and the nbr1 signals . autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized . during starvation , the nutrient - responsive kinase mtor is inhibited and a cell survival mechanism is upregulated that includes activating autophagy to reuse internal resources [ 7 , 23 , 24 ] . using mln0128 to inhibit mtor activity and mimic starvation - induced autophagy , an increase was observed in lc3b puncta levels using tyramide - based signal amplification technology . the increased signal of the tsa method enabled the quantitative analysis of lc3b expression by an automated image analysis method . this was not possible with the traditional ihc method because of low specific signal and high background staining with the standard ihc . a downstream marker of the mtor pathway , ps6 , was used to monitor inhibition of mtor following mln0128 treatment . in response to mtor inhibition , autophagy was activated and soluble lc3 ( lc3b - i ) was conjugated to form lc3-pe ( lc3b - ii ) which participates in the formation of autophagosomes . detecting lc3b by immunoblotting or immunofluorescence in addition to direct mtor inhibition , hypoxic conditions in the tumor microenvironment are also capable of rapidly inducing autophagy via induction of the hypoxia - inducible factor-1 ( hif-1 ) leading to mtor inhibition [ 9 , 10 ] . it is common to observe a central core of necrosis in a rapidly growing xenograft tumor where overexpression of carbonic anhydrase ix ( caix ) has been detected in hypoxic regions of tumors and the caix ihc assay serves as a biomarker of hypoxia in many solid tumors . to understand the relationship between hypoxia and mtor and autophagy pathway regulation , a colocalization analysis of caix with ps6 and lc3b was performed . a colocalization study with ps6 showed low ps6 levels in the same area that was positive for caix , demonstrating an inverse correlation between hypoxia and mtor activity . further investigation using caix costaining with lc3b showed that there was higher level of lc3b in the caix - positive region , indicating hypoxia - induced autophagy in the hypoxic regions of the xenograft tumor . a doxycycline - inducible atg7shrna model was used to study the dynamic range of the amp hq lc3b assay . autophagy ubl conjugation is a key feature of autophagosome formation and is mediated by the e1 enzyme atg7 . as a result of atg7 knockdown , a significant decrease in the number and size of the lc3b puncta this was accompanied by an increase in the level of the adaptor proteins nbr1 and p62 . using standard ihc and tsa methods , the accumulation of p62 and nbr1 , respectively , was clearly detected . this data also showed a large dynamic range in lc3b and nbr1 levels in a panel of xenograft tumors , suggesting that the two markers were useful tools to support the intertumor assessment of autophagy pathway status . in conclusion , with this tsa technology , autophagy markers can be monitored in xenograft tumors which will be helpful in understanding the mechanism of autophagy regulation in tumor development .

cytochrome p450s ( cyp450s ) are a multigene family of enzymes involved in oxidative metabolic activation and detoxification of many compounds . cyp450s act on endogenous substrates introducing oxidative , peroxidative and reductive changes into small molecules of various chemical structures . cyp450s also metabolize exogenous drugs , environmental chemicals and pollutants , resulting in either detoxification or generation of toxic metabolites which contribute to cancer risks and/or other toxic effects . the most common reaction catalyzed by cyps is a mono - oxygenase reaction : rh + o2 + 2h + 2e roh + h2o , where rh indicates an organic substrate . in this general reaction , one atom of oxygen is inserted into the substrate ( rh ) and a new functional group ( roh ) is consequently created , while the other oxygen atom is reduced to water . conserved expression of most cyp orthologs strongly supports similar evolutionary conserved functions in mice and humans during development . the best example of such a role lies in cyp1b1 , where deleterious mutations result in abnormal development of the trabecular meshwork ( tm ) in both mouse and human eyes . human cyp1b1 consists of three exons(371 , 1044 and 3707 bp in length ) and two introns ( 390 and 3032 bp ) spanning 8.5 kb of genomic dna ( genbank accession no . its coding region starts at the 5-end of the second exon and ends with the last exon . conservatively , mouse cyp1b1 gene exhibits a structure with three exons and two introns as well . the open reading frame ( 1.63 kb ) is encoded by exon 2 ( starting at the second nucleotide ; 1042 kb ) and 16% of exon 3 ( 3.78 kb ) . introns 1 and 2 are composed of 376 bp and 2591 bp , respectively . in humans , cyp1b1 is expressed in many adult and fetal extrahepatic tissues , including most of the parenchymal and stromal tissues from brain , kidney , prostate , breast , cervix , uterus , ovary , lymph nodes , heart , placenta , lung and skeletal muscle . , cyp1b1 also exhibits a tissue specific pattern of expression . during early stages of murine development cyp1b1 mrna constitutive cyp1b1 expression is seen in endocrine - regulated tissues such as mammary gland , uterus and prostate . human cyp1b1 and mouse cyp1b1 is believed to be important in ocular development and function due to its conserved expression in both murine and human eyes . its expression level is also higher in fetal than adult eyes , and it is differentially expressed throughout the eye . also , the relative transcript level of human cyp1b1 and mouse cyp1b1 in the eye is fivefold higher than other p450s further supporting the importance of cyp1b1 in normal eye development . the stages of fetal and postnatal development of the human anterior chamber have been determined . by the 15 fetal week , the main anlage of the presumptive chamber angle tissues are formed and separated into corneoscleral and ciliary - iris regions . around the 22 to 24 fetal weeks , ciliary muscle and ciliary processes develop . as the prospective schlemm 's canal grows , endothelial vacuoles appear within its lining endothelium . the corneoscleral trabeculae undergo a longitudinal orientation with deposition of collagen in the trabecular cores and formation of trabecular sheets and the iris is elongated . from the 24 to the 36 fetal weeks the corneal trabeculae enlarge with trabecular sheets under differentiation , and the uveal trabeculae form cords and sheets . vacuoles become present in both the inner and outer walls of schlemm 's canal . at birth , trabecular sheets are enlarged in the corneal , but the uveal meshwork undergoes collagen formation and intracellular differentiation . vacuoles confined to the inner wall . during postnatal years 1 - 4 , the chamber angle reaches the configuration of an adult eye . further formation of collagen fibers and intraceulluar differentiation are seen in the ciliary body , tm and schlemm 's canal . the juxtacanalicular meshwork becomes distinguished with its narrow inter - trabecular spaces and small trabecular sheets . in general , human cyp1b1 expression is of much higher level in fetuses than in adults . in detail , cyp1b1 has been detected in the fetal primitive ciliary epithelium as early as 26 days post - conception in human embryos , much earlier than anterior chamber development . cyp1b1 staining in the corneal epithelium , keratocytes and iris stromal cells is positive in fetal but not in adult eyes . intense intracytoplasmic cyp1b1 staining is observed in the non - pigmented ciliary epithelium , iris pigmented epithelium and iris dilator muscle both in fetuses and adults . development of the murine eye occurs relatively late in gestation . on embryonic day 15 ( e15 ) extend into the anterior chamber to form the pupillary membrane , and the rest of the mesenchyme resides at the margin of the optic vesicle to form the future tm , the stroma of the iris and the ciliary body . after then , the rudimentary ciliary body begins to grow out of the marginal neural retina and pigment epithelium . most of the anterior chamber structure develops postnatally . in the mouse eye , on postnatal day 1 ( p1 ) , the ciliary process slightly projects anteriorly to the lens . from p1 to p2 , a faster proliferation of the outer ciliary epithelium initiates the formation of the ciliary processes , resulting in a discrete ciliary body by p7 consisting of inner ( non - pigmented ) and outer ( pigmented ) ciliary epithelium . by p7 the iridocorneal angle trabecular beams are immature with minimal inter - trabecular spaces on p10 - 12 . by p14 , the angle recess becomes deep , and schlemm 's canal becomes mature with giant vacuoles . by p18 - 21 different from the age - related decrease of human cyp1b1 in human eyes , mouse cyp1b1 staining during fetal development is much lower than adult eyes . on e12.5 cyp1b1 protein in ocular tissues is not consistently detectable . it is detected diffusely in the lens epithelium influencing the development of the adjacent ciliary body on e15.5 . after p7 , it declines to low levels . before e17.5 , cyp1b1 is barely detectable in the inner ciliary epithelium , however , postnatally , it increases rapidly , reaching adult levels by p28 . in contrast , cyp1b1 mrna is expressed in the outer / pigmented ciliary epithelium at p4 and remains positive during eye development . on p14 , the levels of cyp1b1 protein appear to parallel development of the ciliary body following the appearance of ciliary processes . the above - mentioned observations suggest that cyp1b1 protein expression in the eye appears earlier than anterior chamber development in both humans ( e15 ) and mice ( e15-e16.5 ) . in fact , cyp1b1 is the first gene in the cyp450 family in which mutations are demonstrated to be involved in a primary developmental defect in humans . consistently , it has been reported that loss of cyp1b1 activity results in similar developmental defects of the tm in mice . cyp1b1 1 amino acid sequences are 81% similar among humans , rats and mice , suggesting the importance of this gene in ontogeny . all these facts suggest that cyp1b1 expression plays a key role in the normal development of tm tissue . human cyp1b1 mrna has been detected in an adult human tm cdna library and a human tm cell line . a recent study in our group also revealed positive cyp1b1/cyp1b1 protein staining in human and mouse tm cells . however , other studies found human cyp1b1 and murine cyp1b1 protein as well as mouse cyp1b1 mrna to be absent in normal fetal and adult human or mouse tm tissue . these low levels of cyp1b1 expression in tm tissue in vivo may make its detection difficult . in contrast to the small quantities of cyp1b1 within the tm , abundant cyp1b1/cyp1b1 1 protein is shown in the ciliary body , which is responsible for aqueous humor ( ah ) formation . cyp1b1 may generate biologically active metabolites in situ in the ciliary body , which are then secreted into the ah and diffuse to the tm tissue . this notion is supported by studies that suggest proper embryonic tm development may be disrupted due to lack of crucial metabolites from ciliary body in individuals carrying cyp1b1 mutations . glaucoma , the second leading cause of blindness worldwide , is a type of optic neuropathy leading to progressive damage of the optic nerve together with visual field defects which can further result in loss of central vision if left untreated . it is categorized according to the etiology ( primary and secondary ) , anatomy of the anterior chamber ( open versus closed angle ) , and the time of onset ( congenital , infantile , juvenile and adult ) . under the category of primary developmental glaucoma , primary congenital glaucoma ( pcg ) is reserved for cases in whom the major observable anatomical defect is trabeculodysgenesis , that is , a malformation of the tm . the tm is an ocular tissue around the base of the cornea and near the ciliary body which is responsible for draining ah , a transparent fluid from the anterior chamber and maintaining homeostasis of intraocular pressure ( iop ) . failure of proper development and differentiation of tm leads to decreased ah outflow and increased iop in pcg patients . pcg is the most common form of glaucoma in infants with prevalence varying geographically from a rate of 1:10,000 to 1:20,000 in western countries and 1:2500 in the middle east to 1:1250 in the gypsy subpopulation of slovakia . it is familial in 10 - 40% of cases , with an autosomal recessive trait with high ( 73 - 100% ) penetrance . molecular screening of the pcg families linked to the 2p21 locus has determined that mutations in cyp1b1 are responsible . all cyp450s share a set of conserved structural elements including 4 helix bundles ( d- , i- , l - and the antiparallel e - helices ) , j - helix , k - helix , beta - sheets 1 and 2 , the meander region and the heme - binding region . as shown in figure 1a , modified from sarfarazi and stoilov , cyp1b1 has a 53 residue long membrane - spanning domain located at the n - terminal of the molecule , a 10 residue proline - rich hinge region permitting flexibility between the membrane - spanning and cytoplasmic domain of the molecule , and a 480 residue long cytosolic globular domain which includes a substrate binding region and highly conserved core structures ( ccss ) composed of j - helix , k - helix and heme - binding regions [ figure 1b ] . ( a ) the wild - type is anchored on the membrane of the endoplasmic reticulum . with the help of p450 reductase as a redox partner , cyp450s b , c , e and f stands for b- , c- , e - and f - helices . ( c ) the truncating or missense mutations of cyp1b1 highly affect conserved amino acid residues located predominantly in either the hinge region or the ccs part of the protein . cyp1b1 mutations occur in 87% of familial and 27% of sporadic cases of pcg worldwide . five major groups of mutations in cyp1b1 have been found in pcg : missense mutations , frame shift or truncating mutations , mutations triggering the nonsense - mediated mrna decay of cyp1b1 , and mutations in the promoter or control regions of the gene . critical missense mutations are located either at the n - terminus hinge region or c - terminus ccss [ figure 1c ] . they are expected to interfere with fundamental properties of cyp1b1 including proper folding , heme binding , susbtrate accommodation with the reductase and/or the ability to transfer electrons , thus impairing enzyme functions . molecular dynamics simulations further confirm the potential of cyp1b1 mutations in structural alterations of missense mutations found with high frequency in patients with pcg . w57c affects the position that precedes the last proline residue in the proline - rich hinge . it can impair heme incorporation and reduce enzyme activity by interfering with proper protein folding of cyp1b1 . the g61 is one of the most highly conserved positions in the hinge region , with g61e changing the three - dimensional folding basis of the cyp1b1 molecule . a115p is within the b / b loop at the c - terminal end of b - helix and is close to the heme - binding region . this mutation restricts conformational freedom of the site with a larger substituent from p , and precludes hydrogen - binding interactions due to the absence of amino hydrogen . m132r right in the heme - binding region harms the local h - bond interactions and congests the packing of the heme - binding loop . q144p located at the crucial junction of the i- , c - and b/c - helix breaks the continuity of the c - helix , and thus impairs the structure of the heme - binding region . p193l in the n - cap position of e - helix affects the stability of the e - helix and consequent packing in this region . e229k in the vicinity of substrate binding region affects the local charge distribution and disturbs essential salt bridges . it also disrupts the three - dimensional structures of e- and f - helices leading to severe conformational changes in the mutant protein . s239r located in the f / g loop region is close to the substrate access channel and disrupts the dynamics of substrate access . g365w affects the orientation of j - helix of the ccs responsible for active heme binding . r368h located in between the j- and k - helices weakens the bonds among other close amino acids . it impairs the three - dimensional folding basis of the k - helix of the ccs . as e387 and r390 are landmark residues which are absolutely invariant among all members of cyp450 family , e387k , r390h and r390c disrupt the orientation of k - helix and critically interfere with the formation of stable hemo - protein complex . d374n and p379l are in close proximity to these crucial residues and exert their negative effect in a similar fashion . actually , d374n is one of the three major cyp1b1 mutations in saudi arabian pcg patients , and all patients with r390c homozygous mutation show very severe phenotype with poor prognosis . p437l and r444q impair the structural integrity of the conserved meander region which precedes the heme - binding region . g466 and c470 are conserved in all cyp450 proteins to accommodate the limited volume between the heme - binding loop and b - helix . g466d the large and rigid side of w in r469w precedes the invariant c470 and impairs the conformation of the conserved cysteine pocket . it is also one of the three major cyp1b1 mutations in the saudi arabians pcg patients . other critical mutations involve frame shift mutations in the cyp1b1 open reading frame that introduces premature stop codons upstream of the coding sequences for conserved regions and eliminates multiple essential domains of cyp1b1 . in a cohort study on 146 pcg patients among whom 45 subjects were detected with cyp1b1 mutation , the deleterious effect of frame shift mutations on the protein function resulted in 100% blindness . in conclusion , cyp1b1 mutations identified in patients with pcg most probably interfere with the structures of the heme - binding region , the hinge or ccs region of the cyp1b1 ( 12 ) . different combinations of cyp1b1 mutations may produce various degrees of histologically observed structural alterations in the tm and schlemm 's canal that closely correlate with the severity of disease and the difficulty in achieving iop control . at least part of pharmacological treatments for pcg should be focused on supplementing nutritional molecules , for instance , anti - oxidants , which could have been potentially lost due to lack of cyp1b1 metabolic activity . ah contains h2o2 and superoxide anion which impose oxidative stress on the tm and contribute to the onset of pathogenic alterations in the tm . the concentration of ascorbic acid is about 1 mm in the ah of diurnal mammals , a relatively high concentration that is 20 - 50 times greater than that in the plasma . other antioxidants such as glutathione ( gsh ) and superoxide dismutase ( sod ) isoenzymes ( cu - zn sod , mn sod , and extracellular superoxide dismutase [ ec - sod ] ) are also present in ah to scavenge free radicals . in fact , the ah of human glaucoma patients has a significant reduction in total reactive antioxidant potential ( gsh , ascorbate and tyrosine ) due to oxidative stress in the glaucomatous eye . these results are also consistent with decreased reducing power in the ah of congenital glaucomatous rabbits , which exhibited a 90% reduction in ascorbic acid and a 69% reduction in uric acid , concomitant with increased oxidative stress . besides the antioxidant defense in ah , the tm itself also has a sophisticated self - protection mechanism against reactive oxygen species ( ros ) including sod , catalase and glutathione . the developing tm in pcg patients has been found to be deficient in antioxidant enzymes and more susceptible to h2o2 permeating the tissue cells , ultimately resulting in tm damage and consequent ah outflow resistance . consistently , cyp1b1 tm cells are less viable under h2o2 treatment , indicating an insufficient cellular antioxidant capacity to detoxify ros . furthermore , mitochondrial dna sequence variations associated with oxidative stress , including g10398a in nadh dehydrogenase subunit 2 , a12308 g in trna leu gene and g13708a in nadh dehydrogenase subunit 3 have been reported to be significantly more frequent in pcg patients than in controls . these variations can consequently lower atp levels of tm cells and disable cellular elimination of ros , impairing the growth , development and differentiation of tm tissue . recent studies conducted in our laboratory have demonstrated that the cyp1b1/cyp1b1 activity in the tm directly determines their oxidative status . the tm tissues from cyp1b1mice exhibited increased oxidative stress early in life , and showed ultrastructural defects . these defects were prevented by daily administration of n - acetylcysteine ( nac ) a free radical scavenger to cyp1b1 pups from postnatal day 2 . in addition , the abnormal cellular functions of cyp1b1 tm cells including altered tm cell adhesion , that would influence the proper expansion of intercellular spaces and allowance of the trabecular outflow , were restored by treating the cells with the antioxidant nac . increased oxidative stress due to cyp1b1 deficiency was also responsible for the decreased production of periostin ( postn ) , an extracellular matrix protein revealed to be critical for the structural development and functions of tm . periostin ( postn ) is an extracellular matrix ( ecm ) protein essential for collagen fibril assembly and maturation . cyp1b1 tm tissue was found to express less postn than normal cyp1b1 tm tissue and contained irregularly distributed collagens . likewise , human glaucomatous tm tissues expressed decreased levels of postn as compared to the levels expressed in normal human tm . the present study was first to demonstrate a significant role for postn in the structural integrity of tm tissues . the collagen fibers in postn tm were significantly disrupted , with large empty spaces representing abnormal distribution or even loss of collagen fibers . in addition , due to lack of support from regular collagen fibers , the trabecular cytoplasm became uneven and presented severe distortions . overall , these results substantiate a role for postn in dictating mechanical strength and structural integrity of tm tissue . importantly , antioxidant nac restored the expression of postn in cyp1b1 tm cells . in general , postn expression is regulated by cyp1b1 and cellular oxidative state , and its appropriate expression is essential for ultra - structural development and integrity of tm tissue . uncovering how cyp1b1 deficiency negatively impacts postn expression in tm cells is the subject of ongoing investigation and its results will provide deeper insight into tm development in which postn participates and postn is a marker for assessment of therapy . cytochrome p450s are now recognized as important modulators of vascular function through synthesis and metabolism of various vaso - regulators . however , there are limited known facts regarding the expression and function of cyp1b1 in vascular cells . our laboratory was the first to show expression of cyp1b1 in endothelial cells and perivascular supporting cells prepared from vascular bed of various tissues , and its expression can be induced using an ahr agonist . we have found cyp1b1 expression is critical in maintaining the function of retinal vascular cells including retinal endothelial cells and pericytes ( pc ) , which are essential components of retinal blood vessels . retinas from cyp1b1 mice exhibited reduced vascular density and failed to undergo neovascularization during oxygen - induced ischemic retinopathy . cyp1b1-deficiency in retinal endothelial cells led to decreased migration , attenuated endothelial nitric oxide synthase expression and/or nitric oxide bioavailability , and compromised their capillary morphogenesis . cyp1b1-deficiency in retinal pericytes resulted in altered signaling pathways including sustained activation of nf-b p65 , and compromised endothelial cell and pericytes interaction in vitro . these defects were mainly attributed to increased intracellular oxidative stress in endothelial cell and pericytes , and could be relieved by decreased oxygen level ( 2% ) or addition of nac . summary of altered cellular functions associated with cyp1b1-deficiency in various cells as compared to wild type cells our studies suggest that increased oxidative stress associated with cyp1b1 deficiency results in increased expression of thrombospondin-2 , a member of the thrombospondin family of matricellular protein with antiangiogenic activity , in both endothelial cells and pericytes . we have recently shown that expression of thrombospondin-2 is responsible for sustained activation of nf-b and production of inflammatory mediators in retinal pericytes , with adverse effects on pericytes and endothelial cells function . thus , modulation of vascular cell oxidative state by cyp1b1 plays a key role in modulation of vascular function . together our studies establish an important role for cyp1b1 in modulation of intracellular oxidative state . our studies on the vascular and tm cells from cyp1b1 mice established a significant role for cyp1b1 expression in modulation of oxidative state . although the exact identity of ros remains elusive , we believe these are oxygenated products of polyunsaturated fatty acids , which are normally metabolized by cyp1b1 relieving intracellular oxidative stress . while cyp1b1 tissues neutralize the endogenous generation of ros to some degrees , cyp1b1 deficiency may deprive tissues from its ability to counteract ros , rendering them less protected and much more susceptible to the onset of oxidative stress - related diseases such as pcg . our data has suggested that the contribution of nadph - oxidase , xanthine - oxidase , lipoxygenases , mitochondria or glutathione levels to ros generation and oxidative stress in these cells is minimal . the nature of these ros generated with cyp1b1 deficiency and how they are regulated by oxygen levels is another important area of future research . many studies have revealed cyp1b1 as a critical candidate gene in pcg , which is directly related to tm integrity . the detailed mechanism of how lack of cyp1b1 expression and/or activity contributes to pcg has remained under debate . the most recent study from our group has shown that cyp1b1 is an essential modulator of cellular oxidative state in the absence of which , increased oxidative stress results in decreased postn production and tm maldevelopment . however the novel finding of postn as an important regulator of tm tissue development , and the inter - relationship between cyp1b1 , oxidative stress and postn expression , may provide an innovative biomarker for detection and treatment of pcg . this knowledge also provides the rationale for using genetic counseling and antioxidants as an adjunct therapy along with conventional treatments to tackle this disease .

cytochrome p450 1b1 ( cyp1b1 ) belongs to the cyp450 superfamily of heme - binding mono - oxygenases which catalyze oxidation of various endogenous and exogenous substrates . the expression of cyp1b1 plays an important role in the modulation of development and functions of the trabecular meshwork ( tm ) . mutations in cyp1b1 have been reported in patients with primary congenital glaucoma ( pcg ) . mice lacking cyp1b1 also exhibit developmental defects in the tm similar to those reported in congenital glaucoma patients . however , how cyp1b1 deficiency contributes to tm dysgenesis remains unknown . in the present review , we will address the significance of cyp1b1 expression and/or its function in anterior segment development . cyp1b1-deficient ( cyp1b1/ ) mice are discussed as a promising model for an oxidative stress - induced model of pcg , in which cyp1b1 activity is revealed as an important modulator of oxidative homeostasis contributing to the development and structural function of the tm . this conclusion suggests a possible clinical intervention for individuals who are genetically at high risk of developing pcg .

INTRODUCTION None None OXIDATIVE STRESS AND GLAUCOMA PERIOSTIN AND DEVELOPMENT OF TM TISSUE None CONCLUDING REMARKS AND FUTURE PERSPECTIVE

cyp450s act on endogenous substrates introducing oxidative , peroxidative and reductive changes into small molecules of various chemical structures . the most common reaction catalyzed by cyps is a mono - oxygenase reaction : rh + o2 + 2h + 2e roh + h2o , where rh indicates an organic substrate . conserved expression of most cyp orthologs strongly supports similar evolutionary conserved functions in mice and humans during development . the best example of such a role lies in cyp1b1 , where deleterious mutations result in abnormal development of the trabecular meshwork ( tm ) in both mouse and human eyes . in humans , cyp1b1 is expressed in many adult and fetal extrahepatic tissues , including most of the parenchymal and stromal tissues from brain , kidney , prostate , breast , cervix , uterus , ovary , lymph nodes , heart , placenta , lung and skeletal muscle . , cyp1b1 also exhibits a tissue specific pattern of expression . during early stages of murine development cyp1b1 mrna constitutive cyp1b1 expression is seen in endocrine - regulated tissues such as mammary gland , uterus and prostate . human cyp1b1 and mouse cyp1b1 is believed to be important in ocular development and function due to its conserved expression in both murine and human eyes . also , the relative transcript level of human cyp1b1 and mouse cyp1b1 in the eye is fivefold higher than other p450s further supporting the importance of cyp1b1 in normal eye development . the stages of fetal and postnatal development of the human anterior chamber have been determined . by the 15 fetal week , the main anlage of the presumptive chamber angle tissues are formed and separated into corneoscleral and ciliary - iris regions . the corneoscleral trabeculae undergo a longitudinal orientation with deposition of collagen in the trabecular cores and formation of trabecular sheets and the iris is elongated . from the 24 to the 36 fetal weeks the corneal trabeculae enlarge with trabecular sheets under differentiation , and the uveal trabeculae form cords and sheets . vacuoles confined to the inner wall . in detail , cyp1b1 has been detected in the fetal primitive ciliary epithelium as early as 26 days post - conception in human embryos , much earlier than anterior chamber development . cyp1b1 staining in the corneal epithelium , keratocytes and iris stromal cells is positive in fetal but not in adult eyes . intense intracytoplasmic cyp1b1 staining is observed in the non - pigmented ciliary epithelium , iris pigmented epithelium and iris dilator muscle both in fetuses and adults . development of the murine eye occurs relatively late in gestation . on embryonic day 15 ( e15 ) extend into the anterior chamber to form the pupillary membrane , and the rest of the mesenchyme resides at the margin of the optic vesicle to form the future tm , the stroma of the iris and the ciliary body . after then , the rudimentary ciliary body begins to grow out of the marginal neural retina and pigment epithelium . most of the anterior chamber structure develops postnatally . in the mouse eye , on postnatal day 1 ( p1 ) , the ciliary process slightly projects anteriorly to the lens . from p1 to p2 , a faster proliferation of the outer ciliary epithelium initiates the formation of the ciliary processes , resulting in a discrete ciliary body by p7 consisting of inner ( non - pigmented ) and outer ( pigmented ) ciliary epithelium . it is detected diffusely in the lens epithelium influencing the development of the adjacent ciliary body on e15.5 . before e17.5 , cyp1b1 is barely detectable in the inner ciliary epithelium , however , postnatally , it increases rapidly , reaching adult levels by p28 . on p14 , the levels of cyp1b1 protein appear to parallel development of the ciliary body following the appearance of ciliary processes . the above - mentioned observations suggest that cyp1b1 protein expression in the eye appears earlier than anterior chamber development in both humans ( e15 ) and mice ( e15-e16.5 ) . in fact , cyp1b1 is the first gene in the cyp450 family in which mutations are demonstrated to be involved in a primary developmental defect in humans . consistently , it has been reported that loss of cyp1b1 activity results in similar developmental defects of the tm in mice . all these facts suggest that cyp1b1 expression plays a key role in the normal development of tm tissue . however , other studies found human cyp1b1 and murine cyp1b1 protein as well as mouse cyp1b1 mrna to be absent in normal fetal and adult human or mouse tm tissue . these low levels of cyp1b1 expression in tm tissue in vivo may make its detection difficult . in contrast to the small quantities of cyp1b1 within the tm , abundant cyp1b1/cyp1b1 1 protein is shown in the ciliary body , which is responsible for aqueous humor ( ah ) formation . cyp1b1 may generate biologically active metabolites in situ in the ciliary body , which are then secreted into the ah and diffuse to the tm tissue . glaucoma , the second leading cause of blindness worldwide , is a type of optic neuropathy leading to progressive damage of the optic nerve together with visual field defects which can further result in loss of central vision if left untreated . it is categorized according to the etiology ( primary and secondary ) , anatomy of the anterior chamber ( open versus closed angle ) , and the time of onset ( congenital , infantile , juvenile and adult ) . under the category of primary developmental glaucoma , primary congenital glaucoma ( pcg ) is reserved for cases in whom the major observable anatomical defect is trabeculodysgenesis , that is , a malformation of the tm . the tm is an ocular tissue around the base of the cornea and near the ciliary body which is responsible for draining ah , a transparent fluid from the anterior chamber and maintaining homeostasis of intraocular pressure ( iop ) . failure of proper development and differentiation of tm leads to decreased ah outflow and increased iop in pcg patients . pcg is the most common form of glaucoma in infants with prevalence varying geographically from a rate of 1:10,000 to 1:20,000 in western countries and 1:2500 in the middle east to 1:1250 in the gypsy subpopulation of slovakia . molecular screening of the pcg families linked to the 2p21 locus has determined that mutations in cyp1b1 are responsible . all cyp450s share a set of conserved structural elements including 4 helix bundles ( d- , i- , l - and the antiparallel e - helices ) , j - helix , k - helix , beta - sheets 1 and 2 , the meander region and the heme - binding region . as shown in figure 1a , modified from sarfarazi and stoilov , cyp1b1 has a 53 residue long membrane - spanning domain located at the n - terminal of the molecule , a 10 residue proline - rich hinge region permitting flexibility between the membrane - spanning and cytoplasmic domain of the molecule , and a 480 residue long cytosolic globular domain which includes a substrate binding region and highly conserved core structures ( ccss ) composed of j - helix , k - helix and heme - binding regions [ figure 1b ] . ( a ) the wild - type is anchored on the membrane of the endoplasmic reticulum . with the help of p450 reductase as a redox partner , cyp450s b , c , e and f stands for b- , c- , e - and f - helices . ( c ) the truncating or missense mutations of cyp1b1 highly affect conserved amino acid residues located predominantly in either the hinge region or the ccs part of the protein . cyp1b1 mutations occur in 87% of familial and 27% of sporadic cases of pcg worldwide . five major groups of mutations in cyp1b1 have been found in pcg : missense mutations , frame shift or truncating mutations , mutations triggering the nonsense - mediated mrna decay of cyp1b1 , and mutations in the promoter or control regions of the gene . they are expected to interfere with fundamental properties of cyp1b1 including proper folding , heme binding , susbtrate accommodation with the reductase and/or the ability to transfer electrons , thus impairing enzyme functions . molecular dynamics simulations further confirm the potential of cyp1b1 mutations in structural alterations of missense mutations found with high frequency in patients with pcg . w57c affects the position that precedes the last proline residue in the proline - rich hinge . it can impair heme incorporation and reduce enzyme activity by interfering with proper protein folding of cyp1b1 . the g61 is one of the most highly conserved positions in the hinge region , with g61e changing the three - dimensional folding basis of the cyp1b1 molecule . a115p is within the b / b loop at the c - terminal end of b - helix and is close to the heme - binding region . this mutation restricts conformational freedom of the site with a larger substituent from p , and precludes hydrogen - binding interactions due to the absence of amino hydrogen . m132r right in the heme - binding region harms the local h - bond interactions and congests the packing of the heme - binding loop . q144p located at the crucial junction of the i- , c - and b/c - helix breaks the continuity of the c - helix , and thus impairs the structure of the heme - binding region . p193l in the n - cap position of e - helix affects the stability of the e - helix and consequent packing in this region . e229k in the vicinity of substrate binding region affects the local charge distribution and disturbs essential salt bridges . it also disrupts the three - dimensional structures of e- and f - helices leading to severe conformational changes in the mutant protein . s239r located in the f / g loop region is close to the substrate access channel and disrupts the dynamics of substrate access . g365w affects the orientation of j - helix of the ccs responsible for active heme binding . it impairs the three - dimensional folding basis of the k - helix of the ccs . actually , d374n is one of the three major cyp1b1 mutations in saudi arabian pcg patients , and all patients with r390c homozygous mutation show very severe phenotype with poor prognosis . p437l and r444q impair the structural integrity of the conserved meander region which precedes the heme - binding region . g466 and c470 are conserved in all cyp450 proteins to accommodate the limited volume between the heme - binding loop and b - helix . g466d the large and rigid side of w in r469w precedes the invariant c470 and impairs the conformation of the conserved cysteine pocket . it is also one of the three major cyp1b1 mutations in the saudi arabians pcg patients . other critical mutations involve frame shift mutations in the cyp1b1 open reading frame that introduces premature stop codons upstream of the coding sequences for conserved regions and eliminates multiple essential domains of cyp1b1 . in conclusion , cyp1b1 mutations identified in patients with pcg most probably interfere with the structures of the heme - binding region , the hinge or ccs region of the cyp1b1 ( 12 ) . different combinations of cyp1b1 mutations may produce various degrees of histologically observed structural alterations in the tm and schlemm 's canal that closely correlate with the severity of disease and the difficulty in achieving iop control . at least part of pharmacological treatments for pcg should be focused on supplementing nutritional molecules , for instance , anti - oxidants , which could have been potentially lost due to lack of cyp1b1 metabolic activity . ah contains h2o2 and superoxide anion which impose oxidative stress on the tm and contribute to the onset of pathogenic alterations in the tm . the concentration of ascorbic acid is about 1 mm in the ah of diurnal mammals , a relatively high concentration that is 20 - 50 times greater than that in the plasma . in fact , the ah of human glaucoma patients has a significant reduction in total reactive antioxidant potential ( gsh , ascorbate and tyrosine ) due to oxidative stress in the glaucomatous eye . these results are also consistent with decreased reducing power in the ah of congenital glaucomatous rabbits , which exhibited a 90% reduction in ascorbic acid and a 69% reduction in uric acid , concomitant with increased oxidative stress . besides the antioxidant defense in ah , the tm itself also has a sophisticated self - protection mechanism against reactive oxygen species ( ros ) including sod , catalase and glutathione . furthermore , mitochondrial dna sequence variations associated with oxidative stress , including g10398a in nadh dehydrogenase subunit 2 , a12308 g in trna leu gene and g13708a in nadh dehydrogenase subunit 3 have been reported to be significantly more frequent in pcg patients than in controls . these variations can consequently lower atp levels of tm cells and disable cellular elimination of ros , impairing the growth , development and differentiation of tm tissue . recent studies conducted in our laboratory have demonstrated that the cyp1b1/cyp1b1 activity in the tm directly determines their oxidative status . the tm tissues from cyp1b1mice exhibited increased oxidative stress early in life , and showed ultrastructural defects . in addition , the abnormal cellular functions of cyp1b1 tm cells including altered tm cell adhesion , that would influence the proper expansion of intercellular spaces and allowance of the trabecular outflow , were restored by treating the cells with the antioxidant nac . increased oxidative stress due to cyp1b1 deficiency was also responsible for the decreased production of periostin ( postn ) , an extracellular matrix protein revealed to be critical for the structural development and functions of tm . the present study was first to demonstrate a significant role for postn in the structural integrity of tm tissues . overall , these results substantiate a role for postn in dictating mechanical strength and structural integrity of tm tissue . importantly , antioxidant nac restored the expression of postn in cyp1b1 tm cells . in general , postn expression is regulated by cyp1b1 and cellular oxidative state , and its appropriate expression is essential for ultra - structural development and integrity of tm tissue . uncovering how cyp1b1 deficiency negatively impacts postn expression in tm cells is the subject of ongoing investigation and its results will provide deeper insight into tm development in which postn participates and postn is a marker for assessment of therapy . cytochrome p450s are now recognized as important modulators of vascular function through synthesis and metabolism of various vaso - regulators . however , there are limited known facts regarding the expression and function of cyp1b1 in vascular cells . our laboratory was the first to show expression of cyp1b1 in endothelial cells and perivascular supporting cells prepared from vascular bed of various tissues , and its expression can be induced using an ahr agonist . we have found cyp1b1 expression is critical in maintaining the function of retinal vascular cells including retinal endothelial cells and pericytes ( pc ) , which are essential components of retinal blood vessels . retinas from cyp1b1 mice exhibited reduced vascular density and failed to undergo neovascularization during oxygen - induced ischemic retinopathy . these defects were mainly attributed to increased intracellular oxidative stress in endothelial cell and pericytes , and could be relieved by decreased oxygen level ( 2% ) or addition of nac . summary of altered cellular functions associated with cyp1b1-deficiency in various cells as compared to wild type cells our studies suggest that increased oxidative stress associated with cyp1b1 deficiency results in increased expression of thrombospondin-2 , a member of the thrombospondin family of matricellular protein with antiangiogenic activity , in both endothelial cells and pericytes . we have recently shown that expression of thrombospondin-2 is responsible for sustained activation of nf-b and production of inflammatory mediators in retinal pericytes , with adverse effects on pericytes and endothelial cells function . thus , modulation of vascular cell oxidative state by cyp1b1 plays a key role in modulation of vascular function . together our studies establish an important role for cyp1b1 in modulation of intracellular oxidative state . our studies on the vascular and tm cells from cyp1b1 mice established a significant role for cyp1b1 expression in modulation of oxidative state . although the exact identity of ros remains elusive , we believe these are oxygenated products of polyunsaturated fatty acids , which are normally metabolized by cyp1b1 relieving intracellular oxidative stress . while cyp1b1 tissues neutralize the endogenous generation of ros to some degrees , cyp1b1 deficiency may deprive tissues from its ability to counteract ros , rendering them less protected and much more susceptible to the onset of oxidative stress - related diseases such as pcg . our data has suggested that the contribution of nadph - oxidase , xanthine - oxidase , lipoxygenases , mitochondria or glutathione levels to ros generation and oxidative stress in these cells is minimal . the nature of these ros generated with cyp1b1 deficiency and how they are regulated by oxygen levels is another important area of future research . many studies have revealed cyp1b1 as a critical candidate gene in pcg , which is directly related to tm integrity . the detailed mechanism of how lack of cyp1b1 expression and/or activity contributes to pcg has remained under debate . the most recent study from our group has shown that cyp1b1 is an essential modulator of cellular oxidative state in the absence of which , increased oxidative stress results in decreased postn production and tm maldevelopment . however the novel finding of postn as an important regulator of tm tissue development , and the inter - relationship between cyp1b1 , oxidative stress and postn expression , may provide an innovative biomarker for detection and treatment of pcg . this knowledge also provides the rationale for using genetic counseling and antioxidants as an adjunct therapy along with conventional treatments to tackle this disease .

the crystal structures of materials are traditionally investigated by the long - range phenomenon of bragg diffraction and described by unit cells that represent the average structure . atoms in the structure are described in terms of positions and thermal vibrations that are time averaged over all unit cells . the structure of a functional material controls its properties . in cases where the composition necessarily imposes disorder on the structure , it is challenging to develop a complete picture of that structure over the different length scales on which this disorder can manifest itself . this has become clear in recent investigations of functional electroceramics such as the relaxor ferroelectric perovskite pmn - pt , where the disorder between mg and nb is not resolved crystallographically in the average structure but clearly contributes to dynamic and static polarization phenomena as revealed by single crystal diffuse scattering measurements . similarly pb(zr , ti)o3 ( pzt ) , which has applications as piezo sensors , actuators , sonars , and transducers , displays structural heterogeneity that is critical for the formation of the morphotropic phase boundary that produces its properties but to which bragg diffraction is surprisingly insensitive . perovskites with bi cations on the a site are potential alternatives to these pb - based functional materials due to the 6s lone pair that is common to both cations . the hybridization between bi 6s and oxygen 2p states stabilizes the off - center displacement of bi cations and produces ferroelectricity . apart from the well - studied antiferromagnetic ferroelectric bifeo3 , most bi - based perovskites require high pressure ( hp ) synthesis because of the difficulty of accommodating the small bication on the 12-coordinate a site , e.g. , bimno3 and bi2mnnio6 . it is thus of interest to expand the number of materials with bi on the perovskite a site that are accessible under ambient pressure synthesis conditions . this can be achieved by increasing the number of cations on the b site and thus the ability of this site to adapt to the coordination requirements of bi , as in biti3/8fe2/8mg3/8o3 or bi2mn4/3ni2/3o6 ( bmn ) , the subject of the present study , which has two different b site cations with three oxidation states ( mn , mn , and ni ) . as the multiple b site cations are not completely ordered in the average structure , an understanding of the chemical factors contributing to the unexpected stability of these systems requires local structural information . given the importance of short - range structural motifs in controlling the functional behavior of lead - based electroceramics , this local structure information is also needed to understand the properties of a site bi perovskites . bmn is incommensurate at room temperature ( rt ) , as evidenced by the presence of satellite reflections in the powder diffraction pattern , which are produced by modulations that occur beyond the length scale of the incommensurate subcell . these modulations can be in the form of positional or occupational perturbations to the atoms in the subcell and can result in a more diverse range of local environments than a traditional unit cell description . incommensurate structures are frequently visualized using an approximant incommensurate supercell , which is a multiple of the subcell where the period of the modulations is near integer values . the incommensurate structure of bmn has been determined in the centrosymmetric space group ( ibmm(00 , 0-0)mm.ss ) . the incommensurate subcell consists of a 2ap 2ap 2ap expansion of an orthorhombic perovskite structure ( ap denotes cubic perovskite subcell lattice parameter , 3.8 ) where bi displacements along the 100 direction cancel to give a nonpolar subcell structure . the subcell is modulated by positional and occupational modulations of the atoms in higher dimensional space along the 100 and 010 directions , which correspond to ideal cubic perovskite directions 110p and 001p , respectively ( figure 1 ) . for consistency , throughout this work we will relate all crystallographic directions back to those of the ideal cubic perovskite unit cell and denote these as hklp . the modulation in the bmn structure is described by two modulation vectors , q1 = a * ( = 0.4930(3 ) ) and q2 = b * ( = 0.4210(7 ) ) , which define a fourth and fifth dimension and occur along 110p and 001p directions , respectively . displacive modulations of the a site give rise to a wide range of bi environments and induce locally polar regions along 110p from the nonpolar subcell which cancel over longer length scales to produce long - range antiferroelectric displacements ; the result is that the approximant incommensurate supercell ( 712ap 38ap 2ap ) has almost no polarization ( 2 c cm ) . the short - range structural behavior along the locally polar 110p direction is therefore of great interest , as this controls the key physical property . a phase transition is observed upon heating at 200 c where the long - range incommensurate modulations are lost . the long - range average high temperature ( ht , measured here at 300 c ) structure is commensurate and antiferrodistortive ( gdfeo3 structure , space group pcmn in the orthorhombic setting consistent with the other structures discussed here , unit cell size 2ap 2ap 2ap ) and has a single regular a site environment . ( a ) subcell of the commensurate average crystallographic structure p21mn at rt and an rmc supercell obtained by multiplicity of 5 4 10 subcells . ( b ) subcell of the incommensurate structure ( ibmm(00 , 0-0)mm.ss ) and a supercell of comparable size to the rmc supercell . ( c ) representation of bi2(mn4/3ni2/3)o6 ( bi shown as pink - purple spheres , b cation sites as light blue spheres ) within pseudo - cubic ( dark blue box ) , commensurate average p21mn ( purple box ) , and incommensurate orthorhombic ibmm(green box ) unit cells with corresponding pseudo - cubic directions polar 110p , and nonpolar 110p and 001p . ( d ) subcell of the commensurate average crystallographic structure pcmn at ht and the rmc supercell obtained by multiplicity 10 7 10 subcells . all three structures , p21mn , ibmm , and pcmn , and their supercells have the same unit cell axis settings . both the incommensurate ( rt ) and commensurate ( ht ) structures of bmn described above are derived from diffraction phenomena and represent a long - range average of the true local structure that may not adequately describe the diversity of short - range structural features that can be responsible for the observed chemical stability and physical properties . in order to determine the true local structure of a material , one has to reduce the reliance on bragg diffraction and make use of diffuse scattering that lies in the background of a diffraction pattern . the simultaneous analysis of bragg and diffuse scattering data is known as total scattering analysis , described in detail in supporting information section s1 . we employ the reverse monte carlo ( rmc ) optimization of the positions of atoms within a large supercell of the average structure to fit the conventional bragg scattering together with the pair distribution function ( pdf ) which describes the probability of two atoms being separated by a distance r. pdf studies have established the local structures of pb - based perovskite materials , where condensation of static polar nanoregions ( pnr ) at high temperature has been identified in relaxor ferroelectrics such as pb(mg1/3ta2/3)o3 . the investigation of lead - free ferroelectric materials revealed significant differences between the average and local structures of importance in understanding the ferroelectric properties . in contrast to the pb systems , the local structures and short - range correlations of pure bi a site perovskites have not been extensively studied . in bimno3 , pdf analysis showed the existence of ferroelectric domains larger than 100 . in biti3/8fe2/8mg3/8o3 , the local structure revealed local correlations of bi displacements producing monoclinic domains that could not be deduced from the average rhombohedral structure . pdf analysis also contributed to the understanding of the stability of pure bi a site perovskite materials by revealing the local bonding environments at the three b cations , which are all disordered onto one site in the average structure . bmn is more complex than electroceramics previously studied by pdf methods , as it has an incommensurate structure at room temperature . pdf studies on incommensurate structures as a class are rare ; pdf analysis of incommensurate charge - density waves in cete2 revealed significantly larger amplitudes of local atomic distortions in the te coordination environment than in the crystallographic structure . in order to resolve the local structure and local polar nature of bmn in both the incommensurate rt and commensurate ht phases , the rmc method was applied to neutron total scattering data and the resulting models were analyzed to yield local bonding information and atomic correlations . the emerging structural picture is distinct from any reported in lead - based materials and demonstrates the need to combine both average and local structures to understand the chemical control of polar behavior in bi - based electroceramics . the samples were prepared by a standard ceramic method described by claridge et al . neutron total scattering data were collected at general materials diffractometer ( gem ) at the isis pulsed spallation neutron source . rt and ht data at 300 c were collected for 920 and 1135 a h , respectively , over the same scattering vector range , 0.3 q 50 . the high scattering vector accessible on gem produces good real - space resolution in the pdf data ( r ( 2/qmax ) ; so for qmax = 50 , r 0.13 ) . all of the supercells used in the rmc analysis were approximate cubes containing 16 000 atoms . the starting model for the rt structure was based on the commensurate 22ap 4ap 2ap subcell in the polar orthorhombic space group p21mn , previously identified as the best commensurate approximation to the incommensurate structure . the gdfeo3-related structure was used as the starting model for the ht analysis . at the start of the rmc refinement , atom types were assigned randomly to b sites consistent with the fractional occupancies of the parent models , i.e. , 2:1 mn / ni for the rt and ht models . models of the incommensurate structure of the same size as the rmc models were derived by taking appropriate sized sections of an approximate supercell derived from the incommensurate structure ; atom types were assigned randomly to b sites in a manner consistent with the occupational probability from the modulated model . the relationships between these models can be found in figure 1 , and the generation of the models is described in more detail in supporting information section s2 . because of the underdetermined nature of the pdf refinements , it is generally necessary to apply some chemical constraints to the model in order to obtain meaningful results ; here we used bond valence sum ( bvs ) constraints on the metal oxygen distances for rt and ht models and an interatomic potential for ni o octahedra in the rt model . for the rt structure , the rmc refinement was started with only swapping of b site atoms allowed , followed by a second refinement step in which the b site atom swapping was replaced by atomic translation moves . during the rmc refinement of the ht structure , simultaneous atom translations and swapping of b site cations were performed . rmc runs with and without data ( referred to as data - free refinements in what follows ) were performed in order to confirm that these did not prejudice the overall result , as set out in more detail in supporting information section s2 . rmc refinement of the rt and ht starting configurations ( described above ) gave very good fits to the observed bragg profile , total scattering structure factor f(q ) , and the pdf g(r ) ( figures 2 and 3 , respectively ) . the pdf of the rmc - refined local structure at rt was compared with the pdf calculated from the supercells of the incommensurate average structure generated using jana in order to highlight the differences in description of the bonding between the present local structure refinement and the reported average incommensurate structure . the fit of the incommensurate structure to the observed g(r ) data is shown in figure 2d ; it is clear that the incommensurate average structure does not adequately describe the observed local structure in the g(r ) data . the extra information derived from the local structure description in several key aspects of structural understanding is discussed below . average bond distances for both the local structure and the incommensurate average structure were estimated by fitting gaussian functions to the observed partial pair distribution functions , gij(r ) , which represents distribution of bond distances for every pair of atom types , as described in eq 4 ( supporting information section s1 ) . comparison between measured and calculated functions of one of the rmc models of the local structure of bi2mn4/3ni2/3o6 at rt : ( a ) bragg profile data ; ( b ) total correlation function g(r ) ; ( c ) total scattering structure factor f(q ) . the total correlation function , g(r ) , of the incommensurate structure is compared to the observed data in ( d ) . comparison between measured and calculated functions of one of the rmc models of the local structure of bi2mn4/3ni2/3o6at ht : ( a ) bragg profile data ; ( b ) total correlation function g(r ) ; ( c ) total scattering structure factor f(q ) . o(r ) between the average incommensurate model and the rmc local structure showed similar bi o distances for both models ( table 1 ) . o distances at rt ( figure 4a ) in both the rmc local structure and the average incommensurate model produce three main groups of bond distances ( 2.252(5 ) , 2.685(2 ) , and 3.151(9 ) for the rmc local structure and 2.241(13 ) , 2.43(3 ) , and 2.99(3 ) for the average incommensurate structure ) . these peaks are entirely absent in the data - free rmc refinements and so are a result of the experimental data rather than any applied restraints . the area of the first strong peak in the rmc local structure ( table 1 ) indicates that bi atoms move toward the four nearest oxide neighbors to create short bi comparison of the groups of bi o bond distances between the refined rmc local structure and the models calculated from the average incommensurate structure indicates a broader range of bi o bond distances for the rmc models . o distributions in the rmc local structure are attributed to thermal motion , whereas the average incommensurate model includes thermal motion as time averaged thermal ellipsoids around an average atomic position . however , closer inspection of gbi o(r ) of the 10 individual incommensurate models studied shows two different shapes of distribution ( figure 4b ) ; half of the models showed a sharp first peak , and the other half showed a smoother distribution that was fitted to two gaussian peaks . the presence of two types of gbi the smoother distribution , defined as incommensurate models part 1 ( figure 4b top ) , gives bi o bond distances of 2.386(4 ) and 3.007(3 ) with a ratio of 5.82:6.18 for the 12 nearest neighbors . this is consistent with antiparallel displacement of bi cations along 110p which would ideally give rise to five short , two medium , and five long bi o bonds ; we do not observe the two medium bonds as distinct peaks because they will lie beneath the two main observed peaks in gbi the sharp peak in the second type of distribution , defined as incommensurate models part 2 ( figure 4b bottom ) , is likely due to maxima in the modulations that add constructively to produce a narrow distribution of short bi o bonds which is not representative of the true local structure , which is observed by rmc . the application of incommensurate modulations to atomic positions produces a discontinuous distribution of bi o distances due to local minima and maxima in the modulated displacements . o(r ) distribution ( figure 4a ) contains features from both types of distribution ( part 1 and part 2 ) and as such makes determining average bond lengths from this data misleading . a representation of the most common bi local environment determined through rmc modeling is shown in figure 4e and figure 4f . the bi cation is clearly displaced from the center of the coordinating oxygen atoms , with the displacement predominantly along 110p . distances for the average incommensurate model and the rmc local structure were obtained by fitting gaussian functions to the observed partial pair distributions functions ( figure 3 ) , while distances listed for the commensurate ( ht ) phase are the 12 nearest bi approximate ratio of bond distances ( summed to 12 oxygen coordinate system ) obtained from the integrated areas of peaks in gbi o(r ) for the average incommensurate model and the rmc local structure model in the rt and ht structures . o(r ) derived from the rmc local structure and average incommensurate model at rt ; ( b ) two general different shapes of gbi o(r)derived from different incommensurate models at rt ; ( c , d ) comparison of ( c ) gbi b(r ) , respectively , for the rmc local structure at rt and ht . representation of the local structure of the most common bi cation displacement ( purple sphere ) from the oxygen centroid ( indicated as a black sphere ) , shown relative to the oxygen positions from the crystallographic average structure ( red spheres ) viewed down ( e ) 110p and ( f ) 001p . the results are based on the mode value of the displacements in the rmc models with the most significant displacement of biof 0.283 along nonpolar 110p and smaller displacements of 0.099 and 0.052 along polar 110p and nonpolar 001p directions , respectively . the local structure at ht is consistent with the average commensurate crystallographic structure , with a comparable range of bi o(r ) is significantly broader and less well separated at ht than at rt ( figure 4c ) , as expected because of increased thermal motion at ht . the shortest bond length increases from 2.252(5 ) at rt to 2.307(3 ) at ht , and the longest bond length increases from 3.151(9 ) at rt to 3.234(9 ) at ht . the intermediate bond length decreases slightly from 2.685(2 ) at rt to 2.611(2 ) at ht . the bications are expected to be sensitive to the local b site occupancy . inspection of the distribution of bond distances between bi and b site cations ( figure 4d ) shows that bi atoms prefer to move toward ni neighbors and away from manganese cations , which is in agreement with expectations based on electrostatics , although correlations between these displacements are weak and short - range ( see section s3 and figure s1 in supporting information ) . these differences are shown by the respective distances : bi mn 3.412(1 ) and bi furthermore the trend of bond distances between bi and b site cations is preserved at ht where bi mn and bi ni distances of 3.358(14 ) and 3.224(12 ) , respectively , are observed ( table s1 in supporting information ) . data - free refinements revealed a similar trend but to a much reduced extent ( rt bi ni 3.316(7 ) ) , indicating that while this trend may in part be a result of the bvs restraint , the result is dominated by the observed data . the local bonding at the mn and ni sites is revealed by the partial pair distributions , gb o(r ) ( figure 5 ) , which were analyzed by fitting gaussian functions to the observed distributions . the average incommensurate model , where three b cations are disordered onto one site , has a relatively narrow distribution of b o bond distances ; the rt local structure shows a significantly broader distribution for both b sites . o pairs in the rt rmc refinement yields a symmetric gni o(r ) and an average bond distance of 2.075(1) , which is in agreement with those reported for bi0.53+bi0.55+nio3 . the gmn the mn bvs distribution of the rt rmc local structure models shows a unimodal gaussian distribution around an average oxidation state of + 3.43 , indicating that in the local structure models the mn adopts an oxygen coordination environment between that expected for mn and mn . however , the ideal mn o bond lengths for six - coordinate mn and mn from bvs calculations vary only slightly ( 1.90 and 2.01 , respectively ) , and so differences in charge alone can not account for the large range of observed bond lengths . instead , we attribute the broad asymmetric gmn o(r ) distribution to the presence of both undistorted mn and jahn teller ( j t ) distorted mn . mn typically displays six bond distances of approximately 1.9 , while mn with j t phenomena produce lattice distortions giving four short ( 1.921.96 ) and two long bond distances ( 2.112.16 ) , herein referred to as 4:2 j t , as observed in lamno3 or a site doped la1xcaxmno3 and la1xsrxmno3 . however , in the bismuth - based perovskite bi1xcaxmno3 there is a different pattern of two short bonds ( 1.9 ) , two long equatorial bonds ( 2.1 ) , and two medium apical bonds ( 1.95 ) , herein referred to as 2:2:2 j t feature is also observable in bimno3 , where the bond length distribution is broader ( 1.8 , 2.0 , and 2.2 ) . in order to determine which type of j we would expect to observe a single gaussian contribution from undistorted mn , and a range of bond distances from mn ( some of which may overlap with mn o distances ) corresponding to one of the j t distortion types ( 4:2 j t or 2:2:2 j t ) . three gaussian functions gave a much better fit to the mn o distribution than two gaussians and gave average bond lengths of 1.859(9 ) , 1.946(11 ) , and 2.146(57 ) ( table 2 ) . the ratio of integrated areas for these different bond lengths normalized to 12 oxygen nearest neighbors ( 6 for mn and 6 for mn ) is approximately 6:3:3 . the identical ratio for two medium and long bond lengths and the better fit of three gaussian functions than two indicate that the mn o bond distances observed in the local structure are more consistent with the 2:2:2 j t distortions observed for both bimno3 and bi1xcaxmno3 . the larger number of short than medium or long bonds ( ratio of 6:3:3 respectively ) may be attributed to the very comparable bond distances for both regular mn and the shortest bond distance in j however , the integrated area for the different bond lengths is approximately 6:3:3 , as opposed to 8:2:2 expected for an idealized case of a regular mn and a 2:2:2 j t mn , indicating that the observed mn o coordination environment is more complex . o(r ) between the average incommensurate model and the rmc local structure at rt . the local structure enables the differentiation between the different b site cations and reveals a broader distribution of bond lengths than for the average incommensurate model . approximate ratio of bond distances ( summed to 12 , rather than the coordination number of 6 , because of the presence of both mn and mn cations ) obtained from the integrated areas of peaks in gb o(r ) for the rmc local structure models at rt . consistent with this , the average structure of the related high pressure phase bi2nimno6 , which is nominally mn and therefore free from j t distortion , also displays a range of mn o distances ( from 1.83(7 ) to 2.10(5) ) that is relatively wide when compared to the narrow distributions of ni t distortions in bi2mn4/3ni2/3o6 due to the presence of mn leads to an even wider range of mn o bond distances than is observed in bi2nimno6 . this may assist in the accommodation of the bi displacements under ambient pressure synthesis conditions of bi2mn4/3ni2/3o6 . the local short - range order of the b site cations was investigated by nearest neighbor calculations of n(r ) ( eq 1 ) , defined as the mean number of atoms surrounding a central atom . it and has been used to find a site cation ordering in caxsr1xtio3.1where 0 is the average number density of the material , cj is the proportion at atom j in the material , and gij is the partial distribution function for atoms i and j. n(r ) was calculated directly from the partial pair distribution function gb b(r ) where the average numbers of neighbors for each pair of b site cations between distances r1 and r2 have been extracted ( figure s3 ) . distributions from the rmc local structure were compared with the results of data - free refinements that displayed completely random distributions . the average number of neighbors differs significantly between the observed and random distributions ( tables 3 and 4 ) for both rt and ht structures . the most pronounced difference in the rt structure are observed for the first coordination sphere of ni cations where the observed nni mn(r ) of 4.36(1 ) are significantly different than for a random distribution ( 2 and 4 , respectively ) ( figure 6a , b ) . the n(r ) calculations represent the average results of 20 rmc models , and the standard errors quoted are based on standard deviations of the results for the 20 models . the values for the first and second neighbors calculated for the 20 individual rmc models were essentially the same for each model and thus produce small standard deviation of 0.01 , which confirms that this deviation from random cation ordering revealed through rmc modeling is a real result of the data . ni nearest neighbor pairs when compared to a random b site distribution to short - range mn / ni cation site ordering . ni(r ) in the first shell and increase in the second are consistent with a bias toward rock salt ordering , which is observed in the related stoichiometric high pressure phase bi2mnnio6 . this b site ordering within a representative section of an rmc model is visualized in figure 7a , where b site cations are colored relative to the numbers of ni / mn in the six nearest neighbors , while gray atoms represent the average 4mn/2ni ratio . the regions of rock - salt - type ordering are clearly visible in the configuration despite the 2:1 mn / ni ratio of bmn being far from the ideal 1:1 ratio required for perfect rock salt ordering . in consequence , there are regions of higher concentrations of mn cations ( blue ) , which are necessary in order to produce the high concentrations of ni cations ( red ) needed to form rock salt ordered regions . the search for short - range ordering past the second nearest neighbor using n(r ) calculations is prevented by overlap of atoms between nearest neighbor shells . n(r)is given for the refined rmc local structure ( with standard errors derived from the standard deviation between the 20 different supercell models ) and a fully disordered b site distribution . comparison between the refined rmc local structure values ( with standard errors ) and the disordered distribution shows comparable results to the rt phase , with an observable preference for ni mn near - neighbor clustering . nb b(r ) ( a ) at rt and ( c ) at ht show noticeable differences in the rmc local structure from those compared for a statistically disordered distribution , ( b ) and ( d ) , respectively . b(r ) from the average incommensurate model ( e ) indicate comparable results to statistically disordered distribution ( f ) . results are also presented in tables 35 ( a ) b site ordering in a representative portion of an rmc model displaying a rock - salt - type ordering pattern ( outlined in a dotted black line ) . b site cations are colored according to the number of the six nearest neighbors of different atom types , with blue and red atoms corresponding to high and low numbers of mn nearest neighbors , respectively , and gray representing the average value of 4mn ( in accordance with the chemical composition bi2mn4/3ni2/3o6 ) . ( b ) approximant cell of incommensurate structure ( 712ap 38ap 2ap ) , viewed down along the nonmodulated 110p axis . b site cations are colored according to fractional occupancy , with blue and red atoms corresponding to high and low occupancy probability of mn cations and gray representing the compositional average mn0.667 . the cell has sufficient size to provide a view of the range of environments and demonstrate the clustering of mn / ni cations due to occupational modulations . the cation ordering motifs within the rt average incommensurate structure was also probed using the same n(r ) calculations . the rmc sized models of the incommensurate structure , where b sites were randomly assigned in a manner consistent with the occupational probability from the modulated model , were used for these the n(r ) calculations . the n(r ) results for this incommensurate model ( table 5 and figure 6e , f ) do not show the rock salt ordering present in the local structure . however , the absence of order in this type of n(r ) calculation does not necessarily mean that there is no cation ordering whatsoever ; different ordering patterns can still result in the random 4mn/2ni nearest neighbor ratio . the rt incommensurate average structure does indeed show nearest neighbor b site ordering ; however , n(r ) calculations are insensitive to this particular ordering pattern . the nature of the ordering is thus different than found for the rmc local structure . figure 7b shows the general pattern of b site ordering in the incommensurate approximant supercell ( 395 147 5.5 ) where polyhedra are colored according to the fractional occupancy of the b site . instead of the rock - salt - type order observed in the rmc local structure , the incommensurate structure displays a more complex mn / ni clustering , forming 2 2 columns of nickel rich and manganese rich octahedral forming a checkerboard pattern . the average structure is refined against modulations of defined periodicity , and the orderings seen in that structure correspond to that periodicity over distances comparable to the wavelength of the modulation . this long - range modulated ordering is not of rock - salt type , being derived from modulation bragg peaks of the type hklmn ( m , n = 1 ) , but it is possible that it is an average over ordered and disordered regions and that these disordered regions may include short - range rock - salt ordering . the pdf analysis thus reveals a distinct short - range ordering of rock salt type to which any average structure analysis is necessarily insensitive . comparison between the incommensurate model values ( with standard errors ) and the disordered distribution shows very comparable results . the short - range rock - salt - type cation order observed in the rmc analysis of the incommensurate rt structure persists at ht in the commensurate structure , with nni mn(r ) for the first nearest neighbor of 1.51(3 ) and 4.49(3 ) ( figure 6c , d ) . this result emphasizes how the single b site in the long - range average ht structure fails to fully describe the true local structure of ht bmn and confirms the information extracted from the more complex rt structure by rmc analysis . here the rmc local structure contains new information on the b site ordering , which is entirely absent from both the long - range incommensurate ( rt ) and commensurate ( ht ) structures . by collapsing rmc models onto the original orthorhombic cells in both the rt and ht structure , details of the atomic displacements the commensurate ht structure ( figure 8b ) contains two clouds of bismuth atoms displaced in opposite directions , which confirms the simple antiferroelectric displacement character of the a site cations in this commensurate structure . the bismuth atoms in the rt structure ( figure 8a ) present a more complex structured distribution . this led us to study displacements of the a site cations from the centroids of their 12-coordinating oxides . given the random nature of a monte carlo refinement and the strong pseudo - tetragonal nature of the starting cell , it is possible for these displacements to refine to either of the available 110p directions . calculations of the total a site cation displacements ( figure 9a ) and those along nonpolar 110p ( figure 9c ) show bimodal displacements , characteristic of the antiferroelectric displacements within bmn and a single gaussian shape along polar 110p and nonpolar 001p ( figure 9b , d ) in both the average incommensurate model and the refined rmc model . the distribution along polar 110p is represented by single broad gaussian shape , due to the presence of both positive and negative displacements of bications . the different magnitudes of displacement in opposite directions produce local polarity along 110p , which cancels out over long range to produce antiferroelectric displacements in the incommensurate average structure . the bi displacement distribution for the rmc model appears symmetric , indicating that the rmc supercell has minimal net polarity , as observed for the approximant incommensurate supercell . the rmc model gives a broader atomic displacement distribution than observed for the average incommensurate model ; this is consistent with the similarly broader rmc model gbi o(r ) distribution and is a consequence of thermal motion in the local structure . projection of the atomic positions of bi2mn4/3ni2/3o6 from the rmc supercell into the original unit cell viewed along 001p for the local structure at ( a ) rt and ( b ) ht . atoms are colored as follows : bi purple , mn blue , ni yellow , and o red . ( a ) comparison of total absolute displacements of the bi cations from the centroid of the coordinating oxygens between the rmc local structure and the average incommensurate model . comparison of the distribution of bi displacements from the oxygen centroid along ( b ) 110p , ( c ) 001p , and ( d ) 110p . the absence of a net bi displacement , and therefore polarity , within the rmc supercell led us to investigate local correlations between the bi displacements . correlations between atomic displacements can be identified through evaluation of the displacement correlation function ( dcf ) , which is defined between two species according to eq 2:2where the ui and uj vectors define displacement of atoms i and j , respectively , while n and r correspond to number of atoms and their separation , respectively . the displacement of each bi , u , was calculated from the centroid of their neighboring 12 oxygen atoms , and dcf was summed over the volume of the rmc supercell . to eliminate the inherent contribution coming from the symmetry of the crystallographic average structure , the dcf of a completely randomized rmc configuration was calculated and subtracted from the calculated dcf , as previously applied for the ferroelectric material biti3/8fe2/8mg3/8o3 . the difference between the rmc configuration and the randomized configuration should provide the real correlations beyond those arising from the average structure in the material . the dcf was calculated at the interatomic separation r between pairs of atoms and can be resolved into distinct planes within the structure and along the polar 110p and the two nonpolar 001p and 110p directions . a positive value means that the displacements of bi atoms separated by distance r are ferroelectrically correlated , whereas negative peaks indicate antiferroelectric correlations . the most intense peaks in the dcf of the rmc model ( figure 10a ) are along 110p with a pattern of both positive and negative peaks . this is an effect of antiparallel displacement shifts between bications along this direction ( figure 10b ) . these antiferroelectric correlations are also observed in the average incommensurate model ( figure 10c ) . pb(r ) calculated for pbzro3(pz ) ( figure 11a ) where pb atoms describe a well - known antiferroelectric pattern of displacements along 110p ( figure 11b ) , demonstrating that the bi cation correlations in the rmc and average incommensurate model of bmn have the same pattern as in antiferroelectric pz along 110p . this feature is retained , with broadening , in the commensurate ht structure ( figure s4 ) . ( a ) differential dcf for bi bi in the rmc local structure model showing antiparallel displacement along 110p , as illustrated in ( b ) . bi for the average incommensurate model , demonstrating general agreement between rmc and incommensurate models . ( a ) differential dcf for pb pb in pz with antiparallel displacement along 110p , as illustrated in ( b ) . the bi bi(r ) for the rt rmc model ( figure 10a ) and the average incommensurate model ( figure 10c ) both demonstrate a pattern of small negative and positive peaks along 110p , indicating weak antiferroelectric correlations that appear because the different direction of displacement for the neighboring atoms is the dominant effect in the dcf . thus , instead of observing polar nanoregions ( with ferroelectric correlations in the dcf ) , we observe antiferroelectric correlations consistent with the long - range modulated average structure . we hypothesize that the polar correlations do exist at rt along 110p within a larger volume rather than having strong correlations between single pairs of atoms as probed by the dcf calculations . in order to test this hypothesis , we looked at the sum of bi displacements , relative to the center of charge of the 12 nearest neighbor oxygens , within a volume . the sum of bi displacements was calculated by taking the b site cations as the origin and summing the displacement of every bi within a given radius , r. an r value of 7.5 and the b site as the origin were selected in order to return 16 bications ( an even number to ensure that net polarity is not inevitable ) and contain a volume approximately equivalent to that of the polar commensurate subcell ; as the bi displacements are relative to the center of charge , this measure is independent of the b site displacements . the sum of the bi displacements along the polar 110p direction was calculated , and the ratio of bi cations displaced in the same direction over all bismuth atoms was evaluated for each atomic shell ( figure 12a , b ) . thus , the ratio expressed in eq 3 with values above 0.5 gives information about atomic correlations.3where represents an atomic shell , s(+ ) corresponds to sum of bications displaced in the same direction along 110p , and (s(+ ) + s( ) ) defines the overall sum of bications displaced in opposite directions along 110p . ( a ) bi cation displacements are summed within a sphere of radius of 7.5 , with b site cations as the origin . ( b ) illustration of b site atomic shells used to sum volumes of bi displacements , showing first ( red circle ) and second ( green circle ) shells . the ratio of bi cations displaced in the same direction along 110p , summed over all bismuth atoms for ( c ) the rmc local structure model at rt and ( e ) the incommensurate model at rt . a random distribution of bi displacements is presented as a gray error bar . supercells with b site cations colored representing positive ( red ) and negative ( blue ) sum of neighboring bi cations along 110p for ( d ) the rmc local structure model at rt and ( f ) the incommensurate model at rt . the rmc model ( d ) shows clusters of oppositely displaced groups of atoms , clearly indicating local ferroelectric correlations between the bi atoms , whereas the incommensurate model ( f ) shows sinusoidal behavior of displaced bi atoms resulting from the incommensurate character of this model . these calculations showed that local correlations of bi cation displacements do indeed exist within a distance of approximately 12 ( figure 12c ) when compared to the randomly distributed bi cations observed in the supercells generated from data - free refinements ( presented as an error bar ) . there is a strong correlation within the first atomic shell with an s(1 ) ratio of 0.71 , which gradually decreases toward the random value of 0.5 , reaching a value of 0.52 ( i.e. , no significant correlation ) for s(8 ) . figure 12d shows an rt rmc supercell with the positive ( red ) and negative ( blue ) atoms representing the sum of bi displacements in opposite directions along 110p ; groups of atoms gathering in clusters are clearly present indicating the local correlations between bi atom displacements . these clusters with oppositely displaced atoms which cancel out to no net polarity within the rmc supercell ( as evidenced by the symmetric distribution of the bi displacement distribution ; cf . figure 9b ) are similar to the polar regions in the long - range approximant supercell of the incommensurate structure which cancel each other to produce long - range average antiferroelectric displacements . the size of the bmn polar domains is not easy to define from the long - range average structure . equivalent s(n ) calculations for bi displacements in the incommensurate structure result in large minima and maxima appearing repeatedly at fixed distances ( figure 12e ) and a checkerboard pattern of polar regions along the modulated directions that form infinite chains along the nonmodulated direction ( figure 12f ) . what we can say from long - range bragg diffraction is that the polar regions are probably at least the size of the polar approximant commensurate subcell ( p21mn , 22ap 4ap 2ap , a = 11.1496 , b = 15.5302 , and c = 5.5092 ) , given this reasonably good fit of this polar structure to the bragg data , and the absence of polarity in an approximate incommensurate supercell ( 712ap 38ap 2ap ) demonstrates that polar regions are at most half this larger size . by calculating s(n ) for bi in rt rmc models of the local structure bmn , we are able for the first time to quantify the size of the polar domains as existing within a domain of 12 , in reasonable agreement with the long axis dimensions of the polar approximant commensurate subcell . as opposed to the average commensurate subcell , which is entirely polar , and the approximate incommensurate supercell , where polarity is modulated in two dimensions , the local structure shows how bi displacements are correlated in three dimensions in real space . local correlations with dimensions of up to 12 persist in the ht phase , where the sum of bi atoms displaced in the same direction along 110p also creates polar clusters ( figure 13 ) . the displacements of bi cations in the average ht commensurate structure completely cancel out ( red columns in figure 13a ) , which is in agreement with this antiferroelectric displacement of bi atoms . the observation of local ferroelectric correlations in the ht phase means that the local displacements of bi cations giving rise to short - range polar regions still exist above the phase transition from the rt incommensurate structure to the ht commensurate structure , despite there being no signs of this polar nature in the long - range average crystallographic structure , suggesting that these correlations are now dynamic . these locally correlated polar regions would be expected below the burns temperature of a relaxor or conventional ferroelectric , consistent with the measurement being performed only about 100 k above the structural transition . weiss behavior is unfortunately not observable by direct measurement , as the material is too lossy in this temperature range because of the mixed manganese valence . ( a ) comparison of the ratio of bi cations displaced in the same direction along 110p over all bismuth atoms between the rmc local structure ( green ) and average commensurate structure ( red ) at ht . ( b ) rmc supercell with b site cations colored according to positive ( red ) or negative ( blue ) sum of neighboring bi cation displacements along 110p . clusters of bi cations displaced in the same direction persist in the ht phase . the availability of local structure information changes the understanding of the chemical stability and physical properties of incommensurate bi2mn4/3ni2/3o6 ( bmn ) in several significant respects . the b site environment at rt is significantly different in the rmc local structure and average incommensurate model . the application of bvs and interatomic potential constraints in the rmc modeling allowed us to differentiate between ni and mn on the b site , which share the same site in the incommensurate average structure . t distortions , gives rise to a wide range of mn o bond lengths that , together with the distinction between bi mn and bi ni distances seen in the local structure , will enable bmn to accommodate the off - center displacement of the bications required to explain its unusual stability to ambient pressure synthesis . inspection of partial pair distribution functions for both the rmc local structure and average incommensurate models showed three preferred bi o bond distances at similar bi o distances that were consistent with antiparallel displacement of bi cations along 110p . for the incommensurate average structure , gbi o(r ) shows two different discontinuous distributions , one of which contained a short bi o bond resulting from maxima in the applied displacive modulations . the local analysis does not include such features and thus produces a chemically preferable description of the bonding at bi . at rt , both incommensurate and rmc models show b site ordering ; however , the nature of the ordering is quite different . the rmc local structure reveals rock - salt - type nearest neighbor ordering , which is adopted by the related stoichiometric high pressure phase bi2mnnio6 , with the additional disorder necessitated by the 2:1 mn / ni ratio of bmn . the incommensurate average structure is insensitive to this and produces mn / ni clustering on the b site corresponding to the periodicity of the modulation . there are thus two ordering motifs present over different length scales , emphasizing that despite the perceived diversity of local atomic environments present within an incommensurate structure description , these atomic displacements and site orderings are a result of long - range modulations . as such , they may not reflect the true local nature of atomic displacements present within the structure and can not contain displacements or orderings not described by the modulation waves used , e.g. , the simple rock - salt - like order on the b site . the same local rock salt ordering is observed for the ht phase , where the average pcnm commensurate structure displays no ordering whatsoever , although the peaks due to the long - range modulated cation order are still visible in the neutron diffraction data . the local structure analysis thus identifies three important chemical features for the stability of the material , namely , the short - range b site ordering , the distribution of bi o distances , and the wide range of b o distances accommodated by different mn charge states and a j t distortion . the coexistence of two distinct b site ordering patterns over different length scales may play a role in stabilizing this bi a site material . the local structure of biti3/8fe2/8mg3/8o3 reveals flexible coordination behavior of ti on the b site , which may play a similar role to the three mn o distances in the preferred j t distortion at mn here , but there is no b site ordering over any length scale in this commensurate system . the local structures of these two examples thus suggest that a range of chemical strategies may be deployed to stabilize bi a site perovskites . calculation of bi displacements showed a bimodal distribution along the nonpolar 110p at rt in both the incommensurate average structure model and the rmc local structure , accounting for the antiferroelectric displacement behavior of bmn . the bi bi displacement correlation function along the same direction shows similar peaks in both rmc and average incommensurate models that are consistent with the antiferroelectric behavior of pbzro3 ; this feature is preserved in the local structure of the ht phase . as recent ferroelectric instability , this observation is consistent with the presence of polar local features in bmn ( i.e. , that bmn may also be close to global ferroelectric instability as observed in high pressure bi2mnnio6 ) and indicates that pz - like systems may be accessible in bi - based perovskites . bi displacements along the polar 110p showed a single gaussian distribution for both the incommensurate average structure model and the rmc local structure model , indicating that overall the models are nonpolar . bi displacements by calculation of the displacement correlation function instead revealed antiferroelectric correlations that would appear to negate the presence of polar regions . instead , polarity is produced on the local scale by different magnitudes of positive and negative displacements along 110p , which generate a net polarity despite the dominant displacement correlation between neighboring bi atoms being antiferroelectric . the true nature of the polar behavior of bmn was revealed by calculating the sum of bi displacements within a volume , which showed that ferroelectric correlations along 110p within the rmc local structure at rt give rise to polar clusters with a size of up to 12 . this local structure analysis allowed the size of the polar domains , which is elusive from long - range average structure treatments , to be quantified for the first time . these local polar domains are preserved at ht , where the long - range average structure shows no signs of this underlying polar nature . recognizing the length - scale of the probe used for structural characterization is very important for the interpretation of the results . average structures derived from bragg diffraction data are an invaluable tool for structure characterization on the long length scale , but frequently important structural characteristics such as short - range order or cooperative displacements can be missed entirely . here total scattering analysis has provided new insights into the incommensurate structure of the atmospheric pressure synthesized bi perovskite bi2mn4/3ni2/3o6 , revealing features of direct relevance for its unusual stability and explaining the structural and functional relationship to better - studied lead - based systems .

the functional properties of materials can arise from local structural features that are not well determined or described by crystallographic methods based on long - range average structural models . the room temperature ( rt ) structure of the bi perovskite bi2mn4/3ni2/3o6 has previously been modeled as a locally polar structure where polarization is suppressed by a long - range incommensurate antiferroelectric modulation . in this study we investigate the short - range local structure of bi2mn4/3ni2/3o6 , determined through reverse monte carlo ( rmc ) modeling of neutron total scattering data , and compare the results with the long - range incommensurate structure description . while the incommensurate structure has equivalent b site environments for mn and ni , the local structure displays a significantly jahn teller distorted environment for mn3 + . the local structure displays the rock - salt - type mn / ni ordering of the related bi2mnnio6 high pressure phase , as opposed to mn / ni clustering observed in the long - range average incommensurate model . rmc modeling reveals short - range ferroelectric correlations between bi3 + cations , giving rise to polar regions that are quantified for the first time as existing within a distance of approximately 12 . these local correlations persist in the commensurate high temperature ( ht ) phase , where the long - range average structure is nonpolar . the local structure thus provides information about cation ordering and b site structural flexibility that may stabilize bi3 + on the a site of the perovskite structure and reveals the extent of the local polar regions created by this cation .

Introduction Experimental Method and Data Analysis Results Conclusions

the crystal structures of materials are traditionally investigated by the long - range phenomenon of bragg diffraction and described by unit cells that represent the average structure . this can be achieved by increasing the number of cations on the b site and thus the ability of this site to adapt to the coordination requirements of bi , as in biti3/8fe2/8mg3/8o3 or bi2mn4/3ni2/3o6 ( bmn ) , the subject of the present study , which has two different b site cations with three oxidation states ( mn , mn , and ni ) . as the multiple b site cations are not completely ordered in the average structure , an understanding of the chemical factors contributing to the unexpected stability of these systems requires local structural information . given the importance of short - range structural motifs in controlling the functional behavior of lead - based electroceramics , this local structure information is also needed to understand the properties of a site bi perovskites . bmn is incommensurate at room temperature ( rt ) , as evidenced by the presence of satellite reflections in the powder diffraction pattern , which are produced by modulations that occur beyond the length scale of the incommensurate subcell . displacive modulations of the a site give rise to a wide range of bi environments and induce locally polar regions along 110p from the nonpolar subcell which cancel over longer length scales to produce long - range antiferroelectric displacements ; the result is that the approximant incommensurate supercell ( 712ap 38ap 2ap ) has almost no polarization ( 2 c cm ) . the long - range average high temperature ( ht , measured here at 300 c ) structure is commensurate and antiferrodistortive ( gdfeo3 structure , space group pcmn in the orthorhombic setting consistent with the other structures discussed here , unit cell size 2ap 2ap 2ap ) and has a single regular a site environment . both the incommensurate ( rt ) and commensurate ( ht ) structures of bmn described above are derived from diffraction phenomena and represent a long - range average of the true local structure that may not adequately describe the diversity of short - range structural features that can be responsible for the observed chemical stability and physical properties . we employ the reverse monte carlo ( rmc ) optimization of the positions of atoms within a large supercell of the average structure to fit the conventional bragg scattering together with the pair distribution function ( pdf ) which describes the probability of two atoms being separated by a distance r. pdf studies have established the local structures of pb - based perovskite materials , where condensation of static polar nanoregions ( pnr ) at high temperature has been identified in relaxor ferroelectrics such as pb(mg1/3ta2/3)o3 . pdf analysis also contributed to the understanding of the stability of pure bi a site perovskite materials by revealing the local bonding environments at the three b cations , which are all disordered onto one site in the average structure . in order to resolve the local structure and local polar nature of bmn in both the incommensurate rt and commensurate ht phases , the rmc method was applied to neutron total scattering data and the resulting models were analyzed to yield local bonding information and atomic correlations . the starting model for the rt structure was based on the commensurate 22ap 4ap 2ap subcell in the polar orthorhombic space group p21mn , previously identified as the best commensurate approximation to the incommensurate structure . the pdf of the rmc - refined local structure at rt was compared with the pdf calculated from the supercells of the incommensurate average structure generated using jana in order to highlight the differences in description of the bonding between the present local structure refinement and the reported average incommensurate structure . the fit of the incommensurate structure to the observed g(r ) data is shown in figure 2d ; it is clear that the incommensurate average structure does not adequately describe the observed local structure in the g(r ) data . average bond distances for both the local structure and the incommensurate average structure were estimated by fitting gaussian functions to the observed partial pair distribution functions , gij(r ) , which represents distribution of bond distances for every pair of atom types , as described in eq 4 ( supporting information section s1 ) . comparison between measured and calculated functions of one of the rmc models of the local structure of bi2mn4/3ni2/3o6 at rt : ( a ) bragg profile data ; ( b ) total correlation function g(r ) ; ( c ) total scattering structure factor f(q ) . o distances at rt ( figure 4a ) in both the rmc local structure and the average incommensurate model produce three main groups of bond distances ( 2.252(5 ) , 2.685(2 ) , and 3.151(9 ) for the rmc local structure and 2.241(13 ) , 2.43(3 ) , and 2.99(3 ) for the average incommensurate structure ) . the area of the first strong peak in the rmc local structure ( table 1 ) indicates that bi atoms move toward the four nearest oxide neighbors to create short bi comparison of the groups of bi o bond distances between the refined rmc local structure and the models calculated from the average incommensurate structure indicates a broader range of bi o bond distances for the rmc models . distances for the average incommensurate model and the rmc local structure were obtained by fitting gaussian functions to the observed partial pair distributions functions ( figure 3 ) , while distances listed for the commensurate ( ht ) phase are the 12 nearest bi approximate ratio of bond distances ( summed to 12 oxygen coordinate system ) obtained from the integrated areas of peaks in gbi o(r ) for the average incommensurate model and the rmc local structure model in the rt and ht structures . representation of the local structure of the most common bi cation displacement ( purple sphere ) from the oxygen centroid ( indicated as a black sphere ) , shown relative to the oxygen positions from the crystallographic average structure ( red spheres ) viewed down ( e ) 110p and ( f ) 001p . the results are based on the mode value of the displacements in the rmc models with the most significant displacement of biof 0.283 along nonpolar 110p and smaller displacements of 0.099 and 0.052 along polar 110p and nonpolar 001p directions , respectively . inspection of the distribution of bond distances between bi and b site cations ( figure 4d ) shows that bi atoms prefer to move toward ni neighbors and away from manganese cations , which is in agreement with expectations based on electrostatics , although correlations between these displacements are weak and short - range ( see section s3 and figure s1 in supporting information ) . the gmn the mn bvs distribution of the rt rmc local structure models shows a unimodal gaussian distribution around an average oxidation state of + 3.43 , indicating that in the local structure models the mn adopts an oxygen coordination environment between that expected for mn and mn . the larger number of short than medium or long bonds ( ratio of 6:3:3 respectively ) may be attributed to the very comparable bond distances for both regular mn and the shortest bond distance in j however , the integrated area for the different bond lengths is approximately 6:3:3 , as opposed to 8:2:2 expected for an idealized case of a regular mn and a 2:2:2 j t mn , indicating that the observed mn o coordination environment is more complex . the local structure enables the differentiation between the different b site cations and reveals a broader distribution of bond lengths than for the average incommensurate model . consistent with this , the average structure of the related high pressure phase bi2nimno6 , which is nominally mn and therefore free from j t distortion , also displays a range of mn o distances ( from 1.83(7 ) to 2.10(5) ) that is relatively wide when compared to the narrow distributions of ni t distortions in bi2mn4/3ni2/3o6 due to the presence of mn leads to an even wider range of mn o bond distances than is observed in bi2nimno6 . it and has been used to find a site cation ordering in caxsr1xtio3.1where 0 is the average number density of the material , cj is the proportion at atom j in the material , and gij is the partial distribution function for atoms i and j. n(r ) was calculated directly from the partial pair distribution function gb b(r ) where the average numbers of neighbors for each pair of b site cations between distances r1 and r2 have been extracted ( figure s3 ) . ni(r ) in the first shell and increase in the second are consistent with a bias toward rock salt ordering , which is observed in the related stoichiometric high pressure phase bi2mnnio6 . the regions of rock - salt - type ordering are clearly visible in the configuration despite the 2:1 mn / ni ratio of bmn being far from the ideal 1:1 ratio required for perfect rock salt ordering . instead of the rock - salt - type order observed in the rmc local structure , the incommensurate structure displays a more complex mn / ni clustering , forming 2 2 columns of nickel rich and manganese rich octahedral forming a checkerboard pattern . this long - range modulated ordering is not of rock - salt type , being derived from modulation bragg peaks of the type hklmn ( m , n = 1 ) , but it is possible that it is an average over ordered and disordered regions and that these disordered regions may include short - range rock - salt ordering . the short - range rock - salt - type cation order observed in the rmc analysis of the incommensurate rt structure persists at ht in the commensurate structure , with nni mn(r ) for the first nearest neighbor of 1.51(3 ) and 4.49(3 ) ( figure 6c , d ) . this result emphasizes how the single b site in the long - range average ht structure fails to fully describe the true local structure of ht bmn and confirms the information extracted from the more complex rt structure by rmc analysis . here the rmc local structure contains new information on the b site ordering , which is entirely absent from both the long - range incommensurate ( rt ) and commensurate ( ht ) structures . the rmc model gives a broader atomic displacement distribution than observed for the average incommensurate model ; this is consistent with the similarly broader rmc model gbi o(r ) distribution and is a consequence of thermal motion in the local structure . ( a ) comparison of total absolute displacements of the bi cations from the centroid of the coordinating oxygens between the rmc local structure and the average incommensurate model . thus , instead of observing polar nanoregions ( with ferroelectric correlations in the dcf ) , we observe antiferroelectric correlations consistent with the long - range modulated average structure . the rmc model ( d ) shows clusters of oppositely displaced groups of atoms , clearly indicating local ferroelectric correlations between the bi atoms , whereas the incommensurate model ( f ) shows sinusoidal behavior of displaced bi atoms resulting from the incommensurate character of this model . these calculations showed that local correlations of bi cation displacements do indeed exist within a distance of approximately 12 ( figure 12c ) when compared to the randomly distributed bi cations observed in the supercells generated from data - free refinements ( presented as an error bar ) . figure 9b ) are similar to the polar regions in the long - range approximant supercell of the incommensurate structure which cancel each other to produce long - range average antiferroelectric displacements . the size of the bmn polar domains is not easy to define from the long - range average structure . what we can say from long - range bragg diffraction is that the polar regions are probably at least the size of the polar approximant commensurate subcell ( p21mn , 22ap 4ap 2ap , a = 11.1496 , b = 15.5302 , and c = 5.5092 ) , given this reasonably good fit of this polar structure to the bragg data , and the absence of polarity in an approximate incommensurate supercell ( 712ap 38ap 2ap ) demonstrates that polar regions are at most half this larger size . by calculating s(n ) for bi in rt rmc models of the local structure bmn , we are able for the first time to quantify the size of the polar domains as existing within a domain of 12 , in reasonable agreement with the long axis dimensions of the polar approximant commensurate subcell . as opposed to the average commensurate subcell , which is entirely polar , and the approximate incommensurate supercell , where polarity is modulated in two dimensions , the local structure shows how bi displacements are correlated in three dimensions in real space . local correlations with dimensions of up to 12 persist in the ht phase , where the sum of bi atoms displaced in the same direction along 110p also creates polar clusters ( figure 13 ) . the observation of local ferroelectric correlations in the ht phase means that the local displacements of bi cations giving rise to short - range polar regions still exist above the phase transition from the rt incommensurate structure to the ht commensurate structure , despite there being no signs of this polar nature in the long - range average crystallographic structure , suggesting that these correlations are now dynamic . the b site environment at rt is significantly different in the rmc local structure and average incommensurate model . the application of bvs and interatomic potential constraints in the rmc modeling allowed us to differentiate between ni and mn on the b site , which share the same site in the incommensurate average structure . t distortions , gives rise to a wide range of mn o bond lengths that , together with the distinction between bi mn and bi ni distances seen in the local structure , will enable bmn to accommodate the off - center displacement of the bications required to explain its unusual stability to ambient pressure synthesis . the rmc local structure reveals rock - salt - type nearest neighbor ordering , which is adopted by the related stoichiometric high pressure phase bi2mnnio6 , with the additional disorder necessitated by the 2:1 mn / ni ratio of bmn . the incommensurate average structure is insensitive to this and produces mn / ni clustering on the b site corresponding to the periodicity of the modulation . , the simple rock - salt - like order on the b site . the same local rock salt ordering is observed for the ht phase , where the average pcnm commensurate structure displays no ordering whatsoever , although the peaks due to the long - range modulated cation order are still visible in the neutron diffraction data . the local structure analysis thus identifies three important chemical features for the stability of the material , namely , the short - range b site ordering , the distribution of bi o distances , and the wide range of b o distances accommodated by different mn charge states and a j t distortion . the local structure of biti3/8fe2/8mg3/8o3 reveals flexible coordination behavior of ti on the b site , which may play a similar role to the three mn o distances in the preferred j t distortion at mn here , but there is no b site ordering over any length scale in this commensurate system . the bi bi displacement correlation function along the same direction shows similar peaks in both rmc and average incommensurate models that are consistent with the antiferroelectric behavior of pbzro3 ; this feature is preserved in the local structure of the ht phase . the true nature of the polar behavior of bmn was revealed by calculating the sum of bi displacements within a volume , which showed that ferroelectric correlations along 110p within the rmc local structure at rt give rise to polar clusters with a size of up to 12 . this local structure analysis allowed the size of the polar domains , which is elusive from long - range average structure treatments , to be quantified for the first time . these local polar domains are preserved at ht , where the long - range average structure shows no signs of this underlying polar nature . here total scattering analysis has provided new insights into the incommensurate structure of the atmospheric pressure synthesized bi perovskite bi2mn4/3ni2/3o6 , revealing features of direct relevance for its unusual stability and explaining the structural and functional relationship to better - studied lead - based systems .

malaria continues to be a major global health problem exposing over 2000 million of the world 's population to varying degrees of malaria risk . of the four parasite species , p. falciparum , p. vivax , p. malariae , and p. ovale that cause malaria , p. falciparum and p. vivax are the two major parasite species seen in the country , india . it is estimated that 1.21.5 million new cases of malaria occur in india each year , 65% caused by p. vivax . in the city of mumbai , p. vivax accounts for 80% of the total malaria cases . despite this burden , p. vivax species remains less well studied . worldwide , the focus of research in malaria has centered around p. falciparum because of the development of resistance by this species to the standard antimalarial drugs and the mortality associated with the species . however , p. vivax is the most widely distributed parasite being the most common cause of malaria imported into many areas in which malaria is not endemic [ 35 ] with an increasing trend of severe and fatal cases and more importantly , the high morbidity associated with recurrence of infection caused by the relapses . despite numerous studies carried out to evaluate antirelapse potential of existing and new antimalarials such as tafenoquine , and its combinations [ 68 ] , primaquine ( pq ) , an 8-aminoquinoline , remains at present the only agent available worldwide to prevent relapses . unfortunately , there has been emergence of pq nonresponsiveness in patients with p. vivax who received world health organization ( who ) recommended antirelapse regimen , 15 mg / day for 14 days of primaquine [ 1114 ] . despite the surge of resistance monitoring , although protocols to culture the species ex vivo have been developed [ 1517 ] , they remain unsuited for routine assays . thus , well - conducted clinical trials still remain the mainstay and an essential component of p. vivax resistance monitoring . however , in clinical trials , identification of relapses gets further confounded by the inability to distinguish relapses , that is , pq treatment failures , from reinfections , that is , treatment successes which clinically or parasitologically manifest during the follow - up period on account of the infection with fresh mosquito bite . even though distinguishing recrudescence from reinfection has been largely overcome for p. falciparum by use of genotyping methods , thus , the primary objective of the present clinical study was to assess the efficacy of various pq regimens as antirelapse treatment along with no pq regimen for p. vivax malaria patients in mumbai , india , by monitoring the recurrence rate as well as distinguishing the recurrence as relapse or reinfection using three different methods . the secondary objectives of the study were to monitor the safety and find the concordance between the results of the three methods used to differentiate relapse and reinfection and the diversity of the types of strains prevalent in the samples of the study population . the institutional ethics committee approval and patient 's informed consent were obtained prior to enrollment in the study . it was an open label , randomized , controlled , parallel group , assessor blind study comparing antirelapse efficacy of 3 regimens of pq , 15 mg / day 14 days ( group b ) , 30 mg / day 7 days ( group c ) , and 30 mg / day 14 days ( group d ) along with a group of p. vivax patients who did not receive pq ( group a ) . patients suspected to be suffering from malaria were referred to malaria out - patient department ( opd ) , for microscopic diagnosis of malaria using peripheral blood smear stained using giemsa stain . patients diagnosed as positive for plasmodium vivax malaria , willing to participate in the study , and fulfilling inclusion exclusion criteria of the study were enrolled in the study . inclusion criteria were as follows : adult patients , male and female ( equal or over the age of 18 years);peripheral blood smear diagnosis of plasmodium vivax;willing to undergo hospitalization for the entire duration of primaquine treatment;willing to provide informed consent;willing to undergo investigations and come for regular followup;normal g6pd;hemoglobin greater or equal to 10 gm / dl . adult patients , male and female ( equal or over the age of 18 years ) ; peripheral blood smear diagnosis of plasmodium vivax ; willing to undergo hospitalization for the entire duration of primaquine treatment ; willing to provide informed consent ; willing to undergo investigations and come for regular followup ; hemoglobin greater or equal to 10 gm / dl . exclusion criteria were as follows : mixed infection with plasmodium falciparum;pregnancy and lactation;evidence of significant hepatic , renal , or cardiac disease as diagnosed by history , clinical examination , and laboratory tests whenever necessary;any other condition which would interfere with patient 's participation in the study or compliance with the treatment . mixed infection with plasmodium falciparum ; pregnancy and lactation ; evidence of significant hepatic , renal , or cardiac disease as diagnosed by history , clinical examination , and laboratory tests whenever necessary ; any other condition which would interfere with patient 's participation in the study or compliance with the treatment . all the patients enrolled in the study were given the standard who recommended treatment regimen of chloroquine ( cq ) . the treatment was initiated on day 1 with 10 mg / kg of cq . subsequently , patient received 10 mg / kg and 5 mg / kg of cq on days 2 and 3 , respectively . on day 4 , as per the randomization , they received one of the three pq regimens : 15 mg / day 14 days ( b ) , 30 mg / day 7 days ( c ) , or 30 mg / day 14 days ( d ) . the patients who were not eligible to receive pq for a number of reasons such as hg < 10 gm / dl , g6pd deficiency , pregnancy , and lactation formed the no pq group ( a ) . the primary efficacy outcome of the study was the number of patients showing recurrence , relapse , and reinfection of p. vivax infection . the secondary outcome , safety , was assessed by monitoring the adverse events observed in patients . the adverse event evaluation was carried out by clinicians by monitoring the adverse events ( symptoms , signs ) that were not present at baseline or worsened during the study . the secondary outcome also included comparison of number of patients classified as relapse and reinfection by the three methods to find out the concordance between the methods used and the genetic diversity observed based on pcr sequencing method . the pq effect was assessed by regular followup once in a month , after the first 28 days for cq sensitivity monitoring , by examination of peripheral blood smear until next 6 months . the peripheral smear examination was also done whenever patients developed fever within the 6-month follow - up period . patients showing reappearance of p. vivax parasitemia between 1 to 6 months were considered as cases of recurrence . the cases of recurrence were classified as relapse or reinfection based on the three methods , the month of recurrence , and the two genotyping methods : pcr - rflp and pcr sequencing . the recurrence was classified as relapse if it occurred in the period between january and june ( low transmission season ) and reinfection if it occurred in the period between july and december ( active transmission season ) . this method of genotyping involved use of p. vivax msp3 ( pvmsp3 ) and msp3 ( pvmsp3 ) as polymorphic regions . the protocols followed were adopted from the available literature [ 22 , 23 ] . the restriction enzyme ( re ) banding patterns observed on the agarose gel for the paired samples were matched visually to classify them as relapse if it was the same and reinfection if it was different . the results of both the genes used ( pvmsp3 or pvmsp3 ) were pooled for the purposes of genotyping data analysis . when the rflp pattern for pvmsp3 and pvmsp3 indicated contradictory results , meaning one showing same pattern while the other showing different rflp pattern for the same paired sample , the parasite strains were considered as same strains thus categorizing the paired sample as relapse . the basis for this consideration was that it was unlikely to not see polymorphism , if present , for the polymorphic sites used . thus the detection of same parasite with such a polymorphic region indicates presence of same parasite clones in the paired sample which means relapse . the presence of different clones as indicated with another re probably points to either the presence of multiple clones in one of the samples or reactivation of one of the clones from primary infection . this was carried out by selecting the polymorphic region of p. vivax msp1 ( pvmsp1 ) gene . the method was performed at one of the commercial laboratories , namely , chromous biotech pvt . the parasite dna was amplified and the pcr product was sequenced using abi big - dye terminator cycle sequencing kit in abi3100 sequencing machine . the amino acid sequences obtained from different samples were aligned using multiple alignment software ( clustal w ) . based on the variation obtained , our previous study has shown that patients not treated with pq show the recurrence of p. vivax in 1520% of patients [ 14 , 26 ] . also , the 15 mg / d 14 days regimen of pq is associated with 5% recurrence rate of vivax malaria of which 2% are true relapses . in the present study , it was to be assessed if 30 mg / day 7 days and 30 mg / day 14 days , which contain equivalent / higher dose of pq , will have either equivalent ( 5% ) or 0% recurrence rate . therefore the sample size of 120 per group at 5% significance and 80% power of the study were initially considered adequate to detect the difference . however , eventually , we expected that the difference could be lower and also malaria being public health issue , we took larger sample size . a simple , computer generated randomization scheme was used for the randomization of patients into the three pq regimen groups . the patients fulfilling the inclusion exclusion criteria and consenting to participate in the study were enrolled in the study . the blood spots were blotted on the filter paper ( pretreatment sample ) for genotyping analysis . all the patients enrolled in the study were hospitalized either for 10 or 17 days depending on the pq group they were randomized to and then treated with standard who recommended treatment regimen of cq for 3 days , followed by pq as per randomization . all the patients were given follow - up card at the time of discharge and were asked to follow up once a month for subsequent six months and also as when they get fever . in order to get maximum follow - up data and reduce drop outs due to lost to followup active followup was also done when the laboratory assistant visited the homes of the patients and made blood smears , thus avoiding the need for the patient to visit the hospital . any of these patients diagnosed to be positive for p. vivax during the period between 1 to 6 months of followup were considered cases of recurrence . these cases were readmitted and 3 ml of blood was collected again for hemoglobin measurement and five drops of blood were blotted on a filter paper ( posttreatment sample ) . the paired pre- and posttreatment samples were subjected to genotyping analysis to distinguish recurrence as relapse or reinfection . the flow chart given at the end shows the schematic presentation of the plan of the study ( figure 1 ) . although the study was not blinded in terms of treatment administration , the person seeing the slides and carrying out other outcome assessments were blinded to the treatment group by coding the samples . chi - square test was applied to find the differences between the 4 groups for baseline characteristics . the total number and percentages of patients showing recurrence of p. vivax malaria were expressed as a percent of the total number of patients who completed the 6-month followup of the study period . these percentages were compared amongst groups using the chi - square or fisher 's test as appropriate . the number of paired samples available were subjected to classification of relapse and reinfection based on month of recurrence and genotyping ( pcr - rflp , pcr sequencing ) . owing to the nonavailability of some of the paired samples for genotyping as well as nonamplification of some of the samples on account of the genotyping technique flaws , genotyping results were not available for all the samples . to compensate for the loss of data due to reasons listed , relapse and reinfection rates were extrapolated to total number of patients showing recurrence . the data of pcr sequencing were used to depict the diversity of different genotypes prevalent in the samples studied . the number of patients in each group who had adverse events or discontinued medication due to adverse events was expressed as a percent of the total . the percentages were compared using the chi - square or fisher 's test as appropriate . for the purpose of the study , any adverse events ( irrespective of cause - effect relationship ) which were not present at baseline or worsened after baseline were taken for statistical analysis . overall 4215 ( 8.15% ) cases of p. vivax were diagnosed in the total of 51,709 suspected cases screened , over a period from august 01 to feberuary 04 . out of 4215 patients , 1556 patients were part of the study and 1,242 completed the 6-month followup , ( group a = 305 , group b = 322 , group c = 298 , and group d = 317 ) . there was no difference in the baseline demographic characteristics of the study population ( table 1 ) . all the 1556 patients ( except 1 who was discontinued cq due to ae ) in 4 groups ( a , b , c , and d ) showed clearance of parasites by day 6 from the initiation of treatment , without subsequent reappearance until d28 , showing that all p. vivax strains were sensitive to chloroquine . there were a total of 127 patients who showed recurrence of p. vivax malaria , giving the cumulative recurrence incidence of 10% ( 127/1242 ) . the number of patients showing recurrence of vivax parasitemia were 50 ( 16.39% ) , 26 ( 8.07% ) , 30 ( 10.07% ) , and 21 ( 6.62% ) in groups a , b , c , and d , respectively . the rate of recurrence varied significantly in the 4 groups ( p = 0.004 ) . the number of patients showing recurrence was higher in patients who did not receive pq as compared to those who received different regimens of pq ( table 2 ) . ( 2 ) differentiation of recurrence as relapse or reinfection ( 2.1 ) as per the month of recurrence . the relapse rate was 6.89% , 1.55% , 4% , and 3.85% and the reinfection rate was 9.51% , 6.52% , 6% , and 5.13% in groups a , b , c , and d , respectively . there was significant difference in the relapse rate in 4 groups ( p = 0.009 ) . the relapse rate was higher in no pq group ( 6.89% ) as compared to the relapse rate in three pq groups ( 1.55% , 4% , and 3.85% ) ( table 2 ) . ( i ) pcr - rflp : pvmsp3 and pvmsp3. the relapse rate was 8.2% , 2% , 4.58% , and 3.68% and the reinfection rate was 8.2% , 5.82% , 5.49% , and 2.9% for groups a , b , c , and d , respectively . there was significant difference in the relapse rate in 4 groups ( p = 0.007 ) ( table 2 ) . the relapse rate was 2.73% , 1.47% , 1.55% , and 1.53% while the reinfection rate was 13.66% , 6.60% , 8.52% , and 5.09% in groups a , b , c , and d , respectively ( table 2 ) . out of 1556 patients , 9 ( 0.57% ) patients ( a = 1 , b = 0 , c = 4 , and d = 4 ) reported adverse events during the cq treatment . the number of aes reported was significantly different in the 4 groups ( p = 0.033 ) ( table 3 ) . the aes seen were itching ( 5 ) , burning of palms ( 1 ) , abdominal pain ( 1 ) , loose motion ( 1 ) , and heat boils ( 1 ) . of these 9 aes , 1 ae of itching and mucous membrane on lips lead to discontinuation of cq on d3 . out of 1556 patients , 31 ( 1.99% ) patients ( a = 3 , b = 5 , c = 10 , and d = 13 ) reported adverse events during pq treatment ( after completion of cq treatment ) . the number of aes was significantly different in the 4 groups ( p = 0.006 ) ( table 3 ) . the aes seen were nausea ( 1 ) acidity ( 02 ) , weakness ( 1 ) , itching ( 09 ) , acidity plus itching ( 01 ) , abdominal pain ( 04 ) , acidity plus abdominal pain ( 01 ) , epigastric pain ( 02 ) , epigastric burning ( 01 ) , weakness plus abdominal pain ( 01 ) , vomiting ( 01 ) , mucocutaneous lesions on left leg and gluteal region ( 01 ) , boils on forehead and back with pus ( 01 ) , boils rash - maculopapular - all over body ( 01 ) , swelling of upper lips , legs , and palms with itching ( 01 ) , severe pruritus ( 01 ) , morbilliform rash in fingers ( 01 ) , and boils inside the mouth and tongue ( 01 ) . of these 31 patients , aes in 11 patients ( a = na , b = 2 , c = 2 , and d = 7 ) lead to discontinuation of pq treatment . the number of aes leading to discontinuation of pq treatment differed significantly in the 4 groups studied ( p = 0.003 ) ( table 3 ) . these aes were itching ( 03 ) , mucocutaneous lesions on the lower lip and gluteal region ( 01 ) , formation of boils on forehead and back with pus ( 01 ) , abdominal pain ( 01 ) , acidity plus abdominal pain ( 01 ) , morbilliform rash in fingers ( 01 ) , epigastric pain ( 01 ) , boils inside the mouth and tongue ( 01 ) , and vomiting ( 01 ) . the number of drop outs due to aes in 4 groups was significantly different ( p = 0.001 ) . the number of aes was significantly higher in group d as compared to other groups ( table 3 ) . there were 33 paired samples for which the results from all the three methods were available to assess the concordance . the concordance between all the three methods was 45% whereby all the three methods classified the case of recurrence as relapse or recurrence in the same way . while relatively high concordance was noted between pcr - rflp and pcr sequencing ( 57.58% ) , good concordance was also noted between the month of recurrence and pcr - rflp ( 48.48% ) but little concordance between month of recurrence and pcr sequencing method ( 15.15% ) ( table 4 ) . the rates of relapse and reinfection identified by the method of month of recurrence and pcr - rflp were found to be in agreement . while pcr sequencing results were bit different than the results of both these methods of classification used ( table 2 ) . fifty - two genetic subtypes were identified in a total of 143 isolates ( paired primary and recurrence samples ) analyzed . the present comparative study carried out to assess safety and efficacy of different pq regimens as antirelapse treatment identified a higher recurrence rate in the patients who did not receive pq treatment as compared to the patients who received different pq regimens . when the cases of recurrence were subjected to the methods differentiating relapse and reinfection , majority of them were found to be reinfection cases . however , significantly higher adverse events in higher dose of pq , group d ( 30 mg 14 days ) , leading to significantly higher drop outs were noted . discordance was noted in the results of the methods used to differentiate cases of recurrence as relapse or reinfection . the study also documented a lot of genetic diversity in the types of genotype identified using the pcr sequencing method of genotyping indicating the diversity of type of p. vivax strains prevalent in the population ( geographic area ) studied . the low recurrence rate found in the patients receiving different pq regimens as compared to patients not receiving pq ( 16.3% ) is indeed a good indicator of pq effect and thus it is used as antirelapse treatment . the earlier study in the same population has documented recurrence rate of 9.2% in patients not treated with pq . the rates of recurrence as high as 40% in india and 31% in pakistan have been documented in previous studies for patients not given pq treatment . for example , the study in the same population indicated recurrence rate of 4.6% for the 15 mg 14 days pq regimen . this recurrence rate seems to have doubled over a period as found in the current study . diminished effect of pq , given in the standard dose of 15 mg 14 days , has already been reported in earlier studies such as smoak et al . which reported recurrence rate as high as 43% in somalia army troops while bunnag et al . on the other hand , leslie et al . reported pretty low recurrence rates ( 1.8% ) for this regimen . in order to increase the effectiveness of pq , use of higher dosages of pq has been tried including a latest case report being successfully treated with high dose pq as high as 45 mg once weekly for 8 weeks at 3rd instance ; after the failure of 15 mg / d for 2 weeks at 1st instance and 7.5 mg 4 times daily for 2 weeks at 2nd instance for radical cure of vivax malaria . though there are not many studies assessing high dose pq , a study administering higher pq regimen did not show 100% effectiveness , reporting 89% cure rate for the 30 mg / day 7 days pq regimen similar to the recurrence rate of 10.07% observed in the current study . the recurrence of p. vivax at even higher dose pq ( 30 mg / day for 14 days ) has been documented earlier [ 30 , 31 ] . several factors including the type of prevalent genetic strains of vivax as well as the varied length of follow - up period used in the studies might explain the variations observed in these studies . the period of followup is an important consideration for observing the radical cure of p. vivax . with longer follow - up period , the chance of detecting more positive cases increases . thus , the comparison of findings from different studies require due consideration of the follow - up period which might vary among studies . also , the major limitation in assessing the effect of pq as antirelapse is the confounding effect of reinfection in the area where malaria is endemic . several of these earlier studies lack the data on pcr or genotyping corrected relapse rates . the present study used 3 methods to differentiate the cases of recurrence as relapse or reinfection in order to better assess the antirelapse effect of pq . it was noted that , although recurrence rates were high , when these cases of recurrence were classified as relapse or reinfection using different methods , relapse rates observed were much lower . there is not much research involving genotyping of paired samples to compare with the present study . however , the pcr corrected rates of relapse observed in the current study were comparable to our earlier study reporting the true relapse ( pcr corrected ) rate of 2% ( pcr - sscp ) for 15 mg / day 14 days as compared to 1.47% ( pcr sequencing ) and 2% ( pcr - rflp ) noted in the present study . the relapse and reinfection rates varied between the three methods used in the present study leading to discordance in the results of the three methods used to differentiate the cases of recurrence . there could be several reasons for this discordance ranging from the varying sensitivity and specificity of the method , the polymorphic markers used and certain unresolved issues like infection with multiple clones . for example , pcr sequencing technique could detect higher reinfection rate because it can differentiate the two isolates more precisely as compared to pcr - rflp technique . the recent genotyping study done to find the genetic diversity of p. vivax in kolkata , india , by kim et al . , using three polymorphic markers ( pvcs , pvmsp1 , and pvmsp3 ) concluded that genotyping protocols used by them may be useful for differentiating reinfection from relapse and recrudescence in studies assessing antimalarial drug efficacy . two of these polymorphic markers were used in the present study . however , if the patient is getting infected with multiple clones , any method or test may not reliably differentiate relapse and reinfection . thus differentiation of relapse and reinfection remains a major roadblock in assessing efficacy of antirelapse activity of antimalarials . in addition to the limitation of the methods itself , the study by imwong et al . has revealed heterologous reactivation of hypnozoites leading to recurrence , even in the patients from area where reinfection was ruled out . in such a case , observation of different genotypes in the recurrence sample does not indicate reinfection . if this is the case , then all efforts invested in differentiating the recurrence on the basis of genotyping technique seem to be in vain . this drawback of the technique , when mixed clones are present at the time of primary infection , has also been acknowledged previously by kirchgatter and del portillo . probably , cloning experiments , which were not part of the current study , might provide some insight into the presence or absence of multiple clones in the given samples . it is also observed that the technique used for genotyping plays a very crucial role . pcr sequencing being the standard technique , faces the challenge of studying the products with higher base pairs , need of sequencer , while pcr - rflp although being easy to perform with less infrastructure and also cost effective , is not precise . apart from visual inaccuracies , even the insufficient pcr amplification product and amplification errors may hinder the appropriate matching and in turn interpretation of relapse and reinfection based on rflp pattern . as far as selection of marker for genotyping is concerned , it is the highly polymorphic gene that is preferred . however , if the region to be studied is highly polymorphic then it leads to amplification problem due to the polymorphism existing in the primer binding region . we used msp-3 gene family for rflp genotyping on account of the polymorphism documented with this gene . however , recent research also documents extensive recombination as well as gene conversion in this gene . this means , p. vivax genotype identified at the instance of recurrence , even though may belong to the original vivax genotype present at the time of first episode , might have undergone recombination leading to identification of different genotype at the time of recurrence classifying it as reinfection . however , for the current study we can assume this effect to be constant across the four groups compared making the final interpretations still valid . further studies in various other geographic areas are needed for taking decision on policy change , even if that has to be made . in the present the reasons of nonamplification could be varied viz ; absence of or less amount of dna in the sample , presence of pcr inhibitors in the isolated dna , polymorphism present in the primer binding site itself ( high diversity observed in the polymorphic regions studied ) . the pcr sequencing ( msp1 ) used in the study did involve different primer design for some of the nonamplifying samples ( data not shown ) . studying multiple polymorphic regions and their respective primers may yield more data ( as seen in the present study , few samples not amplifying with msp3 ; showed amplification with msp3 and vice a versa ) . the study also determined the extent of polymorphism in p. vivax in mumbai , india as a part of the pcr sequencing method employed for genotyping analysis . this was important , as the data regarding the sequence variations among p. vivax indian field isolates is scanty . the results showed 52 genetic subtypes in a total of 143 isolates ( paired primary and recurrence samples ) analyzed and a quite high pvmsp-1 polymorphism as analyzed by pcr sequencing method . the analysis and studying sequence of more than one polymorphic region would yield even more p. vivax sub - types . the simultaneous use of more than one genetic marker in this kind of study may enhance the knowledge of genetic diversity existing in the parasite populations . nevertheless , high genetic diversity has been reported with the use of one msp-1 gene and therefore , use of this marker is justified for the purposes of genotyping . the genetic diversity of p. vivax has also been demonstrated earlier in other geographic areas . understanding the extent of polymorphism in msp-1 and the resulting genetic diversity in p. vivax populations could help in implementing malaria control activities , being a crucial step for the development of a malaria vaccine although the significance of the diverse polymorphisms and their prevalence is unknown . from this study it is evident that , although the incidence of probable resistance to various pq regimens , used in the present study , is fairly low at this point ( relapse rate ranging from 1.47% to 4.58% using different methods of classification of recurrence ) , it is anticipated that resistance is likely to increase and therefore , continuous monitoring of the therapy with pq is warranted . one should keep in mind the propagation of the resistant strain which may lead to increased relapse rate in near future . also , this is an indication of declining efficacy of pq which could be due to increasing resistance to the drug . one of the major limitation of the study is the nonrandomization of the no pq group due to ethical reasons which forced us to select the patients for this group based on those who did not receive pq because of the clinical reasons such as hg < 10 gm / dl ; g6pd deficiency , pregnancy , lactation , and so forth . however , we do not believe these variables might have had any effect on the recurrence rate . additionally , even though we used this group to compare with rest of the pq regimens tested , the comparative number of recurrence within 3 pq groups presented in the table are self - explanatory . the number thus presented in the study may be an underestimation as the late relapses may not have been adequately captured . the longer follow - up period pose challenge of more drop outs on account of data lost to followup . additionally , because this was a comparative study it can be assumed that the longer followup may not influence the comparison even with increase in the number of patients showing recurrence . the study used month of recurrence as one of the method of classifying recurrence as relapse or reinfection . while a case may be made for relapses during low - transmission season , no case at all may be made for classifying recurrences as reinfection during the transmission season . however , typically a trend in increase in cases of malaria is noted during the monsoon season in mumbai , the geographic area where the study was carried out . in view of this scenario our objective of including this method in the current study was to basically compile the possible methods of differentiation and present an analysis of the data even though it may not be an accurate method . this variable might have helped in calculating per kg dose of the pq that patients received . the low total pq dose per kilogram of body weight [ 28 , 30 , 38 ] as well as genetic mutations in cyp2d6 may influence the effective pq dose available and low dose leading to relapse . however , as per who recommendations , per kg pq dose is 0.25 mg base / kg body weight . considering the average body weight of indian subjects ranging between 50 and 60 kg , the regimens used in the current study would yield the recommended per kg dose thus over ruling the confounding effect of the weight . also , the prevalence of cyp2d6 polymorphism is reported to be as low as 3.36% . additionally higher pq dose included in the present study and the recurrence and relapse rates observed in this group likely overcome the confounding effect of these factors . several factors related to parasite variants , host factors and the epidemiological factors including vector control , transmission dynamics ; the limitations of the existing methods to identify true relapses can influence and confound the analysis of true resistance to pq . this indicates that in the absence of reliable genotyping methods , the recurrence monitoring can be a good surrogate marker to compare provided it is appropriate to assume the constant reinfection rate in the given geographical area where the study population is residing . the study documented statistically significant higher recurrence rate in patients not given pq as compared to patients receiving different pq regimens . there was discordance in the methods ( namely , month of recurrences , rflp , or sequencing ) used to differentiate relapse from reinfection . the lack of concordance in the methods used has implications on the interpretation of the results and therefore warrants caution . using the available methods , the relapse rate ranging from 1.47% to 4.58% was noted in the patients receiving different pq regimens . considering that the paired samples classified as reinfection by rflp or sequencing could be the cases of relapse , the probable resistance to pq could be even higher indicating the decreased effect of pq to eradicate liver forms of p. vivax , hypnozoites . the study found that pq regimens used to be safe without any major or unexpected adverse events , although statistically significantly higher adverse events were noted in the higher dose pq ( 30 mg 14 days ) leading to significantly higher drop outs due to discontinuation of the treatment . the study also documented lots of genetic diversity in the types of genotype identified using the pcr sequencing method of genotyping , indicating diversity of type of p. vivax strains prevalent in the population ( geographic area ) studied .

background . efficacy of standard dose of primaquine ( pq ) as antirelapse for p. vivax has decreased . we aimed to assess efficacy of different pq regimens . methods . it was an open label , randomized , controlled , parallel group , assessor blind study comparing antirelapse efficacy of 3 pq regimens ( b = 15 mg / day 14 days , c = 30 mg / day 7 days , and d = 30 mg / day 14 days ) with no pq group ( a ) in p. vivax patients . paired primary and recurrence samples were subjected to 3 methods : ( i ) month of recurrence and genotyping , ( ii ) by pcr - rflp , and ( iii ) pcr sequencing , to differentiate relapse and reinfection . the rates of recurrence relapse and reinfection were compared . methods were compared for concordance between them . results . the recurrence rate was 16.39% , 8.07% , 10.07% , and 6.62% in groups a , b , c , and d , respectively ( p = 0.004 ) . the relapse rate was 6.89% , 1.55% , 4% , and 3.85% as per the month of recurrence ; 8.2% , 2% , 4.58% , and 3.68% ( p = 0.007 ) as per pcr - rflp ; and 2.73% , 1.47% , 1.55% , and 1.53% as per pcr sequencing for groups a , b , c , and d , respectively . the concordance between methods was low , 45% . conclusion . the higher recurrence rate in no pq as compared to pq groups documents pq antirelapse activity . regimens tested were safe . however , probable resistance to pq warrants continuous monitoring and low concordance and limitations in the methods warrant caution in interpreting .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Limitations 6. Conclusions

of the four parasite species , p. falciparum , p. vivax , p. malariae , and p. ovale that cause malaria , p. falciparum and p. vivax are the two major parasite species seen in the country , india . in the city of mumbai , p. vivax accounts for 80% of the total malaria cases . however , p. vivax is the most widely distributed parasite being the most common cause of malaria imported into many areas in which malaria is not endemic [ 35 ] with an increasing trend of severe and fatal cases and more importantly , the high morbidity associated with recurrence of infection caused by the relapses . despite numerous studies carried out to evaluate antirelapse potential of existing and new antimalarials such as tafenoquine , and its combinations [ 68 ] , primaquine ( pq ) , an 8-aminoquinoline , remains at present the only agent available worldwide to prevent relapses . unfortunately , there has been emergence of pq nonresponsiveness in patients with p. vivax who received world health organization ( who ) recommended antirelapse regimen , 15 mg / day for 14 days of primaquine [ 1114 ] . even though distinguishing recrudescence from reinfection has been largely overcome for p. falciparum by use of genotyping methods , thus , the primary objective of the present clinical study was to assess the efficacy of various pq regimens as antirelapse treatment along with no pq regimen for p. vivax malaria patients in mumbai , india , by monitoring the recurrence rate as well as distinguishing the recurrence as relapse or reinfection using three different methods . the secondary objectives of the study were to monitor the safety and find the concordance between the results of the three methods used to differentiate relapse and reinfection and the diversity of the types of strains prevalent in the samples of the study population . it was an open label , randomized , controlled , parallel group , assessor blind study comparing antirelapse efficacy of 3 regimens of pq , 15 mg / day 14 days ( group b ) , 30 mg / day 7 days ( group c ) , and 30 mg / day 14 days ( group d ) along with a group of p. vivax patients who did not receive pq ( group a ) . patients diagnosed as positive for plasmodium vivax malaria , willing to participate in the study , and fulfilling inclusion exclusion criteria of the study were enrolled in the study . exclusion criteria were as follows : mixed infection with plasmodium falciparum;pregnancy and lactation;evidence of significant hepatic , renal , or cardiac disease as diagnosed by history , clinical examination , and laboratory tests whenever necessary;any other condition which would interfere with patient 's participation in the study or compliance with the treatment . mixed infection with plasmodium falciparum ; pregnancy and lactation ; evidence of significant hepatic , renal , or cardiac disease as diagnosed by history , clinical examination , and laboratory tests whenever necessary ; any other condition which would interfere with patient 's participation in the study or compliance with the treatment . subsequently , patient received 10 mg / kg and 5 mg / kg of cq on days 2 and 3 , respectively . on day 4 , as per the randomization , they received one of the three pq regimens : 15 mg / day 14 days ( b ) , 30 mg / day 7 days ( c ) , or 30 mg / day 14 days ( d ) . the patients who were not eligible to receive pq for a number of reasons such as hg < 10 gm / dl , g6pd deficiency , pregnancy , and lactation formed the no pq group ( a ) . the primary efficacy outcome of the study was the number of patients showing recurrence , relapse , and reinfection of p. vivax infection . the secondary outcome also included comparison of number of patients classified as relapse and reinfection by the three methods to find out the concordance between the methods used and the genetic diversity observed based on pcr sequencing method . the cases of recurrence were classified as relapse or reinfection based on the three methods , the month of recurrence , and the two genotyping methods : pcr - rflp and pcr sequencing . the recurrence was classified as relapse if it occurred in the period between january and june ( low transmission season ) and reinfection if it occurred in the period between july and december ( active transmission season ) . the restriction enzyme ( re ) banding patterns observed on the agarose gel for the paired samples were matched visually to classify them as relapse if it was the same and reinfection if it was different . based on the variation obtained , our previous study has shown that patients not treated with pq show the recurrence of p. vivax in 1520% of patients [ 14 , 26 ] . also , the 15 mg / d 14 days regimen of pq is associated with 5% recurrence rate of vivax malaria of which 2% are true relapses . in the present study , it was to be assessed if 30 mg / day 7 days and 30 mg / day 14 days , which contain equivalent / higher dose of pq , will have either equivalent ( 5% ) or 0% recurrence rate . all the patients enrolled in the study were hospitalized either for 10 or 17 days depending on the pq group they were randomized to and then treated with standard who recommended treatment regimen of cq for 3 days , followed by pq as per randomization . any of these patients diagnosed to be positive for p. vivax during the period between 1 to 6 months of followup were considered cases of recurrence . the paired pre- and posttreatment samples were subjected to genotyping analysis to distinguish recurrence as relapse or reinfection . the number of paired samples available were subjected to classification of relapse and reinfection based on month of recurrence and genotyping ( pcr - rflp , pcr sequencing ) . to compensate for the loss of data due to reasons listed , relapse and reinfection rates were extrapolated to total number of patients showing recurrence . the data of pcr sequencing were used to depict the diversity of different genotypes prevalent in the samples studied . out of 4215 patients , 1556 patients were part of the study and 1,242 completed the 6-month followup , ( group a = 305 , group b = 322 , group c = 298 , and group d = 317 ) . all the 1556 patients ( except 1 who was discontinued cq due to ae ) in 4 groups ( a , b , c , and d ) showed clearance of parasites by day 6 from the initiation of treatment , without subsequent reappearance until d28 , showing that all p. vivax strains were sensitive to chloroquine . the number of patients showing recurrence of vivax parasitemia were 50 ( 16.39% ) , 26 ( 8.07% ) , 30 ( 10.07% ) , and 21 ( 6.62% ) in groups a , b , c , and d , respectively . the rate of recurrence varied significantly in the 4 groups ( p = 0.004 ) . the number of patients showing recurrence was higher in patients who did not receive pq as compared to those who received different regimens of pq ( table 2 ) . ( 2 ) differentiation of recurrence as relapse or reinfection ( 2.1 ) as per the month of recurrence . the relapse rate was 6.89% , 1.55% , 4% , and 3.85% and the reinfection rate was 9.51% , 6.52% , 6% , and 5.13% in groups a , b , c , and d , respectively . there was significant difference in the relapse rate in 4 groups ( p = 0.009 ) . the relapse rate was higher in no pq group ( 6.89% ) as compared to the relapse rate in three pq groups ( 1.55% , 4% , and 3.85% ) ( table 2 ) . ( i ) pcr - rflp : pvmsp3 and pvmsp3. the relapse rate was 8.2% , 2% , 4.58% , and 3.68% and the reinfection rate was 8.2% , 5.82% , 5.49% , and 2.9% for groups a , b , c , and d , respectively . there was significant difference in the relapse rate in 4 groups ( p = 0.007 ) ( table 2 ) . the relapse rate was 2.73% , 1.47% , 1.55% , and 1.53% while the reinfection rate was 13.66% , 6.60% , 8.52% , and 5.09% in groups a , b , c , and d , respectively ( table 2 ) . out of 1556 patients , 9 ( 0.57% ) patients ( a = 1 , b = 0 , c = 4 , and d = 4 ) reported adverse events during the cq treatment . the number of aes reported was significantly different in the 4 groups ( p = 0.033 ) ( table 3 ) . out of 1556 patients , 31 ( 1.99% ) patients ( a = 3 , b = 5 , c = 10 , and d = 13 ) reported adverse events during pq treatment ( after completion of cq treatment ) . the number of aes was significantly different in the 4 groups ( p = 0.006 ) ( table 3 ) . of these 31 patients , aes in 11 patients ( a = na , b = 2 , c = 2 , and d = 7 ) lead to discontinuation of pq treatment . the number of aes leading to discontinuation of pq treatment differed significantly in the 4 groups studied ( p = 0.003 ) ( table 3 ) . the number of drop outs due to aes in 4 groups was significantly different ( p = 0.001 ) . there were 33 paired samples for which the results from all the three methods were available to assess the concordance . the concordance between all the three methods was 45% whereby all the three methods classified the case of recurrence as relapse or recurrence in the same way . while relatively high concordance was noted between pcr - rflp and pcr sequencing ( 57.58% ) , good concordance was also noted between the month of recurrence and pcr - rflp ( 48.48% ) but little concordance between month of recurrence and pcr sequencing method ( 15.15% ) ( table 4 ) . the rates of relapse and reinfection identified by the method of month of recurrence and pcr - rflp were found to be in agreement . fifty - two genetic subtypes were identified in a total of 143 isolates ( paired primary and recurrence samples ) analyzed . the present comparative study carried out to assess safety and efficacy of different pq regimens as antirelapse treatment identified a higher recurrence rate in the patients who did not receive pq treatment as compared to the patients who received different pq regimens . when the cases of recurrence were subjected to the methods differentiating relapse and reinfection , majority of them were found to be reinfection cases . however , significantly higher adverse events in higher dose of pq , group d ( 30 mg 14 days ) , leading to significantly higher drop outs were noted . discordance was noted in the results of the methods used to differentiate cases of recurrence as relapse or reinfection . the study also documented a lot of genetic diversity in the types of genotype identified using the pcr sequencing method of genotyping indicating the diversity of type of p. vivax strains prevalent in the population ( geographic area ) studied . the low recurrence rate found in the patients receiving different pq regimens as compared to patients not receiving pq ( 16.3% ) is indeed a good indicator of pq effect and thus it is used as antirelapse treatment . the earlier study in the same population has documented recurrence rate of 9.2% in patients not treated with pq . the rates of recurrence as high as 40% in india and 31% in pakistan have been documented in previous studies for patients not given pq treatment . for example , the study in the same population indicated recurrence rate of 4.6% for the 15 mg 14 days pq regimen . diminished effect of pq , given in the standard dose of 15 mg 14 days , has already been reported in earlier studies such as smoak et al . in order to increase the effectiveness of pq , use of higher dosages of pq has been tried including a latest case report being successfully treated with high dose pq as high as 45 mg once weekly for 8 weeks at 3rd instance ; after the failure of 15 mg / d for 2 weeks at 1st instance and 7.5 mg 4 times daily for 2 weeks at 2nd instance for radical cure of vivax malaria . though there are not many studies assessing high dose pq , a study administering higher pq regimen did not show 100% effectiveness , reporting 89% cure rate for the 30 mg / day 7 days pq regimen similar to the recurrence rate of 10.07% observed in the current study . the recurrence of p. vivax at even higher dose pq ( 30 mg / day for 14 days ) has been documented earlier [ 30 , 31 ] . also , the major limitation in assessing the effect of pq as antirelapse is the confounding effect of reinfection in the area where malaria is endemic . the present study used 3 methods to differentiate the cases of recurrence as relapse or reinfection in order to better assess the antirelapse effect of pq . however , the pcr corrected rates of relapse observed in the current study were comparable to our earlier study reporting the true relapse ( pcr corrected ) rate of 2% ( pcr - sscp ) for 15 mg / day 14 days as compared to 1.47% ( pcr sequencing ) and 2% ( pcr - rflp ) noted in the present study . the relapse and reinfection rates varied between the three methods used in the present study leading to discordance in the results of the three methods used to differentiate the cases of recurrence . for example , pcr sequencing technique could detect higher reinfection rate because it can differentiate the two isolates more precisely as compared to pcr - rflp technique . however , if the patient is getting infected with multiple clones , any method or test may not reliably differentiate relapse and reinfection . thus differentiation of relapse and reinfection remains a major roadblock in assessing efficacy of antirelapse activity of antimalarials . in such a case , observation of different genotypes in the recurrence sample does not indicate reinfection . pcr sequencing being the standard technique , faces the challenge of studying the products with higher base pairs , need of sequencer , while pcr - rflp although being easy to perform with less infrastructure and also cost effective , is not precise . this means , p. vivax genotype identified at the instance of recurrence , even though may belong to the original vivax genotype present at the time of first episode , might have undergone recombination leading to identification of different genotype at the time of recurrence classifying it as reinfection . the study also determined the extent of polymorphism in p. vivax in mumbai , india as a part of the pcr sequencing method employed for genotyping analysis . the results showed 52 genetic subtypes in a total of 143 isolates ( paired primary and recurrence samples ) analyzed and a quite high pvmsp-1 polymorphism as analyzed by pcr sequencing method . understanding the extent of polymorphism in msp-1 and the resulting genetic diversity in p. vivax populations could help in implementing malaria control activities , being a crucial step for the development of a malaria vaccine although the significance of the diverse polymorphisms and their prevalence is unknown . from this study it is evident that , although the incidence of probable resistance to various pq regimens , used in the present study , is fairly low at this point ( relapse rate ranging from 1.47% to 4.58% using different methods of classification of recurrence ) , it is anticipated that resistance is likely to increase and therefore , continuous monitoring of the therapy with pq is warranted . also , this is an indication of declining efficacy of pq which could be due to increasing resistance to the drug . one of the major limitation of the study is the nonrandomization of the no pq group due to ethical reasons which forced us to select the patients for this group based on those who did not receive pq because of the clinical reasons such as hg < 10 gm / dl ; g6pd deficiency , pregnancy , lactation , and so forth . however , we do not believe these variables might have had any effect on the recurrence rate . additionally , even though we used this group to compare with rest of the pq regimens tested , the comparative number of recurrence within 3 pq groups presented in the table are self - explanatory . the study used month of recurrence as one of the method of classifying recurrence as relapse or reinfection . however , as per who recommendations , per kg pq dose is 0.25 mg base / kg body weight . additionally higher pq dose included in the present study and the recurrence and relapse rates observed in this group likely overcome the confounding effect of these factors . several factors related to parasite variants , host factors and the epidemiological factors including vector control , transmission dynamics ; the limitations of the existing methods to identify true relapses can influence and confound the analysis of true resistance to pq . this indicates that in the absence of reliable genotyping methods , the recurrence monitoring can be a good surrogate marker to compare provided it is appropriate to assume the constant reinfection rate in the given geographical area where the study population is residing . the study documented statistically significant higher recurrence rate in patients not given pq as compared to patients receiving different pq regimens . there was discordance in the methods ( namely , month of recurrences , rflp , or sequencing ) used to differentiate relapse from reinfection . the lack of concordance in the methods used has implications on the interpretation of the results and therefore warrants caution . using the available methods , the relapse rate ranging from 1.47% to 4.58% was noted in the patients receiving different pq regimens . considering that the paired samples classified as reinfection by rflp or sequencing could be the cases of relapse , the probable resistance to pq could be even higher indicating the decreased effect of pq to eradicate liver forms of p. vivax , hypnozoites . the study found that pq regimens used to be safe without any major or unexpected adverse events , although statistically significantly higher adverse events were noted in the higher dose pq ( 30 mg 14 days ) leading to significantly higher drop outs due to discontinuation of the treatment . the study also documented lots of genetic diversity in the types of genotype identified using the pcr sequencing method of genotyping , indicating diversity of type of p. vivax strains prevalent in the population ( geographic area ) studied .

a total of 121 older outpatients with type 2 diabetes in naples , italy , were included from 2007 to 2009 . frequency and duration of walking and other leisure - time physical activities were assessed by interview . exclusion criteria , assessed with self - report and medical records , included treatment with steroids or nonsteroidal anti - inflammatory drugs , acute concurrent illness during the 3-month period preceding the investigation , and cerebrovascular diseases . clinical characteristics of patients data are means sd , n , or n ( % ) . subcutaneous interstitial glucose levels were monitored on an ambulatory basis over a period of 3 consecutive days by using continuous subcutaneous glucose monitoring ( csgm ) ( glucoday ; a. menarini diagnostics , florence , italy ) as described previously ( 10 ) . the sensor was inserted on day 0 and removed on day 3 at mid - morning . the data were downloaded to a computer for evaluation of glucose variations , but calculations of glucose variations were limited to data obtained on days 1 and 2 to avoid bias due to both insertion and removal of the sensor and , thus , to insufficient stabilization of the monitoring system . the characteristic glucose pattern of each patient was calculated by averaging the profiles obtained on study days 1 and 2 . standardized meal tests with 24-h sampling comprising three mixed meals were performed on days 1 , 2 , and 3 . after an overnight fast , patients received medications at 0700 h and consumed breakfast 30 min after treatment . lunch and dinner were provided 5 and 10 h after the beginning of breakfast , respectively . the standardized breakfast contained 419 kcal ( 57% carbohydrate , 17% protein , and 26% fat ) , lunch contained 692 kcal ( 66% carbohydrate , 16% protein , and 18% fat ) , and dinner contained 507 kcal ( 41% carbohydrate , 26% protein , and 32% fat ) . before enrollment , all patients underwent carotid ultrasound examination and magnetic resonance imaging ( mri ) for the screening of carotid atherosclerosis and white matter lesions , significant signs of cortical or subcortical atrophy . patients with alterations in mri scans such as white matter lesions or cortical or subcortical atrophy were considered lost to follow - up analysis because morphological brain lesions as evidenced by mri might affect cognitive functioning independently of the changes in metabolic control . all mri evaluations were made by physicians not involved in the study and blinded to the study design . in brief , all patients were investigated in the supine position with the head slightly turned from the sonographer . the carotid arteries were carefully examined for wall changes from different longitudinal and transverse views . the common carotid artery , the carotid bulb , and the internal and external carotid arteries were examined in all subjects . a region 1.5 cm proximal to the carotid bifurcation was identified , and the intimal - media thickness of the far wall was evaluated as the distance between the luminal - intimal interface and the medial - adventitial interface . one transversal and two longitudinal measurements of intimal - media thickness were obtained from 10 contiguous sites at 1-mm intervals , and the average of the 10 measurements were used for the analysis . all ultrasound measurements were performed before enrollment by two trained investigators who were unaware of the study protocol . blood samples for the assay of insulin were collected in edta tubes , and plasma was immediately aliquoted in eppendorf vials and stored at 80c until thawed for assays . plasma insulin was determined by a commercial double - antibody , solid - phase radioimmunoassay ( sorin biomedica , milan , italy ) ( intra - assay coefficient of variation 3.1 + 0.3% ; cross - reactivity vs. proinsulin 0.9% ) . serum glucose , serum lipid , and serum lipoprotein were quantified from fresh samples drawn after participants had been fasting for at least 12 h. the serum glucose level was determined by an enzymatic colorimetric assay using a modified glucose oxidase - peroxidase method ( roche diagnostics , mannheim , germany ) and a roche - hitachi 917 analyzer . commercial enzymatic tests were used for determining serum total and hdl cholesterol and triglyceride ( roche diagnostics ) levels . the mean amplitude of glycemic excursions ( mage ) , which has been described by service et al . ( 11 ) , was used in the present study for assessing glucose fluctuations during 24 h. in particular , we used the glucose profiles obtained from continuous glucose monitoring system data on study days 1 and 2 , i.e. , from continuous monitoring for 48 h. this parameter was designed to quantify major swings of glycemia and to exclude minor ones . for this reason , only increases of > 1 sd of the mean glycemic values were taken into account . calculation of the mage was obtained by measuring the arithmetic mean of the differences between consecutive peaks and nadirs ; measurement in the peak - to - nadir or nadir - to - peak direction was determined by the first qualifying excursion . the measurement of this parameter , which has been proved to be independent of mean glycemia , is of particular interest because the greater the mage , the higher the glycemic instability ( 12 ) . twenty - four to 48 h before csgm , global cognitive function was assessed with the mini - mental state examination ( mmse ) corrected for educational levels of patients ( 13 ) . this cognitive test covers many cognitive skills , and scores range from 0 to 30 . the trail making test ( tmt ) is a visuomotor speeded task that consists of two parts : tmt - a and tmt - b . tmt - a , a visual scanning test , requires one to draw a line connecting consecutive numbers from 1 to 25 . tmt - b adds cognitive flexibility to tmt - a and requires one to draw a line connecting numbers and letters in alternating sequence ( 14 ) . although time - to - completion scores are typically used to examine aspects of attention and executive function , the difference between the two scores ( tmt - b tmt - a [ diff b - a ] ) provides a measure of cognitive efficiency ( 15 ) . the wechsler adult intelligence scale revised digit span is a measure of mental tracking as well as of brief storage and mental manipulation ( 15 ) . the backward digit span ( dsp backward ) requires the participant listen to increasingly longer lists of digits presented for immediate recall in the reverse order presented , whereas the forward digit span ( dsp forward ) requires immediate recall in the exact order presented . the verbal fluency test requires participants to generate as many words as possible in 1 min for a given letter ( f , a , s ) excluding nouns and variations of the same word ( 16 ) . the first - stage screening was based on the mmse . according to italian standards ( 17 ) , participants with an age- and education - adjusted mmse score of > 26 were considered to not have dementia and those with a score of 26 were directed to the second - stage screening that was performed by a geriatrician and a psychologist with long - term experience in the evaluation of older individuals with cognitive problems . the diagnosis of dementia syndrome , independent of etiology was established using a standard evaluation protocol based on dsm - iv criteria ( 18 ) . plasma insulin and triglycerides were log transformed for data analyses and back transformed for data presentation . we calculated the number of patients required for the study to reject the null hypothesis 99% of the time ( i.e. , with a one - tailed type ii error rate of 0.01 ) when r was 0.80 with a two - tailed type i error at the 0.05 level of significance . because this calculation led to a sample size of at least 110 , the number of required patients was set at 121 . a cluster analysis , using the squared sum of z scores , showed whether an overall value obtained by clustering attention and executive function test results was associated with mage . to create such a cluster analysis , we created a cognition composite score of attention and executive functions , as sum of the z scores of tmt - a , tmt - b , diff b - a , dsp a z score indicates the position of an individual value of a variable in the total distribution of the variable in the population and is calculated as follows : ( individual value the analysis transforms the individual test scores to z scores , summing these and restandardizing this sum . multiple regression models were used to explore the relationship between mage and cognition ( cognition composite score and mmse score ) independently of several covariates . the effect of therapy in patients categorized for number of antidiabetes therapies was assessed by anova , and ptrend was calculated . blood samples for the assay of insulin were collected in edta tubes , and plasma was immediately aliquoted in eppendorf vials and stored at 80c until thawed for assays . plasma insulin was determined by a commercial double - antibody , solid - phase radioimmunoassay ( sorin biomedica , milan , italy ) ( intra - assay coefficient of variation 3.1 + 0.3% ; cross - reactivity vs. proinsulin 0.9% ) . serum glucose , serum lipid , and serum lipoprotein were quantified from fresh samples drawn after participants had been fasting for at least 12 h. the serum glucose level was determined by an enzymatic colorimetric assay using a modified glucose oxidase - peroxidase method ( roche diagnostics , mannheim , germany ) and a roche - hitachi 917 analyzer . commercial enzymatic tests were used for determining serum total and hdl cholesterol and triglyceride ( roche diagnostics ) levels . the mean amplitude of glycemic excursions ( mage ) , which has been described by service et al . ( 11 ) , was used in the present study for assessing glucose fluctuations during 24 h. in particular , we used the glucose profiles obtained from continuous glucose monitoring system data on study days 1 and 2 , i.e. , from continuous monitoring for 48 h. this parameter was designed to quantify major swings of glycemia and to exclude minor ones . for this reason , only increases of > 1 sd of the mean glycemic values were taken into account . calculation of the mage was obtained by measuring the arithmetic mean of the differences between consecutive peaks and nadirs ; measurement in the peak - to - nadir or nadir - to - peak direction was determined by the first qualifying excursion . the measurement of this parameter , which has been proved to be independent of mean glycemia , is of particular interest because the greater the mage , the higher the glycemic instability ( 12 ) . twenty - four to 48 h before csgm , global cognitive function was assessed with the mini - mental state examination ( mmse ) corrected for educational levels of patients ( 13 ) . this cognitive test covers many cognitive skills , and scores range from 0 to 30 . the trail making test ( tmt ) is a visuomotor speeded task that consists of two parts : tmt - a and tmt - b . tmt - a , a visual scanning test , requires one to draw a line connecting consecutive numbers from 1 to 25 . tmt - b adds cognitive flexibility to tmt - a and requires one to draw a line connecting numbers and letters in alternating sequence ( 14 ) . although time - to - completion scores are typically used to examine aspects of attention and executive function , the difference between the two scores ( tmt - b tmt - a [ diff b - a ] ) provides a measure of cognitive efficiency ( 15 ) . the wechsler adult intelligence scale revised digit span is a measure of mental tracking as well as of brief storage and mental manipulation ( 15 ) . the backward digit span ( dsp backward ) requires the participant listen to increasingly longer lists of digits presented for immediate recall in the reverse order presented , whereas the forward digit span ( dsp forward ) requires immediate recall in the exact order presented . the verbal fluency test requires participants to generate as many words as possible in 1 min for a given letter ( f , a , s ) excluding nouns and variations of the same word ( 16 ) . the first - stage screening was based on the mmse . according to italian standards ( 17 ) , participants with an age- and education - adjusted mmse score of > 26 were considered to not have dementia and those with a score of 26 were directed to the second - stage screening that was performed by a geriatrician and a psychologist with long - term experience in the evaluation of older individuals with cognitive problems . the diagnosis of dementia syndrome , independent of etiology was established using a standard evaluation protocol based on dsm - iv criteria ( 18 ) . all data are expressed as means sd . plasma insulin and triglycerides were log transformed for data analyses and back transformed for data presentation . we calculated the number of patients required for the study to reject the null hypothesis 99% of the time ( i.e. , with a one - tailed type ii error rate of 0.01 ) when r was 0.80 with a two - tailed type i error at the 0.05 level of significance . because this calculation led to a sample size of at least 110 , the number of required patients was set at 121 . a cluster analysis , using the squared sum of z scores , showed whether an overall value obtained by clustering attention and executive function test results was associated with mage . to create such a cluster analysis , we created a cognition composite score of attention and executive functions , as sum of the z scores of tmt - a , tmt - b , diff b - a , dsp a z score indicates the position of an individual value of a variable in the total distribution of the variable in the population and is calculated as follows : ( individual value the analysis transforms the individual test scores to z scores , summing these and restandardizing this sum . multiple regression models were used to explore the relationship between mage and cognition ( cognition composite score and mmse score ) independently of several covariates . the effect of therapy in patients categorized for number of antidiabetes therapies was assessed by anova , and ptrend was calculated . the study group had mean sd age of 78 6.7 years ( range 8865 years ) , a1c of 7.9 0.3% , and fpg of 153 10.3 mg / dl . a slightly higher number of patients were women , with modest overweight . according to age most of them were affected by comorbidity ( cvd that was not stroke - related ; 13 reported hypercholesterolemia and 30 reported previous hypertension ) ( table 1 ) . no patients examined had carotid plaque > 70% ( four patients had carotid plaque at 2025% without significant alterations in blood flow ) on a carotid ultrasound examination or white matter lesions or significant signs of cortical or subcortical atrophy with an mri scan ( data not show ) . all patients were treated with oral antidiabetes agents : glyburide alone ( 1015 mg / day in 19 patients ) or metformin alone ( 1,700 mg / day in 38 patients ) or a combination of metformin ( 1,700 mg / day ) and glyburide ( 15 mg / day ) ( in 51 patients ) or a combination of metformin ( 1,700 mg / day ) and thiazolidinediones ( 4 mg / day ) ( in 13 patients ) . the mage over 24 h obtained from the continuous glucose monitoring system in the 121 patients was 71 19 mg / dl and the 24-h glycemic value was 176 45 mg / dl . mage was significantly correlated with mmse ( r = 0.83 p < 0.001 ) and with the cognition composite score ( r = 0.68 , p < 0.001 ) ( fig . 1 ) . this relationship persisted after adjustment for the main anthropometric ( bmi and waist - to - hip ratio [ whr ] ) , metabolic ( fpg and 2-h ppg and a1c ) , and/or vascular ( mean arterial blood pressure ) covariables ( data not shown ) . no correlation between mage and fasting insulin was observed ( r = 0.02 , p = 0.1 ) . the independent effects of mage on both the cognition composite and mmse scores were tested in three different multiple linear regression models ( table 2 ) having , respectively , age , sex , bmi , whr , drug intake , physical activity , and mage ( model 1 ) , model 1 + systolic and diastolic blood pressure ( model 2 ) , and model 1 + model 2 + a1c , fasting and postprandial glucose ( model 3 ) as covariates . in the more complex model 3 ( table 2 ) , mage was associated with mmse and cognition composite score ( independently of age , sex , bmi , whr , medication , physical activity , mean arterial blood pressure , fpg , ppg , and a1c ) . this last model explained 77 and 44% variability of mmse and cognition composite score , respectively , whereas mage per se explained 24 and 26% variability of mmse and cognition , respectively . among fpg , ppg , and a1c , only ppg has been found to be independently associated with cognitive function ( table 2 , model 3 ) . all of the analyses performed gave the same results , even after adjustment for education level ( data not shown ) . linear multivariate analyses with mmse and composite score as dependent variable for mmse : r = 0.73 ( model 1 ) ; r = 0.73 ( model 2 ) ; r = 0.77 ( model 3 ) . for composite score : r = 0.40 ( model 1 ) ; r = 0.41 ( model 2 ) ; r = 0.44 ( model 3 ) . sbp , systolic blood pressure ; dbp , diastolic blood pressure . to assess the impact of antidiabetes therapy , all patients were categorized in three groups according to number of antidiabetes drugs used . no differences were observed in the mean values of mage according to antidiabetes treatment : glyburide , 70.2 29 mg / dl ; metformin , 70.1 18 mg / dl ; metformin plus glyburide , 71.7 16 ; or metformin plus thiazolidinediones , 73.8 23 . no differences in ppg , a1c , cognitive composite score , and mmse were found among the study groups , thus showing a null impact of therapy on mmse score and cognition composite score ( data not shown ) . mage was significantly correlated with mmse ( r = 0.83 p < 0.001 ) and with the cognition composite score ( r = 0.68 , p < 0.001 ) ( fig . this relationship persisted after adjustment for the main anthropometric ( bmi and waist - to - hip ratio [ whr ] ) , metabolic ( fpg and 2-h ppg and a1c ) , and/or vascular ( mean arterial blood pressure ) covariables ( data not shown ) . no correlation between mage and fasting insulin was observed ( r = 0.02 , p = 0.1 ) . the independent effects of mage on both the cognition composite and mmse scores were tested in three different multiple linear regression models ( table 2 ) having , respectively , age , sex , bmi , whr , drug intake , physical activity , and mage ( model 1 ) , model 1 + systolic and diastolic blood pressure ( model 2 ) , and model 1 + model 2 + a1c , fasting and postprandial glucose ( model 3 ) as covariates . in the more complex model 3 ( table 2 ) , mage was associated with mmse and cognition composite score ( independently of age , sex , bmi , whr , medication , physical activity , mean arterial blood pressure , fpg , ppg , and a1c ) . this last model explained 77 and 44% variability of mmse and cognition composite score , respectively , whereas mage per se explained 24 and 26% variability of mmse and cognition , respectively . among fpg , ppg , and a1c , only ppg has been found to be independently associated with cognitive function ( table 2 , model 3 ) . all of the analyses performed gave the same results , even after adjustment for education level ( data not shown ) . linear multivariate analyses with mmse and composite score as dependent variable for mmse : r = 0.73 ( model 1 ) ; r = 0.73 ( model 2 ) ; r = 0.77 ( model 3 ) . for composite score : r = 0.40 ( model 1 ) ; r = 0.41 ( model 2 ) ; r = 0.44 ( model 3 ) . to assess the impact of antidiabetes therapy , all patients were categorized in three groups according to number of antidiabetes drugs used . no differences were observed in the mean values of mage according to antidiabetes treatment : glyburide , 70.2 29 mg / dl ; metformin , 70.1 18 mg / dl ; metformin plus glyburide , 71.7 16 ; or metformin plus thiazolidinediones , 73.8 23 . no differences in ppg , a1c , cognitive composite score , and mmse were found among the study groups , thus showing a null impact of therapy on mmse score and cognition composite score ( data not shown ) . our study shows that impairment of cognitive performance in older type 2 diabetic patients may be associated with daily acute glucose fluctuations . in particular , mage excursions were strongly correlated with cognitive functioning , and this relationship was independent of the main markers of sustained hyperglycemia ( a1c , ppg , and fpg ) . even though a number of studies have investigated and compared the roles of the different glycemic indexes participating in diabetic cognitive disorders ( 24 ) , accurate assessment of their respective contributions is still being debated . by using different methods , including epidemiological ( 2 ) , interventional ( 19 ) , or pathophysiological studies ( 5 ) , several authors have demonstrated that a1c is certainly an independent risk factor of decline in cognitive performance in type 2 diabetes . for instance , it has been established that chronic hyperglycemia induces an overproduction of superoxide which , after reacting with nitric oxide , produces subsequent nitrosative stress with generation of metabolic derivatives such as peroxynitrite and nitrotyrosine ( 20 ) . the toxicity of these substances can lead to neuronal damages and , furthermore , to a decline in cognitive performance . in this context , daily glucose fluctuations , more generally , glucose swings such as peaks and troughs , exhibited a more specific triggering effect on oxidative stress than chronic sustained hyperglycemia ( 21 ) . thus , peaks , usually corresponding to maximum values after meals , and troughs , observed over interprandial periods , could lead to continuous production of oxidative stress , impairing cognitive functioning . oxidative stress caused by free radicals damages the endothelial cells in the blood vessels , promotes lipid peroxidation , and plays a central role in pathogenesis of cerebral complications of type 2 diabetes . various studies have shown that increased oxidative stress can lead to microvascular cerebral diseases , e.g. , stroke , cerebral hemorrhage , and brain infarction . the reason for high risk of microvascular cerebral diseases is that brain consumes 20% of the oxygen consumed by the body and has a low concentration of antioxidants and high content of unsaturated fatty acids and catecholamines that are easily oxidized , making it more vulnerable to oxidative damage than any other organ in the body , and this may predispose diabetic patients to development of cognitive impairment ( 22 ) . because the glycemic fluctuations as estimated from mage indexes reflect both upward and downward glucose changes , whereas a1c , ppg , and fpg values are only markers of upward variations , there is a reason to hypothesize that mage indexes are wider integrators of glycemic variations than the a1c , ppg , and fpg values . our results , showing that mage values were associated with cognitive functioning independently of the main markers of glycemic control , we therefore suggest that acute glycemic excursions should be integrated into glycemic disorders that are larger than chronic hyperglycemia , i.e. , into rapid glucose swings including declines from relatively high glucose levels during postprandial periods to low values or even to asymptomatic hypoglycemia , as observed over interprandial periods . as a consequence , low glycemic levels in type 2 diabetes might also evoke a decline in cognitive performance . indeed , because the appropriate concentration of glucose may have a pivotal role on metabolic activity in the brain , the rapid glucose swings from relatively high glucose levels during postprandial periods to low values or even to asymptomatic hypoglycemia and its associated neuroglycopenia may contribute largely to a more rapid decline of metabolic activity in the brain ( 23 ) thus , glucose variations over time , fluctuating from hyperglycemic peaks to glucose nadirs , may affect cognitive function in older individuals , increasing oxidative damage as well as altering the appropriate brain supply of glucose . these observations provide a possible explanation for the independent role of mage on cognitive function with respect to ppg , fpg , and a1c . in summary , the present study demonstrates a significant relationship between acute glucose swings and cognitive performance impairment . a weakness in the present study is its cross - sectional , observational nature , and it is therefore difficult to draw causal relationships . however , because the glycemic disorders are risk factors for mild cognitive impairment and both vascular dementia ( 15 ) and alzheimer disease , the present data open the field for conducting interventional studies with the aim of treating glycemic disorders not only by reducing a1c and mean hyperglycemia ( 24 ) but also by flattening acute glucose fluctuations .

objectivethe mean amplitude of glycemic excursions ( mage ) is a significant determinant of overall metabolic control as well as increased risk for diabetes complications . older individuals with type 2 diabetes are more likely to have moderate cognitive deficits and structural changes in brain tissue . considering that poor metabolic control is considered a deranging factor for cognitive performance in diabetic patients , we evaluated whether the contributions of mage to cognitive status in older patients with type 2 diabetes were independent from the main markers of glycemic control , such as sustained chronic hyperglycemia ( a1c ) , postprandial glycemia ( ppg ) , and fasting plasma glucose ( fpg).research design and methodsin 121 older patients with type 2 diabetes , 48-h continuous subcutaneous glucose monitoring ( csgm ) were assessed . mage and ppg were evaluated during csgm . the relationship of mage to performance on cognitive tests was assessed , with adjustment for age , glycemic control markers , and other determinants of cognitive status . the cognitive tests were a composite score of executive and attention functioning and the mini mental status examination ( mmse).resultsmage was significantly correlated with mmse ( r = 0.83 ; p < 0.001 ) and with cognition composite score ( r = 0.68 ; p < 0.001 ) . moreover , mage was associated with the mmse ( p < 0.001 ) and cognition composite score ( p < 0.001 ) independently of age , sex , bmi , waist - to - hip ( whr ) ratio , drug intake , physical activity , mean arterial blood pressure , fpg , ppg , and a1c.conclusionsmage during a daily period was associated with an impairment of cognitive functioning independent of a1c , fpg , and ppg . the present data suggest that interventional trials in older patients with type 2 diabetes should target not only a1c , ppg , and fpg but also daily acute glucose swings .

RESEARCH DESIGN AND METHODS Laboratory measurements Assessment of glycemic instability Assessment of cognitive functions Statistical analysis RESULTS Relationships among the cognition composite score, MMSE score, and markers of diabetic control Relationship between glucose fluctuation (MAGE) and therapy for diabetes Conclusions

a total of 121 older outpatients with type 2 diabetes in naples , italy , were included from 2007 to 2009 . frequency and duration of walking and other leisure - time physical activities were assessed by interview . subcutaneous interstitial glucose levels were monitored on an ambulatory basis over a period of 3 consecutive days by using continuous subcutaneous glucose monitoring ( csgm ) ( glucoday ; a. menarini diagnostics , florence , italy ) as described previously ( 10 ) . standardized meal tests with 24-h sampling comprising three mixed meals were performed on days 1 , 2 , and 3 . the standardized breakfast contained 419 kcal ( 57% carbohydrate , 17% protein , and 26% fat ) , lunch contained 692 kcal ( 66% carbohydrate , 16% protein , and 18% fat ) , and dinner contained 507 kcal ( 41% carbohydrate , 26% protein , and 32% fat ) . patients with alterations in mri scans such as white matter lesions or cortical or subcortical atrophy were considered lost to follow - up analysis because morphological brain lesions as evidenced by mri might affect cognitive functioning independently of the changes in metabolic control . in brief , all patients were investigated in the supine position with the head slightly turned from the sonographer . the common carotid artery , the carotid bulb , and the internal and external carotid arteries were examined in all subjects . a region 1.5 cm proximal to the carotid bifurcation was identified , and the intimal - media thickness of the far wall was evaluated as the distance between the luminal - intimal interface and the medial - adventitial interface . one transversal and two longitudinal measurements of intimal - media thickness were obtained from 10 contiguous sites at 1-mm intervals , and the average of the 10 measurements were used for the analysis . serum glucose , serum lipid , and serum lipoprotein were quantified from fresh samples drawn after participants had been fasting for at least 12 h. the serum glucose level was determined by an enzymatic colorimetric assay using a modified glucose oxidase - peroxidase method ( roche diagnostics , mannheim , germany ) and a roche - hitachi 917 analyzer . commercial enzymatic tests were used for determining serum total and hdl cholesterol and triglyceride ( roche diagnostics ) levels . the mean amplitude of glycemic excursions ( mage ) , which has been described by service et al . ( 11 ) , was used in the present study for assessing glucose fluctuations during 24 h. in particular , we used the glucose profiles obtained from continuous glucose monitoring system data on study days 1 and 2 , i.e. calculation of the mage was obtained by measuring the arithmetic mean of the differences between consecutive peaks and nadirs ; measurement in the peak - to - nadir or nadir - to - peak direction was determined by the first qualifying excursion . twenty - four to 48 h before csgm , global cognitive function was assessed with the mini - mental state examination ( mmse ) corrected for educational levels of patients ( 13 ) . this cognitive test covers many cognitive skills , and scores range from 0 to 30 . the trail making test ( tmt ) is a visuomotor speeded task that consists of two parts : tmt - a and tmt - b . although time - to - completion scores are typically used to examine aspects of attention and executive function , the difference between the two scores ( tmt - b tmt - a [ diff b - a ] ) provides a measure of cognitive efficiency ( 15 ) . the wechsler adult intelligence scale revised digit span is a measure of mental tracking as well as of brief storage and mental manipulation ( 15 ) . according to italian standards ( 17 ) , participants with an age- and education - adjusted mmse score of > 26 were considered to not have dementia and those with a score of 26 were directed to the second - stage screening that was performed by a geriatrician and a psychologist with long - term experience in the evaluation of older individuals with cognitive problems . the diagnosis of dementia syndrome , independent of etiology was established using a standard evaluation protocol based on dsm - iv criteria ( 18 ) . , with a one - tailed type ii error rate of 0.01 ) when r was 0.80 with a two - tailed type i error at the 0.05 level of significance . a cluster analysis , using the squared sum of z scores , showed whether an overall value obtained by clustering attention and executive function test results was associated with mage . to create such a cluster analysis , we created a cognition composite score of attention and executive functions , as sum of the z scores of tmt - a , tmt - b , diff b - a , dsp a z score indicates the position of an individual value of a variable in the total distribution of the variable in the population and is calculated as follows : ( individual value the analysis transforms the individual test scores to z scores , summing these and restandardizing this sum . multiple regression models were used to explore the relationship between mage and cognition ( cognition composite score and mmse score ) independently of several covariates . the effect of therapy in patients categorized for number of antidiabetes therapies was assessed by anova , and ptrend was calculated . blood samples for the assay of insulin were collected in edta tubes , and plasma was immediately aliquoted in eppendorf vials and stored at 80c until thawed for assays . serum glucose , serum lipid , and serum lipoprotein were quantified from fresh samples drawn after participants had been fasting for at least 12 h. the serum glucose level was determined by an enzymatic colorimetric assay using a modified glucose oxidase - peroxidase method ( roche diagnostics , mannheim , germany ) and a roche - hitachi 917 analyzer . commercial enzymatic tests were used for determining serum total and hdl cholesterol and triglyceride ( roche diagnostics ) levels . the mean amplitude of glycemic excursions ( mage ) , which has been described by service et al . ( 11 ) , was used in the present study for assessing glucose fluctuations during 24 h. in particular , we used the glucose profiles obtained from continuous glucose monitoring system data on study days 1 and 2 , i.e. calculation of the mage was obtained by measuring the arithmetic mean of the differences between consecutive peaks and nadirs ; measurement in the peak - to - nadir or nadir - to - peak direction was determined by the first qualifying excursion . the measurement of this parameter , which has been proved to be independent of mean glycemia , is of particular interest because the greater the mage , the higher the glycemic instability ( 12 ) . twenty - four to 48 h before csgm , global cognitive function was assessed with the mini - mental state examination ( mmse ) corrected for educational levels of patients ( 13 ) . the trail making test ( tmt ) is a visuomotor speeded task that consists of two parts : tmt - a and tmt - b . although time - to - completion scores are typically used to examine aspects of attention and executive function , the difference between the two scores ( tmt - b tmt - a [ diff b - a ] ) provides a measure of cognitive efficiency ( 15 ) . the wechsler adult intelligence scale revised digit span is a measure of mental tracking as well as of brief storage and mental manipulation ( 15 ) . the first - stage screening was based on the mmse . according to italian standards ( 17 ) , participants with an age- and education - adjusted mmse score of > 26 were considered to not have dementia and those with a score of 26 were directed to the second - stage screening that was performed by a geriatrician and a psychologist with long - term experience in the evaluation of older individuals with cognitive problems . the diagnosis of dementia syndrome , independent of etiology was established using a standard evaluation protocol based on dsm - iv criteria ( 18 ) . a cluster analysis , using the squared sum of z scores , showed whether an overall value obtained by clustering attention and executive function test results was associated with mage . to create such a cluster analysis , we created a cognition composite score of attention and executive functions , as sum of the z scores of tmt - a , tmt - b , diff b - a , dsp a z score indicates the position of an individual value of a variable in the total distribution of the variable in the population and is calculated as follows : ( individual value the analysis transforms the individual test scores to z scores , summing these and restandardizing this sum . multiple regression models were used to explore the relationship between mage and cognition ( cognition composite score and mmse score ) independently of several covariates . the effect of therapy in patients categorized for number of antidiabetes therapies was assessed by anova , and ptrend was calculated . the study group had mean sd age of 78 6.7 years ( range 8865 years ) , a1c of 7.9 0.3% , and fpg of 153 10.3 mg / dl . a slightly higher number of patients were women , with modest overweight . no patients examined had carotid plaque > 70% ( four patients had carotid plaque at 2025% without significant alterations in blood flow ) on a carotid ultrasound examination or white matter lesions or significant signs of cortical or subcortical atrophy with an mri scan ( data not show ) . all patients were treated with oral antidiabetes agents : glyburide alone ( 1015 mg / day in 19 patients ) or metformin alone ( 1,700 mg / day in 38 patients ) or a combination of metformin ( 1,700 mg / day ) and glyburide ( 15 mg / day ) ( in 51 patients ) or a combination of metformin ( 1,700 mg / day ) and thiazolidinediones ( 4 mg / day ) ( in 13 patients ) . the mage over 24 h obtained from the continuous glucose monitoring system in the 121 patients was 71 19 mg / dl and the 24-h glycemic value was 176 45 mg / dl . mage was significantly correlated with mmse ( r = 0.83 p < 0.001 ) and with the cognition composite score ( r = 0.68 , p < 0.001 ) ( fig . this relationship persisted after adjustment for the main anthropometric ( bmi and waist - to - hip ratio [ whr ] ) , metabolic ( fpg and 2-h ppg and a1c ) , and/or vascular ( mean arterial blood pressure ) covariables ( data not shown ) . no correlation between mage and fasting insulin was observed ( r = 0.02 , p = 0.1 ) . the independent effects of mage on both the cognition composite and mmse scores were tested in three different multiple linear regression models ( table 2 ) having , respectively , age , sex , bmi , whr , drug intake , physical activity , and mage ( model 1 ) , model 1 + systolic and diastolic blood pressure ( model 2 ) , and model 1 + model 2 + a1c , fasting and postprandial glucose ( model 3 ) as covariates . in the more complex model 3 ( table 2 ) , mage was associated with mmse and cognition composite score ( independently of age , sex , bmi , whr , medication , physical activity , mean arterial blood pressure , fpg , ppg , and a1c ) . this last model explained 77 and 44% variability of mmse and cognition composite score , respectively , whereas mage per se explained 24 and 26% variability of mmse and cognition , respectively . among fpg , ppg , and a1c , only ppg has been found to be independently associated with cognitive function ( table 2 , model 3 ) . all of the analyses performed gave the same results , even after adjustment for education level ( data not shown ) . linear multivariate analyses with mmse and composite score as dependent variable for mmse : r = 0.73 ( model 1 ) ; r = 0.73 ( model 2 ) ; r = 0.77 ( model 3 ) . for composite score : r = 0.40 ( model 1 ) ; r = 0.41 ( model 2 ) ; r = 0.44 ( model 3 ) . sbp , systolic blood pressure ; dbp , diastolic blood pressure . no differences in ppg , a1c , cognitive composite score , and mmse were found among the study groups , thus showing a null impact of therapy on mmse score and cognition composite score ( data not shown ) . mage was significantly correlated with mmse ( r = 0.83 p < 0.001 ) and with the cognition composite score ( r = 0.68 , p < 0.001 ) ( fig . this relationship persisted after adjustment for the main anthropometric ( bmi and waist - to - hip ratio [ whr ] ) , metabolic ( fpg and 2-h ppg and a1c ) , and/or vascular ( mean arterial blood pressure ) covariables ( data not shown ) . no correlation between mage and fasting insulin was observed ( r = 0.02 , p = 0.1 ) . the independent effects of mage on both the cognition composite and mmse scores were tested in three different multiple linear regression models ( table 2 ) having , respectively , age , sex , bmi , whr , drug intake , physical activity , and mage ( model 1 ) , model 1 + systolic and diastolic blood pressure ( model 2 ) , and model 1 + model 2 + a1c , fasting and postprandial glucose ( model 3 ) as covariates . in the more complex model 3 ( table 2 ) , mage was associated with mmse and cognition composite score ( independently of age , sex , bmi , whr , medication , physical activity , mean arterial blood pressure , fpg , ppg , and a1c ) . this last model explained 77 and 44% variability of mmse and cognition composite score , respectively , whereas mage per se explained 24 and 26% variability of mmse and cognition , respectively . among fpg , ppg , and a1c , only ppg has been found to be independently associated with cognitive function ( table 2 , model 3 ) . all of the analyses performed gave the same results , even after adjustment for education level ( data not shown ) . linear multivariate analyses with mmse and composite score as dependent variable for mmse : r = 0.73 ( model 1 ) ; r = 0.73 ( model 2 ) ; r = 0.77 ( model 3 ) . for composite score : r = 0.40 ( model 1 ) ; r = 0.41 ( model 2 ) ; r = 0.44 ( model 3 ) . no differences were observed in the mean values of mage according to antidiabetes treatment : glyburide , 70.2 29 mg / dl ; metformin , 70.1 18 mg / dl ; metformin plus glyburide , 71.7 16 ; or metformin plus thiazolidinediones , 73.8 23 . no differences in ppg , a1c , cognitive composite score , and mmse were found among the study groups , thus showing a null impact of therapy on mmse score and cognition composite score ( data not shown ) . our study shows that impairment of cognitive performance in older type 2 diabetic patients may be associated with daily acute glucose fluctuations . in particular , mage excursions were strongly correlated with cognitive functioning , and this relationship was independent of the main markers of sustained hyperglycemia ( a1c , ppg , and fpg ) . even though a number of studies have investigated and compared the roles of the different glycemic indexes participating in diabetic cognitive disorders ( 24 ) , accurate assessment of their respective contributions is still being debated . by using different methods , including epidemiological ( 2 ) , interventional ( 19 ) , or pathophysiological studies ( 5 ) , several authors have demonstrated that a1c is certainly an independent risk factor of decline in cognitive performance in type 2 diabetes . for instance , it has been established that chronic hyperglycemia induces an overproduction of superoxide which , after reacting with nitric oxide , produces subsequent nitrosative stress with generation of metabolic derivatives such as peroxynitrite and nitrotyrosine ( 20 ) . in this context , daily glucose fluctuations , more generally , glucose swings such as peaks and troughs , exhibited a more specific triggering effect on oxidative stress than chronic sustained hyperglycemia ( 21 ) . thus , peaks , usually corresponding to maximum values after meals , and troughs , observed over interprandial periods , could lead to continuous production of oxidative stress , impairing cognitive functioning . oxidative stress caused by free radicals damages the endothelial cells in the blood vessels , promotes lipid peroxidation , and plays a central role in pathogenesis of cerebral complications of type 2 diabetes . , stroke , cerebral hemorrhage , and brain infarction . the reason for high risk of microvascular cerebral diseases is that brain consumes 20% of the oxygen consumed by the body and has a low concentration of antioxidants and high content of unsaturated fatty acids and catecholamines that are easily oxidized , making it more vulnerable to oxidative damage than any other organ in the body , and this may predispose diabetic patients to development of cognitive impairment ( 22 ) . because the glycemic fluctuations as estimated from mage indexes reflect both upward and downward glucose changes , whereas a1c , ppg , and fpg values are only markers of upward variations , there is a reason to hypothesize that mage indexes are wider integrators of glycemic variations than the a1c , ppg , and fpg values . our results , showing that mage values were associated with cognitive functioning independently of the main markers of glycemic control , we therefore suggest that acute glycemic excursions should be integrated into glycemic disorders that are larger than chronic hyperglycemia , i.e. , into rapid glucose swings including declines from relatively high glucose levels during postprandial periods to low values or even to asymptomatic hypoglycemia , as observed over interprandial periods . as a consequence , low glycemic levels in type 2 diabetes might also evoke a decline in cognitive performance . indeed , because the appropriate concentration of glucose may have a pivotal role on metabolic activity in the brain , the rapid glucose swings from relatively high glucose levels during postprandial periods to low values or even to asymptomatic hypoglycemia and its associated neuroglycopenia may contribute largely to a more rapid decline of metabolic activity in the brain ( 23 ) thus , glucose variations over time , fluctuating from hyperglycemic peaks to glucose nadirs , may affect cognitive function in older individuals , increasing oxidative damage as well as altering the appropriate brain supply of glucose . these observations provide a possible explanation for the independent role of mage on cognitive function with respect to ppg , fpg , and a1c . in summary , the present study demonstrates a significant relationship between acute glucose swings and cognitive performance impairment . a weakness in the present study is its cross - sectional , observational nature , and it is therefore difficult to draw causal relationships . however , because the glycemic disorders are risk factors for mild cognitive impairment and both vascular dementia ( 15 ) and alzheimer disease , the present data open the field for conducting interventional studies with the aim of treating glycemic disorders not only by reducing a1c and mean hyperglycemia ( 24 ) but also by flattening acute glucose fluctuations .

an expression vector ( phse traf1 ) for traf1 transgenic mice was generated by cloning the flu epitope bamhi 3 site of the ph se-3 expression cassette ( 38 , 39 ) . phse-3 expression cassette with an h-2k promoter and an igh enhancer was previously described to express the transgene both in immature thymocytes and in mature t cells ( 38 , 39 ) . transgene dna was isolated by xhoi digestion and microinjected into ( c57bl/6 cba / j)f2 embryos . transgenic founders were identified by southern blot analysis of tail dna and transgenic lines were established by backcrossing with either c57bl/6 or balb / c mice to yield vsag7 h-2 or h-2 mice . mice transgenic for an tcr specific for h-2d and the lymphocyte choriomeningitis virus ( lcmv ) glycoprotein ( gp)derived peptide amino acids 3341 ( gp33 , the major lcmv epitope in the h-2 haplotype ) were previously described ( 13 , 38 , 39)and crossed with traf1 transgenic mice . mabs , rat anti - cd8 ( 53 - 6.7 ; fitc conjugated ) , and anti - va2 ( b20.1 ; pe conjugated ) were purchased from pharmingen ( san diego , ca ) . for induction of apoptosis in vitro , lymph node t cells from traf1 transgenic and negative littermates were treated with con a ( 5 g / ml ) for 48 h , washed with 10 mg / ml -methylmannoside , and incubated in 50 iu / ml il-2 to predispose to apoptosis as previously described ( 12 ) . cells ( 50,000/well ) with > 97% -tcr were then cultured for 48 h in anti - cd3 ab ( 145 - 2c11)coated plates in triplicate . viable cells were then quantified by propidium iodide ( pi ) exclusion using a facscaliber with cellquest software . specific cell survival ( % ) was determined from the percentages of viable ( pi - negative ) cells and it equals 100 ( % survival after cd3 cross - linking / % survival without cd3 cross - linking ) . to induce apoptosis of antigen - specific t cells in tcr transgenic mice , mice were treated with a single intrafootpad injection of gp33 peptide ( 30 g ) . tcr transgenic cd8 cells from traf1 transgenic mice and negative littermates in regional lymph nodes were analyzed 3 and 4 d after intrafootpad injection of gp33 peptide . draining popliteal and inguinal lymph node cells were taken from tcr transgenic mice ( h-2 ) either untreated ( day 0 ) or 2 d after a single intrafootpad injection of 30 g gp33 peptide and incubated in imdm supplemented with 10% fcs , 5 10 m 2-mercaptoethanol , penicillin , and streptomycin ( complete imdm ) for 24 h ( day 3 ) or 48 h ( day 4 ) as described ( 13 ) . the percentages of surviving cells were determined by flow cytometry ( facscan ) by gating for lymphocytes based on forward and side scatter . for induction of tolerance by high dose peptide antigens in non - tcr transgenic mice , h-2 traf1 transgenic or negative littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide or buffered salt solution in ifa as previously described ( 40 , 41 ) . the mice were infected on day 16 and tested on day 24 as previously described ( 41 , 42 ) . for induction of apoptosis by lcmv , 10 tcr transgenic spleen cells from traf1/tcr double - transgenic or tcr single - transgenic littermates were injected intravenously in c57bl/6 mice as previously described ( 42 ) . at the same time , the mice were infected with lcmv we strain . for in vivo expansion , the viral dose was 200 pfu and analysis was 7 d after infection ; for in vivo deletion , the viral dose was 10 pfu and analysis was 4 and 7 d after infection . tcr transgenic t cells were determined by double staining with cd8- and v2-specific antibodies and flow cytometry analysis by gating for lymphocytes based on forward/ side scatters . t cells isolated from lymph nodes using t cell enrichment columns ( biotex laboratories , inc . , edmonton , canada ) were also induced to proliferate by titrating them in triplicate into anti - tcr ab ( h57 - 597 ) coated tissue culture wells containing mitomycin c treated splenocytes ( 5 10/well ) as previously described ( 43 ) . [ h]thymidine was added 3 d later for 812 h. the culture was harvested and analyzed as previously described ( 43 ) . responder spleen cells ( 10/well ) from traf1/tcr doubletransgenic or tcr single - transgenic littermates were cultured with 2 10 20 gy - irradiated stimulator macrophages previously labeled with 10 nm gp33 in 96-well flat - bottomed microtiter plates at 37c , 5% co2 in complete imdm . after 3 d , the cultures were pulsed for 16 h with 1 ci of [ h]thymidine , harvested , and counted on a matrix 96 direct counter ( canberra packard ) . cytotoxicity by tcr transgenic spleen cells was tested in duplicate cultures and serial dilutions in 96-well round - bottomed plates in 5-h cr ( nen dupont , boston , ma ) release assays using el4 target cells ( h-2 ) previously incubated with 10 nm gp33 peptide for 1 h at 37c . the supernatant of the cytotoxicity cultures was counted by a cobra ii gamma counter ( canberra packard ) . an expression vector ( phse traf1 ) for traf1 transgenic mice was generated by cloning the flu epitope bamhi 3 site of the ph se-3 expression cassette ( 38 , 39 ) . phse-3 expression cassette with an h-2k promoter and an igh enhancer was previously described to express the transgene both in immature thymocytes and in mature t cells ( 38 , 39 ) . transgene dna was isolated by xhoi digestion and microinjected into ( c57bl/6 cba / j)f2 embryos . transgenic founders were identified by southern blot analysis of tail dna and transgenic lines were established by backcrossing with either c57bl/6 or balb / c mice to yield vsag7 h-2 or h-2 mice . mice transgenic for an tcr specific for h-2d and the lymphocyte choriomeningitis virus ( lcmv ) glycoprotein ( gp)derived peptide amino acids 3341 ( gp33 , the major lcmv epitope in the h-2 haplotype ) were previously described ( 13 , 38 , 39)and crossed with traf1 transgenic mice . mabs , rat anti - cd8 ( 53 - 6.7 ; fitc conjugated ) , and anti - va2 ( b20.1 ; pe conjugated ) were purchased from pharmingen ( san diego , ca ) . for induction of apoptosis in vitro , lymph node t cells from traf1 transgenic and negative littermates were treated with con a ( 5 g / ml ) for 48 h , washed with 10 mg / ml -methylmannoside , and incubated in 50 iu / ml il-2 to predispose to apoptosis as previously described ( 12 ) . cells ( 50,000/well ) with > 97% -tcr were then cultured for 48 h in anti - cd3 ab ( 145 - 2c11)coated plates in triplicate . viable cells were then quantified by propidium iodide ( pi ) exclusion using a facscaliber with cellquest software . . specific cell survival ( % ) was determined from the percentages of viable ( pi - negative ) cells and it equals 100 ( % survival after cd3 cross - linking / % survival without cd3 cross - linking ) . to induce apoptosis of antigen - specific t cells in tcr transgenic mice , mice were treated with a single intrafootpad injection of gp33 peptide ( 30 g ) . tcr transgenic cd8 cells from traf1 transgenic mice and negative littermates in regional lymph nodes were analyzed 3 and 4 d after intrafootpad injection of gp33 peptide . draining popliteal and inguinal lymph node cells were taken from tcr transgenic mice ( h-2 ) either untreated ( day 0 ) or 2 d after a single intrafootpad injection of 30 g gp33 peptide and incubated in imdm supplemented with 10% fcs , 5 10 m 2-mercaptoethanol , penicillin , and streptomycin ( complete imdm ) for 24 h ( day 3 ) or 48 h ( day 4 ) as described ( 13 ) . the percentages of surviving cells were determined by flow cytometry ( facscan ) by gating for lymphocytes based on forward and side scatter . for induction of tolerance by high dose peptide antigens in non - tcr transgenic mice , h-2 traf1 transgenic or negative littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide or buffered salt solution in ifa as previously described ( 40 , 41 ) . the mice were infected on day 16 and tested on day 24 as previously described ( 41 , 42 ) . for induction of apoptosis by lcmv , 10 tcr transgenic spleen cells from traf1/tcr double - transgenic or tcr single - transgenic littermates were injected intravenously in c57bl/6 mice as previously described ( 42 ) . at the same time , the mice were infected with lcmv we strain . for in vivo expansion , the viral dose was 200 pfu and analysis was 7 d after infection ; for in vivo deletion , the viral dose was 10 pfu and analysis was 4 and 7 d after infection . tcr transgenic t cells were determined by double staining with cd8- and v2-specific antibodies and flow cytometry analysis by gating for lymphocytes based on forward/ side scatters . t cells isolated from lymph nodes using t cell enrichment columns ( biotex laboratories , inc . , edmonton , canada ) were also induced to proliferate by titrating them in triplicate into anti - tcr ab ( h57 - 597 ) coated tissue culture wells containing mitomycin c treated splenocytes ( 5 10/well ) as previously described ( 43 ) . [ h]thymidine was added 3 d later for 812 h. the culture was harvested and analyzed as previously described ( 43 ) . responder spleen cells ( 10/well ) from traf1/tcr doubletransgenic or tcr single - transgenic littermates were cultured with 2 10 20 gy - irradiated stimulator macrophages previously labeled with 10 nm gp33 in 96-well flat - bottomed microtiter plates at 37c , 5% co2 in complete imdm . after 3 d , the cultures were pulsed for 16 h with 1 ci of [ h]thymidine , harvested , and counted on a matrix 96 direct counter ( canberra packard ) . cytotoxicity by tcr transgenic spleen cells was tested in duplicate cultures and serial dilutions in 96-well round - bottomed plates in 5-h cr ( nen dupont , boston , ma ) release assays using el4 target cells ( h-2 ) previously incubated with 10 nm gp33 peptide for 1 h at 37c . the supernatant of the cytotoxicity cultures was counted by a cobra ii gamma counter ( canberra packard ) . to examine the potential role of traf1 during antigen - induced deletion of mature t cells that are mediated in part by tnfrs , we have generated transgenic mice that overexpress traf1 in various subsets of t cells . levels of traf1 expression in thymus or spleen from traf1 transgenic mice exceeded those seen in control animals by 5- to 20-fold when determined by northern or western blot analysis ( fig . 1 , a and b ) . two of these transgenic lines ( traf1.tg-11 and traf1.tg-10 ) were used for further analysis . flow cytometry analysis of thymocytes from 6- to 8-wk - old traf1 transgenic or negative littermates did not indicate any significant difference in the total number of thymocytes ( data not shown ) . moreover , the percentage of double - negative ( cd4cd8 ) , double - positive ( cd4cd8 ) , and single - positive ( cd4 or cd8 ) thymocytes in traf1 transgenic mice was comparable to that in negative littermates ( data not shown ) . the numbers and ratios of t and b cells or cd4 and cd8 t cells from spleen or lymph nodes of traf1 transgenic mice were also comparable to those in negative littermates ( data not shown ) , suggesting that traf1 overexpression did not dramatically affect lymphocyte development . when purified t cells from traf1 transgenic and negative littermates were tested for their ability to divide in response to anti - tcr antibody ( h57 - 597 ) in vitro , t cells from traf1 transgenic and negative littermates responded similarly to antitcr ab ( fig . these results demonstrate that traf1 overexpression did not affect tcr - mediated proliferation of mature t cells in vitro . to examine whether tcr - mediated mature t cell death from traf1 transgenic mice was affected in vitro , resting lymph node cells from traf1 transgenic and negative littermates were primed for apoptosis by con a and il-2 treatment as previously described ( 12 ) . when the tcr cd3 complex was cross - linked , t cells from control mice underwent massive cell death as previously shown ( 12 ) ( fig . however , the percentage of t cells undergoing anti - cd3induced death from traf1.tg-10 mice was significantly lower than that of t cells from negative littermates ( fig . 2 b ) . percent viable cells of cd8 or cd8 ( cd4 ) t cells as determined from pi - negative cells with and without anti - cd3 stimulation showed a protective effect of traf1 for cd8 t cell death ( fig . no such effect was seen for cd8 ( cd4 ) t cell death ( fig . 2 c ) . because the death of cd8 t cells in this in vitro experimental system is mostly mediated by tnfr2 ( 12 ) , the results strongly suggest that traf1 overexpression antagonized the apoptotic signals exerted by tnfr2 . the results showing that cd8 ( cd4 ) t cell death was not affected by traf1 overexpression indicates that traf1 overexpression specifically inhibited tcr - mediated mature cd8 t cell death at least in this in vitro deletion system . unlike the traf1.tg-10 mice , the death of t cells from traf1 . tg-11 mice triggered by the similar tcr cd3 complex cross - linking in vitro was similar to that of t cells from control mice ( data not shown ) . this is probably because of the difference between these two transgenic lines in the amount of traf1 expressed . to examine further the effect of traf1 overexpression on the induction of apoptosis of cd8 t cells , we have examined the deletion of mature t cells in traf1 transgenic mice using a defined lcmv glycoprotein specific tcr model ( 38 , 39 ) . -tcr transgenic mouse line specific for h-2d and lcmv gp ( residues 3341 , gp33 ) ( 38 , 39 ) was bred with traf1 transgenic mice . owing to the high mortality of traf1.tg-10 mice with yet to be determined reasons , only mice from the traf1.tg-11 line were used in these experiments . development and selection of lcmv - specific -tcrs in tcr traf1 double - transgenic mice was normal when compared with that in control tcr single - transgenic littermates ( data not shown ) . when splenocytes from -tcr traf1 double - transgenic mice were stimulated with gp33 , the degree of proliferation was comparable to splenocytes from -tcr single - transgenic mice ( fig . traf1 double - transgenic mice showed cytotoxicity to target cells presenting gp33 peptides comparable to -tcr single - transgenic mice ( fig . 3 b ) . 2 a ) and confirm that traf1 overexpression did not affect t cell development , the proliferative response , or the effector function of t cells . to test whether traf1 overexpression affected the antigen - induced apoptosis of mature t cells , 30 g of gp33 peptides was injected into the hind footpad . this protocol induces cell death of antigen - specific t cells in the draining lymph nodes ( 13 ) . when transgenic tcr t cells were analyzed from the draining lymph nodes of tcr singletransgenic mice , antigen - induced cell death of transgenic tcr t cells was clearly observed after peptide treatment as previously described ( 13 ) ( fig . traf1 double - transgenic mice injected with gp33 peptide , the death of transgenic tcr t cells was significantly reduced compared with -tcr single - transgenic mice ( fig . 3 c ) . these results suggested that traf1 overexpression hampered a signaling pathway required for the antigen - induced apoptosis of cd8 t cells . additional experiments involving adoptive transfers of transgenic tcr t cells along with antigen were performed to determine the effect of traf1 overexpression on the deletion of cd8 t cells . as previously shown , when transgenic tcr t cells specific for lcmv gp33 were transferred to c57bl/6 mice with a high dose of lcmv , a vigorous expansion of transgenic t cells was followed by a rapid decline due to the induction of cell death ( 42 ) ( fig . 3 d ) . when the same experiment was performed with transgenic tcr t cells from tcr traf1 double - transgenic mice , the deletion of transgenic tcr t cells was significantly lower ( fig . 3 d ) , indicating that traf1 overexpression inhibited the deletion of cd8 t cells induced by high dose antigen treatment . consistent with in vitro proliferation experiments , traf1 overexpression did not affect expansion of transgenic tcr t cells by a low dose of lcmv during adoptive transfer ( 42 ) ( fig . traf1 overexpression also inhibited the induction of tolerance to lcmv glycoprotein by high dose antigen treatment in non - tcr transgenic mice . when mice were infected with lcmv , both traf1 transgenic and negative littermates mounted an efficient ctl response ( fig . when control mice were pretreated three times intraperitoneally with 500 g of gp33 in ifa , the induction of ctl activities against lcmv was completely abrogated as previously described ( 40 , 41 ) ( fig . 4 b ) . in contrast , traf1 transgenic mice mounted significant lcmv - specific ctl activities even after they were pretreated three times intraperitoneally with 500 g gp33 in ifa ( fig . 4 c ) . these results indicate that traf1 overexpression did not affect ctl activation but inhibited antigen - induced t cell death . in contrast with the deletion of mature t cells induced by a peptide antigen , traf1 overexpression did not inhibit superantigen - induced deletion of mature t cells . when traf1 transgenic mice were treated three times intraperitoneally with 100 g of staphyloccocal enterotoxin b ( seb ) , the deletion of either cd4 or cd8 t cells from traf1 transgenic mice was comparable to that of t cells from control mice ( data not shown ) . this suggests that traf1 overexpression inhibits only specific signaling cascades required for deletion of mature t cells in vivo , rather than acting as a nonspecific anti - apoptotic signal . complex and multiple pathways were shown to operate in the induction of apoptosis in mature t cells . for example , the involvement of fas(cd95 ) has been shown for deletion of cd4 t cells in the 2b4 tcr transgenic mice system but both fas(cd95 ) and tnfrs for deletion of cd4 t cells in the hnt tcr transgenic mice system ( 10 , 11 ) . therefore , it is possible that different experimental systems involving various tcr mhc ag complexes with different affinities / avidities may result in the utilization of multiple pathways to mediate cell death . this may explain why traf1 overexpression inhibits the induction of apoptosis in mature cd8 t cells by a peptide antigen ( i.e. , gp33 ) but not by a superantigen ( seb ) . in summary , the results in this study showed that traf1 overexpression inhibits the antigen - induced deletion of activated cd8 t cells in vivo and in vitro . although we have failed to show that traf1 overexpression did not affect cd4 t cells , this does not exclude the possibility that traf1 can play a role , which needs to be determined , in cd4 t cell death in some experimental systems . nevertheless , the results provide evidence for the biological role of traf1 as a regulator of the antigen - induced apoptotic signals in mature t cells . traf1 is a component of the tnfr2 signaling complex ( 24 ) and traf1 overexpression inhibited antigeninduced apoptosis of mature cd8 t cells in an in vitro system , in which anti - tcr induced apoptosis of cd8 t cells is mediated by the tnfr2 signaling complex ( 12 ) . this suggests that the signal intermediates for induction of apoptosis by the tnfr2 involves traf1 . whether tnfr2-mediated signals can induce cell death has been controversial due to various contradicting results ( 7 , 8) . experiments described here showing that the overexpression of traf1 , a tnfr2-proximal signaling component , inhibits the induction of apoptosis support the idea that proximal signals induced by the tnfr2 are directly involved in the tcr - mediated cell death of mature cd8 t cells . the precise mechanism that traf1 overexpression inhibits the induction of apoptosis is not clear at this point . because traf1 ( and also other traf proteins ) does not contain any known catalytic domain , it is most likely that traf1 overexpression inhibited the tnfr2-mediated apoptosis by altering the constituents of the tnfr2 signaling complex . it has been recently shown that nf-b activation acts as an anti - apoptotic signal during tnf - induced apoptosis ( 4447 ) . it is not likely that traf1 overexpression affected nf-b mediated anti - apoptotic signals because it does not influence the tnfr - mediated nf-b activation in transient transfection experiments ( 3436 ) and also in activated t cells from traf1 transgenic mice ( data not shown ) . one plausible mechanism is that traf1 overexpression may increase the recruitment of anti - apoptotic protein(s ) such as c - iaps to the tnfr2 signaling complex ( 37 ) . another possibility is that traf1 overexpression may inhibit the recruitment or aggregation of proapoptotic signal transducers to the tnfr2 signaling complex . it is also possible that traf1 may mediate an as yet to be determined anti - apoptotic signal during antigen - induced cell death of mature t cells . this anti - apoptotic property of traf1 may contribute to cell transformation by lmp protein of ebv , which triggers aggregation of several traf proteins , including traf1 ( 30 ) . in addition to establishing that traf1 is a component of the tnfr - proximal signaling intermediate involved in the regulation of apoptosis , our results also provide in vivo evidence supporting the postulate that traf proteins are likely to exert pleiotropic signals ( cell proliferation or apoptosis ) depending on the various combination of traf proteins in a given cell type ( 25 , 37 ) . future identification of downstream signal transducers that bridge traf proteins to the apoptotic machinery such as caspases will be important to understand the detailed molecular mechanisms of how apoptotic signals are regulated by many members of the tnfr family . transgenic mice overexpressing traf1 . ( a ) northern blot analysis of traf1 expression . total rna was isolated from thymocytes and splenocytes , and analyzed by northern blot analysis using a traf1 cdna probe as previously described ( 32 ) . the amount of total rna loaded in each lane was similar based on the etbr - stained rrnas ( data not shown ) . upon longer exposure of the autoradiogram , low level of traf1 mrna was detected both in thymocytes and splenocytes from control mice ( data not shown ) . cell lysates were purified from thymocytes and splenocytes , and analyzed by western blot analysis using an anti - epitope mab ( 12ca5 ) as previously described ( 32 ) . western blot analysis with anti - traf1 polyclonal abs showed similar results ( data not shown ) . in vitro proliferation and apoptosis of mature t cells . ( a ) traf1 overexpression does not affect tcr - mediated proliferation of mature t cells in vitro . t cells were isolated from lymph nodes of control ( open circle ) and traf1.tg-10 transgenic ( closed circle ) littermates using t cell enrichment columns ( biotex laboratories , inc . ) and cultured in triplicate into anti - tcr ab ( h57 - 597 ) coated - tissue culture wells containing mitomycin c treated splenocytes ( 5 10/well ) . [ h]thymidine was added 3 d later for 812 h. the culture was harvested and analyzed as described in materials and methods . the data are from one of several similar experiments using t cells from nontransgenic and traf1.tg-10 transgenic mice littermates ( h-2 ) . similar results were obtained when t cells from traf1.tg-11 mice were used ( data not shown ) . ( b and c ) traf1 overexpression inhibits tcr - mediated apoptosis of cd8 t cells in vitro . a representative experiment showing the flow cytometric plots displaying propidium iodide ( pi ) fluorescence versus forward light scatter for traf1 transgenic and negative littermates after 48 h with il-2 in either uncoated wells ( medium ) or wells coated with anti - cd3 ( 10 g / ml ) is shown in b. viable cells were quantified by pi exclusion using a facscaliber with cellquest software and show low pi fluorescence . the percentages of cell survival in cd8 ( cd4 ) or cd8 t cells were shown as the mean sem from the triplicate samples of three mice from each group . cells , cultured in either uncoated ( medium ) or anti - cd3coated plates as described in b , were recovered from the plates and stained with anti - cd8 ab ( 53 - 6.7 ; fitc conjugated ) . the cd8 staining was used to gate the cells into cd8 and cd8 pools ; control staining showed that virtually all cd8 cells were cd4 . specific cell survival ( % ) was determined from the percentages of viable ( pi - negative ) cells and it equals 100 ( % survival after cd3 cross - linking/% survival without cd3 cross - linking ) . normal proliferation and ctl response , but impaired activation - induced cell death in tcr proliferation of gp33 peptide - specific tcr transgenic lymphocytes from traf1 transgenic mice ( closed symbols ) and negative littermates ( open symbols ) in the presence of unlabeled macrophages ( circles ) or macrophages labeled with gp33 peptide ( squares ) . ( b ) normal virus - specific cytotoxic responses are induced in traf1 transgenic mice . similar cytotoxicity to el4 target cells unlabeled ( circles ) or labeled with gp33 peptide ( squares ) by tcr transgenic spleen cells stimulated for 2 d with gp33 in vitro : traf1 transgenic mice ( closed symbols ) and negative littermates ( open symbols ) . proliferation and cytotoxicity of transgenic t cells were determined as described in materials and methods and one of three representative experiments is shown . increased survival of tcr transgenic cd8 cells from traf1 transgenic mice ( closed symbols ) as opposed to negative littermates ( open symbols ) in regional lymph nodes analyzed 3 and 4 d after intrafootpad injection of gp33 peptide . ( d ) normal expansion but impaired deletion of virus - induced cd8 t cells in vivo . 10 tcr transgenic spleen cells from traf1tcr double - transgenic ( closed bars ) or tcr single - transgenic ( open bars ) littermates were injected intravenously in c57bl/6 mice as described in materials and methods . at the same time , the mice were infected with lcmv we strain . for in vivo expansion , the viral dose was 200 pfu and analysis was 7 d after infection ; for in vivo deletion , the viral dose was 10 pfu and analysis was 4 and 7 d after infection . the data indicate percentages of spleen cells as determined by double staining with cd8- and v2-specific antibodies and flow cytometry analysis by gating for lymphocytes based on forward / side scatters . traf1 overexpression inhibits peptide - induced t cell tolerance in vivo also in non - tcr transgenic mice . ( a ) the lcmv - specific cytotoxic responses are similar in traf1 transgenic and negative littermates . h-2 mice were infected intravenously with 2000 pfu lcmv armstrong strain . on day 8 , spleen cells from traf1 transgenic ( closed symbols ) and traf1 negative ( open symbols ) littermates were tested on gp33-labeled ( squares ) and not labeled ( circles ) el4 target cells in 5-h cr - release assays as described in materials and methods . ( b and c ) impaired deletion of peptide - specific t cells in traf1 transgenic mice . for induction of tolerance , h-2 control ( b ) or traf1 transgenic ( c ) littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide ( squares ) or buffered salt solution ( circles ) in ifa as described in materials and methods . the mice were infected on day 16 and tested on day 24 as in ( a ) .

tumor necrosis factor receptor ( tnfr)associated factor 2 ( traf2 ) and traf1 were found as components of the tnfr2 signaling complex , which exerts multiple biological effects on cells such as cell proliferation , cytokine production , and cell death . in the tnfr2-mediated signaling pathways , traf2 works as a mediator for activation signals such as nf-b , but the role of traf1 has not been previously determined . here we show in transgenic mice that traf1 overexpression inhibits antigen - induced apoptosis of cd8 + t lymphocytes . our results demonstrate a biological role for traf1 as a regulator of apoptotic signals and also support the hypothesis that the combination of traf proteins in a given cell type determines distinct biological effects triggered by members of the tnf receptor superfamily .

Materials and Methods Generation of TRAF1 Transgenic Mice. Mice and Reagents. Induction of Apoptosis and Lymphocyte Survival Assays. Lymphocyte Proliferation and Cytotoxicity Assay. Results and Discussion Figures and Tables

an expression vector ( phse traf1 ) for traf1 transgenic mice was generated by cloning the flu epitope bamhi 3 site of the ph se-3 expression cassette ( 38 , 39 ) . mice transgenic for an tcr specific for h-2d and the lymphocyte choriomeningitis virus ( lcmv ) glycoprotein ( gp)derived peptide amino acids 3341 ( gp33 , the major lcmv epitope in the h-2 haplotype ) were previously described ( 13 , 38 , 39)and crossed with traf1 transgenic mice . mabs , rat anti - cd8 ( 53 - 6.7 ; fitc conjugated ) , and anti - va2 ( b20.1 ; pe conjugated ) were purchased from pharmingen ( san diego , ca ) . to induce apoptosis of antigen - specific t cells in tcr transgenic mice , mice were treated with a single intrafootpad injection of gp33 peptide ( 30 g ) . tcr transgenic cd8 cells from traf1 transgenic mice and negative littermates in regional lymph nodes were analyzed 3 and 4 d after intrafootpad injection of gp33 peptide . draining popliteal and inguinal lymph node cells were taken from tcr transgenic mice ( h-2 ) either untreated ( day 0 ) or 2 d after a single intrafootpad injection of 30 g gp33 peptide and incubated in imdm supplemented with 10% fcs , 5 10 m 2-mercaptoethanol , penicillin , and streptomycin ( complete imdm ) for 24 h ( day 3 ) or 48 h ( day 4 ) as described ( 13 ) . for induction of tolerance by high dose peptide antigens in non - tcr transgenic mice , h-2 traf1 transgenic or negative littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide or buffered salt solution in ifa as previously described ( 40 , 41 ) . after 3 d , the cultures were pulsed for 16 h with 1 ci of [ h]thymidine , harvested , and counted on a matrix 96 direct counter ( canberra packard ) . the supernatant of the cytotoxicity cultures was counted by a cobra ii gamma counter ( canberra packard ) . an expression vector ( phse traf1 ) for traf1 transgenic mice was generated by cloning the flu epitope bamhi 3 site of the ph se-3 expression cassette ( 38 , 39 ) . mice transgenic for an tcr specific for h-2d and the lymphocyte choriomeningitis virus ( lcmv ) glycoprotein ( gp)derived peptide amino acids 3341 ( gp33 , the major lcmv epitope in the h-2 haplotype ) were previously described ( 13 , 38 , 39)and crossed with traf1 transgenic mice . to induce apoptosis of antigen - specific t cells in tcr transgenic mice , mice were treated with a single intrafootpad injection of gp33 peptide ( 30 g ) . tcr transgenic cd8 cells from traf1 transgenic mice and negative littermates in regional lymph nodes were analyzed 3 and 4 d after intrafootpad injection of gp33 peptide . draining popliteal and inguinal lymph node cells were taken from tcr transgenic mice ( h-2 ) either untreated ( day 0 ) or 2 d after a single intrafootpad injection of 30 g gp33 peptide and incubated in imdm supplemented with 10% fcs , 5 10 m 2-mercaptoethanol , penicillin , and streptomycin ( complete imdm ) for 24 h ( day 3 ) or 48 h ( day 4 ) as described ( 13 ) . for induction of tolerance by high dose peptide antigens in non - tcr transgenic mice , h-2 traf1 transgenic or negative littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide or buffered salt solution in ifa as previously described ( 40 , 41 ) . to examine the potential role of traf1 during antigen - induced deletion of mature t cells that are mediated in part by tnfrs , we have generated transgenic mice that overexpress traf1 in various subsets of t cells . levels of traf1 expression in thymus or spleen from traf1 transgenic mice exceeded those seen in control animals by 5- to 20-fold when determined by northern or western blot analysis ( fig . two of these transgenic lines ( traf1.tg-11 and traf1.tg-10 ) were used for further analysis . moreover , the percentage of double - negative ( cd4cd8 ) , double - positive ( cd4cd8 ) , and single - positive ( cd4 or cd8 ) thymocytes in traf1 transgenic mice was comparable to that in negative littermates ( data not shown ) . the numbers and ratios of t and b cells or cd4 and cd8 t cells from spleen or lymph nodes of traf1 transgenic mice were also comparable to those in negative littermates ( data not shown ) , suggesting that traf1 overexpression did not dramatically affect lymphocyte development . these results demonstrate that traf1 overexpression did not affect tcr - mediated proliferation of mature t cells in vitro . to examine whether tcr - mediated mature t cell death from traf1 transgenic mice was affected in vitro , resting lymph node cells from traf1 transgenic and negative littermates were primed for apoptosis by con a and il-2 treatment as previously described ( 12 ) . when the tcr cd3 complex was cross - linked , t cells from control mice underwent massive cell death as previously shown ( 12 ) ( fig . percent viable cells of cd8 or cd8 ( cd4 ) t cells as determined from pi - negative cells with and without anti - cd3 stimulation showed a protective effect of traf1 for cd8 t cell death ( fig . no such effect was seen for cd8 ( cd4 ) t cell death ( fig . because the death of cd8 t cells in this in vitro experimental system is mostly mediated by tnfr2 ( 12 ) , the results strongly suggest that traf1 overexpression antagonized the apoptotic signals exerted by tnfr2 . the results showing that cd8 ( cd4 ) t cell death was not affected by traf1 overexpression indicates that traf1 overexpression specifically inhibited tcr - mediated mature cd8 t cell death at least in this in vitro deletion system . tg-11 mice triggered by the similar tcr cd3 complex cross - linking in vitro was similar to that of t cells from control mice ( data not shown ) . this is probably because of the difference between these two transgenic lines in the amount of traf1 expressed . to examine further the effect of traf1 overexpression on the induction of apoptosis of cd8 t cells , we have examined the deletion of mature t cells in traf1 transgenic mice using a defined lcmv glycoprotein specific tcr model ( 38 , 39 ) . -tcr transgenic mouse line specific for h-2d and lcmv gp ( residues 3341 , gp33 ) ( 38 , 39 ) was bred with traf1 transgenic mice . owing to the high mortality of traf1.tg-10 mice with yet to be determined reasons , only mice from the traf1.tg-11 line were used in these experiments . development and selection of lcmv - specific -tcrs in tcr traf1 double - transgenic mice was normal when compared with that in control tcr single - transgenic littermates ( data not shown ) . when splenocytes from -tcr traf1 double - transgenic mice were stimulated with gp33 , the degree of proliferation was comparable to splenocytes from -tcr single - transgenic mice ( fig . 2 a ) and confirm that traf1 overexpression did not affect t cell development , the proliferative response , or the effector function of t cells . to test whether traf1 overexpression affected the antigen - induced apoptosis of mature t cells , 30 g of gp33 peptides was injected into the hind footpad . this protocol induces cell death of antigen - specific t cells in the draining lymph nodes ( 13 ) . when transgenic tcr t cells were analyzed from the draining lymph nodes of tcr singletransgenic mice , antigen - induced cell death of transgenic tcr t cells was clearly observed after peptide treatment as previously described ( 13 ) ( fig . traf1 double - transgenic mice injected with gp33 peptide , the death of transgenic tcr t cells was significantly reduced compared with -tcr single - transgenic mice ( fig . these results suggested that traf1 overexpression hampered a signaling pathway required for the antigen - induced apoptosis of cd8 t cells . additional experiments involving adoptive transfers of transgenic tcr t cells along with antigen were performed to determine the effect of traf1 overexpression on the deletion of cd8 t cells . when the same experiment was performed with transgenic tcr t cells from tcr traf1 double - transgenic mice , the deletion of transgenic tcr t cells was significantly lower ( fig . 3 d ) , indicating that traf1 overexpression inhibited the deletion of cd8 t cells induced by high dose antigen treatment . traf1 overexpression also inhibited the induction of tolerance to lcmv glycoprotein by high dose antigen treatment in non - tcr transgenic mice . these results indicate that traf1 overexpression did not affect ctl activation but inhibited antigen - induced t cell death . in contrast with the deletion of mature t cells induced by a peptide antigen , traf1 overexpression did not inhibit superantigen - induced deletion of mature t cells . when traf1 transgenic mice were treated three times intraperitoneally with 100 g of staphyloccocal enterotoxin b ( seb ) , the deletion of either cd4 or cd8 t cells from traf1 transgenic mice was comparable to that of t cells from control mice ( data not shown ) . this suggests that traf1 overexpression inhibits only specific signaling cascades required for deletion of mature t cells in vivo , rather than acting as a nonspecific anti - apoptotic signal . for example , the involvement of fas(cd95 ) has been shown for deletion of cd4 t cells in the 2b4 tcr transgenic mice system but both fas(cd95 ) and tnfrs for deletion of cd4 t cells in the hnt tcr transgenic mice system ( 10 , 11 ) . therefore , it is possible that different experimental systems involving various tcr mhc ag complexes with different affinities / avidities may result in the utilization of multiple pathways to mediate cell death . this may explain why traf1 overexpression inhibits the induction of apoptosis in mature cd8 t cells by a peptide antigen ( i.e. in summary , the results in this study showed that traf1 overexpression inhibits the antigen - induced deletion of activated cd8 t cells in vivo and in vitro . although we have failed to show that traf1 overexpression did not affect cd4 t cells , this does not exclude the possibility that traf1 can play a role , which needs to be determined , in cd4 t cell death in some experimental systems . nevertheless , the results provide evidence for the biological role of traf1 as a regulator of the antigen - induced apoptotic signals in mature t cells . traf1 is a component of the tnfr2 signaling complex ( 24 ) and traf1 overexpression inhibited antigeninduced apoptosis of mature cd8 t cells in an in vitro system , in which anti - tcr induced apoptosis of cd8 t cells is mediated by the tnfr2 signaling complex ( 12 ) . this suggests that the signal intermediates for induction of apoptosis by the tnfr2 involves traf1 . whether tnfr2-mediated signals can induce cell death has been controversial due to various contradicting results ( 7 , 8) . experiments described here showing that the overexpression of traf1 , a tnfr2-proximal signaling component , inhibits the induction of apoptosis support the idea that proximal signals induced by the tnfr2 are directly involved in the tcr - mediated cell death of mature cd8 t cells . the precise mechanism that traf1 overexpression inhibits the induction of apoptosis is not clear at this point . because traf1 ( and also other traf proteins ) does not contain any known catalytic domain , it is most likely that traf1 overexpression inhibited the tnfr2-mediated apoptosis by altering the constituents of the tnfr2 signaling complex . it has been recently shown that nf-b activation acts as an anti - apoptotic signal during tnf - induced apoptosis ( 4447 ) . it is not likely that traf1 overexpression affected nf-b mediated anti - apoptotic signals because it does not influence the tnfr - mediated nf-b activation in transient transfection experiments ( 3436 ) and also in activated t cells from traf1 transgenic mice ( data not shown ) . one plausible mechanism is that traf1 overexpression may increase the recruitment of anti - apoptotic protein(s ) such as c - iaps to the tnfr2 signaling complex ( 37 ) . another possibility is that traf1 overexpression may inhibit the recruitment or aggregation of proapoptotic signal transducers to the tnfr2 signaling complex . it is also possible that traf1 may mediate an as yet to be determined anti - apoptotic signal during antigen - induced cell death of mature t cells . this anti - apoptotic property of traf1 may contribute to cell transformation by lmp protein of ebv , which triggers aggregation of several traf proteins , including traf1 ( 30 ) . in addition to establishing that traf1 is a component of the tnfr - proximal signaling intermediate involved in the regulation of apoptosis , our results also provide in vivo evidence supporting the postulate that traf proteins are likely to exert pleiotropic signals ( cell proliferation or apoptosis ) depending on the various combination of traf proteins in a given cell type ( 25 , 37 ) . future identification of downstream signal transducers that bridge traf proteins to the apoptotic machinery such as caspases will be important to understand the detailed molecular mechanisms of how apoptotic signals are regulated by many members of the tnfr family . total rna was isolated from thymocytes and splenocytes , and analyzed by northern blot analysis using a traf1 cdna probe as previously described ( 32 ) . upon longer exposure of the autoradiogram , low level of traf1 mrna was detected both in thymocytes and splenocytes from control mice ( data not shown ) . ( a ) traf1 overexpression does not affect tcr - mediated proliferation of mature t cells in vitro . t cells were isolated from lymph nodes of control ( open circle ) and traf1.tg-10 transgenic ( closed circle ) littermates using t cell enrichment columns ( biotex laboratories , inc . ) the data are from one of several similar experiments using t cells from nontransgenic and traf1.tg-10 transgenic mice littermates ( h-2 ) . ( b and c ) traf1 overexpression inhibits tcr - mediated apoptosis of cd8 t cells in vitro . a representative experiment showing the flow cytometric plots displaying propidium iodide ( pi ) fluorescence versus forward light scatter for traf1 transgenic and negative littermates after 48 h with il-2 in either uncoated wells ( medium ) or wells coated with anti - cd3 ( 10 g / ml ) is shown in b. viable cells were quantified by pi exclusion using a facscaliber with cellquest software and show low pi fluorescence . normal proliferation and ctl response , but impaired activation - induced cell death in tcr proliferation of gp33 peptide - specific tcr transgenic lymphocytes from traf1 transgenic mice ( closed symbols ) and negative littermates ( open symbols ) in the presence of unlabeled macrophages ( circles ) or macrophages labeled with gp33 peptide ( squares ) . ( b ) normal virus - specific cytotoxic responses are induced in traf1 transgenic mice . similar cytotoxicity to el4 target cells unlabeled ( circles ) or labeled with gp33 peptide ( squares ) by tcr transgenic spleen cells stimulated for 2 d with gp33 in vitro : traf1 transgenic mice ( closed symbols ) and negative littermates ( open symbols ) . ( d ) normal expansion but impaired deletion of virus - induced cd8 t cells in vivo . traf1 overexpression inhibits peptide - induced t cell tolerance in vivo also in non - tcr transgenic mice . on day 8 , spleen cells from traf1 transgenic ( closed symbols ) and traf1 negative ( open symbols ) littermates were tested on gp33-labeled ( squares ) and not labeled ( circles ) el4 target cells in 5-h cr - release assays as described in materials and methods . ( b and c ) impaired deletion of peptide - specific t cells in traf1 transgenic mice . for induction of tolerance , h-2 control ( b ) or traf1 transgenic ( c ) littermates were injected intraperitoneally on days 0 , 3 , and 6 with 500 g gp33 peptide ( squares ) or buffered salt solution ( circles ) in ifa as described in materials and methods .

all reagents , except for those stated otherwise , were purchased from sigma - aldrich co ( st . louis , mo ) . antibodies for skp2 were obtained from us biological inc ( swampscott , mass ) and invitrogen ( carlsbad , calif ) . mouse anti - p27 and anti - p21 antibodies were from bd transduction laboratories ( oxford , united kingdom ) . rabbit anti - phospho - rb and mouse anti - bromodeoxyuridine ( brdu ) antibodies were bought from cell signaling technology ( danvers , mass ) and mp biomedicals ( solon , ohio ) , respectively . all secondary antibodies were purchased from dako ( burlingame , calif ) and novared from vector laboratories ( burlingame , calif ) . genotypes of mice were determined by polymerase change reaction ( pcr ) of tail tip dna . male sprague - dawley ( sd ) rats were obtained from charles river ( margate , united kingdom ) or biolasco taiwan co ltd ( taiwan ) . the housing and care of the animals and all procedures used adhered to the guidelines and regulations of the united kingdom animal ( scientific procedures ) act 1986 , united kingdom , and mackay memorial hospital , taiwan . male skp2 and skp2 mice ( 20 - 30 grams ) were anesthetized by inhalation of isoflurane and oxygen ( 0.2 l / minute ) . all animals were killed 28 days later and perfusion fixed with 4% formaldehyde / phosphate buffered saline ( pbs ) for 7 minutes before excision of ligated carotid arteries . sections were stained with elastic van gieson ( evg ) and hematoxylin and eosin ( h&e ) stain and neointimal areas , defined as the area of tissue within the internal elastic lamina , quantified using image pro analysis software ( mediacybernetics , bethesda , md ) . vessel diameter ( distance between the external elastic lamellae ) and medial thickness ( distance between the internal and external elastic lamellae ) were measured 300 - 400 m distal to the ligation point . balloon injury of left common carotid artery was performed as described by clowes et al . briefly , male sd rats ( 400 grams ) were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride ( 100 mg / kg ) and xylazine ( 10 mg / kg ) . the bifurcation of the left common carotid artery was surgically exposed , and a 2f arterial embolectomy balloon catheter ( edwards lifesciences co , irvine , calif ) was introduced through an arteriotomy on left external carotid artery . the balloon was inflated with saline and pulled back with rotation through common carotid artery three times . the injured common carotid arteries were then subjected to adenovirus - mediated gene delivery . for cell proliferation assay , animals were injected subcutaneously with brdu solution ( 25 mg / kg ) at 17 , 9 , and 1 hour before euthanasia . the procedures of filament de - endothelialization of rat common carotid artery were modified from the methods developed by ma reidy 's group . male sd rats were anesthetized and the left common carotid arteries were approached as described in balloon injury . the endothelium was then denuded from a carotid artery by a 5 - 0 nylon loop . briefly , the nylon loop was introduced into the left external carotid artery through a trocar . the loop was then slowly pulled back along the common carotid artery with constant rotation for 30 seconds . the trocar was retrieved and the artery underwent subsequent adenoviral infection for specific gene expression . recombinant replication - defective adenoviruses encoding wild - type skp2 ( ad : wt - skp2 ) , dominant - negative skp2 ( ad : dn - skp2 ) , and -galactosidase ( ad:gal ) have been described previously . to infect rat common carotid artery with adenoviruses for gene expression , both proximal end of common carotid artery and internal carotid artery were temporarily clamped and an aliquot ( 100 l ) of adenovirus ( 1 10 pfu / ml)/pbs solution was infused into the filament- or balloon - injured common carotid artery through the external carotid artery . after ligation of external carotid artery , the clamps were removed and the blood flow was restored . the infection rate using this protocol was 40% of medial smooth muscle cells according to the extent of -galactosidase expression ( data not shown ) . rat carotid arteries were perfused fixed in formalin and cut into 0.3-cm - long pieces ( three pieces per artery ) before being embedded in paraffin wax . each paraffin block containing three pieces of the same carotid artery was cut into 4-m - thick sections so that each section had three arterial rings representing arterial lesion 0.3 cm apart in the actual artery . for analysis of cell proliferation ( brdu ) and cell - cycle regulators ( hyperphosphorylated rb , p27 , p21 , and skp2 ) , all cells in each arterial ring ( 300 - 450 cells per ring ) were counted and three arterial rings were analyzed for each carotid artery . the number of positive cells was expressed as a percentage of total cell number calculated by counting hematoxylin counterstained nuclei . cell apoptosis was assessed by terminal deoxynucleotidyl transferase biotin - dutp nick end labeling ( tunel ) method using dna fragmentation kit ( calbiochem ) according to the manufacturer 's instruction . briefly , arterial sections were incubated with terminal deoxynucleotidyl transferase ( tdt ) end - labeling cocktail containing tdt , biotin - dutp , and tdt buffer . the dnase i ( 0.375 kunitz unit/l ) treated arterial rings were served as positive control . total cell nuclei visualized by hoescht 33258 and nuclei containing cleaved dna ( fitc - labeled ) cells were counted . for the evaluation of neointimal thickening , arterial sections were subjected to h&e staining , followed by neointimal ( the tissue within the ring of internal elastic lamina ) and medial areas measured using image pro software system ( mediacybernetics ) . the extents of neointimal thickening were presented as intima / media ( i / m ) ratios . unpaired two - tailed t test was adopted to test the significance , if appropriate . where indicated , data was analyzed using nonparametric mann - whitney test . a p value genotypes of mice were determined by polymerase change reaction ( pcr ) of tail tip dna . male sprague - dawley ( sd ) rats were obtained from charles river ( margate , united kingdom ) or biolasco taiwan co ltd ( taiwan ) . the housing and care of the animals and all procedures used adhered to the guidelines and regulations of the united kingdom animal ( scientific procedures ) act 1986 , united kingdom , and mackay memorial hospital , taiwan . male skp2 and skp2 mice ( 20 - 30 grams ) were anesthetized by inhalation of isoflurane and oxygen ( 0.2 l / minute ) . all animals were killed 28 days later and perfusion fixed with 4% formaldehyde / phosphate buffered saline ( pbs ) for 7 minutes before excision of ligated carotid arteries . sections were stained with elastic van gieson ( evg ) and hematoxylin and eosin ( h&e ) stain and neointimal areas , defined as the area of tissue within the internal elastic lamina , quantified using image pro analysis software ( mediacybernetics , bethesda , md ) . vessel diameter ( distance between the external elastic lamellae ) and medial thickness ( distance between the internal and external elastic lamellae ) were measured 300 - 400 m distal to the ligation point . balloon injury of left common carotid artery was performed as described by clowes et al . briefly , male sd rats ( 400 grams ) were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride ( 100 mg / kg ) and xylazine ( 10 mg / kg ) . the bifurcation of the left common carotid artery was surgically exposed , and a 2f arterial embolectomy balloon catheter ( edwards lifesciences co , irvine , calif ) was introduced through an arteriotomy on left external carotid artery . the balloon was inflated with saline and pulled back with rotation through common carotid artery three times . the injured common carotid arteries were then subjected to adenovirus - mediated gene delivery . for cell proliferation assay , animals were injected subcutaneously with brdu solution ( 25 mg / kg ) at 17 , 9 , and 1 hour before euthanasia . the procedures of filament de - endothelialization of rat common carotid artery were modified from the methods developed by ma reidy 's group . male sd rats were anesthetized and the left common carotid arteries were approached as described in balloon injury . the endothelium was then denuded from a carotid artery by a 5 - 0 nylon loop . briefly , the nylon loop was introduced into the left external carotid artery through a trocar . the loop was then slowly pulled back along the common carotid artery with constant rotation for 30 seconds . the trocar was retrieved and the artery underwent subsequent adenoviral infection for specific gene expression . recombinant replication - defective adenoviruses encoding wild - type skp2 ( ad : wt - skp2 ) , dominant - negative skp2 ( ad : dn - skp2 ) , and -galactosidase ( ad:gal ) have been described previously . to infect rat common carotid artery with adenoviruses for gene expression , both proximal end of common carotid artery and internal carotid artery were temporarily clamped and an aliquot ( 100 l ) of adenovirus ( 1 10 pfu / ml)/pbs solution was infused into the filament- or balloon - injured common carotid artery through the external carotid artery . after ligation of external carotid artery , the clamps were removed and the blood flow was restored . the infection rate using this protocol was 40% of medial smooth muscle cells according to the extent of -galactosidase expression ( data not shown ) . rat carotid arteries were perfused fixed in formalin and cut into 0.3-cm - long pieces ( three pieces per artery ) before being embedded in paraffin wax . each paraffin block containing three pieces of the same carotid artery was cut into 4-m - thick sections so that each section had three arterial rings representing arterial lesion 0.3 cm apart in the actual artery . for analysis of cell proliferation ( brdu ) and cell - cycle regulators ( hyperphosphorylated rb , p27 , p21 , and skp2 ) , all cells in each arterial ring ( 300 - 450 cells per ring ) were counted and three arterial rings were analyzed for each carotid artery . the number of positive cells was expressed as a percentage of total cell number calculated by counting hematoxylin counterstained nuclei . cell apoptosis was assessed by terminal deoxynucleotidyl transferase biotin - dutp nick end labeling ( tunel ) method using dna fragmentation kit ( calbiochem ) according to the manufacturer 's instruction . briefly , arterial sections were incubated with terminal deoxynucleotidyl transferase ( tdt ) end - labeling cocktail containing tdt , biotin - dutp , and tdt buffer . the dnase i ( 0.375 kunitz unit/l ) treated arterial rings were served as positive control . total cell nuclei visualized by hoescht 33258 and nuclei containing cleaved dna ( fitc - labeled ) cells were counted . for the evaluation of neointimal thickening , arterial sections were subjected to h&e staining , followed by neointimal ( the tissue within the ring of internal elastic lamina ) and medial areas measured using image pro software system ( mediacybernetics ) . the extents of neointimal thickening were presented as intima / media ( i / m ) ratios . unpaired two - tailed t test was adopted to test the significance , if appropriate . where indicated , data was analyzed using nonparametric mann - whitney test . to demonstrate the overall effect of skp2 gene deletion on the neointimal thickening , skp2 and skp2 mice were subjected to common carotid arterial ligation , which induces arterial remodeling , vsmc proliferation , and neointimal thickening . compared with wild - type skp2 mice ( n = 11 ) , skp2 mice ( n = 8) developed significantly smaller neointimal areas ( 130,371 29,054 vs 52,903 35,291 m ; mann - whitney test , p = .026 ) 28 days after common carotid ligation ( fig 1 ) . however , vessel diameter ( 309.5 19.5 m vs 384.8 40.6 m , p = .086 for wild - type and skp2-null , respectively ) and medial wall thickness ( 40.6 2.1 m vs 41.0 2.2 m , p = .9 ) was not significantly different between wild - type and skp2-null mice . further mechanistic studies in the mouse model were impossible because of the small number of longitudinal sections that could be cut from each lesion and the difficulties of breeding additional skp2-null mice due to low fertility and fecundity . to more specifically elucidate the local effect of skp2 on vsmc proliferation and neointimal thickening , and to probe more deeply into the mechanisms , we used adenovirus - mediated delivery of either wild - type skp2 or dn - skp2 in balloon- or filament - injured rat carotid arteries , respectively . dn - skp2 is an f - box - deleted skp2 mutant , which preserves substrate - binding ability but loses its ability to form scf complex , and , therefore , inhibits skp2 function by competing with endogenous skp2 . to test whether local inhibition of skp2 function also reduces vsmc proliferation in vivo , dn - skp2 was expressed in balloon - injured rat carotid arteries using an adenoviral vector . the dn - skp2 expression significantly enhanced medial vsmc p27 expression ( from 15.7 1.3% to 24.1 2.5% of medial vsmc , p = .012 , n = 6 and 5 , respectively ) but not p21 expression ( from 20.5 3.2% to 30.2 dn - skp2 also inhibited vsmc proliferation measured as brdu index ( from 37.3 2.5% to 17.0 3.2% , p < .01 , n = 6 and 5 , respectively ) and hyper - phosphorylation of rb protein ( from 43.5 4.5% to 25.9 2.8% of medial vsmc , p = .012 ) , a marker of g1-s phase transition , 4 days after injury , compared with vessels infected with a -galactosidase expressing control adenovirus ( fig 2 ) . in addition , subsequent neointimal thickening 14 days postinjury was also significantly reduced by dn - skp2 ( i / m ratios , from 1.58 0.20 to 0.49 0.21 , p < .01 , n = 5 ) ( fig 2 ) . importantly , frequency of apoptosis , detected by tunel staining ( fig e1 , online only ) , was not increased by expression of dominant - negative skp2 ( less than 0.15% in both ad : gal and ad : dn - skp2-infected vessels ) . this data demonstrates that skp2 activity is required for maximal vsmc proliferation and neointimal lesion development in vivo . we next sought to determine if increased skp2 activity alone , in the absence of a large medial injury inflicted by balloon angioplasty , can induce vsmc proliferation and neointimal thickening in vivo . to this end , we used adenoviral - delivery of wild - type skp2 to carotids subjected to filament de - endothelialization . unlike balloon injury , filament de - endothelialization results in minor medial injury , minimal vsmc proliferation , and thus , a limited thickness of neointima . compared with filament - injured carotids infected with control adenovirus ( ad:gal ) , those infected with wild - type skp2 adenovirus ( ad : wt - skp2 ) had substantially increased skp2 expression ( 5 1% compared to 43 5% of medial vsmc , p < .01 ) , accompanied by reduced expression of p27 ( from 18.4 5.4% to 0.6 0.3% , p = .016 , n = 4 and 6 , respectively ) but not p21 ( from 25.8 4.8 to 18.0 2.4% , p = .26 ) . wild - type skp2 also enhanced vsmc proliferation measured by brdu incorporation ( 6 2% to 32 5% , p < .01 , n = 4 and 6 , respectively ) and hyper - phosphorylation of rb ( from 9.5 2.1% to 28.3 5.5% , p = .015 ) 4 days after injury ( fig 3 ) . eight days after filament injury , ad : wt - skp2-infected carotids gave rise to significantly higher i / m ratios than those infected with ad:gal ( 0.11 0.03 compared to 0.42 0.09 , p = .024 , n = 4 and 5 , respectively ) ( fig 4 ) . although the role of skp2 in the oncogenesis is well recognized , its role in the regulation of vsmc proliferation is less established . the current studies directly demonstrate for the first time that skp2 function is involved in injury - induced vsmc proliferation and neointima development in vivo . furthermore , we demonstrate that skp2 over - expression is sufficient to drive both proliferation and lesion development after endothelial denudation in the absence of major medial injury . these experiments implicate skp2 as a novel target for the treatment of vasculoproliferative diseases , such as vascular restenosis after percutaneous vascular intervention . numerous factors , such as growth factors , inflammatory mediators , extracellular matrix attachment , cell - cell contact , and cyclic nucleotides are involved in the regulation of vsmc proliferation . nevertheless , the cell - cycle is the final machinery to integrate and orchestrate intricate upstream signals to delicately tune vsmc proliferation . recent encouraging clinical reports show that stents coated with rapamycin or paclitaxel , which target g1-s or g2-m transition , respectively , prevent the occurrence of restenosis after coronary intervention . this strongly supports the notion that the cell - cycle , the common final pathway for vsmc proliferation , is an ideal target for the treatment of vascular restenosis and , by implication , other vasculoproliferative diseases . our previous studies established skp2 as an important mediator in the final common pathway for vsmc proliferation in vitro . for example , we showed that positive mediators for vsmc proliferation ( growth factors and matrix contact ) increase skp2 levels , whereas negative regulators ( cyclic nucleotides ) decrease them . these effects are mediated through regulation of focal adhesion kinase , phosphoinositide 3-kinase , and rac1 signaling pathways . dn - skp2 blocks the stimulation of vsmc proliferation after growth factor addition , whereas overexpression of native skp2 reverses the inhibitory effects of cyclic - nucleotides . in vivo we also demonstrated that skp2 expression levels are strikingly enhanced after balloon injury with a time course that perfectly mirrors that of vsmc proliferation . moreover , adventitial application of forskolin ( an activator of endogenous cyclic amp levels ) inhibited skp2 expression , vsmc proliferation , and neointima formation . . justified by this correlative evidence , we directly tested for the first time whether skp2 is necessary and sufficient for vsmc proliferation and neointima formation in vivo after vascular injury in mice and rats . initially , we demonstrated that neointima formation after carotid ligation was significantly attenuated in skp2 mice , compared with that in skp2 mice . although skp2 mice tend to be smaller than their wild - type counterparts we found that carotid diameter and medial thickness was not different and no gross vessel abnormalities were observed at the time of surgery . impaired lesion formation in these mice is consistent with previous reports of reduced rates of cellular proliferation . it is important to note that impaired lesion formation in skp2 mice could be explained in part by actions of skp2 in other cells , such as bone marrow progenitor cells , inflammatory cells , fibroblasts , and endothelial cells , all of which may interact with vsmc and contribute significantly to the final neointimal thickening . to more specifically pinpoint the effects of skp2 inhibition on local vsmc and neointimal thickening , dn - skp2 was expressed in balloon - injured rat carotid arteries via adenovirus - mediated gene transfer . the expression of dn - skp2 resulted in elevated levels of the cdki p27 , which is normally rapidly downregulated soon after vascular injury . these observations are consistent with the hypothesis that injury - induced expression of skp2 is responsible for the downregulation of p27 after vascular injury . downregulation of p27 is thought to be an important step in removing the brake on proliferation , because forced expression of p27 inhibits s - phase entry and neointima formation after balloon injury and p27-null mice display a hyper - proliferative phenotype and exaggerated responses to vascular injury . we showed here that expression of dn - skp2 prevented downregulation of p27 and resulted in significantly reduced levels of hyperphosphorylated rb protein , a marker of g1-s transition , reduced rates of vsmc proliferation and smaller neointimal lesions . expression of dn - skp2 did not , however , significantly effect the expression of p21 , even though our previous data demonstrated that skp2 regulates p21 levels in vsmc in vitro . however , p21 regulation seems to be more complicated than that of p27 in these cells . the p21 levels actually increase in response to mitogen stimulation , coordinately with increases in skp2 , which functions to maintain p21 within growth permissive levels . this complex pattern of regulation may account for the lack of a significant change in p21 in this study . importantly , apoptosis rates were not increased in vessels expressing dn - skp2 , indicating that reduced lesion formation was due to inhibition of proliferation rather than increased cell death . likewise , expression of dn - skp2 in vsmc in vitro does not result in increased rate of apoptosis ( unpublished data ) . taken together with our observations from skp2 mice , our data demonstrates that injury - induced expression of skp2 is a major mechanism regulating p27 levels and is essential for vsmc proliferation and the final neointimal thickening . we previously demonstrated that exogenous expression of skp2 is able to force vsmc to enter s - phase in the uninjured rat aorta in organ cultures . in the present study , we asked whether increased skp2 expression would be sufficient to induce vsmc proliferation and neointimal lesion development , in the absence of major medial injury in vivo . consistent with previous reports , we showed that filament de - endothelialization of rat carotid artery caused minimal levels of vsmc proliferation and neointimal thickening , and only a small increase in skp2 expression . however , forced expression of wild - type skp2 resulted in a remarkable reduction of p27 expression and a transmedial increase of brdu - labeled cells . this demonstrates that skp2 alone is sufficient to promote p27 downregulation and vsmc proliferation in the absence of major medial injury . taken together , our data provides the first direct and robust evidence that skp2 plays an important role in promoting vsmc proliferation and neointimal thickening in vivo in response to vascular injury . these results suggest that skp2 may represent an attractive target for future gene or pharmacologic - based therapies to combat vasculoproliferative diseases . targeting skp2 in this way may be more effective than approaches designed to block the activity of single downstream cell - cycle regulators , such as p27 , because skp2 regulates the levels and activity of multiple cell - cycle proteins . however , more work is required to elucidate the role and regulation of skp2 in other vascular cells , particularly endothelial cells , before this may become a reality . conception and design : mb , ananalysis and interpretation : yw , mbdata collection : yw , gs , ks , kin , kn , hy , mbwriting the article : yw , an , mbcritical revision of the article : yw , mbfinal approval of the article : mbstatistical analysis : yw , mbobtained funding : mb , anoverall responsibility : mb conception and design : mb , an analysis and interpretation : yw , mb data collection : yw , gs , ks , kin , kn , hy , mb writing the article : yw , an , mb critical revision of the article : yw , mb final approval of the article : mb statistical analysis : yw , mb obtained funding : mb , an overall responsibility : mb fig e1 , online onlytunel analysis of apoptosis in ad:gal and ad : dn - skp2 infected rat carotid arteries . tunel analysis of apoptosis in ad:gal and ad : dn - skp2 infected rat carotid arteries .

objectivevascular smooth muscle cell ( vsmc ) proliferation plays an important role in the development of postangioplasty or in - stent restenosis , venous graft failure , and atherosclerosis . our previous work has demonstrated s - phase kinase - associated protein-2 ( skp2 ) , an f - box subunit of scfskp2 ubiquitin ligase , as an important mediator and common final pathway for growth factors , extracellular matrices , and cyclic - nucleotides to regulate vsmc proliferation in vitro . however , whether alteration of skp2 function also regulates vsmc proliferation in vivo and neointimal thickening postvascular injury remains unclear . we investigated the effect of skp2 on vsmc proliferation and neointimal formation in vivo.methods and resultsfirstly , we demonstrated that skp2-null mice developed significantly smaller neointimal areas than wild - type mice after carotid ligation . secondly , to further identify a local rather than a systemic effect of skp2 alteration , we demonstrated that adenovirus - mediated expression of dominant - negative skp2 in the balloon - injured rat carotid artery significantly increased medial p27kip1 levels , inhibited vsmc proliferation , and the subsequent neointimal thickening . lastly , to determine if skp2 alone is sufficient to drive vsmc proliferation and lesion development in vivo , we demonstrated that adenovirus - delivery of wild - type skp2 to the minimally - injured rat carotids is sufficient to downregulate p27kip1 protein levels , enhanced medial vsmc proliferation , and the neointimal thickening.conclusionthis data provides , we believe for the first time , a more comprehensive understanding of skp2 in the regulation of vsmc proliferation and neointimal formation and suggests that skp2 is a promising target in the treatment of vasculoproliferative diseases .

Materials Methods Animals Ligation of mouse common carotid artery Balloon injury of rat common carotid artery Filament de-endothelialization of rat common carotid artery Adenovirus-mediated gene delivery and expression Immunohistochemical analysis Statistical analysis Results Discussion Author contributions Supplementary data

the housing and care of the animals and all procedures used adhered to the guidelines and regulations of the united kingdom animal ( scientific procedures ) act 1986 , united kingdom , and mackay memorial hospital , taiwan . sections were stained with elastic van gieson ( evg ) and hematoxylin and eosin ( h&e ) stain and neointimal areas , defined as the area of tissue within the internal elastic lamina , quantified using image pro analysis software ( mediacybernetics , bethesda , md ) . balloon injury of left common carotid artery was performed as described by clowes et al . the bifurcation of the left common carotid artery was surgically exposed , and a 2f arterial embolectomy balloon catheter ( edwards lifesciences co , irvine , calif ) was introduced through an arteriotomy on left external carotid artery . the balloon was inflated with saline and pulled back with rotation through common carotid artery three times . the injured common carotid arteries were then subjected to adenovirus - mediated gene delivery . for cell proliferation assay , animals were injected subcutaneously with brdu solution ( 25 mg / kg ) at 17 , 9 , and 1 hour before euthanasia . the procedures of filament de - endothelialization of rat common carotid artery were modified from the methods developed by ma reidy 's group . briefly , the nylon loop was introduced into the left external carotid artery through a trocar . the trocar was retrieved and the artery underwent subsequent adenoviral infection for specific gene expression . recombinant replication - defective adenoviruses encoding wild - type skp2 ( ad : wt - skp2 ) , dominant - negative skp2 ( ad : dn - skp2 ) , and -galactosidase ( ad:gal ) have been described previously . to infect rat common carotid artery with adenoviruses for gene expression , both proximal end of common carotid artery and internal carotid artery were temporarily clamped and an aliquot ( 100 l ) of adenovirus ( 1 10 pfu / ml)/pbs solution was infused into the filament- or balloon - injured common carotid artery through the external carotid artery . after ligation of external carotid artery , the clamps were removed and the blood flow was restored . the infection rate using this protocol was 40% of medial smooth muscle cells according to the extent of -galactosidase expression ( data not shown ) . rat carotid arteries were perfused fixed in formalin and cut into 0.3-cm - long pieces ( three pieces per artery ) before being embedded in paraffin wax . each paraffin block containing three pieces of the same carotid artery was cut into 4-m - thick sections so that each section had three arterial rings representing arterial lesion 0.3 cm apart in the actual artery . for analysis of cell proliferation ( brdu ) and cell - cycle regulators ( hyperphosphorylated rb , p27 , p21 , and skp2 ) , all cells in each arterial ring ( 300 - 450 cells per ring ) were counted and three arterial rings were analyzed for each carotid artery . for the evaluation of neointimal thickening , arterial sections were subjected to h&e staining , followed by neointimal ( the tissue within the ring of internal elastic lamina ) and medial areas measured using image pro software system ( mediacybernetics ) . the extents of neointimal thickening were presented as intima / media ( i / m ) ratios . the housing and care of the animals and all procedures used adhered to the guidelines and regulations of the united kingdom animal ( scientific procedures ) act 1986 , united kingdom , and mackay memorial hospital , taiwan . sections were stained with elastic van gieson ( evg ) and hematoxylin and eosin ( h&e ) stain and neointimal areas , defined as the area of tissue within the internal elastic lamina , quantified using image pro analysis software ( mediacybernetics , bethesda , md ) . the bifurcation of the left common carotid artery was surgically exposed , and a 2f arterial embolectomy balloon catheter ( edwards lifesciences co , irvine , calif ) was introduced through an arteriotomy on left external carotid artery . the balloon was inflated with saline and pulled back with rotation through common carotid artery three times . the injured common carotid arteries were then subjected to adenovirus - mediated gene delivery . for cell proliferation assay , animals were injected subcutaneously with brdu solution ( 25 mg / kg ) at 17 , 9 , and 1 hour before euthanasia . the procedures of filament de - endothelialization of rat common carotid artery were modified from the methods developed by ma reidy 's group . male sd rats were anesthetized and the left common carotid arteries were approached as described in balloon injury . the endothelium was then denuded from a carotid artery by a 5 - 0 nylon loop . briefly , the nylon loop was introduced into the left external carotid artery through a trocar . recombinant replication - defective adenoviruses encoding wild - type skp2 ( ad : wt - skp2 ) , dominant - negative skp2 ( ad : dn - skp2 ) , and -galactosidase ( ad:gal ) have been described previously . to infect rat common carotid artery with adenoviruses for gene expression , both proximal end of common carotid artery and internal carotid artery were temporarily clamped and an aliquot ( 100 l ) of adenovirus ( 1 10 pfu / ml)/pbs solution was infused into the filament- or balloon - injured common carotid artery through the external carotid artery . after ligation of external carotid artery , the clamps were removed and the blood flow was restored . the infection rate using this protocol was 40% of medial smooth muscle cells according to the extent of -galactosidase expression ( data not shown ) . each paraffin block containing three pieces of the same carotid artery was cut into 4-m - thick sections so that each section had three arterial rings representing arterial lesion 0.3 cm apart in the actual artery . for analysis of cell proliferation ( brdu ) and cell - cycle regulators ( hyperphosphorylated rb , p27 , p21 , and skp2 ) , all cells in each arterial ring ( 300 - 450 cells per ring ) were counted and three arterial rings were analyzed for each carotid artery . briefly , arterial sections were incubated with terminal deoxynucleotidyl transferase ( tdt ) end - labeling cocktail containing tdt , biotin - dutp , and tdt buffer . for the evaluation of neointimal thickening , arterial sections were subjected to h&e staining , followed by neointimal ( the tissue within the ring of internal elastic lamina ) and medial areas measured using image pro software system ( mediacybernetics ) . to demonstrate the overall effect of skp2 gene deletion on the neointimal thickening , skp2 and skp2 mice were subjected to common carotid arterial ligation , which induces arterial remodeling , vsmc proliferation , and neointimal thickening . compared with wild - type skp2 mice ( n = 11 ) , skp2 mice ( n = 8) developed significantly smaller neointimal areas ( 130,371 29,054 vs 52,903 35,291 m ; mann - whitney test , p = .026 ) 28 days after common carotid ligation ( fig 1 ) . however , vessel diameter ( 309.5 19.5 m vs 384.8 40.6 m , p = .086 for wild - type and skp2-null , respectively ) and medial wall thickness ( 40.6 2.1 m vs 41.0 2.2 m , p = .9 ) was not significantly different between wild - type and skp2-null mice . further mechanistic studies in the mouse model were impossible because of the small number of longitudinal sections that could be cut from each lesion and the difficulties of breeding additional skp2-null mice due to low fertility and fecundity . to more specifically elucidate the local effect of skp2 on vsmc proliferation and neointimal thickening , and to probe more deeply into the mechanisms , we used adenovirus - mediated delivery of either wild - type skp2 or dn - skp2 in balloon- or filament - injured rat carotid arteries , respectively . dn - skp2 is an f - box - deleted skp2 mutant , which preserves substrate - binding ability but loses its ability to form scf complex , and , therefore , inhibits skp2 function by competing with endogenous skp2 . to test whether local inhibition of skp2 function also reduces vsmc proliferation in vivo , dn - skp2 was expressed in balloon - injured rat carotid arteries using an adenoviral vector . the dn - skp2 expression significantly enhanced medial vsmc p27 expression ( from 15.7 1.3% to 24.1 2.5% of medial vsmc , p = .012 , n = 6 and 5 , respectively ) but not p21 expression ( from 20.5 3.2% to 30.2 dn - skp2 also inhibited vsmc proliferation measured as brdu index ( from 37.3 2.5% to 17.0 3.2% , p < .01 , n = 6 and 5 , respectively ) and hyper - phosphorylation of rb protein ( from 43.5 4.5% to 25.9 2.8% of medial vsmc , p = .012 ) , a marker of g1-s phase transition , 4 days after injury , compared with vessels infected with a -galactosidase expressing control adenovirus ( fig 2 ) . in addition , subsequent neointimal thickening 14 days postinjury was also significantly reduced by dn - skp2 ( i / m ratios , from 1.58 0.20 to 0.49 0.21 , p < .01 , n = 5 ) ( fig 2 ) . importantly , frequency of apoptosis , detected by tunel staining ( fig e1 , online only ) , was not increased by expression of dominant - negative skp2 ( less than 0.15% in both ad : gal and ad : dn - skp2-infected vessels ) . this data demonstrates that skp2 activity is required for maximal vsmc proliferation and neointimal lesion development in vivo . we next sought to determine if increased skp2 activity alone , in the absence of a large medial injury inflicted by balloon angioplasty , can induce vsmc proliferation and neointimal thickening in vivo . to this end , we used adenoviral - delivery of wild - type skp2 to carotids subjected to filament de - endothelialization . unlike balloon injury , filament de - endothelialization results in minor medial injury , minimal vsmc proliferation , and thus , a limited thickness of neointima . compared with filament - injured carotids infected with control adenovirus ( ad:gal ) , those infected with wild - type skp2 adenovirus ( ad : wt - skp2 ) had substantially increased skp2 expression ( 5 1% compared to 43 5% of medial vsmc , p < .01 ) , accompanied by reduced expression of p27 ( from 18.4 5.4% to 0.6 0.3% , p = .016 , n = 4 and 6 , respectively ) but not p21 ( from 25.8 4.8 to 18.0 2.4% , p = .26 ) . wild - type skp2 also enhanced vsmc proliferation measured by brdu incorporation ( 6 2% to 32 5% , p < .01 , n = 4 and 6 , respectively ) and hyper - phosphorylation of rb ( from 9.5 2.1% to 28.3 5.5% , p = .015 ) 4 days after injury ( fig 3 ) . although the role of skp2 in the oncogenesis is well recognized , its role in the regulation of vsmc proliferation is less established . the current studies directly demonstrate for the first time that skp2 function is involved in injury - induced vsmc proliferation and neointima development in vivo . furthermore , we demonstrate that skp2 over - expression is sufficient to drive both proliferation and lesion development after endothelial denudation in the absence of major medial injury . these experiments implicate skp2 as a novel target for the treatment of vasculoproliferative diseases , such as vascular restenosis after percutaneous vascular intervention . numerous factors , such as growth factors , inflammatory mediators , extracellular matrix attachment , cell - cell contact , and cyclic nucleotides are involved in the regulation of vsmc proliferation . nevertheless , the cell - cycle is the final machinery to integrate and orchestrate intricate upstream signals to delicately tune vsmc proliferation . this strongly supports the notion that the cell - cycle , the common final pathway for vsmc proliferation , is an ideal target for the treatment of vascular restenosis and , by implication , other vasculoproliferative diseases . our previous studies established skp2 as an important mediator in the final common pathway for vsmc proliferation in vitro . for example , we showed that positive mediators for vsmc proliferation ( growth factors and matrix contact ) increase skp2 levels , whereas negative regulators ( cyclic nucleotides ) decrease them . these effects are mediated through regulation of focal adhesion kinase , phosphoinositide 3-kinase , and rac1 signaling pathways . dn - skp2 blocks the stimulation of vsmc proliferation after growth factor addition , whereas overexpression of native skp2 reverses the inhibitory effects of cyclic - nucleotides . in vivo we also demonstrated that skp2 expression levels are strikingly enhanced after balloon injury with a time course that perfectly mirrors that of vsmc proliferation . moreover , adventitial application of forskolin ( an activator of endogenous cyclic amp levels ) inhibited skp2 expression , vsmc proliferation , and neointima formation . justified by this correlative evidence , we directly tested for the first time whether skp2 is necessary and sufficient for vsmc proliferation and neointima formation in vivo after vascular injury in mice and rats . initially , we demonstrated that neointima formation after carotid ligation was significantly attenuated in skp2 mice , compared with that in skp2 mice . although skp2 mice tend to be smaller than their wild - type counterparts we found that carotid diameter and medial thickness was not different and no gross vessel abnormalities were observed at the time of surgery . impaired lesion formation in these mice is consistent with previous reports of reduced rates of cellular proliferation . it is important to note that impaired lesion formation in skp2 mice could be explained in part by actions of skp2 in other cells , such as bone marrow progenitor cells , inflammatory cells , fibroblasts , and endothelial cells , all of which may interact with vsmc and contribute significantly to the final neointimal thickening . to more specifically pinpoint the effects of skp2 inhibition on local vsmc and neointimal thickening , dn - skp2 was expressed in balloon - injured rat carotid arteries via adenovirus - mediated gene transfer . these observations are consistent with the hypothesis that injury - induced expression of skp2 is responsible for the downregulation of p27 after vascular injury . downregulation of p27 is thought to be an important step in removing the brake on proliferation , because forced expression of p27 inhibits s - phase entry and neointima formation after balloon injury and p27-null mice display a hyper - proliferative phenotype and exaggerated responses to vascular injury . we showed here that expression of dn - skp2 prevented downregulation of p27 and resulted in significantly reduced levels of hyperphosphorylated rb protein , a marker of g1-s transition , reduced rates of vsmc proliferation and smaller neointimal lesions . expression of dn - skp2 did not , however , significantly effect the expression of p21 , even though our previous data demonstrated that skp2 regulates p21 levels in vsmc in vitro . likewise , expression of dn - skp2 in vsmc in vitro does not result in increased rate of apoptosis ( unpublished data ) . taken together with our observations from skp2 mice , our data demonstrates that injury - induced expression of skp2 is a major mechanism regulating p27 levels and is essential for vsmc proliferation and the final neointimal thickening . we previously demonstrated that exogenous expression of skp2 is able to force vsmc to enter s - phase in the uninjured rat aorta in organ cultures . in the present study , we asked whether increased skp2 expression would be sufficient to induce vsmc proliferation and neointimal lesion development , in the absence of major medial injury in vivo . consistent with previous reports , we showed that filament de - endothelialization of rat carotid artery caused minimal levels of vsmc proliferation and neointimal thickening , and only a small increase in skp2 expression . however , forced expression of wild - type skp2 resulted in a remarkable reduction of p27 expression and a transmedial increase of brdu - labeled cells . this demonstrates that skp2 alone is sufficient to promote p27 downregulation and vsmc proliferation in the absence of major medial injury . taken together , our data provides the first direct and robust evidence that skp2 plays an important role in promoting vsmc proliferation and neointimal thickening in vivo in response to vascular injury . these results suggest that skp2 may represent an attractive target for future gene or pharmacologic - based therapies to combat vasculoproliferative diseases . however , more work is required to elucidate the role and regulation of skp2 in other vascular cells , particularly endothelial cells , before this may become a reality . conception and design : mb , ananalysis and interpretation : yw , mbdata collection : yw , gs , ks , kin , kn , hy , mbwriting the article : yw , an , mbcritical revision of the article : yw , mbfinal approval of the article : mbstatistical analysis : yw , mbobtained funding : mb , anoverall responsibility : mb conception and design : mb , an analysis and interpretation : yw , mb data collection : yw , gs , ks , kin , kn , hy , mb writing the article : yw , an , mb critical revision of the article : yw , mb final approval of the article : mb statistical analysis : yw , mb obtained funding : mb , an overall responsibility : mb fig e1 , online onlytunel analysis of apoptosis in ad:gal and ad : dn - skp2 infected rat carotid arteries .

interventional radiology ( ir ) has evolved rapidly in past two decades with advancement in imaging technology . there has also been significant improvement and development of more sophisticated hardware with the help of which it is possible to target any vascular territory in the body and carry out therapeutic procedures by minimally invasive technique . the head and neck region is unique in terms of the critical anatomy and vital neurovascular structures involved . it needs detailed knowledge of the head and neck vascular anatomy and special technique to perform vascular ir procedure in extra cranial head and neck ( echn ) . the pathologies where ir can offer minimally invasive treatment include vascular malformations , tumor embolization , and echn bleeding . the slow flow type of venous and lymphatic malformations respond very well to percutaneous sclerotherapy with sclerosing agents like sodium tetradecyl sulfate ( sts ) , polidocanol sulfate , or ethanol [ figure 1 ] . other abnormalities like plunging ranula , sialocele , and other benign cysts also respond well to percutaneous sclerotherapy . ( c , d ) images showing intralesional distribution of sclerosant mixture after percutaneous sclerotherapy . ( e ) pretreatment , ( f ) posttreatment photograph shows regression of the lesion . courtesy : dr uday limaye , inr division , kemh , mumbai high flow arteriovenous malformations ( avm ) of echn present with disfigurement or episodes of bleeding . they need to be managed by multi specialty team where ir has an important role to embolize the abnormal vascularity , which can be followed by surgery to completely remove the lesion . when avm is extensive with involvement of bones and soft tissues , multiple sessions of embolization by transarterial route or direct puncture using liquid embolizing agents like onyx [ ev3 , irvine , usa ] or n - bca ( n - butyl-2-cynoacrylate ) glue [ bbraun , aesculap , germany ] would be required [ figure 2 ] . in cases where transarterial approach is precluded due to previous surgical ligation of external carotid artery ( eca ) , , transarterial embolization can still be performed by gaining vascular access into one of the superficial eca branch after surgical dissection [ figure 3 ] . percutaneous embolization of scalp avm 34-year - old female patient with history of pulsatile swelling on her forehead . dsa images of left ica ( a ) and left eca ( b ) shows forehead avm with feeders from the supraorbital branch of ophthalmic artery and branches from superficial temporal artery ( sta ) . ( d , e ) radiograph shows glue cast ( white arrow ) conforming to the avm . courtesy : dr uday limaye , inr division , kemh , mumbai embolization of facial avm . a 24-year - old female presented with facial disfigurement on the right side and bleeding from pinna with previous history of surgery for facial avm . ( a ) reformatted ct angiography , ( b ) volume rendered ( vr ) image shows extensive soft tissue avm with tortuous dilated vascular channels . dsa images of right ica ( c ) and vertebral artery ( d ) show feeders from the ophthalmic artery and cervical branches of the vertebral artery ( white arrow ) . ( e ) surgical dissection of superficial temporal artery was done to gain vascular access ( arrow ) and embolized using onyx injection ( f ) radiograph shows the cast of onyx ( dotted arrow ) conforming to the avm feeder that was embolized approximately , 75 - 95% of patients with low flow venous malformation show good results with ethanol or sts while poor outcome is seen with diffuse avm . echn tumors need embolization procedure for some highly vascular tumors like juvenile nasopharyngeal angiofibroma ( jna ) and glomus tumors either preoperatively or to stop an episode of bleeding . similarly , other tumors like hypervascular metastases , schwannomas , plasmacytomas , chordomas , and hemangiopericytomas may also need preoperative embolization . benign vascular lesions like hemangiomas can be effectively treated by embolization and surgery may be avoided [ figure 4 ] . a 21-year - old female with a large slow flow vascular malformation of the tongue base presented with dysphagia and difficulty in speech . ( a ) axial , ( b ) sagittal t2w images shows a hyperintense lesion ( arrow ) in the base of tongue causing marked narrowing of oropharynx ( arrow head ) . ( c ) arterial , ( d ) venous phase of lingual artery ( arrow ) angiogram shows progressively increasing and persistent vascular blush in the lesion ( arrow ) suggestive of slow flow vascular malformation . direct puncture sclerotherapy was performed through percutaneous ( e ) under sonographic guidance and transoral ( f ) route ( black arrow ) using sts . using expandable suspending laryngoscope , the base of tongue can be optimally visualized and direct puncture of the lesion can be performed by transoral route . ( g ) axial , ( h ) sagittal fat suppressed contrast t1w images done after 6 weeks shows significant reduction in the size and enhancement of residual malformation ( arrow ) with good clinical improvement in symptoms preoperative embolization of echn tumors is frequently performed by transarterial route . preoperative embolization helps to prevent blood loss during surgery and provides a clean field for the surgical resection [ figure 5 ] . during transarterial tumor embolization , the feeders from eca are super selectively catheterized using micro catheter to prevent nontarget embolization . commonly used embolizing materials are particulate embolizing agents like polyvinyl alcohol ( pva ) particles , [ cook incorporated , bloomington , usa ] , embospheres [ biosphere medical sa , paris , france ] in the size range of 100300 microns . the aim of embolization is to inject the particles in the arteriolar capillary bed of tumor parenchyma , so that collateral circulation does not develop rapidly . coil embolization of feeding arteries is not routinely performed when open surgical resection is planned . however , as endoscopic resection of skull base tumors is being increasingly performed , proximal coil embolization of the feeding artery ( e.g. , internal maxillary artery coiling in case of jna ) after particulate embolization of tumor bed provides additional protection from possible avulsion of the vessel and intraoperative bleed . a 16-year - old girl presented with tinnitus and reduced hearing on the left side . ( a ) contrast enhanced mr image shows an intensely enhancing mass ( white arrow ) in the middle ear . ( b ) left eca angiogram shows intense tumor blush ( arrow head ) predominantly supplied by ascending pharyngeal artery which were embolized using pva particles . ( c ) post embolization eca angiogram shows significant reduction in the tumor blush tumors involving the skull base frequently derive blood supply from ophthalmic or petrous branches of internal carotid artery ( ica ) . the ica branches are often difficult to cannulate and there is considerable risk of reflux of the embolic agent with nontarget embolization if transarterial embolization of the ica feeders are attempted . the territory supplied by ica can be then be safely embolized by directly puncturing the tumor and injecting liquid embolizing agents like nbca glue or onyx [ figure 6 ] . the approach could be either percutaneous , transnasal , or transoral depending on the location of tumor . though in most cases preoperative embolization is done electively a day or two prior to surgery , it can also be performed intraoperatively by direct puncture of tumor under vision and embolization under fluoroscopic guidance [ figure 7 ] . ( a ) preembolization right eca angiogram shows intense tumor blush ( black arrow ) predominantly supplied by the internal maxillary artery ( b ) postembolization eca angiogram shows no tumor blush . ica angiogram after embolization of the external carotid artery feeders shows extensive tumor blush ( arrow ) supplied by cavernous and ophthalmic branches of ica . ( d ) the tumor territory supplied by ica was devascularized by direct puncture ( arrow head ) and glue embolization . note the coils ( dotted arrows ) placed in multiple feeding arteries of eca after embolizing the tumor bed using pva . ( e ) radiograph shows glue cast ( white arrow ) conforming to the tumor blush supplied by ica feeders . ( f ) postembolization ica angiogram shows marked reduction in tumor blush with minimal residue ( dotted arrow ) . estimated blood loss during surgery was less than 500 cc and did not receive blood transfusion intraoperative direct puncture embolization of jna . ( a ) post eca embolization , ica angiogram shows a small residual tumor blush ( arrow ) , which was not embolized . ( b ) due to excessive bleeding during surgery , intraoperative direct puncture ( arrow ) and glue embolization was done under fluoroscopic guidance . postintraoperative embolization , complete surgical resection was achieved without significant bleed extreme care should be taken to prevent the embolizing agent from entering ophthalmic artery or vital circulation of brain . the interventional radiologist should be aware about the potentially dangerous anastomosis between the eca and ica and should carefully watch for opening of these collaterals during echn embolization . in situations where internal carotid artery is encased by the tumor , surgical resection would be difficult with high risk of injury to the artery . in such cases a balloon test occlusion ( bto ) is performed to assess the ipsilateral cerebral circulation from opposite carotid or vertebra - basilar circulation . if patient tolerates bto , the involved carotid artery can be safely occluded using coils or detachable balloons [ figure 8 ] . ( a ) common carotid angiogram shows ligated external carotid artery ( long arrow ) , with residual tumor being supplied by cavernous branches of ica ( block arrow ) . ( b ) post ica occlusion , common carotid angiogram shows no tumor blush with coil mass in the ica . patient had tolerated the balloon test occlusion ( bto ) and intraoperative estimated blood loss was < 500 cc overall , preoperative embolization is a safe procedure and serious complications occur in less than 2% patients that includes facial numbness , mucosal necrosis , blindness , or cerebrovascular accident . preoperative embolization is not only cost effective , it reduces the morbidity by reducing blood loss and operative time , shortens the hospital stay while reducing the rate of tumor recurrence . bleeding in echn manifests as epistaxis , hematemesis , or bleeding from an open wound or ulcer . various causes of bleed include craniofacial injury , infections , tumors , surgery , and vascular abnormalities while medical causes like hypertension and anticoagulation therapy [ figure 9 ] . ( a ) dsa image shows abnormal blush ( arrow ) at the site of tracheotomy site with feeders from the inferior thyroid artery . ( c ) inferior thyroid artery feeders were embolized using 300 micon pva particles followed by coil embolization ( dotted arrow ) . ( d ) postembolization check subclavian angiogram shows complete resolution of abnormal blush treatment is often variable and multidisciplinary approach is required for successful outcome . preprocedure computed tomography ( ct ) angiography helps in identifying the likely source and cause of bleed while also exquisitely depicting the anatomy . subclavian , carotid , and vertebral artery angiograms are obtained to identify and target the culprit vessel . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . difficult postoperative anatomy , radiation effects , infections preclude safe , and effective surgery in these patients . angioembolization has been proven to be effective with minimal risk of complications in a few reported series . goal of endovascular therapy is complete devascularization of the tumor by embolizing the tumor bed . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. pva / embospheres of size 100 - 300 micron ) [ figure 10 ] . a 54year - old male suffering for inoperatble carcinoma tongue presented with persistent tumor bleed in spite of receiving hemostatic radiotherapy . ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( c ) axial , ( d ) sagittal cone beam ct images of the lingual artery ( dotted arrow ) angiogram confirms tumor blush ( white arrow ) . ( e ) lingual artery angiogram shows tumor blush ( black arrow ) , which was embolized . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . carotid blow out patient usually presents with neck swelling or bleed from nose or mouth which could be sentinel or massive . carotid blow out can be accurately diagnosed by ct angiography and definitive treatment can be planned by knowing the exact size and location . the blow out can be seen in common carotid , internal , or eca and imaging would reveal either a pseudoaneurysm or an active bleed . the clinical presentation and imaging findings demands the endovascular treatment to be performed on an emergency or semi - emergency basis . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . this not only achieves immediate hemostasis but also maintains patency of lumen and blood flow to the brain . patients requiring stent graft placements need to be administered with antiplatelets before and after the procedure . however , if placement of stent graft becomes technically difficult , the diseased carotid artery can be occluded after confirming adequate cross flow from the contralateral carotid or vertebrobasilar circulation by bto [ figure 13 ] . rarely , in case of uncontrolled massive bleeding after skull base surgery , failed attempt to deploy stent graft mandates immediate occlusion of carotid artery to prevent death from exsanguination [ figure 14 ] . an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( c ) carotid angiogram after stent graft placement shows exclusion of the aneurysm and maintained forward flow within the carotid artery courtesy : dr uday limaye , inr division , kemh , mumbai a 54-year - old male patient with history of radiotherapy to metastatic neck nodes presented with recurrent bouts of hematemesis . ( a ) ct angiogram sagittal reformatted image shows an irregular pseudoaneurysm ( arrow head ) at the carotid bifurcation with non visualization of eca origin . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . ( c ) angiogram after stent - graft placement shows complete exclusion of pseudoaneurysm from the circulation and maintained distal flow in ica a 12-year - old girl presented with total ophthalmoplegia and epistaxis . cect of brain ( a ) and dsa of right ica ( b ) show an aneurysm ( arrow , arrowhead ) in the cavernous portion of ica . ( c ) right ica occluded using detachable balloon ( white arrow ) and coils ( black arrow ) after bto . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( a ) dsa , ( b ) vr image of left ica shows a pseudoaneurysm ( white arrow ) arising from the cavernous portion . ( c ) right ica , ( d ) vertebral angiogram shows poor cross circulation across the circle of willis during bto . in view of torrential bleeding and failed attempt to deploy a covered stent , the left ica was occluded ( e ) using coils ( arrowhead ) to prevent exsanguination . ( f ) plain ct brain done after 24 hours shows fully evolved cerebral infarct ( dotted arrow ) blow out of eca or its branch is usually treated by embolizing the culprit vessel both proximal and distal to the pseudoaneurysm [ figure 15 ] . the carotid artery can be permanently occluded using coils or balloons and approximately 15 - 20% would develop cerebral ischemia . a middle - aged man presented with progressive neck swelling after attempted biopsy . ( a ) axial , ( b ) sagittal ct angiography images show two partially thrombosed pseudoaneurysm ( arrow ) arising from the right carotid artery . ( c , d ) dsa image shows the pseudoaneurysms ( arrow ) with no opacification of the ica and faint distal run - off of eca branches ( arrowhead ) . ( e , f ) the pseudo aneurysms were treated by coiling ( dotted arrow ) and occluding eca as well as cca . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( a ) left carotid angiogram shows fistulous communication ( arrow ) between eca and internal jugular vein . note the poor opacification of rest of eca ( b ) lateral radiograph of neck shows a detachable balloon ( arrow ) deployed at the site of fistula . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less . the slow flow type of venous and lymphatic malformations respond very well to percutaneous sclerotherapy with sclerosing agents like sodium tetradecyl sulfate ( sts ) , polidocanol sulfate , or ethanol [ figure 1 ] . other abnormalities like plunging ranula , sialocele , and other benign cysts also respond well to percutaneous sclerotherapy . ( c , d ) images showing intralesional distribution of sclerosant mixture after percutaneous sclerotherapy . ( e ) pretreatment , ( f ) posttreatment photograph shows regression of the lesion . courtesy : dr uday limaye , inr division , kemh , mumbai high flow arteriovenous malformations ( avm ) of echn present with disfigurement or episodes of bleeding . they need to be managed by multi specialty team where ir has an important role to embolize the abnormal vascularity , which can be followed by surgery to completely remove the lesion . when avm is extensive with involvement of bones and soft tissues , multiple sessions of embolization by transarterial route or direct puncture using liquid embolizing agents like onyx [ ev3 , irvine , usa ] or n - bca ( n - butyl-2-cynoacrylate ) glue [ bbraun , aesculap , germany ] would be required [ figure 2 ] . in cases where transarterial approach is precluded due to previous surgical ligation of external carotid artery ( eca ) , , transarterial embolization can still be performed by gaining vascular access into one of the superficial eca branch after surgical dissection [ figure 3 ] . percutaneous embolization of scalp avm 34-year - old female patient with history of pulsatile swelling on her forehead . dsa images of left ica ( a ) and left eca ( b ) shows forehead avm with feeders from the supraorbital branch of ophthalmic artery and branches from superficial temporal artery ( sta ) . ( d , e ) radiograph shows glue cast ( white arrow ) conforming to the avm . courtesy : dr uday limaye , inr division , kemh , mumbai embolization of facial avm . a 24-year - old female presented with facial disfigurement on the right side and bleeding from pinna with previous history of surgery for facial avm . ( a ) reformatted ct angiography , ( b ) volume rendered ( vr ) image shows extensive soft tissue avm with tortuous dilated vascular channels . dsa images of right ica ( c ) and vertebral artery ( d ) show feeders from the ophthalmic artery and cervical branches of the vertebral artery ( white arrow ) . ( e ) surgical dissection of superficial temporal artery was done to gain vascular access ( arrow ) and embolized using onyx injection ( f ) radiograph shows the cast of onyx ( dotted arrow ) conforming to the avm feeder that was embolized approximately , 75 - 95% of patients with low flow venous malformation show good results with ethanol or sts while poor outcome is seen with diffuse avm . echn tumors need embolization procedure for some highly vascular tumors like juvenile nasopharyngeal angiofibroma ( jna ) and glomus tumors either preoperatively or to stop an episode of bleeding . similarly , other tumors like hypervascular metastases , schwannomas , plasmacytomas , chordomas , and hemangiopericytomas may also need preoperative embolization . benign vascular lesions like hemangiomas can be effectively treated by embolization and surgery may be avoided [ figure 4 ] . direct puncture sclerotherapy of tongue base slow flow vascular malformation . a 21-year - old female with a large slow flow vascular malformation of the tongue base presented with dysphagia and difficulty in speech . ( a ) axial , ( b ) sagittal t2w images shows a hyperintense lesion ( arrow ) in the base of tongue causing marked narrowing of oropharynx ( arrow head ) . ( c ) arterial , ( d ) venous phase of lingual artery ( arrow ) angiogram shows progressively increasing and persistent vascular blush in the lesion ( arrow ) suggestive of slow flow vascular malformation . direct puncture sclerotherapy was performed through percutaneous ( e ) under sonographic guidance and transoral ( f ) route ( black arrow ) using sts . using expandable suspending laryngoscope , the base of tongue can be optimally visualized and direct puncture of the lesion can be performed by transoral route . ( g ) axial , ( h ) sagittal fat suppressed contrast t1w images done after 6 weeks shows significant reduction in the size and enhancement of residual malformation ( arrow ) with good clinical improvement in symptoms preoperative embolization of echn tumors is frequently performed by transarterial route . preoperative embolization helps to prevent blood loss during surgery and provides a clean field for the surgical resection [ figure 5 ] . during transarterial tumor embolization , the feeders from eca are super selectively catheterized using micro catheter to prevent nontarget embolization . commonly used embolizing materials are particulate embolizing agents like polyvinyl alcohol ( pva ) particles , [ cook incorporated , bloomington , usa ] , embospheres [ biosphere medical sa , paris , france ] in the size range of 100300 microns . the aim of embolization is to inject the particles in the arteriolar capillary bed of tumor parenchyma , so that collateral circulation does not develop rapidly . coil embolization of feeding arteries is not routinely performed when open surgical resection is planned . however , as endoscopic resection of skull base tumors is being increasingly performed , proximal coil embolization of the feeding artery ( e.g. , internal maxillary artery coiling in case of jna ) after particulate embolization of tumor bed provides additional protection from possible avulsion of the vessel and intraoperative bleed . a 16-year - old girl presented with tinnitus and reduced hearing on the left side . ( a ) contrast enhanced mr image shows an intensely enhancing mass ( white arrow ) in the middle ear . ( b ) left eca angiogram shows intense tumor blush ( arrow head ) predominantly supplied by ascending pharyngeal artery which were embolized using pva particles . ( c ) post embolization eca angiogram shows significant reduction in the tumor blush tumors involving the skull base frequently derive blood supply from ophthalmic or petrous branches of internal carotid artery ( ica ) . the ica branches are often difficult to cannulate and there is considerable risk of reflux of the embolic agent with nontarget embolization if transarterial embolization of the ica feeders are attempted . the territory supplied by ica can be then be safely embolized by directly puncturing the tumor and injecting liquid embolizing agents like nbca glue or onyx [ figure 6 ] . the approach could be either percutaneous , transnasal , or transoral depending on the location of tumor . though in most cases preoperative embolization is done electively a day or two prior to surgery , it can also be performed intraoperatively by direct puncture of tumor under vision and embolization under fluoroscopic guidance [ figure 7 ] . transarterial and percutaneous embolization of recurrent juvenile nasopharyngeal angiofibroma ( jna ) . a 16-year - old boy with recurrent jna ( a ) preembolization right eca angiogram shows intense tumor blush ( black arrow ) predominantly supplied by the internal maxillary artery ( b ) postembolization eca angiogram shows no tumor blush . ( c ) ica angiogram after embolization of the external carotid artery feeders shows extensive tumor blush ( arrow ) supplied by cavernous and ophthalmic branches of ica . ( d ) the tumor territory supplied by ica was devascularized by direct puncture ( arrow head ) and glue embolization . note the coils ( dotted arrows ) placed in multiple feeding arteries of eca after embolizing the tumor bed using pva . ( e ) radiograph shows glue cast ( white arrow ) conforming to the tumor blush supplied by ica feeders . ( f ) postembolization ica angiogram shows marked reduction in tumor blush with minimal residue ( dotted arrow ) . estimated blood loss during surgery was less than 500 cc and did not receive blood transfusion intraoperative direct puncture embolization of jna . ( a ) post eca embolization , ica angiogram shows a small residual tumor blush ( arrow ) , which was not embolized . ( b ) due to excessive bleeding during surgery , intraoperative direct puncture ( arrow ) and glue embolization was done under fluoroscopic guidance . postintraoperative embolization , complete surgical resection was achieved without significant bleed extreme care should be taken to prevent the embolizing agent from entering ophthalmic artery or vital circulation of brain . the interventional radiologist should be aware about the potentially dangerous anastomosis between the eca and ica and should carefully watch for opening of these collaterals during echn embolization . in situations where internal carotid artery is encased by the tumor , surgical resection would be difficult with high risk of injury to the artery . in such cases a balloon test occlusion ( bto ) is performed to assess the ipsilateral cerebral circulation from opposite carotid or vertebra - basilar circulation . if patient tolerates bto , the involved carotid artery can be safely occluded using coils or detachable balloons [ figure 8 ] . ( a ) common carotid angiogram shows ligated external carotid artery ( long arrow ) , with residual tumor being supplied by cavernous branches of ica ( block arrow ) . ( b ) post ica occlusion , common carotid angiogram shows no tumor blush with coil mass in the ica . patient had tolerated the balloon test occlusion ( bto ) and intraoperative estimated blood loss was < 500 cc overall , preoperative embolization is a safe procedure and serious complications occur in less than 2% patients that includes facial numbness , mucosal necrosis , blindness , or cerebrovascular accident . preoperative embolization is not only cost effective , it reduces the morbidity by reducing blood loss and operative time , shortens the hospital stay while reducing the rate of tumor recurrence . bleeding in echn manifests as epistaxis , hematemesis , or bleeding from an open wound or ulcer . various causes of bleed include craniofacial injury , infections , tumors , surgery , and vascular abnormalities while medical causes like hypertension and anticoagulation therapy [ figure 9 ] . ( a ) dsa image shows abnormal blush ( arrow ) at the site of tracheotomy site with feeders from the inferior thyroid artery . ( c ) inferior thyroid artery feeders were embolized using 300 micon pva particles followed by coil embolization ( dotted arrow ) . ( d ) postembolization check subclavian angiogram shows complete resolution of abnormal blush treatment is often variable and multidisciplinary approach is required for successful outcome . preprocedure computed tomography ( ct ) angiography helps in identifying the likely source and cause of bleed while also exquisitely depicting the anatomy . subclavian , carotid , and vertebral artery angiograms are obtained to identify and target the culprit vessel . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . difficult postoperative anatomy , radiation effects , infections preclude safe , and effective surgery in these patients . angioembolization has been proven to be effective with minimal risk of complications in a few reported series . goal of endovascular therapy is complete devascularization of the tumor by embolizing the tumor bed . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. pva / embospheres of size 100 - 300 micron ) [ figure 10 ] . a 54year - old male suffering for inoperatble carcinoma tongue presented with persistent tumor bleed in spite of receiving hemostatic radiotherapy . ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( c ) axial , ( d ) sagittal cone beam ct images of the lingual artery ( dotted arrow ) angiogram confirms tumor blush ( white arrow ) . ( e ) lingual artery angiogram shows tumor blush ( black arrow ) , which was embolized . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . carotid blow out patient usually presents with neck swelling or bleed from nose or mouth which could be sentinel or massive . carotid blow out can be accurately diagnosed by ct angiography and definitive treatment can be planned by knowing the exact size and location . the blow out can be seen in common carotid , internal , or eca and imaging would reveal either a pseudoaneurysm or an active bleed . the clinical presentation and imaging findings demands the endovascular treatment to be performed on an emergency or semi - emergency basis . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . this not only achieves immediate hemostasis but also maintains patency of lumen and blood flow to the brain . patients requiring stent graft placements need to be administered with antiplatelets before and after the procedure . however , if placement of stent graft becomes technically difficult , the diseased carotid artery can be occluded after confirming adequate cross flow from the contralateral carotid or vertebrobasilar circulation by bto [ figure 13 ] . rarely , in case of uncontrolled massive bleeding after skull base surgery , failed attempt to deploy stent graft mandates immediate occlusion of carotid artery to prevent death from exsanguination [ figure 14 ] . an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( c ) carotid angiogram after stent graft placement shows exclusion of the aneurysm and maintained forward flow within the carotid artery courtesy : dr uday limaye , inr division , kemh , mumbai a 54-year - old male patient with history of radiotherapy to metastatic neck nodes presented with recurrent bouts of hematemesis . ( a ) ct angiogram sagittal reformatted image shows an irregular pseudoaneurysm ( arrow head ) at the carotid bifurcation with non visualization of eca origin . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . ( c ) angiogram after stent - graft placement shows complete exclusion of pseudoaneurysm from the circulation and maintained distal flow in ica a 12-year - old girl presented with total ophthalmoplegia and epistaxis . cect of brain ( a ) and dsa of right ica ( b ) show an aneurysm ( arrow , arrowhead ) in the cavernous portion of ica . ( c ) right ica occluded using detachable balloon ( white arrow ) and coils ( black arrow ) after bto . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( a ) dsa , ( b ) vr image of left ica shows a pseudoaneurysm ( white arrow ) arising from the cavernous portion . ( c ) right ica , ( d ) vertebral angiogram shows poor cross circulation across the circle of willis during bto . in view of torrential bleeding and failed attempt to deploy a covered stent , the left ica was occluded ( e ) using coils ( arrowhead ) to prevent exsanguination . ( f ) plain ct brain done after 24 hours shows fully evolved cerebral infarct ( dotted arrow ) blow out of eca or its branch is usually treated by embolizing the culprit vessel both proximal and distal to the pseudoaneurysm [ figure 15 ] . the carotid artery can be permanently occluded using coils or balloons and approximately 15 - 20% would develop cerebral ischemia . a middle - aged man presented with progressive neck swelling after attempted biopsy . ( a ) axial , ( b ) sagittal ct angiography images show two partially thrombosed pseudoaneurysm ( arrow ) arising from the right carotid artery . ( c , d ) dsa image shows the pseudoaneurysms ( arrow ) with no opacification of the ica and faint distal run - off of eca branches ( arrowhead ) . ( e , f ) the pseudo aneurysms were treated by coiling ( dotted arrow ) and occluding eca as well as cca . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( a ) left carotid angiogram shows fistulous communication ( arrow ) between eca and internal jugular vein . note the poor opacification of rest of eca ( b ) lateral radiograph of neck shows a detachable balloon ( arrow ) deployed at the site of fistula . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . difficult postoperative anatomy , radiation effects , infections preclude safe , and effective surgery in these patients . angioembolization has been proven to be effective with minimal risk of complications in a few reported series . goal of endovascular therapy is complete devascularization of the tumor by embolizing the tumor bed . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. pva / embospheres of size 100 - 300 micron ) [ figure 10 ] . a 54year - old male suffering for inoperatble carcinoma tongue presented with persistent tumor bleed in spite of receiving hemostatic radiotherapy . ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( c ) axial , ( d ) sagittal cone beam ct images of the lingual artery ( dotted arrow ) angiogram confirms tumor blush ( white arrow ) . ( e ) lingual artery angiogram shows tumor blush ( black arrow ) , which was embolized . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . carotid blow out patient usually presents with neck swelling or bleed from nose or mouth which could be sentinel or massive . carotid blow out can be accurately diagnosed by ct angiography and definitive treatment can be planned by knowing the exact size and location . the blow out can be seen in common carotid , internal , or eca and imaging would reveal either a pseudoaneurysm or an active bleed . the clinical presentation and imaging findings demands the endovascular treatment to be performed on an emergency or semi - emergency basis . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . this not only achieves immediate hemostasis but also maintains patency of lumen and blood flow to the brain . patients requiring stent graft placements need to be administered with antiplatelets before and after the procedure . however , if placement of stent graft becomes technically difficult , the diseased carotid artery can be occluded after confirming adequate cross flow from the contralateral carotid or vertebrobasilar circulation by bto [ figure 13 ] . rarely , in case of uncontrolled massive bleeding after skull base surgery , failed attempt to deploy stent graft mandates immediate occlusion of carotid artery to prevent death from exsanguination [ figure 14 ] an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( c ) carotid angiogram after stent graft placement shows exclusion of the aneurysm and maintained forward flow within the carotid artery courtesy : dr uday limaye , inr division , kemh , mumbai a 54-year - old male patient with history of radiotherapy to metastatic neck nodes presented with recurrent bouts of hematemesis . ( a ) ct angiogram sagittal reformatted image shows an irregular pseudoaneurysm ( arrow head ) at the carotid bifurcation with non visualization of eca origin . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . ( c ) angiogram after stent - graft placement shows complete exclusion of pseudoaneurysm from the circulation and maintained distal flow in ica a 12-year - old girl presented with total ophthalmoplegia and epistaxis . cect of brain ( a ) and dsa of right ica ( b ) show an aneurysm ( arrow , arrowhead ) in the cavernous portion of ica . ( c ) right ica occluded using detachable balloon ( white arrow ) and coils ( black arrow ) after bto . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( a ) dsa , ( b ) vr image of left ica shows a pseudoaneurysm ( white arrow ) arising from the cavernous portion . ( c ) right ica , ( d ) vertebral angiogram shows poor cross circulation across the circle of willis during bto . in view of torrential bleeding and failed attempt to deploy a covered stent , the left ica was occluded ( e ) using coils ( arrowhead ) to prevent exsanguination . ( f ) plain ct brain done after 24 hours shows fully evolved cerebral infarct ( dotted arrow ) blow out of eca or its branch is usually treated by embolizing the culprit vessel both proximal and distal to the pseudoaneurysm [ figure 15 ] . the carotid artery can be permanently occluded using coils or balloons and approximately 15 - 20% would develop cerebral ischemia . a middle - aged man presented with progressive neck swelling after attempted biopsy . ( a ) axial , ( b ) sagittal ct angiography images show two partially thrombosed pseudoaneurysm ( arrow ) arising from the right carotid artery . ( c , d ) dsa image shows the pseudoaneurysms ( arrow ) with no opacification of the ica and faint distal run - off of eca branches ( arrowhead ) . ( e , f ) the pseudo aneurysms were treated by coiling ( dotted arrow ) and occluding eca as well as cca . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( a ) left carotid angiogram shows fistulous communication ( arrow ) between eca and internal jugular vein . note the poor opacification of rest of eca ( b ) lateral radiograph of neck shows a detachable balloon ( arrow ) deployed at the site of fistula . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less .

medicine is an ever changing field and interventional radiology ( ir ) procedures are becoming increasingly popular because of high efficacy and its minimally invasive nature of the procedure . management of disease processes in the extra cranial head and neck ( echn ) has always been a challenge due to the complex anatomy of the region . cross sectional imaging of the echn has grown and evolved tremendously and occupies a pivotal and integral position in the clinical management of variety of head and neck pathologies . advances in angiographic technologies including flat panel detector systems , biplane , and 3-dimensional rotational angiography have consolidated and expanded the role of ir in the management of various echn pathologies . the echn is at cross roads between the origins of great vessels and the cerebral vasculature . thorough knowledge of functional and technical aspects of neuroangiography is essential before embarking on head and neck vascular interventions . the vessels of the head and neck can be involved by infectious and inflammatory conditions , get irradiated during radiotherapy and injured due to trauma or iatrogenic cause . the echn is also a common site for various hypervascular neoplasms and vascular malformations , which can be treated with endovascular and percutaneous embolization . this pictorial essay provides a review of variety of echn pathologies which were managed by various ir procedures using different approaches .

Introduction Vascular malformations Tumor embolization Management of head and neck bleeding Bleeding from head and neck malignancy Carotid artery blow out

interventional radiology ( ir ) has evolved rapidly in past two decades with advancement in imaging technology . there has also been significant improvement and development of more sophisticated hardware with the help of which it is possible to target any vascular territory in the body and carry out therapeutic procedures by minimally invasive technique . the head and neck region is unique in terms of the critical anatomy and vital neurovascular structures involved . it needs detailed knowledge of the head and neck vascular anatomy and special technique to perform vascular ir procedure in extra cranial head and neck ( echn ) . the pathologies where ir can offer minimally invasive treatment include vascular malformations , tumor embolization , and echn bleeding . other abnormalities like plunging ranula , sialocele , and other benign cysts also respond well to percutaneous sclerotherapy . courtesy : dr uday limaye , inr division , kemh , mumbai high flow arteriovenous malformations ( avm ) of echn present with disfigurement or episodes of bleeding . they need to be managed by multi specialty team where ir has an important role to embolize the abnormal vascularity , which can be followed by surgery to completely remove the lesion . in cases where transarterial approach is precluded due to previous surgical ligation of external carotid artery ( eca ) , , transarterial embolization can still be performed by gaining vascular access into one of the superficial eca branch after surgical dissection [ figure 3 ] . percutaneous embolization of scalp avm 34-year - old female patient with history of pulsatile swelling on her forehead . ( d , e ) radiograph shows glue cast ( white arrow ) conforming to the avm . a 21-year - old female with a large slow flow vascular malformation of the tongue base presented with dysphagia and difficulty in speech . ( a ) axial , ( b ) sagittal t2w images shows a hyperintense lesion ( arrow ) in the base of tongue causing marked narrowing of oropharynx ( arrow head ) . ( c ) arterial , ( d ) venous phase of lingual artery ( arrow ) angiogram shows progressively increasing and persistent vascular blush in the lesion ( arrow ) suggestive of slow flow vascular malformation . using expandable suspending laryngoscope , the base of tongue can be optimally visualized and direct puncture of the lesion can be performed by transoral route . ( g ) axial , ( h ) sagittal fat suppressed contrast t1w images done after 6 weeks shows significant reduction in the size and enhancement of residual malformation ( arrow ) with good clinical improvement in symptoms preoperative embolization of echn tumors is frequently performed by transarterial route . preoperative embolization helps to prevent blood loss during surgery and provides a clean field for the surgical resection [ figure 5 ] . commonly used embolizing materials are particulate embolizing agents like polyvinyl alcohol ( pva ) particles , [ cook incorporated , bloomington , usa ] , embospheres [ biosphere medical sa , paris , france ] in the size range of 100300 microns . the aim of embolization is to inject the particles in the arteriolar capillary bed of tumor parenchyma , so that collateral circulation does not develop rapidly . however , as endoscopic resection of skull base tumors is being increasingly performed , proximal coil embolization of the feeding artery ( e.g. ( c ) post embolization eca angiogram shows significant reduction in the tumor blush tumors involving the skull base frequently derive blood supply from ophthalmic or petrous branches of internal carotid artery ( ica ) . the ica branches are often difficult to cannulate and there is considerable risk of reflux of the embolic agent with nontarget embolization if transarterial embolization of the ica feeders are attempted . ica angiogram after embolization of the external carotid artery feeders shows extensive tumor blush ( arrow ) supplied by cavernous and ophthalmic branches of ica . ( a ) post eca embolization , ica angiogram shows a small residual tumor blush ( arrow ) , which was not embolized . ( b ) due to excessive bleeding during surgery , intraoperative direct puncture ( arrow ) and glue embolization was done under fluoroscopic guidance . various causes of bleed include craniofacial injury , infections , tumors , surgery , and vascular abnormalities while medical causes like hypertension and anticoagulation therapy [ figure 9 ] . subclavian , carotid , and vertebral artery angiograms are obtained to identify and target the culprit vessel . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . difficult postoperative anatomy , radiation effects , infections preclude safe , and effective surgery in these patients . goal of endovascular therapy is complete devascularization of the tumor by embolizing the tumor bed . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( e ) lingual artery angiogram shows tumor blush ( black arrow ) , which was embolized . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( c ) carotid angiogram after stent graft placement shows exclusion of the aneurysm and maintained forward flow within the carotid artery courtesy : dr uday limaye , inr division , kemh , mumbai a 54-year - old male patient with history of radiotherapy to metastatic neck nodes presented with recurrent bouts of hematemesis . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( c , d ) dsa image shows the pseudoaneurysms ( arrow ) with no opacification of the ica and faint distal run - off of eca branches ( arrowhead ) . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less . other abnormalities like plunging ranula , sialocele , and other benign cysts also respond well to percutaneous sclerotherapy . ( e ) pretreatment , ( f ) posttreatment photograph shows regression of the lesion . they need to be managed by multi specialty team where ir has an important role to embolize the abnormal vascularity , which can be followed by surgery to completely remove the lesion . in cases where transarterial approach is precluded due to previous surgical ligation of external carotid artery ( eca ) , , transarterial embolization can still be performed by gaining vascular access into one of the superficial eca branch after surgical dissection [ figure 3 ] . percutaneous embolization of scalp avm 34-year - old female patient with history of pulsatile swelling on her forehead . a 21-year - old female with a large slow flow vascular malformation of the tongue base presented with dysphagia and difficulty in speech . using expandable suspending laryngoscope , the base of tongue can be optimally visualized and direct puncture of the lesion can be performed by transoral route . ( g ) axial , ( h ) sagittal fat suppressed contrast t1w images done after 6 weeks shows significant reduction in the size and enhancement of residual malformation ( arrow ) with good clinical improvement in symptoms preoperative embolization of echn tumors is frequently performed by transarterial route . preoperative embolization helps to prevent blood loss during surgery and provides a clean field for the surgical resection [ figure 5 ] . commonly used embolizing materials are particulate embolizing agents like polyvinyl alcohol ( pva ) particles , [ cook incorporated , bloomington , usa ] , embospheres [ biosphere medical sa , paris , france ] in the size range of 100300 microns . the aim of embolization is to inject the particles in the arteriolar capillary bed of tumor parenchyma , so that collateral circulation does not develop rapidly . ( a ) contrast enhanced mr image shows an intensely enhancing mass ( white arrow ) in the middle ear . ( c ) post embolization eca angiogram shows significant reduction in the tumor blush tumors involving the skull base frequently derive blood supply from ophthalmic or petrous branches of internal carotid artery ( ica ) . the ica branches are often difficult to cannulate and there is considerable risk of reflux of the embolic agent with nontarget embolization if transarterial embolization of the ica feeders are attempted . transarterial and percutaneous embolization of recurrent juvenile nasopharyngeal angiofibroma ( jna ) . various causes of bleed include craniofacial injury , infections , tumors , surgery , and vascular abnormalities while medical causes like hypertension and anticoagulation therapy [ figure 9 ] . subclavian , carotid , and vertebral artery angiograms are obtained to identify and target the culprit vessel . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( c ) axial , ( d ) sagittal cone beam ct images of the lingual artery ( dotted arrow ) angiogram confirms tumor blush ( white arrow ) . ( e ) lingual artery angiogram shows tumor blush ( black arrow ) , which was embolized . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . the clinical presentation and imaging findings demands the endovascular treatment to be performed on an emergency or semi - emergency basis . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . however , if placement of stent graft becomes technically difficult , the diseased carotid artery can be occluded after confirming adequate cross flow from the contralateral carotid or vertebrobasilar circulation by bto [ figure 13 ] . an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( c ) carotid angiogram after stent graft placement shows exclusion of the aneurysm and maintained forward flow within the carotid artery courtesy : dr uday limaye , inr division , kemh , mumbai a 54-year - old male patient with history of radiotherapy to metastatic neck nodes presented with recurrent bouts of hematemesis . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . cect of brain ( a ) and dsa of right ica ( b ) show an aneurysm ( arrow , arrowhead ) in the cavernous portion of ica . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( c , d ) dsa image shows the pseudoaneurysms ( arrow ) with no opacification of the ica and faint distal run - off of eca branches ( arrowhead ) . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less . endovascular treatment is increasingly being preferred in the management of bleeding from echn malignancies as compared with traditional open surgical approach and ligation of involved vessels . difficult postoperative anatomy , radiation effects , infections preclude safe , and effective surgery in these patients . goal of endovascular therapy is complete devascularization of the tumor by embolizing the tumor bed . this can be achieved by superselective catheterization of target vessels by microcatheter and embolizing with particulate embolic agents ( e.g. ( a ) plain , ( b ) postcontrast axial t1w images show a large enhancing lesion ( white arrow ) in the tongue . ( c ) axial , ( d ) sagittal cone beam ct images of the lingual artery ( dotted arrow ) angiogram confirms tumor blush ( white arrow ) . ( f ) postembolization angiogram shows significant reduction in the tumor blush with cessation of the tumor bleed . carotid artery in echn is vulnerable to rupture by trauma , infection , malignancy , and iatrogenic injury . radiotherapy to head and neck malignancy increases the risk of carotid blow out by 7.6fold . posttraumatic and iatrogenic vascular injuries can be potentially life threatening though carotid injury is seen in less than 1% of blunt head and neck trauma . carotid blow out can be accurately diagnosed by ct angiography and definitive treatment can be planned by knowing the exact size and location . the blow out can be seen in common carotid , internal , or eca and imaging would reveal either a pseudoaneurysm or an active bleed . the clinical presentation and imaging findings demands the endovascular treatment to be performed on an emergency or semi - emergency basis . the common carotid and internal carotid artery blow out can be treated by endovascular placement of covered stent [ figures 11 and 12 ] . this not only achieves immediate hemostasis but also maintains patency of lumen and blood flow to the brain . however , if placement of stent graft becomes technically difficult , the diseased carotid artery can be occluded after confirming adequate cross flow from the contralateral carotid or vertebrobasilar circulation by bto [ figure 13 ] . rarely , in case of uncontrolled massive bleeding after skull base surgery , failed attempt to deploy stent graft mandates immediate occlusion of carotid artery to prevent death from exsanguination [ figure 14 ] an elderly male presented with a bleeding ulcerated pulsatile swelling in the neck following trauma . ( a ) photograph showing ulcerated swelling with blood clot in the neck ( b ) dsa of left carotid artery shows a large pseudoaneurysm arising from cca . ( b ) dsa of left cca confirms pseudoaneurysm in the region of carotid bulb ( arrow ) . ( d)postembolization left carotid angiogram shows opacification of the right cerebral hemisphere branches across the circle of willis a case of nasoethmoidal carcinoma who had intraoperative injury to the left internal carotid artery during endoscopic surgery . ( f ) plain ct brain done after 24 hours shows fully evolved cerebral infarct ( dotted arrow ) blow out of eca or its branch is usually treated by embolizing the culprit vessel both proximal and distal to the pseudoaneurysm [ figure 15 ] . the carotid artery can be permanently occluded using coils or balloons and approximately 15 - 20% would develop cerebral ischemia . no neurological deficit seen postprocedure at times , penetrating injuries could result in the formation of complex arterio - venous fistulae , which can be effectively treated by minimally invasive endovascular means using balloons and coils for flow modification [ figure 16 ] . a 15-year - old boy presented with swelling in the left side of the neck with history of injury 3years back . ( c ) postembolization carotid angiogram shows complete absence of fistula and well opacified eca branches courtesy : dr uday limaye , inr division , kemh , mumbai overall , ir can provide a wide spectrum of procedures for the effective management of various vascular ailments involving the head and neck region with a minimally invasive approach , where recovery of patient is faster and morbidity significantly less .

the online version of this article ( doi:10.1007/s00134 - 010 - 2087-y ) contains supplementary material , which is available to authorized users . acute kidney injury ( aki ) is a common complication of critical illness and carries high mortality despite significant advances in medical care [ 1 , 2 ] . this apparent lack of improvement may result from the use of more aggressive medical and surgical interventions in an ever - ageing population . on the other hand , potentially effective therapeutic interventions for aki may currently fail because they are applied late in the course of injury after an obvious increase of serum creatinine ( scr ) is observed . due to the delayed rise in scr following injury , recent efforts have focused on identification of an early and reliable biomarker of kidney injury [ 5 , 6 ] . cystatin c ( cyc ) is a 13-kda , nonglycosylated basic protein , produced at a constant rate by all nucleated cells . it is freely filtered by glomeruli and catabolized in tubules . in high - risk patients , serum cyc ( scyc ) moreover , although cyc is normally not detected in urine , it has been found in urine of patients with tubular disease , suggesting that it is a tubular marker [ 813 ] . one drawback of use of cyc at present is a lack of recognition of its potential value for use in the general critical care setting , in which the population is heterogeneous and aki etiology and timing are often unclear . in the present study in a heterogeneous intensive care unit ( icu ) population we collected serial samples of scyc and ucyc , and determined the first day of aki based on the rifle classification system . we hypothesized that these markers predict aki 12 days earlier than the rifle criteria in patients developing aki after entry , and that these biomarkers predict the need for renal replacement therapy ( rrt ) on the first day of aki . the protocol was approved by the institutional review board of all participating institutions , and written informed consent was obtained from all patients or their authorized representatives . the study was a prospective observational cohort study in adult patients with expected duration of mechanical ventilation of at least 48 h , and/or expected length of icu stay of at least 72 h. patients were enrolled within 48 h of icu admission . detailed methods ( e.g. , exclusion criteria ) are described in electronic supplementary material ( esm ) file 1 . demographic data , admission diagnosis , acute physiology and chronic health evaluation ( apache ) ii , and simplified acute physiology score ( saps ) ii scores were documented upon icu admittance [ 16 , 17 ] . the patients were scored daily for aki using the creatinine and urine output criteria of the rifle classification system for aki . to define the baseline renal function we compared the premorbid scr within 1 year prior to icu admission with the scr at icu admission . if the premorbid scr was unavailable , it was estimated by solving the modification of diet in renal disease ( mdrd ) equation as recommended by the acute dialysis quality initiative ( adqi ) working group . the premorbid scr was not estimated in patients with history of kidney insufficiency . in these patients , the first day of aki was termed day 0 , while the two days prior to this day were termed day 1 and day 2 , respectively . blood and urine sampling for cyc measurements were performed on inclusion , day 1 and alternate days until the start of rrt , or icu discharge . samples were centrifuged at 1,500 g for 10 min at 4c . cystatin c was measured with an n latex cystatin c test kit , a particle - enhanced immunonephelometric method , on a bn prospec analyzer ( dade behring , leusden , the netherlands ) . we normalized urinary excretion of cyc for millimoles of urinary creatinine ( ucyccorr ) to compensate for differences in urine flow rate . the primary endpoint was the first rifle event ( risk , injury or failure ) . data were analyzed using the statistical package for the social sciences ( spss ) version 17.0 ( spss , chicago il , usa ) for windows . continuous variables are expressed as mean standard deviation ( sd ) or median with interquartile range . cystatin c levels below the detection limit of 0.05 mg / l were considered to be 0.025 mg / l . normally distributed variables were compared using one - way analysis of variance with bonferroni s correction for multiple comparisons . for significant findings , post hoc wallis one - way analysis of variance was used to compare non - normally distributed variables . all testing was two - tailed , and p < 0.05 was considered statistically significant . the protocol was approved by the institutional review board of all participating institutions , and written informed consent was obtained from all patients or their authorized representatives . the study was a prospective observational cohort study in adult patients with expected duration of mechanical ventilation of at least 48 h , and/or expected length of icu stay of at least 72 h. patients were enrolled within 48 h of icu admission . detailed methods ( e.g. , exclusion criteria ) are described in electronic supplementary material ( esm ) file 1 . demographic data , admission diagnosis , acute physiology and chronic health evaluation ( apache ) ii , and simplified acute physiology score ( saps ) ii scores were documented upon icu admittance [ 16 , 17 ] . the patients were scored daily for aki using the creatinine and urine output criteria of the rifle classification system for aki . to define the baseline renal function we compared the premorbid scr within 1 year prior to icu admission with the scr at icu admission . if the premorbid scr was unavailable , it was estimated by solving the modification of diet in renal disease ( mdrd ) equation as recommended by the acute dialysis quality initiative ( adqi ) working group . the premorbid scr was not estimated in patients with history of kidney insufficiency . in these patients , the first day of aki was termed day 0 , while the two days prior to this day were termed day 1 and day 2 , respectively . blood and urine sampling for cyc measurements were performed on inclusion , day 1 and alternate days until the start of rrt , or icu discharge . samples were centrifuged at 1,500 g for 10 min at 4c . cystatin c was measured with an n latex cystatin c test kit , a particle - enhanced immunonephelometric method , on a bn prospec analyzer ( dade behring , leusden , the netherlands ) . urea and creatinine levels were measured by standard clinical chemical methods . we normalized urinary excretion of cyc for millimoles of urinary creatinine ( ucyccorr ) to compensate for differences in urine flow rate . the primary endpoint was the first rifle event ( risk , injury or failure ) . data were analyzed using the statistical package for the social sciences ( spss ) version 17.0 ( spss , chicago il , usa ) for windows . continuous variables are expressed as mean standard deviation ( sd ) or median with interquartile range . cystatin c levels below the detection limit of 0.05 mg / l were considered to be 0.025 mg / l . normally distributed variables were compared using one - way analysis of variance with bonferroni s correction for multiple comparisons . for significant findings , post hoc wallis one - way analysis of variance was used to compare non - normally distributed variables . all testing was two - tailed , and p < 0.05 was considered statistically significant . we enrolled 170 patients from april 2006 to november 2007 and excluded 19 patients during the analysis because of incomplete sampling , leaving 151 patients for analysis . one hundred thirty - three patients ( 88% ) were included on the first day of icu admission , and 18 patients ( 12% ) were included the next day of admission . we defined three groups : group 0 ( n = 60 ) , never developed aki , serving as controls ; group 1 ( n = 35 ) , developed aki after admission ; and group 2 ( n = 56 ) , presented with aki at admission . aki developed after a median of 2 [ 12 ] days ( range 17 days ) in group 1 : 17 patients ( 49% ) were classified for aki by the creatinine criteria , 14 patients ( 38% ) by the urine criteria , and in 3 patients ( 9% ) the creatinine and urine scores were identical . in group 2 , 28 patients ( 50% ) had aki at admission by the creatinine criteria , 13 patients ( 23% ) by the urine criteria , and in 17 patients the creatinine and urine scores were identical . table 1 compares the baseline clinical data of the three groups , while renal and outcome data are shown in table 2 . the maximum rifle class was higher in group 2 compared with group 1 . in group 1 the maximum rifle class was achieved by the creatinine criteria in 21 patients ( 60% ) , by the urine criteria in 8 patients ( 23% ) , and in 6 patients ( 17% ) the creatinine and urine aki classification were identical . in group 2 the maximum rifle class was achieved by the creatinine criteria in 24 patients ( 43% ) , by the urine criteria in 13 patients ( 23% ) , and in 17 ( 30% ) patients the creatinine and urine aki classification were identical.table 1baseline characteristics grouped according to aki statusgroup 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)page ( years)59.2 16.168.4 15.474.6 9.3<0.001male ( % ) 39 ( 65)24 ( 69)34 ( 62)0.81bmi ( kg / m)24.6 4.326.5 3.829.6 12.80.11apache ii18.5 9.919.6 8.324.2 12.90.016saps ii37.2 13.344.1 14.247.6 16.40.01comorbidities ( % ) hypertension15 ( 26)14 ( 40)24 ( 44)0.12 diabetes7 ( 12)5 ( 14)8 ( 15)0.92 ckd2 ( 3)1 ( 3)6 ( 11)0.17 tsh ( mu / l)0.40 ( 0.180.87)0.42 ( 0.251.0)0.41 ( 0.211.20)0.83 ft4 ( pmol / l)13.2 3.512.8 3.3)13.6 3.60.67admission category ( % ) 0.68 medical29 ( 48.3)17 ( 48.6)23 ( 41.8 ) surgical31 ( 51.7)18 ( 51.4)33 ( 59)primary diagnosis ( % ) 0.21 cardiovascular failure2 ( 3.3)4 ( 11.4)4 ( 7.1 ) cerebrovascular event2 ( 3.3)00 hemorrhagic shock7 ( 11.7)2 ( 5.7)6 ( 10.7 ) multiple trauma4 ( 6.7)2 ( 5.7)1 ( 1.8 ) elective major surgery1 ( 1.7)1 ( 2.9)5 ( 8.9 ) respiratory failure23 ( 38.3)11 ( 31.4)14 ( 25 ) septic shock20 ( 33.3)13 ( 37.1)26 ( 46.6)aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormonetable 2renal characteristics and outcome grouped according to aki status group 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)ppremorbid scr ( mol / l ) actual92 30 ( n = 37)91 25 ( n = 28)83 23 ( n = 37)0.31 estimated88 1286 1283 110.05study inclusion scr ( mol / l)72 ( 6194)87 ( 7687)139 ( 97211)<0.001 scyc ( mg / l)0.80 ( 0.651.04)0.91 ( 0.811.17)1.14 ( 0.881.94)<0.001 ucyc ( mg / l)0.16 ( 0.0251.47)0.18 ( 0.070.68)0.59 ( 0.103.47)0.46ucyccorr ( g / molcr)0.03 ( 0.010.30)0.04 ( 0.010.13)0.13 ( 0.010.67)0.47maximum rifle class ( % ) < 0.001 risk27 ( 77)22 ( 39.3 ) injury5 ( 14.3)14 ( 25 ) failure3 ( 8.6)20 ( 35.8 ) cvvh ( % ) 1 ( 1.7)4 ( 11.4)10 ( 17.6)0.01icu treatment ( days)5 ( 38)8 ( 518)6 ( 39)0.002icu mortality ( % ) 04 ( 11.4)11 ( 19.6)0.002hospital mortality ( % ) 4 ( 6.7)6 ( 17.1)18 ( 32.1)0.002aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unitbaseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admissionbaseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation baseline characteristics grouped according to aki status aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormone renal characteristics and outcome grouped according to aki status aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unit baseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admission baseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation cystatin c was measured in 582 blood samples ( 3.8 3.2 samples / patient ) and 569 urine samples ( 3.8 3.1 samples / patient ) . cystatin c level was below the detection limit in 171 ( 30% ) urine samples . urinary cyc levels also increased with increasing rifle class ; however , the difference between rifle risk and rifle injury was not statistically significant.table 3cystatin c per rifle classnoneriskinjuryfailurepserum samples number3991093531 cyc ( mg / l)1.15 ( 1.051.25)1.34 ( 1.221.45)1.75 ( 1.591.92)2.31 ( 2.092.52)<0.001urine samples number3911044034 cyc ( mg / l)1.02 ( 0.541.51)2.36 ( 1.523.19)2.77 ( 1.434.12)5.87 ( 4.247.51)<0.001 cyccorr ( g / mol cr)0.43 ( 0.280.59)0.44 ( 0.210.68)0.34 ( 0.040.72)0.79 ( 0.321.24)0.46values are mean and 95% confidence interval . the p value represents the difference among groups by linear mixed modelsscyc serum cystatin c , ucyc urine cystatin c , ucyccorr ucyc normalized for urinary creatinine cystatin c per rifle class values are mean and 95% confidence interval . the p value represents the difference among groups by linear mixed models scyc serum cystatin c , ucyc urine cystatin c , ucyccorr ucyc normalized for urinary creatinine figure 1 compares the longitudinal trend of scr , scyc , and ucyc starting from 2 days prior to aki ( group 1 ) with the non - aki trend ( group 0 ) . on day 2 , median scyc was significantly higher ( 0.93 versus 0.80 mg / l , p = 0.01 ) , but not on day 1 ( 0.98 versus 0.86 mg / l , p = 0.08 ) . on the 2 days prior to aki , ucyc levels did not differ significantly between group 1 and group 0 ( median 0.13 [ 0.0250.88 ] mg / l versus median 0.16 [ 0.0251.50 ] mg / l , p = 0.89 , and median 0.14 [ 0.0250.37 ] mg / l versus median 0.17 [ 0.0250.84 ] mg / l , p = 0.57 , day 2 and day 1 , respectively ) . the diagnostic performance for scyc was fair on day 2 [ area under the curve ( auc ) 0.72 ] and poor on day 1 ( auc 0.62 ) , while ucyc had no diagnostic value on day 2 and day 1 ( fig . 2 ) . the test characteristics of scyc at various cutoff levels are shown in esm table 1.fig . 1time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . the number of patients investigated is shown in italics at each time point . * p < 0.05 compared with the non - aki groupfig . 2receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of acute kidney injury ( aki ) . upper panels 2 days prior to aki ( n = 71 , disease prevalence 0.20 ) ; lower panels 1 day prior to aki . ucyccorr urine cystatin c normalized for urinary creatinine time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . values are mean and standard error of the means . the number of patients investigated is shown in italics at each time point . * p < 0.05 compared with the non - aki group receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of acute kidney injury ( aki ) . upper panels 2 days prior to aki ( n = 71 , disease prevalence 0.20 ) ; lower panels 1 day prior to aki . ucyccorr urine cystatin c normalized for urinary creatinine fourteen ( 15% ) out of 91 aki patients received continuous venovenous hemofiltration ( cvvh ) : 4 patients from group 1 , and 10 patients from group 2 . median duration between day 0 and initiation of cvvh was 1.5 [ 04 ] days . in comparison with the non - cvvh patients , the aki patients requiring cvvh had significantly higher apache ii scores on admission ( 28 [ 2133 ] versus 19 [ 1427 ] , p = 0.01 ) and produced less urine ( 740 [ 4001,088 ] ml / day versus 1,660 [ 1,0282,918 ] ml / day , p = 0.001 ) . systemic and urinary cyc determined on day 0 were poor predictors for the need for rrt ( auc 0.66 , fig . 3).fig . 3receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . area under the curve ( auc ) is depicted in each panel receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . we enrolled 170 patients from april 2006 to november 2007 and excluded 19 patients during the analysis because of incomplete sampling , leaving 151 patients for analysis . one hundred thirty - three patients ( 88% ) were included on the first day of icu admission , and 18 patients ( 12% ) were included the next day of admission . we defined three groups : group 0 ( n = 60 ) , never developed aki , serving as controls ; group 1 ( n = 35 ) , developed aki after admission ; and group 2 ( n = 56 ) , presented with aki at admission . aki developed after a median of 2 [ 12 ] days ( range 17 days ) in group 1 : 17 patients ( 49% ) were classified for aki by the creatinine criteria , 14 patients ( 38% ) by the urine criteria , and in 3 patients ( 9% ) the creatinine and urine scores were identical . in group 2 , 28 patients ( 50% ) had aki at admission by the creatinine criteria , 13 patients ( 23% ) by the urine criteria , and in 17 patients the creatinine and urine scores were identical . table 1 compares the baseline clinical data of the three groups , while renal and outcome data are shown in table 2 . the maximum rifle class was higher in group 2 compared with group 1 . in group 1 the maximum rifle class was achieved by the creatinine criteria in 21 patients ( 60% ) , by the urine criteria in 8 patients ( 23% ) , and in 6 patients ( 17% ) the creatinine and urine aki classification were identical . in group 2 the maximum rifle class was achieved by the creatinine criteria in 24 patients ( 43% ) , by the urine criteria in 13 patients ( 23% ) , and in 17 ( 30% ) patients the creatinine and urine aki classification were identical.table 1baseline characteristics grouped according to aki statusgroup 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)page ( years)59.2 16.168.4 15.474.6 9.3<0.001male ( % ) 39 ( 65)24 ( 69)34 ( 62)0.81bmi ( kg / m)24.6 4.326.5 3.829.6 12.80.11apache ii18.5 9.919.6 8.324.2 12.90.016saps ii37.2 13.344.1 14.247.6 16.40.01comorbidities ( % ) hypertension15 ( 26)14 ( 40)24 ( 44)0.12 diabetes7 ( 12)5 ( 14)8 ( 15)0.92 ckd2 ( 3)1 ( 3)6 ( 11)0.17 tsh ( mu / l)0.40 ( 0.180.87)0.42 ( 0.251.0)0.41 ( 0.211.20)0.83 ft4 ( pmol / l)13.2 3.512.8 3.3)13.6 3.60.67admission category ( % ) 0.68 medical29 ( 48.3)17 ( 48.6)23 ( 41.8 ) surgical31 ( 51.7)18 ( 51.4)33 ( 59)primary diagnosis ( % ) 0.21 cardiovascular failure2 ( 3.3)4 ( 11.4)4 ( 7.1 ) cerebrovascular event2 ( 3.3)00 hemorrhagic shock7 ( 11.7)2 ( 5.7)6 ( 10.7 ) multiple trauma4 ( 6.7)2 ( 5.7)1 ( 1.8 ) elective major surgery1 ( 1.7)1 ( 2.9)5 ( 8.9 ) respiratory failure23 ( 38.3)11 ( 31.4)14 ( 25 ) septic shock20 ( 33.3)13 ( 37.1)26 ( 46.6)aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormonetable 2renal characteristics and outcome grouped according to aki status group 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)ppremorbid scr ( mol / l ) actual92 30 ( n = 37)91 25 ( n = 28)83 23 ( n = 37)0.31 estimated88 1286 1283 110.05study inclusion scr ( mol / l)72 ( 6194)87 ( 7687)139 ( 97211)<0.001 scyc ( mg / l)0.80 ( 0.651.04)0.91 ( 0.811.17)1.14 ( 0.881.94)<0.001 ucyc ( mg / l)0.16 ( 0.0251.47)0.18 ( 0.070.68)0.59 ( 0.103.47)0.46ucyccorr ( g / molcr)0.03 ( 0.010.30)0.04 ( 0.010.13)0.13 ( 0.010.67)0.47maximum rifle class ( % ) < 0.001 risk27 ( 77)22 ( 39.3 ) injury5 ( 14.3)14 ( 25 ) failure3 ( 8.6)20 ( 35.8 ) cvvh ( % ) 1 ( 1.7)4 ( 11.4)10 ( 17.6)0.01icu treatment ( days)5 ( 38)8 ( 518)6 ( 39)0.002icu mortality ( % ) 04 ( 11.4)11 ( 19.6)0.002hospital mortality ( % ) 4 ( 6.7)6 ( 17.1)18 ( 32.1)0.002aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unitbaseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admissionbaseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation baseline characteristics grouped according to aki status aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormone renal characteristics and outcome grouped according to aki status aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unit baseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admission baseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation cystatin c was measured in 582 blood samples ( 3.8 3.2 samples / patient ) and 569 urine samples ( 3.8 3.1 samples / patient ) . cystatin c level was below the detection limit in 171 ( 30% ) urine samples . urinary cyc levels also increased with increasing rifle class ; however , the difference between rifle risk and rifle injury was not statistically significant.table 3cystatin c per rifle classnoneriskinjuryfailurepserum samples number3991093531 cyc ( mg / l)1.15 ( 1.051.25)1.34 ( 1.221.45)1.75 ( 1.591.92)2.31 ( 2.092.52)<0.001urine samples number3911044034 cyc ( mg / l)1.02 ( 0.541.51)2.36 ( 1.523.19)2.77 ( 1.434.12)5.87 ( 4.247.51)<0.001 cyccorr ( g / mol cr)0.43 ( 0.280.59)0.44 ( 0.210.68)0.34 ( 0.040.72)0.79 ( 0.321.24)0.46values are mean and 95% confidence interval . the p value represents the difference among groups by linear mixed modelsscyc serum cystatin c , ucyc urine cystatin c , ucyccorr ucyc normalized for urinary creatinine cystatin c per rifle class values are mean and 95% confidence interval . the p value represents the difference among groups by linear mixed models scyc serum cystatin c , ucyc urine cystatin c , ucyccorr ucyc normalized for urinary creatinine figure 1 compares the longitudinal trend of scr , scyc , and ucyc starting from 2 days prior to aki ( group 1 ) with the non - aki trend ( group 0 ) . on day l , p = 0.01 ) , but not on day 1 ( 0.98 versus 0.86 mg / l , p = 0.08 ) . serum cyc levels , however , did not rise earlier than scr . on the 2 days prior to aki , ucyc levels did not differ significantly between group 1 and group 0 ( median 0.13 [ 0.0250.88 ] mg / l versus median 0.16 [ 0.0251.50 ] mg / l , p = 0.89 , and median 0.14 [ 0.0250.37 ] mg / l versus median 0.17 [ 0.0250.84 ] mg / l , p = 0.57 , day 2 and day 1 , respectively ) . the diagnostic performance for scyc was fair on day 2 [ area under the curve ( auc ) 0.72 ] and poor on day 1 ( auc 0.62 ) , while ucyc had no diagnostic value on day 2 and day 1 ( fig . 2 ) . the test characteristics of scyc at various cutoff levels are shown in esm table 1.fig . 1time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . values are mean and standard error of the means . the number of patients investigated is shown in italics at each time point . * p < 0.05 compared with the non - aki groupfig . 2receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of acute kidney injury ( aki ) . upper panels 2 days prior to aki ( n = 71 , disease prevalence 0.20 ) ; lower panels 1 day prior to aki . ucyccorr urine cystatin c normalized for urinary creatinine time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . values are mean and standard error of the means . the number of patients investigated is shown in italics at each time point . * p < 0.05 compared with the non - aki group receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of acute kidney injury ( aki ) . upper panels 2 days prior to aki ( n = 71 , disease prevalence 0.20 ) ; lower panels 1 day prior to aki . ucyccorr urine cystatin c normalized for urinary creatinine fourteen ( 15% ) out of 91 aki patients received continuous venovenous hemofiltration ( cvvh ) : 4 patients from group 1 , and 10 patients from group 2 . median duration between day 0 and initiation of cvvh was 1.5 [ 04 ] days . in comparison with the non - cvvh patients , the aki patients requiring cvvh had significantly higher apache ii scores on admission ( 28 [ 2133 ] versus 19 [ 1427 ] , p = 0.01 ) and produced less urine ( 740 [ 4001,088 ] ml / day versus 1,660 [ 1,0282,918 ] ml / day , p = 0.001 ) . systemic and urinary cyc determined on day 0 were poor predictors for the need for rrt ( auc 0.66 , fig . 3).fig . 3receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . area under the curve ( auc ) is depicted in each panel receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . in this prospective multicenter cohort study in a heterogeneous icu population , scyc and ucyc increased with increasing rifle class . approximately one - third of the patients without aki at icu admission developed aki during the icu treatment , and in these patients scyc was significantly higher 2 days prior to aki compared with patients who did not develop aki . the diagnostic performance of scyc was at best fair for day 2 and poor on day 1 . levels of ucyc were not different in patients prior to aki in comparison with patients who did not develop aki and had no diagnostic accuracy for aki . previous studies investigating whether cyc increases before development of aki are limited and their results inconsistent ; however , comparison among studies is hampered by case mix and heterogeneity in study designs [ 7 , 12 , 2024 ] . notably , no two studies used identical definitions for aki , and this definition is critical in biomarker research . [ 20 , 25 , 26 ] recently reported on the predictive performance of cyc in two subanalyses of the earlyarf study . notably , while 3,966 patients were screened for the earlyarf study , only 444 patients ( 11% ) were included in the subanalyses , suggesting selection bias . based on the scr criteria used by the acute kidney injury network definition , 125 ( 28% ) patients had aki on entry , 73 ( 16% ) patients developed aki over the subsequent 7 days ( aki 7d ) , and 246 ( 55% ) patients did not [ 20 , 26 ] . scyc on entry was predictive for sustained aki ( auc 0.80 ) , defined as an increase in scr of at least 50% from baseline for 24 h or longer , but not for aki 7d ( auc 0.65 ) . a subanalysis of sepsis patients showed that only ucyc was predictive of aki within 48 h ( auc 0.71 ) and not scys , while ucyc was not predictive of aki in patients without sepsis ( auc = 0.45 ) . studied 202 patients in a mixed icu , of whom 49 developed aki according to the urine output and/or scr rifle failure criteria . in that study scyc showed excellent predictive value for aki ; however , scyc did not rise earlier than scr . analyzed 85 icu patients at high risk of developing aki and used the creatinine risk criteria of the rifle classification to define aki ( n = 44 ) . in contrast to our findings , scyc was shown to detect aki 12 days earlier than scr ( auc 0.82 and 0.97 on day 2 and day 1 , respectively ) . in the study by haase - fielitz in 100 patients following cardiopulmonary bypass surgery ( cpb ) , 23 patients developed aki , defined as increase in scr of 50% from baseline within the first five postoperative days . in that study , scyc on icu arrival and at 24 h after cpb were good predictors of aki ( auc 0.83 ) . notably , the authors suggested that presence of chronic kidney disease ( ckd ) could affect the diagnostic performance of cyc , because exclusion of the ckd patients reduced the diagnostic performance of scyc . this reduction in diagnostic performance , however , may have been caused by the smaller sample size ( n = 15 ) , rather than by the exclusion of ckd patients . we did not evaluate the effect of ckd , because only one patient had ckd on day 2 and day 1 . in another , small ( n = 30 ) cpb study , 15 patients developed aki defined as 50% or greater increase in scr from baseline within 72 h , and the optimal diagnostic performance for scyc ( auc 0.83 ) was 10 h post cpb . in the cpb study ( n = 72 ) of koyner , 34 patients developed aki defined as scr rise of 25% from baseline within the first 3 days or need of rrt . serum cyc at icu arrival and at 6 h were poor predictors for aki ( auc 0.62 and 0.63 , respectively ) , while urine cyc at icu arrival and at 6 h were fair predictors ( auc 0.69 and 0.72 , respectively ) . in the study by liangos ( n = 103 ) , ucyc measured 2 h post cpb did not predict aki ( n = 13 ) , defined as scr increase of 50% from post cpb to peak value within the first 3 days ( auc 0.50 ) . in a very small study in septic patients ( n = 29 ) no correlation was found between scyc at icu admission and occurrence of aki ( n = 10 ) defined as scr > 267 mol / l or diuresis < 30 ml / h . four small studies in icu patients reported that scyc was superior to scr to identify glomerular filtration rate ( gfr ) < 80 ml / min per 1.73 m [ 2932 ] . the gfr studies , however , can be criticized because they used derivatives for gfr and not the real gold standard based on clearance of exogenous substances . in the present study , 56 ( 37% ) patients had aki at admission , which is similar to a previous aki biomarker study in a heterogeneous icu population . it make little sense to predict aki in patients with aki ; however , in these patients , cyc may help to identify those requiring rrt . unfortunately , in our study both scyc and ucyc determined on day 0 were poor predictors for the need for rrt , although it must be mentioned that rrt was started in 14 patients only . our findings agree with the findings of perianayagam et al . , but disagree with three earlier reports suggesting excellent diagnostic performance of scyc [ 11 , 20 , 23 ] . several factors may explain the conflicting results , including case mix and the reason for starting rrt . in our study , oligo / anuria often triggered rrt , while low gfr probably triggered rrt in the study by herget - rosenthal et al . only five patients fulfilled the composite endpoint ( need for rrt and in - hospital mortality ) . another potential role for biomarkers is to allow differentiation between extrinsic and intrinsic causes of aki ; however , our study was not designed to investigate this issue . nearly 40% of our patients never developed aki , yet some of these patients had increased levels of cyc according to the reference intervals reported in literature . first , the published reference intervals were determined in healthy volunteers with no history of renal disease , and may not apply to our critically ill population . although scyc is less influenced by age , sex , and muscle mass compared with serum creatinine level , it still can be affected by these patient variables . moreover , scyc levels can be affected by levels of glucocorticosteroids , thyroid hormones , and insulin as well as markers of inflammation such as white blood cell count and c reactive protein level . all these factors play an important role in critically ill patients and could affect the reference interval . second , we did not use any protease inhibitor in the storage of the urine to clear all disturbing elements in the urine . however , herget - rosenthal reported good agreement between ucyc measurement with and without stabilization buffer . finally , the consensus rifle definition for aki is based on scr and/or urine output . these are functional parameters and may not be appropriate for detection of injury to the kidney . we can not rule out that increased cyc levels indeed reflect damage to the kidneys which is not recognized by the rifle criteria . notably , in the present study , prevalence of aki was high , and therefore a low scyc of < 0.80 mg / l resulted in high [ negative predictive value ( npv ) 7595% ] despite its low specificity . therefore , in a critically ill population with high prevalence of aki , scyc may be helpful to rule out aki . our study is the first multicenter study investigating whether cyc can predict development of aki in the general critical care setting in which the population is heterogeneous and aki etiology and timing are often unclear . , a small percentage of our patients ( 12% ) were enrolled the day following admission , and specimens were collected on alternate days after the first 2 days . as a result , our sample size was relatively small , particularly on day 2 . missing preadmission scr value is a recognized problem in aki research , which may lead to misclassification of aki incidence [ 41 , 42 ] . on the other hand , use of surrogate measures for baseline renal function avoids selection bias . third , we did not evaluate whether cyc concentrations were affected by other factors ( e.g. , inflammatory state , insulin or hydrocortisone therapy ) . finally , the definition of aki is critical in biomarker research , and the rifle criteria may not be an adequate gold standard for aki . in this relatively small multicenter study in a heterogeneous icu population and using the rifle classification system to define aki , scyc and ucyc were poor biomarkers for aki . moreover , on the first day of aki these biomarkers did not predict the need of rrt . however , scyc < 0.80 mg / l had high npv in our population with high incidence of aki . below is the link to the electronic supplementary material . supplementary material 1 ( doc 78 kb )

purposeto evaluate whether cystatin c in serum ( scyc ) and urine ( ucyc ) can predict early acute kidney injury ( aki ) in a mixed heterogeneous intensive care unit ( icu ) , and also whether these biomarkers can predict the need for renal replacement therapy ( rrt).methodsmulticenter prospective observational cohort study in patients 18 years old and with expected icu stay 72 h. the rifle class for aki was calculated daily , while scyc and ucyc were determined on days 0 , 1 , and alternate days until icu discharge . test characteristics were calculated to assess the diagnostic performance of cyc.resultsone hundred fifty - one patients were studied , and three groups were defined : group 0 ( n = 60 ) , non - aki ; group 1 ( n = 35 ) , aki after admission ; and group 2 ( n = 56 ) , aki at admission . we compared the two days prior to developing aki from group 1 with the first two study days from group 0 . on day 2 , median scyc was significantly higher ( 0.93 versus 0.80 mg / l , p = 0.01 ) , but not on day 1 ( 0.98 versus 0.86 mg / l , p = 0.08 ) . the diagnostic performance for scyc was fair on day 2 [ area under the curve ( auc ) 0.72 ] and poor on day 1 ( auc 0.62 ) . urinary cyc had no diagnostic value on either of the two days prior to aki ( auc < 0.50 ) . rrt was started in 14 patients with aki ; scyc and ucyc determined on day 0 were poor predictors for the need for rrt ( auc 0.66).conclusionsin this study , scyc and ucyc were poor biomarkers for prediction of aki and the need for rrt.electronic supplementary materialthe online version of this article ( doi:10.1007/s00134 - 010 - 2087-y ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Concise methods Patients Definition of acute kidney injury Sampling and measurement of cystatinC Statistical analysis Results Patients Serum and urinary cystatinC levels Discussion Conclusions Electronic supplementary material

the online version of this article ( doi:10.1007/s00134 - 010 - 2087-y ) contains supplementary material , which is available to authorized users . we hypothesized that these markers predict aki 12 days earlier than the rifle criteria in patients developing aki after entry , and that these biomarkers predict the need for renal replacement therapy ( rrt ) on the first day of aki . we defined three groups : group 0 ( n = 60 ) , never developed aki , serving as controls ; group 1 ( n = 35 ) , developed aki after admission ; and group 2 ( n = 56 ) , presented with aki at admission . in group 2 the maximum rifle class was achieved by the creatinine criteria in 24 patients ( 43% ) , by the urine criteria in 13 patients ( 23% ) , and in 17 ( 30% ) patients the creatinine and urine aki classification were identical.table 1baseline characteristics grouped according to aki statusgroup 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)page ( years)59.2 16.168.4 15.474.6 9.3<0.001male ( % ) 39 ( 65)24 ( 69)34 ( 62)0.81bmi ( kg / m)24.6 4.326.5 3.829.6 12.80.11apache ii18.5 9.919.6 8.324.2 12.90.016saps ii37.2 13.344.1 14.247.6 16.40.01comorbidities ( % ) hypertension15 ( 26)14 ( 40)24 ( 44)0.12 diabetes7 ( 12)5 ( 14)8 ( 15)0.92 ckd2 ( 3)1 ( 3)6 ( 11)0.17 tsh ( mu / l)0.40 ( 0.180.87)0.42 ( 0.251.0)0.41 ( 0.211.20)0.83 ft4 ( pmol / l)13.2 3.512.8 3.3)13.6 3.60.67admission category ( % ) 0.68 medical29 ( 48.3)17 ( 48.6)23 ( 41.8 ) surgical31 ( 51.7)18 ( 51.4)33 ( 59)primary diagnosis ( % ) 0.21 cardiovascular failure2 ( 3.3)4 ( 11.4)4 ( 7.1 ) cerebrovascular event2 ( 3.3)00 hemorrhagic shock7 ( 11.7)2 ( 5.7)6 ( 10.7 ) multiple trauma4 ( 6.7)2 ( 5.7)1 ( 1.8 ) elective major surgery1 ( 1.7)1 ( 2.9)5 ( 8.9 ) respiratory failure23 ( 38.3)11 ( 31.4)14 ( 25 ) septic shock20 ( 33.3)13 ( 37.1)26 ( 46.6)aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormonetable 2renal characteristics and outcome grouped according to aki status group 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)ppremorbid scr ( mol / l ) actual92 30 ( n = 37)91 25 ( n = 28)83 23 ( n = 37)0.31 estimated88 1286 1283 110.05study inclusion scr ( mol / l)72 ( 6194)87 ( 7687)139 ( 97211)<0.001 scyc ( mg / l)0.80 ( 0.651.04)0.91 ( 0.811.17)1.14 ( 0.881.94)<0.001 ucyc ( mg / l)0.16 ( 0.0251.47)0.18 ( 0.070.68)0.59 ( 0.103.47)0.46ucyccorr ( g / molcr)0.03 ( 0.010.30)0.04 ( 0.010.13)0.13 ( 0.010.67)0.47maximum rifle class ( % ) < 0.001 risk27 ( 77)22 ( 39.3 ) injury5 ( 14.3)14 ( 25 ) failure3 ( 8.6)20 ( 35.8 ) cvvh ( % ) 1 ( 1.7)4 ( 11.4)10 ( 17.6)0.01icu treatment ( days)5 ( 38)8 ( 518)6 ( 39)0.002icu mortality ( % ) 04 ( 11.4)11 ( 19.6)0.002hospital mortality ( % ) 4 ( 6.7)6 ( 17.1)18 ( 32.1)0.002aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unitbaseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admissionbaseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation baseline characteristics grouped according to aki status aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormone renal characteristics and outcome grouped according to aki status aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unit baseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admission baseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation cystatin c was measured in 582 blood samples ( 3.8 3.2 samples / patient ) and 569 urine samples ( 3.8 3.1 samples / patient ) . on day 2 , median scyc was significantly higher ( 0.93 versus 0.80 mg / l , p = 0.01 ) , but not on day 1 ( 0.98 versus 0.86 mg / l , p = 0.08 ) . on the 2 days prior to aki , ucyc levels did not differ significantly between group 1 and group 0 ( median 0.13 [ 0.0250.88 ] mg / l versus median 0.16 [ 0.0251.50 ] mg / l , p = 0.89 , and median 0.14 [ 0.0250.37 ] mg / l versus median 0.17 [ 0.0250.84 ] mg / l , p = 0.57 , day 2 and day 1 , respectively ) . the diagnostic performance for scyc was fair on day 2 [ area under the curve ( auc ) 0.72 ] and poor on day 1 ( auc 0.62 ) , while ucyc had no diagnostic value on day 2 and day 1 ( fig . 1time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . ucyccorr urine cystatin c normalized for urinary creatinine time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . systemic and urinary cyc determined on day 0 were poor predictors for the need for rrt ( auc 0.66 , fig . area under the curve ( auc ) is depicted in each panel receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . we defined three groups : group 0 ( n = 60 ) , never developed aki , serving as controls ; group 1 ( n = 35 ) , developed aki after admission ; and group 2 ( n = 56 ) , presented with aki at admission . in group 2 the maximum rifle class was achieved by the creatinine criteria in 24 patients ( 43% ) , by the urine criteria in 13 patients ( 23% ) , and in 17 ( 30% ) patients the creatinine and urine aki classification were identical.table 1baseline characteristics grouped according to aki statusgroup 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)page ( years)59.2 16.168.4 15.474.6 9.3<0.001male ( % ) 39 ( 65)24 ( 69)34 ( 62)0.81bmi ( kg / m)24.6 4.326.5 3.829.6 12.80.11apache ii18.5 9.919.6 8.324.2 12.90.016saps ii37.2 13.344.1 14.247.6 16.40.01comorbidities ( % ) hypertension15 ( 26)14 ( 40)24 ( 44)0.12 diabetes7 ( 12)5 ( 14)8 ( 15)0.92 ckd2 ( 3)1 ( 3)6 ( 11)0.17 tsh ( mu / l)0.40 ( 0.180.87)0.42 ( 0.251.0)0.41 ( 0.211.20)0.83 ft4 ( pmol / l)13.2 3.512.8 3.3)13.6 3.60.67admission category ( % ) 0.68 medical29 ( 48.3)17 ( 48.6)23 ( 41.8 ) surgical31 ( 51.7)18 ( 51.4)33 ( 59)primary diagnosis ( % ) 0.21 cardiovascular failure2 ( 3.3)4 ( 11.4)4 ( 7.1 ) cerebrovascular event2 ( 3.3)00 hemorrhagic shock7 ( 11.7)2 ( 5.7)6 ( 10.7 ) multiple trauma4 ( 6.7)2 ( 5.7)1 ( 1.8 ) elective major surgery1 ( 1.7)1 ( 2.9)5 ( 8.9 ) respiratory failure23 ( 38.3)11 ( 31.4)14 ( 25 ) septic shock20 ( 33.3)13 ( 37.1)26 ( 46.6)aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormonetable 2renal characteristics and outcome grouped according to aki status group 0 ( n = 60)group 1 ( n = 35)group 2 ( n = 56)ppremorbid scr ( mol / l ) actual92 30 ( n = 37)91 25 ( n = 28)83 23 ( n = 37)0.31 estimated88 1286 1283 110.05study inclusion scr ( mol / l)72 ( 6194)87 ( 7687)139 ( 97211)<0.001 scyc ( mg / l)0.80 ( 0.651.04)0.91 ( 0.811.17)1.14 ( 0.881.94)<0.001 ucyc ( mg / l)0.16 ( 0.0251.47)0.18 ( 0.070.68)0.59 ( 0.103.47)0.46ucyccorr ( g / molcr)0.03 ( 0.010.30)0.04 ( 0.010.13)0.13 ( 0.010.67)0.47maximum rifle class ( % ) < 0.001 risk27 ( 77)22 ( 39.3 ) injury5 ( 14.3)14 ( 25 ) failure3 ( 8.6)20 ( 35.8 ) cvvh ( % ) 1 ( 1.7)4 ( 11.4)10 ( 17.6)0.01icu treatment ( days)5 ( 38)8 ( 518)6 ( 39)0.002icu mortality ( % ) 04 ( 11.4)11 ( 19.6)0.002hospital mortality ( % ) 4 ( 6.7)6 ( 17.1)18 ( 32.1)0.002aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unitbaseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admissionbaseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation baseline characteristics grouped according to aki status aki acute kidney injury , group 0 no aki , group 1 developed aki after admission , group 2 aki at admission . bmi body mass index , apache acute physiology and chronic health evaluation , saps simplified acute physiology score , ckd chronic kidney disease , tsh thyroid - stimulating hormone renal characteristics and outcome grouped according to aki status aki acute kidney injury , group 0 never aki , group 1 developed aki after admission , group 2 aki at admission , scr serum creatinine , scyc serum cystatin c , ucyc urine cystatin c , ucyccorr urine cystatin c normalized for urinary creatinine , cvvh continuous venovenous hemofiltration , icu intensive care unit baseline serum creatinine based on premorbid serum creatinine within 1 year prior to icu admission baseline serum creatinine based on solving the modification of diet in renal disease ( mdrd ) equation cystatin c was measured in 582 blood samples ( 3.8 3.2 samples / patient ) and 569 urine samples ( 3.8 3.1 samples / patient ) . on day l , p = 0.01 ) , but not on day 1 ( 0.98 versus 0.86 mg / l , p = 0.08 ) . on the 2 days prior to aki , ucyc levels did not differ significantly between group 1 and group 0 ( median 0.13 [ 0.0250.88 ] mg / l versus median 0.16 [ 0.0251.50 ] mg / l , p = 0.89 , and median 0.14 [ 0.0250.37 ] mg / l versus median 0.17 [ 0.0250.84 ] mg / l , p = 0.57 , day 2 and day 1 , respectively ) . the diagnostic performance for scyc was fair on day 2 [ area under the curve ( auc ) 0.72 ] and poor on day 1 ( auc 0.62 ) , while ucyc had no diagnostic value on day 2 and day 1 ( fig . 1time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . ucyccorr urine cystatin c normalized for urinary creatinine time course of serum creatinine , serum cystatin c , and urine cystatin c. time courses are from 2 days prior to acute kidney injury ( aki ) for patients developing aki after entry ( open circles ) , and from entry for the non - aki group ( closed circles ) . systemic and urinary cyc determined on day 0 were poor predictors for the need for rrt ( auc 0.66 , fig . area under the curve ( auc ) is depicted in each panel receiver - operating curves demonstrating the performance of cystatin c ( cyc ) for prediction of the need of renal replacement therapy . unfortunately , in our study both scyc and ucyc determined on day 0 were poor predictors for the need for rrt , although it must be mentioned that rrt was started in 14 patients only .

subjects are mexican american ( both parents and 3 grandparents mexican or of mexican descent ) who are either probands with gdm diagnosed within the previous 5 years and diagnosed in their family members or non - gdm probands with normal glucose levels in pregnancy in the past 5 years . the institutional review boards of participating institutions approved all protocols for betagene , and all participants provided written informed consent before participation . phenotyping was performed on two separate visits to the university of southern california general clinical research center . the first visit consisted of a physical examination , dna collection , and an oral glucose tolerance test ( ogtt ; 75 g ) as previously described ( 3335 ) . participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . plasma glucose is measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) . insulin is measured by two - site immunoenzymometric assay that has < 0.1% crossreactivity with proinsulin and intermediate split products . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . the primary goal of the metsim study was to investigate the effect of genetic variation on risk for type 2 diabetes and cardiovascular disease in a random sample of finnish men ( aged 5070 years ) living in the town of kuopio , eastern finland ( population 95,000 ) . there were 5,327 men from this ongoing population - based study included in this report . the ethics committee of the university of kuopio and in accordance with the helsinki declaration approved the metsim study . phenotyping for metsim was performed in a single visit to the clinical research unit of the university of kuopio . in addition to fasting blood samples for dna , glucose , and insulin , all subjects underwent an ogtt with samples obtained at 30 and 120 min postload . in betagene , gkc 30ga polymorphism ( rs1799884 ) and g6pc2 rs560887 were genotyped using the taqman system ( applied biosystems ) ( 38,39 ) . genotyping discrepancy rate based on blinded duplicates was < 0.1% , and overall genotyping success rates were > 97% for rs1799884 and > 99% for rs560887 . in metsim , gck rs4607517 and g6pc2 rs560887 were genotyped by the homogeneous massextend reaction using the massarray system ( sequenom ) ( 9 ) . in the hapmap ceu data ( 40 ) , which provide a good representation of linkage disequilibrium in finns ( 41 ) , rs4607517 is a perfect proxy for rs1799884 ( d ' = 1.0 , r = 1.0 ) . there were no genotype discrepancies based on > 220 blinded duplicates , and overall genotyping success rates were > 96% for rs4607517 and > 94% for rs560887 . genotype data were tested for deviation from hardy - weinberg equilibrium and for non - mendelian inheritance using pedstats version 0.6.4 ( 42 ) . allele frequencies for betagene samples were estimated using all available data taking into account relatedness using solar version 2.1.4 ( 43,44 ) , whereas metsim frequencies were estimated by gene counting . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . ivgtt glucose and insulin data were analyzed using the minimal model ( minmod millennium version 5.18 ) to derive measures of glucose effectiveness and si ( 45 ) . disposition index , a measure of -cell compensation for insulin resistance , was computed as the product of si and air . we also computed an ogtt - derived measure of -cell compensation as the product of si and 30 insulin ( di30 ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . we recognized that these variants could also underlie variation in other type 2 diabetes related quantitative traits . we tested these snps for association with 2-h glucose , 2-h insulin , si , sg , bmi , and percent of body fat as a series of secondary analyses . we first tested each snp for association with type 2 diabetes related quantitative traits using likelihood ratio testing under a variance components framework as implemented in solar ( 43,44 ) . because families were ascertained through probands based on previous gdm status , we corrected for ascertainment bias by conditioning each model on the proband 's phenotype . each snp was tested for association with quantitative traits assuming a dominant genetic model because of the relatively low minor allele frequencies observed for both snps . all models were adjusted for age , sex , and , where appropriate , percent body fat . p values were corrected for multiple testing to account for the number of traits tested ( four traits in the primary analysis , six traits in the secondary analysis ) . we used pact to control for the number of tests performed and to account for the correlation among the quantitative traits tested for association ( 46 ) . pact compares the observed test statistic with their asymptotic distribution using numeric integration and directly accounts for correlation among both dependent and independent variables . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . we compared a model with age , sex , rs1799884 , and rs560887 as main effects with a second model that included the multiplicative interaction between the two snps . test and performed a bonferroni correction to account for the number of traits tested because pact is not valid for multi - d.f . tests ( 46 ) . however , because many of the quantitative traits examined are correlated and bonferroni assumes independence , the corrected p values we report should be overly conservative . given that gck and g6pc2 work in opposite directions on the glucose cycle , we hypothesized that additivity may best characterize their interaction . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . standard regression methods were used to test for association between genetic variants and type 2 diabetes related traits as metsim participants are unrelated individuals . given that only specific a priori hypotheses were tested in the metsim data , we did not apply a correction for multiple testing . all data for betagene are reported as age- and sex - adjusted means and sd , whereas metsim data are reported as age - adjusted means and sd unless noted otherwise . subjects are mexican american ( both parents and 3 grandparents mexican or of mexican descent ) who are either probands with gdm diagnosed within the previous 5 years and diagnosed in their family members or non - gdm probands with normal glucose levels in pregnancy in the past 5 years . the institutional review boards of participating institutions approved all protocols for betagene , and all participants provided written informed consent before participation . phenotyping was performed on two separate visits to the university of southern california general clinical research center . the first visit consisted of a physical examination , dna collection , and an oral glucose tolerance test ( ogtt ; 75 g ) as previously described ( 3335 ) . participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . subjects are mexican american ( both parents and 3 grandparents mexican or of mexican descent ) who are either probands with gdm diagnosed within the previous 5 years and diagnosed in their family members or non - gdm probands with normal glucose levels in pregnancy in the past 5 years . the institutional review boards of participating institutions approved all protocols for betagene , and all participants provided written informed consent before participation . phenotyping was performed on two separate visits to the university of southern california general clinical research center . the first visit consisted of a physical examination , dna collection , and an oral glucose tolerance test ( ogtt ; 75 g ) as previously described ( 3335 ) . participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . plasma glucose is measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) . insulin is measured by two - site immunoenzymometric assay that has < 0.1% crossreactivity with proinsulin and intermediate split products . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . the primary goal of the metsim study was to investigate the effect of genetic variation on risk for type 2 diabetes and cardiovascular disease in a random sample of finnish men ( aged 5070 years ) living in the town of kuopio , eastern finland ( population 95,000 ) . there were 5,327 men from this ongoing population - based study included in this report . the ethics committee of the university of kuopio and in accordance with the helsinki declaration approved the metsim study . phenotyping for metsim was performed in a single visit to the clinical research unit of the university of kuopio . in addition to fasting blood samples for dna , glucose , and insulin , all subjects underwent an ogtt with samples obtained at 30 and 120 min postload . in betagene , gkc 30ga polymorphism ( rs1799884 ) and g6pc2 rs560887 were genotyped using the taqman system ( applied biosystems ) ( 38,39 ) . genotyping discrepancy rate based on blinded duplicates was < 0.1% , and overall genotyping success rates were > 97% for rs1799884 and > 99% for rs560887 . in metsim , gck rs4607517 and g6pc2 rs560887 were genotyped by the homogeneous massextend reaction using the massarray system ( sequenom ) ( 9 ) . in the hapmap ceu data ( 40 ) , which provide a good representation of linkage disequilibrium in finns ( 41 ) , rs4607517 is a perfect proxy for rs1799884 ( d ' = 1.0 , r = 1.0 ) . there were no genotype discrepancies based on > 220 blinded duplicates , and overall genotyping success rates were > 96% for rs4607517 and > 94% for rs560887 . genotype data were tested for deviation from hardy - weinberg equilibrium and for non - mendelian inheritance using pedstats version 0.6.4 ( 42 ) . allele frequencies for betagene samples were estimated using all available data taking into account relatedness using solar version 2.1.4 ( 43,44 ) , whereas metsim frequencies were estimated by gene counting . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . ivgtt glucose and insulin data were analyzed using the minimal model ( minmod millennium version 5.18 ) to derive measures of glucose effectiveness and si ( 45 ) . disposition index , a measure of -cell compensation for insulin resistance , was computed as the product of si and air . we also computed an ogtt - derived measure of -cell compensation as the product of si and 30 insulin ( di30 ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . we recognized that these variants could also underlie variation in other type 2 diabetes related quantitative traits . we tested these snps for association with 2-h glucose , 2-h insulin , si , sg , bmi , and percent of body fat as a series of secondary analyses . we first tested each snp for association with type 2 diabetes related quantitative traits using likelihood ratio testing under a variance components framework as implemented in solar ( 43,44 ) . because families were ascertained through probands based on previous gdm status , we corrected for ascertainment bias by conditioning each model on the proband 's phenotype . each snp was tested for association with quantitative traits assuming a dominant genetic model because of the relatively low minor allele frequencies observed for both snps . all models were adjusted for age , sex , and , where appropriate , percent body fat . p values were corrected for multiple testing to account for the number of traits tested ( four traits in the primary analysis , six traits in the secondary analysis ) . we used pact to control for the number of tests performed and to account for the correlation among the quantitative traits tested for association ( 46 ) . pact compares the observed test statistic with their asymptotic distribution using numeric integration and directly accounts for correlation among both dependent and independent variables . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . we compared a model with age , sex , rs1799884 , and rs560887 as main effects with a second model that included the multiplicative interaction between the two snps . test and performed a bonferroni correction to account for the number of traits tested because pact is not valid for multi - d.f . however , because many of the quantitative traits examined are correlated and bonferroni assumes independence , the corrected p values we report should be overly conservative . given that gck and g6pc2 work in opposite directions on the glucose cycle , we hypothesized that additivity may best characterize their interaction . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . the primary goal of the metsim study was to investigate the effect of genetic variation on risk for type 2 diabetes and cardiovascular disease in a random sample of finnish men ( aged 5070 years ) living in the town of kuopio , eastern finland ( population 95,000 ) . there were 5,327 men from this ongoing population - based study included in this report . the ethics committee of the university of kuopio and in accordance with the helsinki declaration approved the metsim study . phenotyping for metsim was performed in a single visit to the clinical research unit of the university of kuopio . in addition to fasting blood samples for dna , glucose , and insulin , all subjects underwent an ogtt with samples obtained at 30 and 120 min postload . in betagene , gkc 30ga polymorphism ( rs1799884 ) and g6pc2 rs560887 were genotyped using the taqman system ( applied biosystems ) ( 38,39 ) . genotyping discrepancy rate based on blinded duplicates was < 0.1% , and overall genotyping success rates were > 97% for rs1799884 and > 99% for rs560887 . in metsim , gck rs4607517 and g6pc2 rs560887 were genotyped by the homogeneous massextend reaction using the massarray system ( sequenom ) ( 9 ) . in the hapmap ceu data ( 40 ) , which provide a good representation of linkage disequilibrium in finns ( 41 ) , rs4607517 is a perfect proxy for rs1799884 ( d ' = 1.0 , r = 1.0 ) . there were no genotype discrepancies based on > 220 blinded duplicates , and overall genotyping success rates were > 96% for rs4607517 and > 94% for rs560887 . genotype data were tested for deviation from hardy - weinberg equilibrium and for non - mendelian inheritance using pedstats version 0.6.4 ( 42 ) . allele frequencies for betagene samples were estimated using all available data taking into account relatedness using solar version 2.1.4 ( 43,44 ) , whereas metsim frequencies were estimated by gene counting . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . ivgtt glucose and insulin data were analyzed using the minimal model ( minmod millennium version 5.18 ) to derive measures of glucose effectiveness and si ( 45 ) . disposition index , a measure of -cell compensation for insulin resistance , was computed as the product of si and air . we also computed an ogtt - derived measure of -cell compensation as the product of si and 30 insulin ( di30 ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . we recognized that these variants could also underlie variation in other type 2 diabetes related quantitative traits . we tested these snps for association with 2-h glucose , 2-h insulin , si , sg , bmi , and percent of body fat as a series of secondary analyses . we first tested each snp for association with type 2 diabetes related quantitative traits using likelihood ratio testing under a variance components framework as implemented in solar ( 43,44 ) . because families were ascertained through probands based on previous gdm status , we corrected for ascertainment bias by conditioning each model on the proband 's phenotype . each snp was tested for association with quantitative traits assuming a dominant genetic model because of the relatively low minor allele frequencies observed for both snps . all models were adjusted for age , sex , and , where appropriate , percent body fat . p values were corrected for multiple testing to account for the number of traits tested ( four traits in the primary analysis , six traits in the secondary analysis ) . we used pact to control for the number of tests performed and to account for the correlation among the quantitative traits tested for association ( 46 ) . pact compares the observed test statistic with their asymptotic distribution using numeric integration and directly accounts for correlation among both dependent and independent variables . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . we compared a model with age , sex , rs1799884 , and rs560887 as main effects with a second model that included the multiplicative interaction between the two snps . test and performed a bonferroni correction to account for the number of traits tested because pact is not valid for multi - d.f . however , because many of the quantitative traits examined are correlated and bonferroni assumes independence , the corrected p values we report should be overly conservative . given that gck and g6pc2 work in opposite directions on the glucose cycle , we hypothesized that additivity may best characterize their interaction . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . standard regression methods were used to test for association between genetic variants and type 2 diabetes related traits as metsim participants are unrelated individuals . given that only specific a priori hypotheses were tested in the metsim data , we did not apply a correction for multiple testing . all data for betagene are reported as age- and sex - adjusted means and sd , whereas metsim data are reported as age - adjusted means and sd unless noted otherwise . we report results from 861 betagene individuals in 251 families with available phenotype and genotype data ( table 1 ) . probands , siblings , and cousins were similar in median age , bmi , and percent of body fat , although these characteristics tended to be highest in the gdm probands and lowest in cousins . non - gdm probands were slightly younger and less obese compared with gdm probands , reflecting the fact that betagene participant accrual was ongoing and recruitment of non - gdm probands was slightly lagging to allow for matching as previously described ( 3335 ) . parameters of glucose metabolism tended to be best in the non - gdm probands and worst in the gdm probands . ( g > a ) had a minor allele frequency of 18.0% and was marginally associated with fasting glucose ( table 2 , p = 0.052 ) that modestly increased ( p = 0.021 ) after adjustment for body fat . individuals homozygous for the g allele had a lower average fasting glucose compared with those with at least one a allele ( 5.13 0.52 vs. 5.22 0.59 . however , the association between gck rs1799884 and fasting glucose did not remain significant when corrected for multiple testing . in addition , we did not observe any evidence for association between this snp and measures of insulin secretion ( table 2 ) or other type 2 diabetes related quantitative traits ( table 3 ) , although there was a tendency for mean fasting insulin , 30 insulin , and air to be lower in individuals with at least one rs1799884 a allele ( table 2 ) . univariate tests of associations between genetic variants and fasting glucose and measures of insulin secretion data are unadjusted means sd unless otherwise indicated . univariate tests of associations between genetic variants and additional type 2 diabetes related quantitative traits data are unadjusted means sd unless otherwise indicated . * age- and sex - adjusted p value not corrected for multiple testing . g6pc2 rs560887 ( g > a ) had a minor allele frequency of 14.5% and was nominally associated with fasting glucose ( table 2 , p = 0.015 ) and 30 insulin ( p = 0.0038 ) and marginally associated with air ( p = 0.088 ) . these associations became weaker when body fat was included in the model ( p = 0.059 , p = 0.014 , and p = 0.222 , respectively ) . individuals homozygous for the rs560887 g allele had an average fasting glucose higher than those with at least one a allele ( 5.19 0.52 vs. 5.08 0.61 both 30 insulin and air were , on average , higher in individuals homozygous for the g allele ( table 2 ) . only the association with 30 insulin remained significant after correction for multiple testing ( pact = 0.021 ) . although g6pc2 rs560887 was not associated with most type 2 diabetes related quantitative traits , we did observe an association between this snp and percent body fat ( pact = 0.035 ) , where individuals homozygous for the g allele had higher trait values ( table 3 ) . we found no evidence for association between the multiplicative interaction between gck rs1799884 and g6pc2 rs560887 and type 2 diabetes related quantitative traits ( uncorrected p > 0.26 ) , and additional adjustment for body fat did not change the overall results . in contrast , when we tested whether the additive effects of gck rs1799884 and g6cp2 rs560887 were associated with type 2 diabetes related quantitative traits , we observed association with fasting glucose ( bonferroni p = 0.03 ) and 30 insulin ( bonferroni p = 0.027 ) . bouatia - naji et al . reported a linear relationship between number of glucose - lowering alleles for gck , g6pc2 , and gckr and fasting glucose , which suggested additive effects of these alleles on fasting glucose ( 10 ) . we observed a similar linear relationship ( data not shown ) when we stratified gck and g6pc2 genotypes , consistent with the results of our statistical analysis and the observations of bouatia - naji et al . ( 10 ) . however , given the specificity of these two gene products in regulating the glucose - sensing step in pancreatic -cells , we hypothesized the primary effect of variation in gck and g6pc2 to be at the level of insulin secretion , not directly upon glucose . the presence of an a allele in the gck rs1799884 promoter variant is hypothesized to reduce gck gene expression and result in decreased gck protein ( 23 ) . this should result in modest reductions in glycolytic flux and atp production , resulting in modest reductions in insulin secretion for any level of circulating glucose . g6pc2 rs560887 is located in intron 3 , just 26 bp proximal to exon 4 ( 9,10 ) . the presence of an a allele may result in a g6pc2 isoform with differential activity that favors increased conversion of glucose-6-phosphate to glucose , significantly reducing glycolytic flux and atp generation , resulting in larger reductions in insulin secretion for any glucose level . the balance between the effects of gck and g6pc2 should differentially modulate insulin secretion , depending upon genotype combination as detailed in fig . 1 . hypothesized effect of gck rs1799884 and g6pc2 rs560887 genotype combinations on atp production and insulin secretion in pancreatic -cells . upper portion of the figure shows the glucose - cycling step and the effect of specific alleles in gck and g6pc2 on activity of each enzyme : thick arrow depicts increased activity , thin arrow depicts reduced activity . lower portion shows the specific genotype combinations and the hypothesized effect on atp production and subsequent insulin secretion . when we stratified individuals by gck and g6pc2 genotypes as depicted in fig . 2 , top ; p = 0.0016 ) is observed where on average , 30 insulin decreased 5.6 pmol / l among genotype groups . we do not see a clear linear relationship when the same stratification is used to examine the relationship with fasting glucose ( fig . thus , the linearity between gck and g6pc2 genotype combinations and insulin secretion does not directly translate into linear changes in fasting glucose . we observed a similar dichotomy when we examined fasting glucose and 30 insulin stratified by total number of glucose - raising alleles , as previously performed by bouatia - naji et al . ( 10 ) ; fasting glucose is linear with total number of glucose - raising alleles , whereas 30 insulin is not ( data not shown ) . a : age- and sex - adjusted 30 insulin ( means se ) stratified by gck rs1799884 and g6pc2 rs560887 genotype combination . genotype groups are ordered by hypothesized effect on glucose cycling in the pancreatic -cell ( see text for details ) . b : age- and sex - adjusted fasting plasma glucose ( means se ) stratified by genotype . therefore , we plotted fasting glucose against 30 insulin from betagene stratified by gck and g6pc2 genotypes ( fig . 3 , top ) to better characterize the relationship between 30 insulin and fasting glucose . we noted a different pattern of coordinate changes in fasting glucose and 30 insulin associated with variation in gck as compared with variation in g6pc2 . 3 , circles ) had lower fasting glucose and higher 30 insulin than individuals with at least one a allele ( fig . 3 , squares ) . for g6pc2 rs560887 , individuals homozygous for the g allele ( fig . 3 , open symbols ) had higher fasting glucose and higher 30 insulin than individuals with at least one a allele ( fig . variation in gck led to inverse changes in fasting glucose and 30 insulin , whereas variation in g6pc2 led to parallel changes in these two traits . the 30 insulin vs. fasting plasma glucose stratified by gck rs1799884 and g6pc2 rs560887 genotype . a : shows results from the betagene study . model - predicted age- and sex - adjusted 30 insulin ( means sd ) stratified by genotype is plotted against model- predicted age- and sex - adjusted fasting plasma glucose ( means sd ) . solid symbols represent g6pc2 rs560887 g / a or a / a genotypes and open symbols represent g6pc2 rs560887 g / g genotype . circles represent gck rs1799884 g / g genotype and squares represent gck rs1799884 g / a and a / a genotypes . b : shows results from the metsim study . details are identical to that described for the top panel except that circles and squares represent gck rs4607517 genotypes . given the observation of additivity between gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin in betagene , we attempted to replicate these findings in the metsim sample , in which gck rs4607517 is in perfect linkage disequilibrium with rs1799884 . gck rs4607517 was associated with fasting glucose but not associated with 30 insulin ( table 4 ) . g6pc2 rs560887 was associated with fasting glucose and marginally associated with 30 insulin ( table 4 ) . the multiplicative interaction between gck rs4607517 and g6pc2 rs560887 was weakly associated with fasting glucose ( p = 0.038 ) and marginally associated with 30 insulin ( p = 0.049 ) . test of the joint additive effect of the two snps was significant for both fasting glucose ( p = 3.5 10 ) and 30 insulin ( p = 0.028 ) . the evidence for association between the joint additive effect of both snps and fasting glucose was not affected when bmi was included as an additional covariate ( p = 3.3 10 ) ; the association with 30 insulin became stronger when bmi was included as a covariate ( p = 0.0028 ) . when we examined the relationship between fasting glucose and 30 insulin stratified by gck rs4607517 and g6pc2 rs560887 genotype groupings , an identical pattern of relationship to that seen in betagene was observed ( fig . individuals homozygous for the g allele had lower fasting glucose and higher 30 insulin than individuals with at least one a allele . for g6pc2 rs560887 , individuals homozygous for the g allele had higher fasting glucose and higher 30 insulin than individuals with at least one a allele . univariate tests of associations between genetic variants and fasting glucose and 30 insulin in metsim data are unadjusted means sd unless otherwise indicated . we report results from 861 betagene individuals in 251 families with available phenotype and genotype data ( table 1 ) . probands , siblings , and cousins were similar in median age , bmi , and percent of body fat , although these characteristics tended to be highest in the gdm probands and lowest in cousins . non - gdm probands were slightly younger and less obese compared with gdm probands , reflecting the fact that betagene participant accrual was ongoing and recruitment of non - gdm probands was slightly lagging to allow for matching as previously described ( 3335 ) . parameters of glucose metabolism tended to be best in the non - gdm probands and worst in the gdm probands . ( g > a ) had a minor allele frequency of 18.0% and was marginally associated with fasting glucose ( table 2 , p = 0.052 ) that modestly increased ( p = 0.021 ) after adjustment for body fat . individuals homozygous for the g allele had a lower average fasting glucose compared with those with at least one a allele ( 5.13 0.52 vs. 5.22 0.59 . however , the association between gck rs1799884 and fasting glucose did not remain significant when corrected for multiple testing . in addition , we did not observe any evidence for association between this snp and measures of insulin secretion ( table 2 ) or other type 2 diabetes related quantitative traits ( table 3 ) , although there was a tendency for mean fasting insulin , 30 insulin , and air to be lower in individuals with at least one rs1799884 a allele ( table 2 ) . univariate tests of associations between genetic variants and fasting glucose and measures of insulin secretion data are unadjusted means sd unless otherwise indicated . univariate tests of associations between genetic variants and additional type 2 diabetes related quantitative traits data are unadjusted means sd unless otherwise indicated . * age- and sex - adjusted p value not corrected for multiple testing . g6pc2 rs560887 ( g > a ) had a minor allele frequency of 14.5% and was nominally associated with fasting glucose ( table 2 , p = 0.015 ) and 30 insulin ( p = 0.0038 ) and marginally associated with air ( p = 0.088 ) . these associations became weaker when body fat was included in the model ( p = 0.059 , p = 0.014 , and p = 0.222 , respectively ) . individuals homozygous for the rs560887 g allele had an average fasting glucose higher than those with at least one a allele ( 5.19 0.52 vs. 5.08 0.61 both 30 insulin and air were , on average , higher in individuals homozygous for the g allele ( table 2 ) . only the association with 30 insulin remained significant after correction for multiple testing ( pact = 0.021 ) . although g6pc2 rs560887 was not associated with most type 2 diabetes related quantitative traits , we did observe an association between this snp and percent body fat ( pact = 0.035 ) , where individuals homozygous for the g allele had higher trait values ( table 3 ) . we found no evidence for association between the multiplicative interaction between gck rs1799884 and g6pc2 rs560887 and type 2 diabetes related quantitative traits ( uncorrected p > 0.26 ) , and additional adjustment for body fat did not change the overall results . in contrast , when we tested whether the additive effects of gck rs1799884 and g6cp2 rs560887 were associated with type 2 diabetes related quantitative traits , we observed association with fasting glucose ( bonferroni p = 0.03 ) and 30 insulin ( bonferroni p = 0.027 ) . bouatia - naji et al . reported a linear relationship between number of glucose - lowering alleles for gck , g6pc2 , and gckr and fasting glucose , which suggested additive effects of these alleles on fasting glucose ( 10 ) . we observed a similar linear relationship ( data not shown ) when we stratified gck and g6pc2 genotypes , consistent with the results of our statistical analysis and the observations of bouatia - naji et al . ( 10 ) . however , given the specificity of these two gene products in regulating the glucose - sensing step in pancreatic -cells , we hypothesized the primary effect of variation in gck and g6pc2 to be at the level of insulin secretion , not directly upon glucose . the presence of an a allele in the gck rs1799884 promoter variant is hypothesized to reduce gck gene expression and result in decreased gck protein ( 23 ) . this should result in modest reductions in glycolytic flux and atp production , resulting in modest reductions in insulin secretion for any level of circulating glucose . g6pc2 rs560887 is located in intron 3 , just 26 bp proximal to exon 4 ( 9,10 ) . the presence of an a allele may result in a g6pc2 isoform with differential activity that favors increased conversion of glucose-6-phosphate to glucose , significantly reducing glycolytic flux and atp generation , resulting in larger reductions in insulin secretion for any glucose level . the balance between the effects of gck and g6pc2 should differentially modulate insulin secretion , depending upon genotype combination as detailed in fig . 1 . hypothesized effect of gck rs1799884 and g6pc2 rs560887 genotype combinations on atp production and insulin secretion in pancreatic -cells . upper portion of the figure shows the glucose - cycling step and the effect of specific alleles in gck and g6pc2 on activity of each enzyme : thick arrow depicts increased activity , thin arrow depicts reduced activity . lower portion shows the specific genotype combinations and the hypothesized effect on atp production and subsequent insulin secretion . when we stratified individuals by gck and g6pc2 genotypes as depicted in fig . 2 , top ; p = 0.0016 ) is observed where on average , 30 insulin decreased 5.6 pmol / l among genotype groups . we do not see a clear linear relationship when the same stratification is used to examine the relationship with fasting glucose ( fig . thus , the linearity between gck and g6pc2 genotype combinations and insulin secretion does not directly translate into linear changes in fasting glucose . we observed a similar dichotomy when we examined fasting glucose and 30 insulin stratified by total number of glucose - raising alleles , as previously performed by bouatia - naji et al . ( 10 ) ; fasting glucose is linear with total number of glucose - raising alleles , whereas 30 insulin is not ( data not shown ) . a : age- and sex - adjusted 30 insulin ( means se ) stratified by gck rs1799884 and g6pc2 rs560887 genotype combination . genotype groups are ordered by hypothesized effect on glucose cycling in the pancreatic -cell ( see text for details ) . b : age- and sex - adjusted fasting plasma glucose ( means se ) stratified by genotype . therefore , we plotted fasting glucose against 30 insulin from betagene stratified by gck and g6pc2 genotypes ( fig . 3 , top ) to better characterize the relationship between 30 insulin and fasting glucose . we noted a different pattern of coordinate changes in fasting glucose and 30 insulin associated with variation in gck as compared with variation in g6pc2 . 3 , circles ) had lower fasting glucose and higher 30 insulin than individuals with at least one a allele ( fig . 3 , squares ) . for g6pc2 rs560887 , individuals homozygous for the g allele ( fig . 3 , open symbols ) had higher fasting glucose and higher 30 insulin than individuals with at least one a allele ( fig . variation in gck led to inverse changes in fasting glucose and 30 insulin , whereas variation in g6pc2 led to parallel changes in these two traits . the 30 insulin vs. fasting plasma glucose stratified by gck rs1799884 and g6pc2 rs560887 genotype . a : shows results from the betagene study . model - predicted age- and sex - adjusted 30 insulin ( means sd ) stratified by genotype is plotted against model- predicted age- and sex - adjusted fasting plasma glucose ( means sd ) . solid symbols represent g6pc2 rs560887 g / a or a / a genotypes and open symbols represent g6pc2 rs560887 g / g genotype . circles represent gck rs1799884 g / g genotype and squares represent gck rs1799884 g / a and a / a genotypes . b : shows results from the metsim study . details are identical to that described for the top panel except that circles and squares represent gck rs4607517 genotypes . given the observation of additivity between gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin in betagene , we attempted to replicate these findings in the metsim sample , in which gck rs4607517 is in perfect linkage disequilibrium with rs1799884 . gck rs4607517 was associated with fasting glucose but not associated with 30 insulin ( table 4 ) . g6pc2 rs560887 was associated with fasting glucose and marginally associated with 30 insulin ( table 4 ) . the multiplicative interaction between gck rs4607517 and g6pc2 rs560887 was weakly associated with fasting glucose ( p = 0.038 ) and marginally associated with 30 insulin ( p = 0.049 ) . test of the joint additive effect of the two snps was significant for both fasting glucose ( p = 3.5 10 ) and 30 insulin ( p = 0.028 ) . the evidence for association between the joint additive effect of both snps and fasting glucose was not affected when bmi was included as an additional covariate ( p = 3.3 10 ) ; the association with 30 insulin became stronger when bmi was included as a covariate ( p = 0.0028 ) . when we examined the relationship between fasting glucose and 30 insulin stratified by gck rs4607517 and g6pc2 rs560887 genotype groupings , an identical pattern of relationship to that seen in betagene was observed ( fig . individuals homozygous for the g allele had lower fasting glucose and higher 30 insulin than individuals with at least one a allele . for g6pc2 rs560887 , individuals homozygous for the g allele had higher fasting glucose and higher 30 insulin than individuals with at least one a allele . univariate tests of associations between genetic variants and fasting glucose and 30 insulin in metsim data are unadjusted means sd unless otherwise indicated . our results from mexican americans , replicated in a sample of finnish men , show that variation in both gck and g6pc2 have additive effects on both fasting glucose and insulin secretion . our examination of the relationship between 30 insulin and fasting glucose stratified by gck and g6pc2 genotype reveals that the joint effect of genetic variation in these two genes may be through distinct mechanisms . variation in gck leads to inverse changes in fasting glucose and insulin secretion , consistent with a primary effect on insulin secretion . variation in g6pc2 leads to parallel changes in fasting glucose and insulin secretion , suggesting a primary effect on glucose , with secondary changes in insulin secretion . table 5 summarizes our examination of the effects of gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin . although it is possible that our conclusion of these variants having an additive effect could be driven mainly by g6pc2 rs560887 , the summary in table 5 suggests otherwise . it is clear that in both betagene and metsim , the model testing the joint effect of gck and g6pc2 shows greater significance than the effect of each individual gene variant alone , which suggests both genes are contributing to the effect on fasting glucose and 30 insulin . in support of this observation , when we tested for the additional effect of gck in the presence of g6pc2 we observed marginal or significant effects for fasting glucose ( p = 0.058 for betagene ; p = 0.0009 for metsim ) and 30 insulin ( p = 0.148 for betagene ; p = 0.723 for metsim ) . our a priori hypothesis tested whether the multiplicative interaction between rs1799884 and rs560887 was associated with type 2 diabetes related quantitative traits . in betagene we observed no evidence for a multiplicative interaction ; our analysis favored a joint additive effect of these two loci . however , our relatively small size may limit our ability to exclude a multiplicative interaction . a priori power estimates indicated that a sample size of 600 should have provided 80% power to detect a multiplicative interaction effect that accounted for 1.8% of the variability in fasting glucose or 30 insulin , assuming = 0.01 , minor allele frequencies of 15% for both loci , and dominant genetic models for both loci . thus , betagene can not discount the possibility that a very weak multiplicative interaction exists between these two loci . in fact , the metsim sample , which is 6 times larger than betagene , showed modest evidence for a multiplicative interaction ( table 5 ) suggesting that a weak multiplicative interaction between gck and g6pc2 could be contributing to variation in fasting glucose and 30 insulin . this interaction would need to be replicated in a sample of similar or larger size . summary of modeling analysis data are uncorrected p values for the genetic effect being tested . our initial examination of the additive effect of these two genes was motivated by the fact that when we plotted 30 insulin against gck rs1799884 and g6pc2 rs560887 genotype combinations sorted by the hypothesized biologic effect on insulin secretion , we observed a linear relationship with 30 insulin . recently , pirot et al . ( 47 ) showed in mouse and human islets that g6pc2 protein levels and enzymatic activity increased when islets were incubated in high - glucose media , whereas those for gck did not change , suggesting that g6pc2 activity may adapt to changes in glucose to alter insulin secretion . because insulin secretion was not measured , it is unclear whether the changes observed translated into altered rates of insulin secretion . however , previous studies have demonstrated that manipulation of glucose cycling in insulin - secreting cell lines does result in changes in insulin secretion ( 31,32 ) . for example , iizuka et al . ( 32 ) showed that altering glucose cycling by varying the g6pc2-to - gck ratio leads to reductions in atp production and reduced glucose - stimulated insulin secretion in min-6 cells . taken together , these studies examining the balance between gck and g6pc2 support our hypothesized effects of variation in gck and g6pc2 on insulin secretion ( compared with fig . however , they do not account for the differences we observed in the relationship between fasting glucose and insulin secretion when stratified by these two loci . primary enhancement of -cell sensitivity to glucose should result in increased systemic insulin concentrations in relation to glucose levels . the fact that the linearity in 30 insulin we observed between gck rs1799884 and g6pc2 rs560887 genotype combinations did not extend to fasting glucose ( compared with fig . we confirmed this initial observation by examining the relationship between fasting glucose and 30 insulin , stratifying on gck and g6pc2 genotype ( compared with fig . the presence of the gck rs1799884 a allele resulted in lower 30 insulin and higher fasting glucose , regardless of g6pc2 genotype . the results suggest that variation in gck may alter insulin secretion and fasting glucose in the traditionally envisioned manner where changes in insulin secretion result in reciprocal changes in glucose concentration . however , the presence of a g6pc2 a allele resulted in both lower 30 insulin and fasting glucose , regardless of gck genotype . first , variation in g6pc2 could have a direct and independent effect to regulate hepatic glucose production . this effect is unlikely because the hepatic isoform of glucose-6-phosphatase is encoded by an independent gene . second , variation in g6pc2 could alter a characteristic of insulin secretion that disrupts the normal signaling between the pancreas and other tissues , resulting in both insulin secretion and glucose changing in tandem . specifically , their studies show that in canines with partial pancreas resection or in rats transgenic for human islet amyloid polypeptide , disruption of pulsatile insulin secretion results in hepatic insulin resistance and impaired fasting glucose ( a. matveyenko , personal communication ) . they conclude that the loss of pulsatile insulin secretion results in a loss of efficiency in insulin action at the liver , leading to hepatic insulin resistance and increased glucose output , which subsequently leads to a gradual rise in glucose levels . variation in g6pc2 may disrupt pulsatility in insulin secretion , thereby reducing insulin - signaling efficiency between the pancreas and liver . the small increase in glucose because of hepatic insulin resistance may result in modest increases in absolute insulin secretion . additional studies will be required to test this hypothesis . finally , we observed association between g6pc2 rs560887 and measures of adiposity that have not been previously reported . bmi and body fat were both lower in the presence of the a allele ( compared with table 3 ) , but the biologic mechanism underlying this association is unclear . one possibility is that if variation in g6pc2 results in decreased insulin - signaling efficiency because of the loss of pulsatility and leads to hepatic insulin resistance , then a similar loss of insulin signaling may occur in other tissues , such as adipose . our observed association between variation in g6pc2 and adiposity will require replication in other populations and further study to elucidate the mechanism underlying the association . in conclusion , we observed evidence for an additive effect of variation in gck ( rs1799884 ) and g6pc2 ( rs560887 ) on insulin secretion and fasting glucose . when stratified by genotype , the effect of gck rs1799884 follows an expected pattern of reduced insulin secretion and reciprocal increase in fasting glucose . by contrast , glucose levels and insulin secretion change in parallel with g6pc2 rs560887 , suggesting a primary effect on insulin signaling to regulate hepatic glucose production . our analyses highlight the importance of considering biologic effects in assessing relationships between genotype and quantitative traits . like previous studies , our analyses show that these variants account for only a small fraction of the variability in insulin secretion or glucose . the remaining variability is likely because of the effects of other common genetic variants of both modest and moderate effect and a variety of additional gene - gene and gene - environment interactions . thus , additional studies of samples with detailed phenotyping will be necessary to fully unravel the genetic architecture underlying glucose regulation and diabetes .

objectiveglucokinase ( gck ) and glucose-6-phosphatase catalytic subunit 2 ( g6pc2 ) regulate the glucose - cycling step in pancreatic -cells and may regulate insulin secretion . gck rs1799884 and g6pc2 rs560887 have been independently associated with fasting glucose , but their interaction on glucose - insulin relationships is not well characterized.research design and methodswe tested whether these variants are associated with diabetes - related quantitative traits in mexican americans from the betagene study and attempted to replicate our findings in finnish men from the metabolic syndrome in men ( metsim ) study.resultsrs1799884 was not associated with any quantitative trait ( corrected p > 0.1 ) , whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response ( 30 insulin , corrected p = 0.021 ) . we found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 ( p > 0.26 ) . however , the additive effect of these single nucleotide polymorphisms was associated with fasting glucose ( corrected p = 0.03 ) and 30 insulin ( corrected p = 0.027 ) . this additive association was replicated in metsim ( fasting glucose , p = 3.5 1010 30 insulin , p = 0.028 ) . when we examined the relationship between fasting glucose and 30 insulin stratified by gck and g6pc2 , we noted divergent changes in these quantitative traits for gck but parallel changes for g6pc2 . we observed a similar pattern in metsim.conclusionsour data suggest that variation in gck and g6pc2 have additive effects on both fasting glucose and insulin secretion .

RESEARCH DESIGN AND METHODS The BetaGene Study Subject recruitment. Clinical protocols. Assays. The METSIM Study Subject recruitment. Clinical protocols. Molecular analysis. Data analysis. METSIM Study. RESULTS The BetaGene Study. Replication in the METSIM Study. DISCUSSION

participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . participants with fasting glucose < 126 mg / dl were invited back for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition ( percent of body fat ) and an insulin - modified intravenous glucose tolerance test ( ivgtt ) performed as previously described ( 37 ) . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . given that gck and g6pc2 work in opposite directions on the glucose cycle , we hypothesized that additivity may best characterize their interaction . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . we attempted to replicate our findings in subjects participating in the ongoing metsim study ( 36 ) . we calculated two measures of insulin response to glucose : the difference between the 30 and fasting plasma insulin concentration from ogtt ( 30 insulin ) and the incremental area under the insulin curve for the first 10 min of the ivgtt ( air ) . we initially examined the association between the two single nucleotide polymorphisms ( snps ) and fasting glucose , fasting insulin , 30 insulin , and air given our primary hypothesis that these variants may underlie variation in these specific quantitative traits . next , we performed a test of the association between type 2 diabetes related quantitative traits and the interaction between gck and g6pc2 . given that gck and g6pc2 work in opposite directions on the glucose cycle , we hypothesized that additivity may best characterize their interaction . test to test the joint effect of rs1799884 and rs560887 for association with type 2 diabetes related quantitative traits . ( g > a ) had a minor allele frequency of 18.0% and was marginally associated with fasting glucose ( table 2 , p = 0.052 ) that modestly increased ( p = 0.021 ) after adjustment for body fat . however , the association between gck rs1799884 and fasting glucose did not remain significant when corrected for multiple testing . in addition , we did not observe any evidence for association between this snp and measures of insulin secretion ( table 2 ) or other type 2 diabetes related quantitative traits ( table 3 ) , although there was a tendency for mean fasting insulin , 30 insulin , and air to be lower in individuals with at least one rs1799884 a allele ( table 2 ) . g6pc2 rs560887 ( g > a ) had a minor allele frequency of 14.5% and was nominally associated with fasting glucose ( table 2 , p = 0.015 ) and 30 insulin ( p = 0.0038 ) and marginally associated with air ( p = 0.088 ) . although g6pc2 rs560887 was not associated with most type 2 diabetes related quantitative traits , we did observe an association between this snp and percent body fat ( pact = 0.035 ) , where individuals homozygous for the g allele had higher trait values ( table 3 ) . we found no evidence for association between the multiplicative interaction between gck rs1799884 and g6pc2 rs560887 and type 2 diabetes related quantitative traits ( uncorrected p > 0.26 ) , and additional adjustment for body fat did not change the overall results . in contrast , when we tested whether the additive effects of gck rs1799884 and g6cp2 rs560887 were associated with type 2 diabetes related quantitative traits , we observed association with fasting glucose ( bonferroni p = 0.03 ) and 30 insulin ( bonferroni p = 0.027 ) . reported a linear relationship between number of glucose - lowering alleles for gck , g6pc2 , and gckr and fasting glucose , which suggested additive effects of these alleles on fasting glucose ( 10 ) . we observed a similar linear relationship ( data not shown ) when we stratified gck and g6pc2 genotypes , consistent with the results of our statistical analysis and the observations of bouatia - naji et al . however , given the specificity of these two gene products in regulating the glucose - sensing step in pancreatic -cells , we hypothesized the primary effect of variation in gck and g6pc2 to be at the level of insulin secretion , not directly upon glucose . hypothesized effect of gck rs1799884 and g6pc2 rs560887 genotype combinations on atp production and insulin secretion in pancreatic -cells . upper portion of the figure shows the glucose - cycling step and the effect of specific alleles in gck and g6pc2 on activity of each enzyme : thick arrow depicts increased activity , thin arrow depicts reduced activity . thus , the linearity between gck and g6pc2 genotype combinations and insulin secretion does not directly translate into linear changes in fasting glucose . we observed a similar dichotomy when we examined fasting glucose and 30 insulin stratified by total number of glucose - raising alleles , as previously performed by bouatia - naji et al . ( 10 ) ; fasting glucose is linear with total number of glucose - raising alleles , whereas 30 insulin is not ( data not shown ) . a : age- and sex - adjusted 30 insulin ( means se ) stratified by gck rs1799884 and g6pc2 rs560887 genotype combination . therefore , we plotted fasting glucose against 30 insulin from betagene stratified by gck and g6pc2 genotypes ( fig . we noted a different pattern of coordinate changes in fasting glucose and 30 insulin associated with variation in gck as compared with variation in g6pc2 . variation in gck led to inverse changes in fasting glucose and 30 insulin , whereas variation in g6pc2 led to parallel changes in these two traits . the 30 insulin vs. fasting plasma glucose stratified by gck rs1799884 and g6pc2 rs560887 genotype . given the observation of additivity between gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin in betagene , we attempted to replicate these findings in the metsim sample , in which gck rs4607517 is in perfect linkage disequilibrium with rs1799884 . gck rs4607517 was associated with fasting glucose but not associated with 30 insulin ( table 4 ) . g6pc2 rs560887 was associated with fasting glucose and marginally associated with 30 insulin ( table 4 ) . the multiplicative interaction between gck rs4607517 and g6pc2 rs560887 was weakly associated with fasting glucose ( p = 0.038 ) and marginally associated with 30 insulin ( p = 0.049 ) . test of the joint additive effect of the two snps was significant for both fasting glucose ( p = 3.5 10 ) and 30 insulin ( p = 0.028 ) . the evidence for association between the joint additive effect of both snps and fasting glucose was not affected when bmi was included as an additional covariate ( p = 3.3 10 ) ; the association with 30 insulin became stronger when bmi was included as a covariate ( p = 0.0028 ) . when we examined the relationship between fasting glucose and 30 insulin stratified by gck rs4607517 and g6pc2 rs560887 genotype groupings , an identical pattern of relationship to that seen in betagene was observed ( fig . for g6pc2 rs560887 , individuals homozygous for the g allele had higher fasting glucose and higher 30 insulin than individuals with at least one a allele . univariate tests of associations between genetic variants and fasting glucose and 30 insulin in metsim data are unadjusted means sd unless otherwise indicated . ( g > a ) had a minor allele frequency of 18.0% and was marginally associated with fasting glucose ( table 2 , p = 0.052 ) that modestly increased ( p = 0.021 ) after adjustment for body fat . however , the association between gck rs1799884 and fasting glucose did not remain significant when corrected for multiple testing . in addition , we did not observe any evidence for association between this snp and measures of insulin secretion ( table 2 ) or other type 2 diabetes related quantitative traits ( table 3 ) , although there was a tendency for mean fasting insulin , 30 insulin , and air to be lower in individuals with at least one rs1799884 a allele ( table 2 ) . g6pc2 rs560887 ( g > a ) had a minor allele frequency of 14.5% and was nominally associated with fasting glucose ( table 2 , p = 0.015 ) and 30 insulin ( p = 0.0038 ) and marginally associated with air ( p = 0.088 ) . although g6pc2 rs560887 was not associated with most type 2 diabetes related quantitative traits , we did observe an association between this snp and percent body fat ( pact = 0.035 ) , where individuals homozygous for the g allele had higher trait values ( table 3 ) . we found no evidence for association between the multiplicative interaction between gck rs1799884 and g6pc2 rs560887 and type 2 diabetes related quantitative traits ( uncorrected p > 0.26 ) , and additional adjustment for body fat did not change the overall results . in contrast , when we tested whether the additive effects of gck rs1799884 and g6cp2 rs560887 were associated with type 2 diabetes related quantitative traits , we observed association with fasting glucose ( bonferroni p = 0.03 ) and 30 insulin ( bonferroni p = 0.027 ) . reported a linear relationship between number of glucose - lowering alleles for gck , g6pc2 , and gckr and fasting glucose , which suggested additive effects of these alleles on fasting glucose ( 10 ) . we observed a similar linear relationship ( data not shown ) when we stratified gck and g6pc2 genotypes , consistent with the results of our statistical analysis and the observations of bouatia - naji et al . however , given the specificity of these two gene products in regulating the glucose - sensing step in pancreatic -cells , we hypothesized the primary effect of variation in gck and g6pc2 to be at the level of insulin secretion , not directly upon glucose . hypothesized effect of gck rs1799884 and g6pc2 rs560887 genotype combinations on atp production and insulin secretion in pancreatic -cells . upper portion of the figure shows the glucose - cycling step and the effect of specific alleles in gck and g6pc2 on activity of each enzyme : thick arrow depicts increased activity , thin arrow depicts reduced activity . thus , the linearity between gck and g6pc2 genotype combinations and insulin secretion does not directly translate into linear changes in fasting glucose . we observed a similar dichotomy when we examined fasting glucose and 30 insulin stratified by total number of glucose - raising alleles , as previously performed by bouatia - naji et al . ( 10 ) ; fasting glucose is linear with total number of glucose - raising alleles , whereas 30 insulin is not ( data not shown ) . a : age- and sex - adjusted 30 insulin ( means se ) stratified by gck rs1799884 and g6pc2 rs560887 genotype combination . therefore , we plotted fasting glucose against 30 insulin from betagene stratified by gck and g6pc2 genotypes ( fig . we noted a different pattern of coordinate changes in fasting glucose and 30 insulin associated with variation in gck as compared with variation in g6pc2 . variation in gck led to inverse changes in fasting glucose and 30 insulin , whereas variation in g6pc2 led to parallel changes in these two traits . the 30 insulin vs. fasting plasma glucose stratified by gck rs1799884 and g6pc2 rs560887 genotype . given the observation of additivity between gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin in betagene , we attempted to replicate these findings in the metsim sample , in which gck rs4607517 is in perfect linkage disequilibrium with rs1799884 . gck rs4607517 was associated with fasting glucose but not associated with 30 insulin ( table 4 ) . g6pc2 rs560887 was associated with fasting glucose and marginally associated with 30 insulin ( table 4 ) . the multiplicative interaction between gck rs4607517 and g6pc2 rs560887 was weakly associated with fasting glucose ( p = 0.038 ) and marginally associated with 30 insulin ( p = 0.049 ) . test of the joint additive effect of the two snps was significant for both fasting glucose ( p = 3.5 10 ) and 30 insulin ( p = 0.028 ) . the evidence for association between the joint additive effect of both snps and fasting glucose was not affected when bmi was included as an additional covariate ( p = 3.3 10 ) ; the association with 30 insulin became stronger when bmi was included as a covariate ( p = 0.0028 ) . when we examined the relationship between fasting glucose and 30 insulin stratified by gck rs4607517 and g6pc2 rs560887 genotype groupings , an identical pattern of relationship to that seen in betagene was observed ( fig . univariate tests of associations between genetic variants and fasting glucose and 30 insulin in metsim data are unadjusted means sd unless otherwise indicated . our results from mexican americans , replicated in a sample of finnish men , show that variation in both gck and g6pc2 have additive effects on both fasting glucose and insulin secretion . our examination of the relationship between 30 insulin and fasting glucose stratified by gck and g6pc2 genotype reveals that the joint effect of genetic variation in these two genes may be through distinct mechanisms . variation in gck leads to inverse changes in fasting glucose and insulin secretion , consistent with a primary effect on insulin secretion . variation in g6pc2 leads to parallel changes in fasting glucose and insulin secretion , suggesting a primary effect on glucose , with secondary changes in insulin secretion . table 5 summarizes our examination of the effects of gck rs1799884 and g6pc2 rs560887 on fasting glucose and 30 insulin . although it is possible that our conclusion of these variants having an additive effect could be driven mainly by g6pc2 rs560887 , the summary in table 5 suggests otherwise . it is clear that in both betagene and metsim , the model testing the joint effect of gck and g6pc2 shows greater significance than the effect of each individual gene variant alone , which suggests both genes are contributing to the effect on fasting glucose and 30 insulin . in support of this observation , when we tested for the additional effect of gck in the presence of g6pc2 we observed marginal or significant effects for fasting glucose ( p = 0.058 for betagene ; p = 0.0009 for metsim ) and 30 insulin ( p = 0.148 for betagene ; p = 0.723 for metsim ) . our a priori hypothesis tested whether the multiplicative interaction between rs1799884 and rs560887 was associated with type 2 diabetes related quantitative traits . in betagene we observed no evidence for a multiplicative interaction ; our analysis favored a joint additive effect of these two loci . a priori power estimates indicated that a sample size of 600 should have provided 80% power to detect a multiplicative interaction effect that accounted for 1.8% of the variability in fasting glucose or 30 insulin , assuming = 0.01 , minor allele frequencies of 15% for both loci , and dominant genetic models for both loci . in fact , the metsim sample , which is 6 times larger than betagene , showed modest evidence for a multiplicative interaction ( table 5 ) suggesting that a weak multiplicative interaction between gck and g6pc2 could be contributing to variation in fasting glucose and 30 insulin . our initial examination of the additive effect of these two genes was motivated by the fact that when we plotted 30 insulin against gck rs1799884 and g6pc2 rs560887 genotype combinations sorted by the hypothesized biologic effect on insulin secretion , we observed a linear relationship with 30 insulin . taken together , these studies examining the balance between gck and g6pc2 support our hypothesized effects of variation in gck and g6pc2 on insulin secretion ( compared with fig . however , they do not account for the differences we observed in the relationship between fasting glucose and insulin secretion when stratified by these two loci . the fact that the linearity in 30 insulin we observed between gck rs1799884 and g6pc2 rs560887 genotype combinations did not extend to fasting glucose ( compared with fig . we confirmed this initial observation by examining the relationship between fasting glucose and 30 insulin , stratifying on gck and g6pc2 genotype ( compared with fig . the results suggest that variation in gck may alter insulin secretion and fasting glucose in the traditionally envisioned manner where changes in insulin secretion result in reciprocal changes in glucose concentration . however , the presence of a g6pc2 a allele resulted in both lower 30 insulin and fasting glucose , regardless of gck genotype . in conclusion , we observed evidence for an additive effect of variation in gck ( rs1799884 ) and g6pc2 ( rs560887 ) on insulin secretion and fasting glucose . when stratified by genotype , the effect of gck rs1799884 follows an expected pattern of reduced insulin secretion and reciprocal increase in fasting glucose .

fatty liver represents a common clinical entity that can be divided into alcoholic fatty liver disease ( afld ) , and nonalcoholic fatty liver disease ( nafld ) . both are within the most common causes of chronic liver disease worldwide , and are linked to the rising incidence of obesity , diabetes and coronary heart disease.1 only 3% of individuals with nafld progress to fibrosis or cirrhosis,2 whereas 5 - 15% of subjects with afld develop cirrhosis , despite abstinence.1 the prevalence of alcohol consumption is 90% in men with age between 40 - 49 , 88% of men of all age and 70% of women over 19 years consume alcohol representing 79% of total korean adult population.3 in addition , about 14% of koreans meet dsm - iv ( diagnostic and statistical manual of mental disorders , 4 edition ) criteria4 for alcohol use disorder during their lifetime.5 furthermore , the prevalence of high risk drinking is 15% in korea,6 higher than that in canada ( 9.5% ) , france ( 5.3% ) , and the us ( 8.2% ) as well as the global rate of 11.5%.7 alcohol consumption creates various disturbances in metabolic functions and tissue damage in almost every organ of the body.8 although a light to moderate amount of alcohol is reported to decrease risk of coronary artery disease and cardiovascular mortality,9 significant alcohol consumption is associated with an increased risk of hypertension in a dose - dependent manner.10 furthermore , long - term alcohol consumption may increase the prevalence of central obesity , metabolic syndrome ( ms ) , and cardiovascular disease ( cvd).11,12 excess alcohol increases cvd risk because the beneficial increase in high - density lipoprotein cholesterol ( hdl - c ) is offset by increased blood pressure levels.13 however , despite its high prevalence and the substantial side effects involved , there has been limited research into afld not only in korea but also on the international level.14 alcohol is a causal factor in 60 types of diseases and injuries and a component cause in 200 others.2 however , we have little understanding of the negative consequences of afld , except for its cirrhotic complications . our objective was to determine whether those with afld have an elevated 10-year risk of developing chd , to investigate the relationship between afld and chd risk , and to compare this risk with that for nafld population and to assess non - inferiority of afld compared with nafld for cardiovascular risk . this is a cross - sectional study of the association between afld and chd risk . we performed the retrospective analysis of the data collected in 2010 , at a single center of a region , konkuk university health care center in seoul . the recruitment period of the individuals who visited konkuk university health care center for general health check - up was from january 2010 to december 2010 . they completed the self - administered questionnaire that had been sent by mail several days before and submitted it on the visiting day . the results of the examinations were noticed to the individuals by mailing or phone calls . the health care program included several different types of examinations upon the individuals ' requests . study data included laboratory , physical examination , liver us finding and information of the questionnaire . patients were considered eligible if the three following inclusion criteria were fulfilled : ( a ) age 18 years or older , ( b ) subjects who visited konkuk university hospital health care center between january 2010 and december 2010 and who had undergone liver us , ( c ) individuals who had fulfilled the self - administered questionnaire . out of them , we excluded 167 participants with at least one potential cause of chronic liver disease : 98 subjects with hepatitis b virus , 25 subjects with hepatitis c virus , 9 subjects with some other history of hepatitis . in addition we excluded 35 participants with history of chd . altogether , 10,710 individuals were included in the study . study protocol was approved by the institutional review board of konkuk university hospital at seoul and conformed to the ethical guidelines of the 1975 declaration of helsinki . we reviewed all the medical records performed at health check - up , including anthropometric , laboratory and imaging study . the anthropometric data included systolic and diastolic blood pressure , height , weight , waist circumference . there was also information about participants ' past history , family history , drug history and alcohol , smoking history , all collected by self - administered questionnaire . regarding to alcohol history , the self - administered questionnaire included the following two questions : ( a ) how often do you have a drink containing alcohol , during an average week ( b ) how many units of alcohol have you drunk during a typical occasion over the past 12 months the weekly frequency was evaluated using a closed question comprising six subcategories where patients were to choose one of the following answers : ( a ) once daily ( b ) 5 - 6 times per week ( c ) 3 - 4 times per week ( d ) 1 - 2 times per week ( e ) 2 - 3 times per month ( f ) less than once a month . the standard number of drinks consumed on an average occasion was measured by an open question . we defined significant alcohol consumption based on the threshold of 210 g / week ( men ) and 140 g / week ( women ) and discriminated between nafld and afld accordingly ( 15 ) . a standard drink was defined as having 10 g of ethanol , which comprises 150 ml of wine , 200 ml of beer , 50 ml of spirit , or 50 ml of soju , the most popular alcoholic beverage among koreans.14 the total amount of alcohol consumed per week on average was obtained by multiplying the weekly frequency ( ( a ) 7 for once daily ( b ) 5.5 for 5 - 6 times per week ( c ) 3.5 for 3 - 4 times per week ( d ) 1.5 for 1 - 2 times per week ( e ) 0.75 for 2 - 3 times per month ( f ) 0.25 for less than once a month ) by the consumption amount on a single occasion , at 10 g per drink . laboratory data included aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , gamma glutamyl transpeptidase ( rgt ) , serum total cholesterol , triglyceride , hdl - c , fasting plasma glucose , c - reactive protein and ferritin . hepatic steatosis was diagnosed if he / she had the following components at liver us : ultrasound beam attenuation and poor visualization of intra - hepatic vessel borders and diaphragm , evidence of diffuse hyper - echogenicity of liver relative to kidneys . the intra - observer variability for the ultrasound diagnosis of hepatic steatosis was within 3%.15 we diagnosed nafld by relying on the presence of hepatic steatosis as confirmed by liver us when the following criteria were absent : a ) excess alcohol consumption defined as > 210 g / week in men and > 140 g / week in women based on the kasl definition , b ) causes for secondary hepatic fat accumulation such as steatogenic medications or hereditary disorders c ) positivity for hepatitis b surface antigen or antibody to hepatitis c virus . afld was diagnosed based on a combination of features including ongoing or recent excessive alcohol consumption with hepatic steatosis confirmed by liver us . two radiologists performed us scanning using the philips model ( iu22x matrix ultrasound system , philips ) . definition of metabolic syndrome was based on the world health organization - west pacific region guideline16 and insulin resistance was estimated by homeostasis model assessment of insulin resistance.17 among various framingham - based tools , we used the conventional framingham risk score ( frs ) from wilson et al 199818 to estimate total chd risk at 10-year . for hard chd risk estimation , we used the revised frs form , where atp iii risk factor scoring is incorporated into the 10-year risk assessment tool.19 the frs was calculated based on six coronary risk factors : gender , age , total cholesterol , hdl - c , systolic bp and smoking habit . among these factors , age , bp , and cholesterol levels were categorized according to their values and smoking status was classified as either " current smoker " or " non - smoker " . finally , the corresponding point was assigned to each individual and the total score was used as the individual 's chd risk level.19 quantitative data was expressed as meanstandard deviation ( sd ) , and categorical data as percentages . we used chi - square tests or the mann - whitney test and analysis of variance or kruskal - wallis test for continuous variables . one - way analysis of covariance ( ancova ) was used to compare the means of frs and 10-year risk for total and hard chd between the control and fatty liver group , and between the nafld and afld group after adjustment for the main differences between the groups ( age , sex , metabolic syndrome , smoking and family history of chd ) . all statistical tests were two - sided , and a p value of less than 0.05 was considered to be significant . this is a cross - sectional study of the association between afld and chd risk . we performed the retrospective analysis of the data collected in 2010 , at a single center of a region , konkuk university health care center in seoul . the recruitment period of the individuals who visited konkuk university health care center for general health check - up was from january 2010 to december 2010 . they completed the self - administered questionnaire that had been sent by mail several days before and submitted it on the visiting day . the results of the examinations were noticed to the individuals by mailing or phone calls . the health care program included several different types of examinations upon the individuals ' requests . study data included laboratory , physical examination , liver us finding and information of the questionnaire . patients were considered eligible if the three following inclusion criteria were fulfilled : ( a ) age 18 years or older , ( b ) subjects who visited konkuk university hospital health care center between january 2010 and december 2010 and who had undergone liver us , ( c ) individuals who had fulfilled the self - administered questionnaire . out of them , we excluded 167 participants with at least one potential cause of chronic liver disease : 98 subjects with hepatitis b virus , 25 subjects with hepatitis c virus , 9 subjects with some other history of hepatitis . in addition we excluded 35 participants with history of chd . altogether , 10,710 individuals were included in the study . study protocol was approved by the institutional review board of konkuk university hospital at seoul and conformed to the ethical guidelines of the 1975 declaration of helsinki . we reviewed all the medical records performed at health check - up , including anthropometric , laboratory and imaging study . the anthropometric data included systolic and diastolic blood pressure , height , weight , waist circumference . there was also information about participants ' past history , family history , drug history and alcohol , smoking history , all collected by self - administered questionnaire . regarding to alcohol history , the self - administered questionnaire included the following two questions : ( a ) how often do you have a drink containing alcohol , during an average week ( b ) how many units of alcohol have you drunk during a typical occasion over the past 12 months the weekly frequency was evaluated using a closed question comprising six subcategories where patients were to choose one of the following answers : ( a ) once daily ( b ) 5 - 6 times per week ( c ) 3 - 4 times per week ( d ) 1 - 2 times per week ( e ) 2 - 3 times per month ( f ) less than once a month . the standard number of drinks consumed on an average occasion was measured by an open question . we defined significant alcohol consumption based on the threshold of 210 g / week ( men ) and 140 g / week ( women ) and discriminated between nafld and afld accordingly ( 15 ) . a standard drink was defined as having 10 g of ethanol , which comprises 150 ml of wine , 200 ml of beer , 50 ml of spirit , or 50 ml of soju , the most popular alcoholic beverage among koreans.14 the total amount of alcohol consumed per week on average was obtained by multiplying the weekly frequency ( ( a ) 7 for once daily ( b ) 5.5 for 5 - 6 times per week ( c ) 3.5 for 3 - 4 times per week ( d ) 1.5 for 1 - 2 times per week ( e ) 0.75 for 2 - 3 times per month ( f ) 0.25 for less than once a month ) by the consumption amount on a single occasion , at 10 g per drink . laboratory data included aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , gamma glutamyl transpeptidase ( rgt ) , serum total cholesterol , triglyceride , hdl - c , fasting plasma glucose , c - reactive protein and ferritin . hepatic steatosis was diagnosed if he / she had the following components at liver us : ultrasound beam attenuation and poor visualization of intra - hepatic vessel borders and diaphragm , evidence of diffuse hyper - echogenicity of liver relative to kidneys . the intra - observer variability for the ultrasound diagnosis of hepatic steatosis was within 3%.15 we diagnosed nafld by relying on the presence of hepatic steatosis as confirmed by liver us when the following criteria were absent : a ) excess alcohol consumption defined as > 210 g / week in men and > 140 g / week in women based on the kasl definition , b ) causes for secondary hepatic fat accumulation such as steatogenic medications or hereditary disorders c ) positivity for hepatitis b surface antigen or antibody to hepatitis c virus . afld was diagnosed based on a combination of features including ongoing or recent excessive alcohol consumption with hepatic steatosis confirmed by liver us . two radiologists performed us scanning using the philips model ( iu22x matrix ultrasound system , philips ) . definition of metabolic syndrome was based on the world health organization - west pacific region guideline16 and insulin resistance was estimated by homeostasis model assessment of insulin resistance.17 among various framingham - based tools , we used the conventional framingham risk score ( frs ) from wilson et al 199818 to estimate total chd risk at 10-year . for hard chd risk estimation , we used the revised frs form , where atp iii risk factor scoring is incorporated into the 10-year risk assessment tool.19 the frs was calculated based on six coronary risk factors : gender , age , total cholesterol , hdl - c , systolic bp and smoking habit . among these factors , age , bp , and cholesterol levels were categorized according to their values and smoking status was classified as either " current smoker " or " non - smoker " . finally , the corresponding point was assigned to each individual and the total score was used as the individual 's chd risk level.19 quantitative data was expressed as meanstandard deviation ( sd ) , and categorical data as percentages . we used chi - square tests or the mann - whitney test and analysis of variance or kruskal - wallis test for continuous variables . one - way analysis of covariance ( ancova ) was used to compare the means of frs and 10-year risk for total and hard chd between the control and fatty liver group , and between the nafld and afld group after adjustment for the main differences between the groups ( age , sex , metabolic syndrome , smoking and family history of chd ) . all statistical tests were two - sided , and a p value of less than 0.05 was considered to be significant . statistical analysis of the data was performed using spss version 17.0 ( spss inc . , of 10,710 individuals , 59.4% were men with an average age of 44.5 and a bmi of 23.8 kg / m , for whom the prevalence of metabolic syndrome was 24.1% . relying on liver us , we discriminated 4,142 fatty liver subjects ( 38.7% ) , with the remaining 6,568 ( 61.3% ) constituting the control group . the majority of overall group comparisons showed significant differences , with higher mean values in the population with fatty liver as compared to the control group , except for waist - to - hip ratio ( p=0.08 ) and hdl - c which was higher in the control group ( p<0.001 ) . of the 4,142 subjects with fatty liver , 71.3% ( 2,953 ) had nafld and 28.7% ( 1,189 ) had afld . subjects with afld were more likely to be younger ( 46.2 vs. 44.3 years , p<0.001 ) , men ( 62.4 vs. 92.3% , p<0.001 ) , and were also more likely to be smokers ( 36.8 vs.74.6% , p<0.001 ) , with all these variables showing significant differences compared to the nafld group . subjects with nafld and afld showed comparable results for homa - ir , plasma level of hdl - c , low - density lipoprotein cholesterol ( ldl - c ) , serum high - sensitive c - reactive protein ( hscrp ) and daily physical activity . there were statistical differences in mean blood pressure , fasting plasma glucose , serum ast , alt , and ferritin between the two groups . table 3 shows comparisons of mean 10-year estimated total risk between the control and fatty liver group , and the nafld and afld group . the mean 10-year risk for total chd was higher in the fatty liver group than the matched control group . individuals with afld had a higher 10-year estimated chd risk compared to the control group ( 6.720.12 vs 5.500.04 p<0.001 ) and a comparable risk to those with nafld ( 6.720.12 vs 7.320.07 , p=0.02 ) . the prevalence of total chd risk over 10% ( more than moderate risk ) and over 20% ( high risk ) were significantly higher in the fatty liver group as compared to the control . however , between nafld and afld , we obtained inconsistent results showing a higher prevalence of total chd events greater than 10% in the afld group , whereas total chd over 20% showed no unidirectional tendency ( table 4 ) . of 10,710 individuals , 59.4% were men with an average age of 44.5 and a bmi of 23.8 kg / m , for whom the prevalence of metabolic syndrome was 24.1% . relying on liver us , we discriminated 4,142 fatty liver subjects ( 38.7% ) , with the remaining 6,568 ( 61.3% ) constituting the control group . the majority of overall group comparisons showed significant differences , with higher mean values in the population with fatty liver as compared to the control group , except for waist - to - hip ratio ( p=0.08 ) and hdl - c which was higher in the control group ( p<0.001 ) . of the 4,142 subjects with fatty liver , 71.3% ( 2,953 ) had nafld and 28.7% ( 1,189 ) had afld . subjects with afld were more likely to be younger ( 46.2 vs. 44.3 years , p<0.001 ) , men ( 62.4 vs. 92.3% , p<0.001 ) , and were also more likely to be smokers ( 36.8 vs.74.6% , p<0.001 ) , with all these variables showing significant differences compared to the nafld group . subjects with nafld and afld showed comparable results for homa - ir , plasma level of hdl - c , low - density lipoprotein cholesterol ( ldl - c ) , serum high - sensitive c - reactive protein ( hscrp ) and daily physical activity . there were statistical differences in mean blood pressure , fasting plasma glucose , serum ast , alt , and ferritin between the two groups . table 3 shows comparisons of mean 10-year estimated total risk between the control and fatty liver group , and the nafld and afld group . the mean 10-year risk for total chd was higher in the fatty liver group than the matched control group . individuals with afld had a higher 10-year estimated chd risk compared to the control group ( 6.720.12 vs 5.500.04 p<0.001 ) and a comparable risk to those with nafld ( 6.720.12 vs 7.320.07 , p=0.02 ) . the prevalence of total chd risk over 10% ( more than moderate risk ) and over 20% ( high risk ) were significantly higher in the fatty liver group as compared to the control . however , between nafld and afld , we obtained inconsistent results showing a higher prevalence of total chd events greater than 10% in the afld group , whereas total chd over 20% showed no unidirectional tendency ( table 4 ) . the aim of the current study was to determine whether subjects with afld have an elevated chd risk using frs and to compare it with that for nafld . we found a strong association between afld and chd risk comparable to nafld . in addition , afld was a strong predictor elevated chd risk ( chd risk > 10% ) independent of traditional risk factors . to our knowledge , this is the first attempt to measure chd risk in afld and to compare it with that of nafld . although there are many reports for nafld population on this topic,20,21 few exists on afld population . only study comparing the frequency of coronary artery disease in alcohol - related versus non - alcoholic end - stage liver disease ( esld ) is available in the literature.22 in this study , 420 patients with esld either alcohol - related or non - alcohol related , underwent coronary angiography for candidate screening of liver transplantation . and a significantly higher proportion of patients in the alcohol - related group had coronary artery disease compared to non - alcohol group ( p<0.01 ) . this difference was observed despite these groups otherwise being comparable in terms of age and cad risk factors . their report has some disparities with our results . however the overall study population in this analysis had advanced liver diseases with substantially different baseline metabolic risk profiles when compared to our study population , making it inappropriate to directly compare the results . another research by jh kim and colleagues that examines 265 fatty liver subjects has recently been published providing comparison of carotid intima - media thickness ( cimt ) between individuals with nafld and afld.23 the relationship between increasing cimt and incident cvd events has been clearly established24 and several prospective , population - based studies demonstrated that the presence of carotid plaque is associated with a significantly increased risk for myocardial infarction , stroke and chd death , independent of traditional risk factors.25 in this study , cimt was elevated in subjects with fatty liver in both nafld and afld patients , in agreement with our results . in the current study , we demonstrated the prevalence of both nafld and afld in apparently healthy individuals . the prevalence of nafld based on liver us was 27.6% in our study , consistent with the previous reports between 17 and 46%.26,27 although reference data on the prevalence of afld is limited , there is a study using the population - based fin - d2d survey , in which a 7.0% of prevalence is reported.28 we found a higher prevalence of afld ( 11.1% ) , with this discrepancy possibly due to the high alcohol consumption rate in the korean population . as mentioned earlier , compared to the nafld group , the afld group is comprised individuals with younger age , male sex , current smoking status , central obesity and metabolic syndrome . these findings agree well with several previous reports that excess alcohol consumption is more prevalent in males29 with combined smoking habits . indeed 90% of all alcoholics smoke and alcoholism is ten times more prevalent among smokers than non - smokers.30 however the fact that the prevalence of essential hypertension , and metabolic syndrome is higher in individuals with afld compared to nafld suggests the new possibility that in some afld subjects , the cvd risk may be higher than our expectation and further investigation should be conducted . another important strength of the current article is that it is the first study comparing nafld and afld populations in terms of chd risk and metabolic profiles . there has been accumulating evidence for a strong association between nafld and elevated chd risk over the last years.31 although the pathogenesis behind this link has not been fully elucidated , the contributions of obesity and metabolic syndrome to increased chd events are well known.21,32 by contrast , the association of afld with chd risk had not been investigated . in this study , we have demonstrated a significant increase in 10-year chd risk not only in subjects with nafld , but also those with afld , a close association that was independent of age , sex , homa - ir , or the individual components of metabolic syndrome , implying that afld is a strong independent predictor for developing chd events within 10 years . the current study benefitted from the fact that its results were based on a large population . it is also significant that we compared subjects with nafld and afld in terms of future chd risk by estimating 10 year total chd risk . considering its high alcohol consumption rate , racial difference and genetic variances , the conclusion may not be applicable widely up to other countries . secondly , we diagnosed hepatic steatosis based on liver us instead of histological tissue confirmation . although this is better than using a definition on elevated transaminase as a proxy for the diagnosis , it is still a surrogate for the diagnosis and is not completely sensitive in cases with milder liver steatosis . thus mild steatosis ( mild nafld or afld ) might have been included in the control group . to correct for this , thirdly as this is a large retrospective cohort study , we could only estimate future chd risk using frs based on laboratory and clinical data . additionally , the alcohol consumption was measured by self - reported questionnaire participants so this study may show selection bias . regardless of these limitations , strengths of the present study include the large sample size of healthy male participants with comprehensive measurements of liver , including the direct measurement of estimated cardiovascular risk factors . in summary , subjects with fatty liver had significantly higher estimated 10-year total chd risk compared to the control group , not only for nafld but also for afld . therefore , afld should be considered as a meaningful risk for future chd events and preventive measures should be considered earlier .

background / aimsa close relationship has been established between nonalcoholic fatty liver disease ( nafld ) and an elevated risk of coronary heart disease ( chd ) , but little is known about the association between alcoholic fatty liver disease ( afld ) and chd risk . the aim of this study was to determine whether afld is associated with elevated chd risk.methodswe retrospectively enrolled 10,710 subjects out of 11,469 individuals who visited the konkuk university health care center for a routine health checkup in 2010 . afld was diagnosed made when the usual amount of alcohol consumption exceeded 210 g / week in males and 140 g / week in females for the previous 2 years and when hepatic steatosis was detected by liver ultrasonography . the 10-year risk for chd was estimated using the framingham risk score.resultshepatic steatosis was diagnosed in 4,142 of the 10,710 individuals ( 38.7% ) ; the remainder ( i.e. , n=6,568 ) became the control group . the 4,142 individuals with hepatic steatosis were divided into two groups : nafld ( n=2,953 ) and afld ( n=1,189 ) . the risk of chd was higher in afld ( 6.720.12 ) than in the control group ( 5.500.04 , p<0.001 ) , and comparable to that in nafld ( 7.320.07 , p=0.02).conclusionsindividuals with afld have an elevated 10-year risk of chd that is comparable to those with nafld . therefore , afld should be considered a significant risk for future chd , and preventive measures should be considered earlier .

INTRODUCTION MATERIALS AND METHODS Study design Study samples Clinical and laboratory evaluations Diagnosis of NAFLD and AFLD CHD risk assessment Statistical analysis RESULTS Baseline characteristics NAFLD vs. AFLD Comparison of CHD risk between groups DISCUSSION CONCLUSIONS

fatty liver represents a common clinical entity that can be divided into alcoholic fatty liver disease ( afld ) , and nonalcoholic fatty liver disease ( nafld ) . both are within the most common causes of chronic liver disease worldwide , and are linked to the rising incidence of obesity , diabetes and coronary heart disease.1 only 3% of individuals with nafld progress to fibrosis or cirrhosis,2 whereas 5 - 15% of subjects with afld develop cirrhosis , despite abstinence.1 the prevalence of alcohol consumption is 90% in men with age between 40 - 49 , 88% of men of all age and 70% of women over 19 years consume alcohol representing 79% of total korean adult population.3 in addition , about 14% of koreans meet dsm - iv ( diagnostic and statistical manual of mental disorders , 4 edition ) criteria4 for alcohol use disorder during their lifetime.5 furthermore , the prevalence of high risk drinking is 15% in korea,6 higher than that in canada ( 9.5% ) , france ( 5.3% ) , and the us ( 8.2% ) as well as the global rate of 11.5%.7 alcohol consumption creates various disturbances in metabolic functions and tissue damage in almost every organ of the body.8 although a light to moderate amount of alcohol is reported to decrease risk of coronary artery disease and cardiovascular mortality,9 significant alcohol consumption is associated with an increased risk of hypertension in a dose - dependent manner.10 furthermore , long - term alcohol consumption may increase the prevalence of central obesity , metabolic syndrome ( ms ) , and cardiovascular disease ( cvd).11,12 excess alcohol increases cvd risk because the beneficial increase in high - density lipoprotein cholesterol ( hdl - c ) is offset by increased blood pressure levels.13 however , despite its high prevalence and the substantial side effects involved , there has been limited research into afld not only in korea but also on the international level.14 alcohol is a causal factor in 60 types of diseases and injuries and a component cause in 200 others.2 however , we have little understanding of the negative consequences of afld , except for its cirrhotic complications . our objective was to determine whether those with afld have an elevated 10-year risk of developing chd , to investigate the relationship between afld and chd risk , and to compare this risk with that for nafld population and to assess non - inferiority of afld compared with nafld for cardiovascular risk . this is a cross - sectional study of the association between afld and chd risk . we performed the retrospective analysis of the data collected in 2010 , at a single center of a region , konkuk university health care center in seoul . the recruitment period of the individuals who visited konkuk university health care center for general health check - up was from january 2010 to december 2010 . patients were considered eligible if the three following inclusion criteria were fulfilled : ( a ) age 18 years or older , ( b ) subjects who visited konkuk university hospital health care center between january 2010 and december 2010 and who had undergone liver us , ( c ) individuals who had fulfilled the self - administered questionnaire . out of them , we excluded 167 participants with at least one potential cause of chronic liver disease : 98 subjects with hepatitis b virus , 25 subjects with hepatitis c virus , 9 subjects with some other history of hepatitis . altogether , 10,710 individuals were included in the study . study protocol was approved by the institutional review board of konkuk university hospital at seoul and conformed to the ethical guidelines of the 1975 declaration of helsinki . regarding to alcohol history , the self - administered questionnaire included the following two questions : ( a ) how often do you have a drink containing alcohol , during an average week ( b ) how many units of alcohol have you drunk during a typical occasion over the past 12 months the weekly frequency was evaluated using a closed question comprising six subcategories where patients were to choose one of the following answers : ( a ) once daily ( b ) 5 - 6 times per week ( c ) 3 - 4 times per week ( d ) 1 - 2 times per week ( e ) 2 - 3 times per month ( f ) less than once a month . we defined significant alcohol consumption based on the threshold of 210 g / week ( men ) and 140 g / week ( women ) and discriminated between nafld and afld accordingly ( 15 ) . a standard drink was defined as having 10 g of ethanol , which comprises 150 ml of wine , 200 ml of beer , 50 ml of spirit , or 50 ml of soju , the most popular alcoholic beverage among koreans.14 the total amount of alcohol consumed per week on average was obtained by multiplying the weekly frequency ( ( a ) 7 for once daily ( b ) 5.5 for 5 - 6 times per week ( c ) 3.5 for 3 - 4 times per week ( d ) 1.5 for 1 - 2 times per week ( e ) 0.75 for 2 - 3 times per month ( f ) 0.25 for less than once a month ) by the consumption amount on a single occasion , at 10 g per drink . hepatic steatosis was diagnosed if he / she had the following components at liver us : ultrasound beam attenuation and poor visualization of intra - hepatic vessel borders and diaphragm , evidence of diffuse hyper - echogenicity of liver relative to kidneys . the intra - observer variability for the ultrasound diagnosis of hepatic steatosis was within 3%.15 we diagnosed nafld by relying on the presence of hepatic steatosis as confirmed by liver us when the following criteria were absent : a ) excess alcohol consumption defined as > 210 g / week in men and > 140 g / week in women based on the kasl definition , b ) causes for secondary hepatic fat accumulation such as steatogenic medications or hereditary disorders c ) positivity for hepatitis b surface antigen or antibody to hepatitis c virus . afld was diagnosed based on a combination of features including ongoing or recent excessive alcohol consumption with hepatic steatosis confirmed by liver us . definition of metabolic syndrome was based on the world health organization - west pacific region guideline16 and insulin resistance was estimated by homeostasis model assessment of insulin resistance.17 among various framingham - based tools , we used the conventional framingham risk score ( frs ) from wilson et al 199818 to estimate total chd risk at 10-year . for hard chd risk estimation , we used the revised frs form , where atp iii risk factor scoring is incorporated into the 10-year risk assessment tool.19 the frs was calculated based on six coronary risk factors : gender , age , total cholesterol , hdl - c , systolic bp and smoking habit . finally , the corresponding point was assigned to each individual and the total score was used as the individual 's chd risk level.19 quantitative data was expressed as meanstandard deviation ( sd ) , and categorical data as percentages . one - way analysis of covariance ( ancova ) was used to compare the means of frs and 10-year risk for total and hard chd between the control and fatty liver group , and between the nafld and afld group after adjustment for the main differences between the groups ( age , sex , metabolic syndrome , smoking and family history of chd ) . this is a cross - sectional study of the association between afld and chd risk . we performed the retrospective analysis of the data collected in 2010 , at a single center of a region , konkuk university health care center in seoul . the recruitment period of the individuals who visited konkuk university health care center for general health check - up was from january 2010 to december 2010 . patients were considered eligible if the three following inclusion criteria were fulfilled : ( a ) age 18 years or older , ( b ) subjects who visited konkuk university hospital health care center between january 2010 and december 2010 and who had undergone liver us , ( c ) individuals who had fulfilled the self - administered questionnaire . altogether , 10,710 individuals were included in the study . regarding to alcohol history , the self - administered questionnaire included the following two questions : ( a ) how often do you have a drink containing alcohol , during an average week ( b ) how many units of alcohol have you drunk during a typical occasion over the past 12 months the weekly frequency was evaluated using a closed question comprising six subcategories where patients were to choose one of the following answers : ( a ) once daily ( b ) 5 - 6 times per week ( c ) 3 - 4 times per week ( d ) 1 - 2 times per week ( e ) 2 - 3 times per month ( f ) less than once a month . we defined significant alcohol consumption based on the threshold of 210 g / week ( men ) and 140 g / week ( women ) and discriminated between nafld and afld accordingly ( 15 ) . a standard drink was defined as having 10 g of ethanol , which comprises 150 ml of wine , 200 ml of beer , 50 ml of spirit , or 50 ml of soju , the most popular alcoholic beverage among koreans.14 the total amount of alcohol consumed per week on average was obtained by multiplying the weekly frequency ( ( a ) 7 for once daily ( b ) 5.5 for 5 - 6 times per week ( c ) 3.5 for 3 - 4 times per week ( d ) 1.5 for 1 - 2 times per week ( e ) 0.75 for 2 - 3 times per month ( f ) 0.25 for less than once a month ) by the consumption amount on a single occasion , at 10 g per drink . hepatic steatosis was diagnosed if he / she had the following components at liver us : ultrasound beam attenuation and poor visualization of intra - hepatic vessel borders and diaphragm , evidence of diffuse hyper - echogenicity of liver relative to kidneys . the intra - observer variability for the ultrasound diagnosis of hepatic steatosis was within 3%.15 we diagnosed nafld by relying on the presence of hepatic steatosis as confirmed by liver us when the following criteria were absent : a ) excess alcohol consumption defined as > 210 g / week in men and > 140 g / week in women based on the kasl definition , b ) causes for secondary hepatic fat accumulation such as steatogenic medications or hereditary disorders c ) positivity for hepatitis b surface antigen or antibody to hepatitis c virus . afld was diagnosed based on a combination of features including ongoing or recent excessive alcohol consumption with hepatic steatosis confirmed by liver us . definition of metabolic syndrome was based on the world health organization - west pacific region guideline16 and insulin resistance was estimated by homeostasis model assessment of insulin resistance.17 among various framingham - based tools , we used the conventional framingham risk score ( frs ) from wilson et al 199818 to estimate total chd risk at 10-year . for hard chd risk estimation , we used the revised frs form , where atp iii risk factor scoring is incorporated into the 10-year risk assessment tool.19 the frs was calculated based on six coronary risk factors : gender , age , total cholesterol , hdl - c , systolic bp and smoking habit . finally , the corresponding point was assigned to each individual and the total score was used as the individual 's chd risk level.19 quantitative data was expressed as meanstandard deviation ( sd ) , and categorical data as percentages . one - way analysis of covariance ( ancova ) was used to compare the means of frs and 10-year risk for total and hard chd between the control and fatty liver group , and between the nafld and afld group after adjustment for the main differences between the groups ( age , sex , metabolic syndrome , smoking and family history of chd ) . relying on liver us , we discriminated 4,142 fatty liver subjects ( 38.7% ) , with the remaining 6,568 ( 61.3% ) constituting the control group . the majority of overall group comparisons showed significant differences , with higher mean values in the population with fatty liver as compared to the control group , except for waist - to - hip ratio ( p=0.08 ) and hdl - c which was higher in the control group ( p<0.001 ) . of the 4,142 subjects with fatty liver , 71.3% ( 2,953 ) had nafld and 28.7% ( 1,189 ) had afld . subjects with afld were more likely to be younger ( 46.2 vs. 44.3 years , p<0.001 ) , men ( 62.4 vs. 92.3% , p<0.001 ) , and were also more likely to be smokers ( 36.8 vs.74.6% , p<0.001 ) , with all these variables showing significant differences compared to the nafld group . subjects with nafld and afld showed comparable results for homa - ir , plasma level of hdl - c , low - density lipoprotein cholesterol ( ldl - c ) , serum high - sensitive c - reactive protein ( hscrp ) and daily physical activity . table 3 shows comparisons of mean 10-year estimated total risk between the control and fatty liver group , and the nafld and afld group . the mean 10-year risk for total chd was higher in the fatty liver group than the matched control group . individuals with afld had a higher 10-year estimated chd risk compared to the control group ( 6.720.12 vs 5.500.04 p<0.001 ) and a comparable risk to those with nafld ( 6.720.12 vs 7.320.07 , p=0.02 ) . the prevalence of total chd risk over 10% ( more than moderate risk ) and over 20% ( high risk ) were significantly higher in the fatty liver group as compared to the control . relying on liver us , we discriminated 4,142 fatty liver subjects ( 38.7% ) , with the remaining 6,568 ( 61.3% ) constituting the control group . the majority of overall group comparisons showed significant differences , with higher mean values in the population with fatty liver as compared to the control group , except for waist - to - hip ratio ( p=0.08 ) and hdl - c which was higher in the control group ( p<0.001 ) . of the 4,142 subjects with fatty liver , 71.3% ( 2,953 ) had nafld and 28.7% ( 1,189 ) had afld . subjects with afld were more likely to be younger ( 46.2 vs. 44.3 years , p<0.001 ) , men ( 62.4 vs. 92.3% , p<0.001 ) , and were also more likely to be smokers ( 36.8 vs.74.6% , p<0.001 ) , with all these variables showing significant differences compared to the nafld group . subjects with nafld and afld showed comparable results for homa - ir , plasma level of hdl - c , low - density lipoprotein cholesterol ( ldl - c ) , serum high - sensitive c - reactive protein ( hscrp ) and daily physical activity . table 3 shows comparisons of mean 10-year estimated total risk between the control and fatty liver group , and the nafld and afld group . the mean 10-year risk for total chd was higher in the fatty liver group than the matched control group . individuals with afld had a higher 10-year estimated chd risk compared to the control group ( 6.720.12 vs 5.500.04 p<0.001 ) and a comparable risk to those with nafld ( 6.720.12 vs 7.320.07 , p=0.02 ) . the prevalence of total chd risk over 10% ( more than moderate risk ) and over 20% ( high risk ) were significantly higher in the fatty liver group as compared to the control . however , between nafld and afld , we obtained inconsistent results showing a higher prevalence of total chd events greater than 10% in the afld group , whereas total chd over 20% showed no unidirectional tendency ( table 4 ) . the aim of the current study was to determine whether subjects with afld have an elevated chd risk using frs and to compare it with that for nafld . we found a strong association between afld and chd risk comparable to nafld . in addition , afld was a strong predictor elevated chd risk ( chd risk > 10% ) independent of traditional risk factors . only study comparing the frequency of coronary artery disease in alcohol - related versus non - alcoholic end - stage liver disease ( esld ) is available in the literature.22 in this study , 420 patients with esld either alcohol - related or non - alcohol related , underwent coronary angiography for candidate screening of liver transplantation . another research by jh kim and colleagues that examines 265 fatty liver subjects has recently been published providing comparison of carotid intima - media thickness ( cimt ) between individuals with nafld and afld.23 the relationship between increasing cimt and incident cvd events has been clearly established24 and several prospective , population - based studies demonstrated that the presence of carotid plaque is associated with a significantly increased risk for myocardial infarction , stroke and chd death , independent of traditional risk factors.25 in this study , cimt was elevated in subjects with fatty liver in both nafld and afld patients , in agreement with our results . the prevalence of nafld based on liver us was 27.6% in our study , consistent with the previous reports between 17 and 46%.26,27 although reference data on the prevalence of afld is limited , there is a study using the population - based fin - d2d survey , in which a 7.0% of prevalence is reported.28 we found a higher prevalence of afld ( 11.1% ) , with this discrepancy possibly due to the high alcohol consumption rate in the korean population . indeed 90% of all alcoholics smoke and alcoholism is ten times more prevalent among smokers than non - smokers.30 however the fact that the prevalence of essential hypertension , and metabolic syndrome is higher in individuals with afld compared to nafld suggests the new possibility that in some afld subjects , the cvd risk may be higher than our expectation and further investigation should be conducted . another important strength of the current article is that it is the first study comparing nafld and afld populations in terms of chd risk and metabolic profiles . there has been accumulating evidence for a strong association between nafld and elevated chd risk over the last years.31 although the pathogenesis behind this link has not been fully elucidated , the contributions of obesity and metabolic syndrome to increased chd events are well known.21,32 by contrast , the association of afld with chd risk had not been investigated . in this study , we have demonstrated a significant increase in 10-year chd risk not only in subjects with nafld , but also those with afld , a close association that was independent of age , sex , homa - ir , or the individual components of metabolic syndrome , implying that afld is a strong independent predictor for developing chd events within 10 years . it is also significant that we compared subjects with nafld and afld in terms of future chd risk by estimating 10 year total chd risk . thus mild steatosis ( mild nafld or afld ) might have been included in the control group . in summary , subjects with fatty liver had significantly higher estimated 10-year total chd risk compared to the control group , not only for nafld but also for afld . therefore , afld should be considered as a meaningful risk for future chd events and preventive measures should be considered earlier .

the embl ( european molecular biology laboratory ) nucleotide sequence database ( often referred to as the embl database ) is hosted at the european bioinformatics institute ( ebi ) . it is a comprehensive database of dna and rna sequences that are directly submitted from researchers and genome sequencing groups , and collected from the scientific literature and patent applications . it is produced in an international collaboration with genbank ( ncbi , bethesda , usa ) and ddbj ( the dna data bank of japan , cib , mishima , japan ) . each of the three collaborating groups collects a portion of the total sequence data reported worldwide , and all new and updated database entries are exchanged daily . the amount of sequence data is growing exponentially . as our scientific understanding deepens , the complexity of the related information increases as well . as a result the embl database is managed and maintained using the relational database management system ( dbms ) oracle . it contains over 130 tables and 140 relationships , having around 80 gigabytes ( gb ) of data comprising nearly 10 million objects of primary data and millions of sub - objects called ' features ' . traditionally , the sequences and related information , which have been collected over a long period of time , are made available in flat - file format via ftp , cd - rom , www tools , and so on . the queries through tools such as srs ( sequence retrieval system , a network browser for databanks in molecular biology ) also return data in flat - file format . however , flat files have a number of shortcomings : the format may not be described formally ; it is difficult to represent complex data and relationships , the meaningful units of information ( ' objects ' ) are not represented or handled well ; it is hard to retrieve objects separately ; assembly of objects into bigger aggregates is difficult ; elaborate parsing is often required ; and so on . in general , the current availability of the resources is not matched by a flexible environment to meet individual researchers ' needs . an industry standard , the object management group 's ( omg ) common object request broker architecture ( corba ) , provides platform - independent programming interfaces and models for portable distributed object - oriented computing applications . its independence from programming languages , computing platforms and network protocols provides a solution for developing new applications for querying and distributing biological data , which can also be integrated into existing systems . here we present a corba infrastructure developed at embl - ebi and show that the corba interfaces to the embl database address some of the limitations of the flat - file format and provide an efficient means for accessing and distributing embl data . corba also provides a flexible environment for users to develop application programs ( for example , for sequence analysis or data mining ) . the diversity and structure of biological data complicate their use . to develop a corba server that provides access to our biological data , we need a well - defined object model to model the real - world biological entities , that is , to describe the structure and constraints present in the data , as well as how the data can be accessed and queried . it is a specification of the data in the problem domain , independent of how the actual database is implemented . this model can then be expressed in idl ( interface definition language ) interfaces to the corba server at one end , and mapped to a database schema for the underlying data management and storage at the other end . we use the unified modeling language ( uml ) notation for this model . according to the uml , a model is organized into packages . a package groups classes that are semantically related and a dependency indicates that a package uses classes from another package a class is a descriptor for a set of objects that have similar properties , behavior and relationships to other objects . an attribute can be derived , that is , its value is computed from the values of other attributes . an operation is a procedure attached to a class , describing the behavior of the class . a class can be created by inheriting all attributes and operations from one or more of its superclasses . an association is a description of links or a set of links that specify connections among objects . it can be seen as an association that also has class properties , or as a class that also has association properties . it holds data that are relevant for the association , but for neither of the associated classes alone . a structured ( composite ) data type is represented as a class ( as usual in object - oriented ( oo ) modeling ) . a class is used as an attribute type mainly if its role is solely to bundle simple data into a composite type ( for example , date ) . multi - valued attributes are represented by instances of a parameterized class coll{type } ( for example , coll{string } ) . our object model of the embl database is organised into five main packages , as shown in figure 1 , where each package holds a set of closely related classes with a common purpose . the packages are : sequence info , classes representing biological sequences , general information about these sequences and administrative data associated with database entries ; feature info , classes representing detailed sequence annotation ( known as sequence features ) ; reference info , classes representing bibliographic references that hold information about the sequences ; taxonomy info , classes representing the taxonomy of the organisms from which the sequences were obtained ; location info , classes representing locations on sequences . the database is divided into five main packages : sequence info , all general information about sequences ; feature info , detailed sequence annotation ; reference info , bibliographic references ; taxonomy info , the taxonomy of the organisms from which the sequences were obtained ; location info , representing locations on sequences . there is one additional package , types , which holds classes representing all the special data types used in various parts of the model . each package contains a relatively isolated part of the entire object model , and is a clear candidate for re - use in models for other databases . a full definition of the embl nucleotide sequence database object model can be found in additional data file 1 with the online version of this article . the package sequence info defines class bioseq , which represents biological sequences , and class seqlnfo , which describes general information about these sequences . the biological classes of sequence nsdbseq , for nucleotide sequences , and pidseq , for protein sequences , are subclasses of bioseq . virtualseq and physicalseq are storage classes of sequence , that is , virtual or literal . this package defines class bioseq , which represents biological sequences , and class seqinfo , which describes general information about these sequences . the biological classes of sequence nsdbseq , which is for nucleotide sequences , and pidseq , which is for protein sequences , are subclasses of bioseq . virtualseq and physicalseq are storage classes of sequence , that is , virtual or literal . the definition of some biological entities is prone to change because of the rapid developments in molecular biology . any change made to the structure of the model needs to be propagated to both the idl specification that defines the corba server interfaces and underlying relational schema . to handle this problem , a strategy of using both explicit model and meta model the structure of the model is therefore not affected by changes to the feature definition , which makes it suitable for defining stable idl interfaces . the embl corba server mainly covers sequence info , location info and feature info , which are grouped into a big package that also includes reference info and taxonomy info . on the server side , corba implementation objects access and query the relational database via persistence ( persistence software ) , which acts as a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) , where the distribution details are handled by the orb . corba implementation objects access and query the relational database via persistence , which is a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) where the distribution details are handled by the orb . corba object interfaces together with their operations and type of data are defined in idl . for the orb , clients can be written in any language for which an orb and idl compiler are available , including ada , c , c++ , cobol , commonlisp , eiffel , java , python , peri , smalltalk , tcl , and so on . note that we do not use the new features in corba 2.3 , as orbs that implement corba 2.3 have only become available recently . corba object interfaces together with their operations and type of data are defined in idl . clients can be written in any language for which an orb and idl compiler are available , including ada , c , c++ , cobol , commonlisp , eiffel , java , python , perl , smalltalk , tcl , and so on . the omg idl is corba 's fundamental abstraction mechanism for separating object interfaces from their implementations . it allows object interfaces to be defined in a manner that is independent of any particular programming language . it establishes a contract between client and server that describes the types and object interfaces used by an application . idl definitions focus on object interfaces , the operations supported by those interfaces , and exceptions that might be raised by the operations . as data can only be exchanged between client and server if their types are defined in idl , typically a large part of an idl is concerned with the definition of data types . following the embl data model , the idl definition for the embl server comprises three idl files : nsdb.idl , seqdb.idl and types.idl . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . the seqdb.idl defines the module seqdb that includes three interfaces : bioseq , seqlnfo and feature , which contain more general biological sequence information . the nsdb.idl and seqdb.idl use basic types defined in types.idl . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . these objects are instances of ' data classes ' , which are the results of queries . figure 5 gives the idl specification of interfaces bioseq and seqlnfo in module seqdb , and entrylnfo , nucseq and emblseq in module nsdb ( the full idl definition can be found in the additional data file with the online version of this article ) ) . the interfaces bioseq and seqinfo are defined in module seqdb ; the interfaces entryinfo , nucseq and emblseq are defined in module nsdb . each idl interface is mapped into a class in c++ by the orb 's idl compiler ( in our case , the orb is iona 's orbix ) , and operations are mapped to member functions of the class . for the above interfaces bioseq and seqlnfo , we have two mapped classes as shown in figure 6 the returned values of methods represent attributes of class bioseq defined in the object model , providing information on biological sequences . the class seqinfo has methods : getdescription , getkeywords , getcomments , getdbxrefs and getreferences , representing general information on the sequences . although it can normally be mapped to a namespace , our c++ compiler ( sun 's sparcworks 4.2 ) does not support namespaces . the relationship between classes is shown in figure 7 in uml notation . emblseq inherits from the classes of entrylnfo , seqlnfo and nucseq that in turn inherits from bioseq . emblseq inherits from the classes of entryinfo , seqinfo and nucseq that in turn inherits from bioseq . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . it maps objects to relational rows and manages the objects in a shared cache , called the live object cache . it uses a proprietary object model description that maps classes to tables , objects to rows , attributes to columns and associations to foreign keys . for inheritance relationships ( single inheritance only ) , persistence insists on a so - called horizontal mapping for performance reasons ; that is , in the class hierarchy , only leaf nodes are represented by real database tables ( or views ) . non - leaf class objects are obtained as projections of the leaf class tables . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . in this case , each node in the class hierarchy has its own table , with subclass tables having no superclass attributes ; their primary keys are also foreign keys to the superclass table . as our objects provide read - only access , it is possible to set up relational views that transform our tables into the horizontal object - to - relational mapping that is required by persistence . for example , for the class nsdbseq that is inherited from bioseq , its so - called persistence horizontal object - to - relational mapping ( view ) is shown in figure 8 . this view is built from a number of tables ( or views ) as shown in figure 9 . the corba class emblseq is mapped to the view of nsdbseq . the nsdbseq view in persistence . as the class nsdnseq inherits from the class bioseq , the view of nsdbseq in persistence therefore has attributes defined in both nsdbseq and bioseq , representing information on nucleotide sequences . the view is built from a number of tables ( or views ) of the database . using the data model and schema description , persistence can also offer an automatic generation of an idl specification as well as a complete corba server . we have not used this facility , however , as we want full control over the idl specification . this approach has an advantage in using views even when a one - to - one mapping from idl to embl tables remains . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . we much less frequently require a change on the persistence mapping and code as these can still map the changed tables or data to the same objects at the corba side . the basic model for managing live objects is to cache data instances read from the database , to register their primary key values , and to respond to queries based on the cached data . as tuples are retrieved from the database , they are converted to objects and ' knitted ' together according to the object - model mapping to form a network of in - memory objects . a live object cache maps information from relational tables into objects . accessing and manipulating these objects in the live object cache is faster than querying the relational database , speeding up application performance considerably . there are roughly three types of objects involved here : persistent objects , live objects , and corba objects . here creation of a corba object is called instantiation . when a persistent object is loaded into memory note that the orb 's object adapter , no matter whether a basic object adapter ( boa ) or portable object adapter ( poa ) , only serves as the glue between corba objects and the orb . it is an object that adapts the interface of one object to a different interface expected by a caller and allows the caller to invoke requests on an object without knowing the object 's true interface . although the future corba may include garbage collection , the management of objects is currently at the application developer 's discretion . data kept in the database are only loaded into the cache on an as - needed basis . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . when an operation invocation arrives at the process , orbix orb searches for the target object in the process 's object table . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . objects are created at the clients ' request . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . the basic idea is that we use an object manager to instantiate objects on demand . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . if the called object is already in the pool , it can be used directly . if not if so , the object manager will evict an older instantiated object and then instantiate a new one for the current request . consequently , the older related persistence live objects will also be deleted from the cache and new live objects will be loaded in if requested . one more interesting issue of the evictor pattern is how to choose which object to evict . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . we use a simple lru algorithm to implement the evictor and prove it is effective . when the corba server is up and running , a client , which can be developed using any corba - compliant orb on the user 's preferred environment and language ( for which the orb is available ) at any local or remote machine , can access embl data through these objects using an ior ( interoperable object reference ) or via a naming service . we have published our embl server ior and its naming as ' databases / embl / nsdb / embl ' , which is registered with the naming service . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . it invokes the operation findme provided by emblseq object , which in turn invokes the loader object . once the client has the emblseq object reference , it can then invoke the methods provided by emblseq to get the sequence information defined in seqlnfo . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . it invokes the operation findme provided by emblseq object , which in turn invokes the loader object . once the client has the emblseq object reference , it can then invoke the methods provided by emblseq to get the sequence information defined in seqinfo . , it can be seen that corba interfaces to the embl database provide : meaningful units of information : objects ; encapsulated methods , for example getlength ( ) ; interoperation between objects ; easy access and distribution of data ; and easy to comply with the standard . accessibility to the embl database via the embl corba server can be as fine as any small attribute defined in the embl data model . the embl corba server can also provide a blob object that contains a number of objects . currently the embl server is undergoing a trial with internal and external users . the diversity and structure of biological data complicate their use . to develop a corba server that provides access to our biological data , we need a well - defined object model to model the real - world biological entities , that is , to describe the structure and constraints present in the data , as well as how the data can be accessed and queried . it is a specification of the data in the problem domain , independent of how the actual database is implemented . this model can then be expressed in idl ( interface definition language ) interfaces to the corba server at one end , and mapped to a database schema for the underlying data management and storage at the other end . we use the unified modeling language ( uml ) notation for this model . according to the uml , a model is organized into packages . a package groups classes that are semantically related and a dependency indicates that a package uses classes from another package a class is a descriptor for a set of objects that have similar properties , behavior and relationships to other objects . an attribute can be derived , that is , its value is computed from the values of other attributes . an operation is a procedure attached to a class , describing the behavior of the class . a class can be created by inheriting all attributes and operations from one or more of its superclasses . an association is a description of links or a set of links that specify connections among objects . it can be seen as an association that also has class properties , or as a class that also has association properties . it holds data that are relevant for the association , but for neither of the associated classes alone . a structured ( composite ) data type is represented as a class ( as usual in object - oriented ( oo ) modeling ) . a class is used as an attribute type mainly if its role is solely to bundle simple data into a composite type ( for example , date ) . multi - valued attributes are represented by instances of a parameterized class coll{type } ( for example , coll{string } ) . our object model of the embl database is organised into five main packages , as shown in figure 1 , where each package holds a set of closely related classes with a common purpose . the packages are : sequence info , classes representing biological sequences , general information about these sequences and administrative data associated with database entries ; feature info , classes representing detailed sequence annotation ( known as sequence features ) ; reference info , classes representing bibliographic references that hold information about the sequences ; taxonomy info , classes representing the taxonomy of the organisms from which the sequences were obtained ; location info , classes representing locations on sequences . the database is divided into five main packages : sequence info , all general information about sequences ; feature info , detailed sequence annotation ; reference info , bibliographic references ; taxonomy info , the taxonomy of the organisms from which the sequences were obtained ; location info , representing locations on sequences . there is one additional package , types , which holds classes representing all the special data types used in various parts of the model . each package contains a relatively isolated part of the entire object model , and is a clear candidate for re - use in models for other databases . a full definition of the embl nucleotide sequence database object model can be found in additional data file 1 with the online version of this article . the package sequence info defines class bioseq , which represents biological sequences , and class seqlnfo , which describes general information about these sequences . the biological classes of sequence nsdbseq , for nucleotide sequences , and pidseq , for protein sequences , are subclasses of bioseq . virtualseq and physicalseq are storage classes of sequence , that is , virtual or literal . this package defines class bioseq , which represents biological sequences , and class seqinfo , which describes general information about these sequences . the biological classes of sequence nsdbseq , which is for nucleotide sequences , and pidseq , which is for protein sequences , are subclasses of bioseq . virtualseq and physicalseq are storage classes of sequence , that is , virtual or literal . the definition of some biological entities is prone to change because of the rapid developments in molecular biology . any change made to the structure of the model needs to be propagated to both the idl specification that defines the corba server interfaces and underlying relational schema . to handle this problem , a strategy of using both explicit model and meta model the structure of the model is therefore not affected by changes to the feature definition , which makes it suitable for defining stable idl interfaces . the embl corba server mainly covers sequence info , location info and feature info , which are grouped into a big package that also includes reference info and taxonomy info . the system architecture is shown in figure 3 . on the server side , corba implementation objects access and query the relational database via persistence ( persistence software ) , which acts as a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) , where the distribution details are handled by the orb . system architecture . on the server side , corba implementation objects access and query the relational database via persistence , which is a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) where the distribution details are handled by the orb . corba object interfaces together with their operations and type of data are defined in idl . for the orb , clients can be written in any language for which an orb and idl compiler are available , including ada , c , c++ , cobol , commonlisp , eiffel , java , python , peri , smalltalk , tcl , and so on . note that we do not use the new features in corba 2.3 , as orbs that implement corba 2.3 have only become available recently . corba object interfaces together with their operations and type of data are defined in idl . for the orb , clients can be written in any language for which an orb and idl compiler are available , including ada , c , c++ , cobol , commonlisp , eiffel , java , python , perl , smalltalk , tcl , and so on . the omg idl is corba 's fundamental abstraction mechanism for separating object interfaces from their implementations . it allows object interfaces to be defined in a manner that is independent of any particular programming language . it establishes a contract between client and server that describes the types and object interfaces used by an application . idl definitions focus on object interfaces , the operations supported by those interfaces , and exceptions that might be raised by the operations . as data can only be exchanged between client and server if their types are defined in idl , typically a large part of an idl is concerned with the definition of data types . following the embl data model , the idl definition for the embl server comprises three idl files : nsdb.idl , seqdb.idl and types.idl . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . the seqdb.idl defines the module seqdb that includes three interfaces : bioseq , seqlnfo and feature , which contain more general biological sequence information . the nsdb.idl and seqdb.idl use basic types defined in types.idl . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . these objects are instances of ' data classes ' , which are the results of queries . figure 5 gives the idl specification of interfaces bioseq and seqlnfo in module seqdb , and entrylnfo , nucseq and emblseq in module nsdb ( the full idl definition can be found in the additional data file with the online version of this article ) ) . the interfaces bioseq and seqinfo are defined in module seqdb ; the interfaces entryinfo , nucseq and emblseq are defined in module nsdb . each idl interface is mapped into a class in c++ by the orb 's idl compiler ( in our case , the orb is iona 's orbix ) , and operations are mapped to member functions of the class . for the above interfaces bioseq and seqlnfo , we have two mapped classes as shown in figure 6 . the returned values of methods represent attributes of class bioseq defined in the object model , providing information on biological sequences . the class seqinfo has methods : getdescription , getkeywords , getcomments , getdbxrefs and getreferences , representing general information on the sequences . although it can normally be mapped to a namespace , our c++ compiler ( sun 's sparcworks 4.2 ) does not support namespaces . the relationship between classes is shown in figure 7 in uml notation . emblseq inherits from the classes of entrylnfo , seqlnfo and nucseq that in turn inherits from bioseq . emblseq inherits from the classes of entryinfo , seqinfo and nucseq that in turn inherits from bioseq . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . it maps objects to relational rows and manages the objects in a shared cache , called the live object cache . it uses a proprietary object model description that maps classes to tables , objects to rows , attributes to columns and associations to foreign keys . for inheritance relationships ( single inheritance only ) , persistence insists on a so - called horizontal mapping for performance reasons ; that is , in the class hierarchy , only leaf nodes are represented by real database tables ( or views ) . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . in this case , each node in the class hierarchy has its own table , with subclass tables having no superclass attributes ; their primary keys are also foreign keys to the superclass table . as our objects provide read - only access , it is possible to set up relational views that transform our tables into the horizontal object - to - relational mapping that is required by persistence . for example , for the class nsdbseq that is inherited from bioseq , its so - called persistence horizontal object - to - relational mapping ( view ) is shown in figure 8 . this view is built from a number of tables ( or views ) as shown in figure 9 . the corba class emblseq is mapped to the view of nsdbseq . the nsdbseq view in persistence . as the class nsdnseq inherits from the class bioseq , the view of nsdbseq in persistence therefore has attributes defined in both nsdbseq and bioseq , representing information on nucleotide sequences . the view is built from a number of tables ( or views ) of the database . using the data model and schema description , persistence can also offer an automatic generation of an idl specification as well as a complete corba server . we have not used this facility , however , as we want full control over the idl specification . this approach has an advantage in using views even when a one - to - one mapping from idl to embl tables remains . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . we much less frequently require a change on the persistence mapping and code as these can still map the changed tables or data to the same objects at the corba side . the omg idl is corba 's fundamental abstraction mechanism for separating object interfaces from their implementations . it allows object interfaces to be defined in a manner that is independent of any particular programming language . it establishes a contract between client and server that describes the types and object interfaces used by an application . idl definitions focus on object interfaces , the operations supported by those interfaces , and exceptions that might be raised by the operations . as data can only be exchanged between client and server if their types are defined in idl , typically a large part of an idl is concerned with the definition of data types . following the embl data model , the idl definition for the embl server comprises three idl files : nsdb.idl , seqdb.idl and types.idl . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . the seqdb.idl defines the module seqdb that includes three interfaces : bioseq , seqlnfo and feature , which contain more general biological sequence information . the nsdb.idl and seqdb.idl use basic types defined in types.idl . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . these objects are instances of ' data classes ' , which are the results of queries . figure 5 gives the idl specification of interfaces bioseq and seqlnfo in module seqdb , and entrylnfo , nucseq and emblseq in module nsdb ( the full idl definition can be found in the additional data file with the online version of this article ) ) . the interfaces bioseq and seqinfo are defined in module seqdb ; the interfaces entryinfo , nucseq and emblseq are defined in module nsdb . each idl interface is mapped into a class in c++ by the orb 's idl compiler ( in our case , the orb is iona 's orbix ) , and operations are mapped to member functions of the class . for the above interfaces bioseq and seqlnfo , we have two mapped classes as shown in figure 6 the returned values of methods represent attributes of class bioseq defined in the object model , providing information on biological sequences . the class seqinfo has methods : getdescription , getkeywords , getcomments , getdbxrefs and getreferences , representing general information on the sequences . the module itself is also mapped into a class here . although it can normally be mapped to a namespace , our c++ compiler ( sun 's sparcworks 4.2 ) does not support namespaces . the relationship between classes is shown in figure 7 in uml notation . emblseq inherits from the classes of entrylnfo , seqlnfo and nucseq that in turn inherits from bioseq . emblseq inherits from the classes of entryinfo , seqinfo and nucseq that in turn inherits from bioseq . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . it maps objects to relational rows and manages the objects in a shared cache , called the live object cache . it uses a proprietary object model description that maps classes to tables , objects to rows , attributes to columns and associations to foreign keys . for inheritance relationships ( single inheritance only ) , persistence insists on a so - called horizontal mapping for performance reasons ; that is , in the class hierarchy , only leaf nodes are represented by real database tables ( or views ) . non - leaf class objects are obtained as projections of the leaf class tables . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . in this case , each node in the class hierarchy has its own table , with subclass tables having no superclass attributes ; their primary keys are also foreign keys to the superclass table . as our objects provide read - only access , it is possible to set up relational views that transform our tables into the horizontal object - to - relational mapping that is required by persistence . for example , for the class nsdbseq that is inherited from bioseq , its so - called persistence horizontal object - to - relational mapping ( view ) is shown in figure 8 . this view is built from a number of tables ( or views ) as shown in figure 9 . as the class nsdnseq inherits from the class bioseq , the view of nsdbseq in persistence therefore has attributes defined in both nsdbseq and bioseq , representing information on nucleotide sequences . the view is built from a number of tables ( or views ) of the database . using the data model and schema description , persistence can also offer an automatic generation of an idl specification as well as a complete corba server . we have not used this facility , however , as we want full control over the idl specification . this approach has an advantage in using views even when a one - to - one mapping from idl to embl tables remains . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . we much less frequently require a change on the persistence mapping and code as these can still map the changed tables or data to the same objects at the corba side . the basic model for managing live objects is to cache data instances read from the database , to register their primary key values , and to respond to queries based on the cached data . as tuples are retrieved from the database , they are converted to objects and ' knitted ' together according to the object - model mapping to form a network of in - memory objects . a live object cache maps information from relational tables into objects . accessing and manipulating these objects in the live object cache is faster than querying the relational database , speeding up application performance considerably . there are roughly three types of objects involved here : persistent objects , live objects , and corba objects . here creation of a corba object is called instantiation . when a persistent object is loaded into memory note that the orb 's object adapter , no matter whether a basic object adapter ( boa ) or portable object adapter ( poa ) , only serves as the glue between corba objects and the orb . it is an object that adapts the interface of one object to a different interface expected by a caller and allows the caller to invoke requests on an object without knowing the object 's true interface . although the future corba may include garbage collection , the management of objects is currently at the application developer 's discretion . data kept in the database are only loaded into the cache on an as - needed basis . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . when an operation invocation arrives at the process , orbix orb searches for the target object in the process 's object table . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . the basic idea is that we use an object manager to instantiate objects on demand . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . if the called object is already in the pool , it can be used directly . if not if so , the object manager will evict an older instantiated object and then instantiate a new one for the current request . consequently , the older related persistence live objects will also be deleted from the cache and new live objects will be loaded in if requested . one more interesting issue of the evictor pattern is how to choose which object to evict . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . we use a simple lru algorithm to implement the evictor and prove it is effective . data kept in the database are only loaded into the cache on an as - needed basis . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . when an operation invocation arrives at the process , orbix orb searches for the target object in the process 's object table . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . the basic idea is that we use an object manager to instantiate objects on demand . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . if the called object is already in the pool , it can be used directly . if not if so , the object manager will evict an older instantiated object and then instantiate a new one for the current request . consequently , the older related persistence live objects will also be deleted from the cache and new live objects will be loaded in if requested . one more interesting issue of the evictor pattern is how to choose which object to evict . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . we use a simple lru algorithm to implement the evictor and prove it is effective . when the corba server is up and running , a client , which can be developed using any corba - compliant orb on the user 's preferred environment and language ( for which the orb is available ) at any local or remote machine , can access embl data through these objects using an ior ( interoperable object reference ) or via a naming service . we have published our embl server ior and its naming as ' databases / embl / nsdb / embl ' , which is registered with the naming service . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . it invokes the operation findme provided by emblseq object , which in turn invokes the loader object . once the client has the emblseq object reference , it can then invoke the methods provided by emblseq to get the sequence information defined in seqlnfo . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . it invokes the operation findme provided by emblseq object , which in turn invokes the loader object . once the client has the emblseq object reference , it can then invoke the methods provided by emblseq to get the sequence information defined in seqinfo . , it can be seen that corba interfaces to the embl database provide : meaningful units of information : objects ; encapsulated methods , for example getlength ( ) ; interoperation between objects ; easy access and distribution of data ; and easy to comply with the standard . accessibility to the embl database via the embl corba server can be as fine as any small attribute defined in the embl data model . the embl corba server can also provide a blob object that contains a number of objects . the embl object model provides a basis to develop the corba server . employing persistence maps the object model to the relational schema in the underlying oracle database . to present persistence with the right relations , properly built loaders make use of the technique of ' live object caching ' and enhance the performance . it has been demonstrated that the corba server addresses some problems of the flat - file format and provides a solution to accessing and distributing embl sequence data . it also provides a flexible and scalable environment for users to develop their applications by building clients . the future work will include migrating the implementation of the embl server to comply with the emerging standard - omg standard for biosequences . by omg rules , the ebi , as a co - submitter on the biomolecular sequence analysis ( bsa ) standard , is not fully compatible with the embl idl specification currently used , care will have to be taken to make this transition as easy as possible for existing clients . the following additional data are included with the online version of this article : the embl nucleotide sequence database object model and the embl idl specification . embl nucleotide sequence database object model it contains the idl belonging to the embl corba servers : the home page of the corba work at ebi is . for questions and suggestions , please feel free to contact corba@ebi.ac.uk . general types used throughout the embl corba project ( save as types.idl ) general sequence interfaces ( save as seqdb.idl ) sequence interfaces specialised for embl ( needs seqdb.idl ) ( save as nsdb.idl ) genetic code used for translation of nucleotide sequences in embl ( save as geneticcode.idl ) the idl for the reference server : access to the bibliographic references of embl ( save as publication.idl ) the idl for the taxonomy server : access to the taxonomy information of embl ( save as taxonomy.idl ) meta information of the embl servers ( save as meta.idl ) a file with only ' # include ' statements for the above idl files , for easy compilation ( save as embl.idl ) makefile is provided ; it can be used to compile the idl using one of three commonly used orbs , i.e. , orbacus , visibroker or orbixweb ( save as makefile ) .

background : the embl nucleotide sequence database is a comprehensive database of dna and rna sequences and related information traditionally made available in flat - file format . queries through tools such as srs ( sequence retrieval system ) also return data in flat - file format . flat files have a number of shortcomings , however , and the resources therefore currently lack a flexible environment to meet individual researchers ' needs . the object management group 's common object request broker architecture ( corba ) is an industry standard that provides platform - independent programming interfaces and models for portable distributed object - oriented computing applications . its independence from programming languages , computing platforms and network protocols makes it attractive for developing new applications for querying and distributing biological data.results:a corba infrastructure developed by embl - ebi provides an efficient means of accessing and distributing embl data . the embl object model is defined such that it provides a basis for specifying interfaces in interface definition language ( idl ) and thus for developing the corba servers . the mapping from the object model to the relational schema in the underlying oracle database uses the facilities provided by persistencetm , an object / relational tool . the techniques of developing loaders and ' live object caching ' with persistent objects achieve a smart live object cache where objects are created on demand . the objects are managed by an evictor pattern mechanism.conclusions:the corba interfaces to the embl database address some of the problems of traditional flat - file formats and provide an efficient means for accessing and distributing embl data . corba also provides a flexible environment for users to develop their applications by building clients to our corba servers , which can be integrated into existing systems .

Background Results and discussion EMBL object data model System architecture and CORBA development IDL definition Class relationships Object-relational mapping Object management Creation of live objects Eviction of CORBA objects Accessibility of the EMBL database Conclusions Additional data Supplementary Material

the embl ( european molecular biology laboratory ) nucleotide sequence database ( often referred to as the embl database ) is hosted at the european bioinformatics institute ( ebi ) . it is a comprehensive database of dna and rna sequences that are directly submitted from researchers and genome sequencing groups , and collected from the scientific literature and patent applications . as a result the embl database is managed and maintained using the relational database management system ( dbms ) oracle . traditionally , the sequences and related information , which have been collected over a long period of time , are made available in flat - file format via ftp , cd - rom , www tools , and so on . the queries through tools such as srs ( sequence retrieval system , a network browser for databanks in molecular biology ) also return data in flat - file format . however , flat files have a number of shortcomings : the format may not be described formally ; it is difficult to represent complex data and relationships , the meaningful units of information ( ' objects ' ) are not represented or handled well ; it is hard to retrieve objects separately ; assembly of objects into bigger aggregates is difficult ; elaborate parsing is often required ; and so on . in general , the current availability of the resources is not matched by a flexible environment to meet individual researchers ' needs . an industry standard , the object management group 's ( omg ) common object request broker architecture ( corba ) , provides platform - independent programming interfaces and models for portable distributed object - oriented computing applications . its independence from programming languages , computing platforms and network protocols provides a solution for developing new applications for querying and distributing biological data , which can also be integrated into existing systems . here we present a corba infrastructure developed at embl - ebi and show that the corba interfaces to the embl database address some of the limitations of the flat - file format and provide an efficient means for accessing and distributing embl data . corba also provides a flexible environment for users to develop application programs ( for example , for sequence analysis or data mining ) . to develop a corba server that provides access to our biological data , we need a well - defined object model to model the real - world biological entities , that is , to describe the structure and constraints present in the data , as well as how the data can be accessed and queried . it is a specification of the data in the problem domain , independent of how the actual database is implemented . this model can then be expressed in idl ( interface definition language ) interfaces to the corba server at one end , and mapped to a database schema for the underlying data management and storage at the other end . our object model of the embl database is organised into five main packages , as shown in figure 1 , where each package holds a set of closely related classes with a common purpose . each package contains a relatively isolated part of the entire object model , and is a clear candidate for re - use in models for other databases . a full definition of the embl nucleotide sequence database object model can be found in additional data file 1 with the online version of this article . any change made to the structure of the model needs to be propagated to both the idl specification that defines the corba server interfaces and underlying relational schema . to handle this problem , a strategy of using both explicit model and meta model the structure of the model is therefore not affected by changes to the feature definition , which makes it suitable for defining stable idl interfaces . on the server side , corba implementation objects access and query the relational database via persistence ( persistence software ) , which acts as a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . clients access the corba objects via operation calls through an object request broker ( orb ) , where the distribution details are handled by the orb . corba implementation objects access and query the relational database via persistence , which is a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . clients access the corba objects via operation calls through an object request broker ( orb ) where the distribution details are handled by the orb . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . figure 5 gives the idl specification of interfaces bioseq and seqlnfo in module seqdb , and entrylnfo , nucseq and emblseq in module nsdb ( the full idl definition can be found in the additional data file with the online version of this article ) ) . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . it maps objects to relational rows and manages the objects in a shared cache , called the live object cache . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . the view is built from a number of tables ( or views ) of the database . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . as tuples are retrieved from the database , they are converted to objects and ' knitted ' together according to the object - model mapping to form a network of in - memory objects . accessing and manipulating these objects in the live object cache is faster than querying the relational database , speeding up application performance considerably . it is an object that adapts the interface of one object to a different interface expected by a caller and allows the caller to invoke requests on an object without knowing the object 's true interface . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . when the corba server is up and running , a client , which can be developed using any corba - compliant orb on the user 's preferred environment and language ( for which the orb is available ) at any local or remote machine , can access embl data through these objects using an ior ( interoperable object reference ) or via a naming service . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . , it can be seen that corba interfaces to the embl database provide : meaningful units of information : objects ; encapsulated methods , for example getlength ( ) ; interoperation between objects ; easy access and distribution of data ; and easy to comply with the standard . accessibility to the embl database via the embl corba server can be as fine as any small attribute defined in the embl data model . the embl corba server can also provide a blob object that contains a number of objects . to develop a corba server that provides access to our biological data , we need a well - defined object model to model the real - world biological entities , that is , to describe the structure and constraints present in the data , as well as how the data can be accessed and queried . it is a specification of the data in the problem domain , independent of how the actual database is implemented . this model can then be expressed in idl ( interface definition language ) interfaces to the corba server at one end , and mapped to a database schema for the underlying data management and storage at the other end . our object model of the embl database is organised into five main packages , as shown in figure 1 , where each package holds a set of closely related classes with a common purpose . each package contains a relatively isolated part of the entire object model , and is a clear candidate for re - use in models for other databases . a full definition of the embl nucleotide sequence database object model can be found in additional data file 1 with the online version of this article . any change made to the structure of the model needs to be propagated to both the idl specification that defines the corba server interfaces and underlying relational schema . to handle this problem , a strategy of using both explicit model and meta model the structure of the model is therefore not affected by changes to the feature definition , which makes it suitable for defining stable idl interfaces . on the server side , corba implementation objects access and query the relational database via persistence ( persistence software ) , which acts as a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) , where the distribution details are handled by the orb . on the server side , corba implementation objects access and query the relational database via persistence , which is a middleware between our corba implementation and the oracle database . to allow invoking operations on the objects , the server provides its clients interfaces in omg idl , which is independent of the server implementation . an object 's interface is composed of the operations and types of data that can be passed to and from those operations . clients access the corba objects via operation calls through an object request broker ( orb ) where the distribution details are handled by the orb . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . the returned values of methods represent attributes of class bioseq defined in the object model , providing information on biological sequences . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . it maps objects to relational rows and manages the objects in a shared cache , called the live object cache . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . the view is built from a number of tables ( or views ) of the database . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . the nsdb.idl defines the embl - specific sequences and related information and includes seven interfaces in the module nsdb : entrylnfo , embl , emblseq , nucseq , nucfeature , location and featurelocation . to reflect the accessing and querying of data , operations are defined in such a way that the return values of the operations represent attributes in the embl object data model . figure 5 gives the idl specification of interfaces bioseq and seqlnfo in module seqdb , and entrylnfo , nucseq and emblseq in module nsdb ( the full idl definition can be found in the additional data file with the online version of this article ) ) . for the above interfaces bioseq and seqlnfo , we have two mapped classes as shown in figure 6 the returned values of methods represent attributes of class bioseq defined in the object model , providing information on biological sequences . the embl corba server provides its clients with an object - oriented interface to the embl database . to achieve this , the object model needs to be mapped to the schema of the underlying oracle relational database . persistence , an object / relational tool from persistence software , is a mediator for transforming object operations to relational database calls and vice versa . however , for maximum flexibility , our existing database schema , which is independent and developed prior to the corba development , uses the so - called vertical mapping . the view is built from a number of tables ( or views ) of the database . when the structure of data changes at the database side as a result of the increasing complexity of biological data or the availability of new modeling capability in the database , we need only a change on the underlying views . as tuples are retrieved from the database , they are converted to objects and ' knitted ' together according to the object - model mapping to form a network of in - memory objects . accessing and manipulating these objects in the live object cache is faster than querying the relational database , speeding up application performance considerably . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . we employ orbix 's loader techniques together with persistence 's live object caching to build our loaders to support the creation of objects in the live object cache . loaders are called when an object reference enters an address space via a function findme , and persistence live objects are then loaded . if no live objects available in the live object cache respond to the call , persistence will create a new live object via querying and accessing the relational database . when the server has been running for some period of time , possibly weeks or months , it will have created a number of corba objects , which in turn contain a number of persistence live objects , and will consume the memory space . however , instead of blindly instantiating a new object every time , the object manager checks instantiated objects in the pool that it manages . there are a number of possible strategies , such as least recently used ( lru ) , least frequently used ( lfu ) , evicting the object with the highest memory consumption ( hmc ) , or using a weighted function that chooses an object for evictor based on a combination of factors ( wf ) . when the corba server is up and running , a client , which can be developed using any corba - compliant orb on the user 's preferred environment and language ( for which the orb is available ) at any local or remote machine , can access embl data through these objects using an ior ( interoperable object reference ) or via a naming service . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . the client submits its query with bio - seq - id to the embl object , which is a factory object representing the whole database . , it can be seen that corba interfaces to the embl database provide : meaningful units of information : objects ; encapsulated methods , for example getlength ( ) ; interoperation between objects ; easy access and distribution of data ; and easy to comply with the standard . accessibility to the embl database via the embl corba server can be as fine as any small attribute defined in the embl data model . the embl corba server can also provide a blob object that contains a number of objects . the embl object model provides a basis to develop the corba server . employing persistence maps the object model to the relational schema in the underlying oracle database . to present persistence with the right relations , properly built loaders make use of the technique of ' live object caching ' and enhance the performance . it has been demonstrated that the corba server addresses some problems of the flat - file format and provides a solution to accessing and distributing embl sequence data . it also provides a flexible and scalable environment for users to develop their applications by building clients . the following additional data are included with the online version of this article : the embl nucleotide sequence database object model and the embl idl specification . embl nucleotide sequence database object model it contains the idl belonging to the embl corba servers : the home page of the corba work at ebi is . general types used throughout the embl corba project ( save as types.idl ) general sequence interfaces ( save as seqdb.idl ) sequence interfaces specialised for embl ( needs seqdb.idl ) ( save as nsdb.idl ) genetic code used for translation of nucleotide sequences in embl ( save as geneticcode.idl ) the idl for the reference server : access to the bibliographic references of embl ( save as publication.idl ) the idl for the taxonomy server : access to the taxonomy information of embl ( save as taxonomy.idl ) meta information of the embl servers ( save as meta.idl ) a file with only ' # include ' statements for the above idl files , for easy compilation ( save as embl.idl ) makefile is provided ; it can be used to compile the idl using one of three commonly used orbs , i.e.

anterior uveitis ( au ) is the most common form of intraocular inflammation , and accounts for 50% to 92% of total uveitis cases in most western countries and 28% to 50% of all uveitis cases in some of the asian countries , such as korea , japan , and india ( 1 ) . as the second most common au next to idiopathic hla - b27 negative au , hla - b27 positive au accounts for 18% to 32% of all au cases in western countries and 6% to 13% of all au cases in asia ( 2 ) . in a previous hospital - based study , au accounted for approximately 28.1% of all uveitis cases in korea ( 3 ) , and 35% to 47% of au patients were positive for hla - b27 in other studies ( 4 , 5 ) . hla - b27 positive au is distinguishable from its hla - b27 negative counterpart and is characterized by early onset , male preponderance , frequent association with systemic diseases , and a high tendency for recurrence ( 2 , 6 ) . there have been many comparative studies on the prognosis of hla - b27 positive and negative au with conflicting results . some studies revealed more favorable outcomes for hla - b27 positive au ( 7 , 8) , whereas others reported the opposite ( 9 ) . in addition , an equal ocular outcome was observed for both hla - b27 positive and negative groups in some studies ( 10 - 14 ) . this study was performed to assess the clinical features and prognosis of hla - b27 positive au compared with hla - b27 negative au in a korean population . in addition , the effects of associated systemic disease on hla - b27 positive au were also evaluated . this study was a retrospective case - controlled study , and was approved by institutional review board . the medical records of all patients with au seen at the uveitis service of a university hospital in korea , between january 1996 and december 2006 were reviewed throughout patients ' entire follow - up and those who were tested for the presence of the hla - b27 allele were included in this study . hla - b27 typing was conducted for patients who agreed to perform the test after receiving information on the diagnostic , therapeutic , and prognostic significance of hla - b27 typing . exclusion criteria were patients with an associated primary uveitic condition ( e.g. , fuchs ' heterochromic cyclitis or infectious uveitis ) and other systemic and/or ocular diseases that are likely to be associated with ocular complications and visual prognosis independent of the hla - b27 antigen ( e.g. , sarcoidosis , behcet 's disease , proliferative diabetic retinopathy , retinal vascular obstruction , glaucoma not associated with the present au ) . patients were divided into two groups : those with a negative hla - b27 haplotype who presented with idiopathic au ( group i ) and those who were positive for hla - b27 ( group ii ) . group ii was further divided into two subgroups ; patients with a systemic disease association ( e.g. , spondyloarthropathy , inflammatory bowel disease ) ( group iia ) and those who had no evidence of systemic disease association ( group iib ) . a detailed medical and ophthalmologic history taking , a complete blood count , liver function tests , blood urea nitrogen , serum creatinine , urinalysis , c - reactive protein , anti - streptolysin o , rheumatoid factor , antinuclear antibody , serologic tests for toxoplasmosis and syphilis , human immunodeficiency virus test , hepatitis b and c tests , angiotensin - converting enzyme , and chest radiography were performed at the initial visit for each patient . in patients with previous episodes of uveitis , the total number of previous attacks and the age at their first episode were recorded during patient interviews . the ophthalmological examination performed at every visit included best - corrected visual acuity ( bcva ) , slit - lamp biomicroscopy , tonometry , and indirect ophthalmoscopy ; and systemic disease association was documented in every possible case . fluorescein angiography , optical coherence tomography , and anterior segment and fundus photography were performed when indicated . patients without prior rheumatologic evaluation at the time of inclusion were referred to a rheumatologist , and medical records were reviewed for patients with a previous rheumatologic follow - up . the characteristics of the uveitis were analyzed according to the definitions of the standardization of uveitis nomenclature ( sun ) working group ( 15 ) . au was defined by anterior chamber cell grade of 1 + ( graded according to the sun working group grading scheme ) of acute onset . all patients were treated according to the degree of intraocular inflammation ( cells in the anterior chamber or vitreous cavity or both ) using a stepladder steroid - sparing therapeutic algorithm ( 16 ) . periocular corticosteroids were used for severe , recurrent , or chronic episodes , for cystoid macular edema , and for patients in whom systemic corticosteroids were contraindicated . long - term topical nonsteroidal anti - inflammatory drugs ( nsaids ) were sometimes used to prevent relapse of inflammation in cases of active inflammation recurrence with attempted topical steroid withdrawal . short course systemic corticosteroids were given if the inflammation could not be controlled with previous therapy . the distribution of counts was analyzed using chi - square test and fisher 's exact test where appropriate for the determination of statistical significance . for the comparison of mean values , independent t - test and wilcoxon 's rank sum test , chicago , il , u.s.a . ) was used , and the results were considered to be significant at p values of less than 0.05 . eighty - two korean patients were included in the present study , and 27 patients ( 40 eyes ) tested hla - b27 negative and suffered from au of unknown etiology ( group i ) . fifty - five hla - b27 positive patients ( 88 eyes ) ( group ii ) were further divided into 29 ( 52.7% ) patients ( 47 eyes ) with systemic disease association ( group iia ) and 26 ( 47.3% ) patients ( 41 eyes ) with no systemic disease association ( group iib ) . the median follow - up period for all 82 patients was 36.5 months ( interquartile range , 16.0 - 75.3 months ) . there was no obvious male or female predominance in group i ( male : female ratio , 1.08:1 ) , whereas there were approximately three times more male patients in group ii ( male : female ratio , 2.9:1 ) , and this difference between groups i and ii was statistically significant ( p=0.040 ) . among the two subgroups of the hla - b27 positive group , group iia ( male : female ratio , 8.7:1 ) had a significantly higher male : female ratio than did group iib ( male : female ratio , 1.4:1 ) ( p=0.007 ) . the age of onset of au was statistically similar between hla - b27 negative and positive au groups ( p=0.919 ) , and the age of onset of group iia was significantly younger than that of group iib ( p=0.015 ) . hla - b27 positive au showed more severe anterior chamber inflammation than its hla - b27 negative counterpart . the mean grade of anterior chamber cells and the rate of hypopyon formation were significantly higher in the eyes of group ii than in those of group i ( p=0.006 and 0.034 , respectively ) ; hypopyon occurred almost exclusively in hla - b27 positive eyes ( table 2 ) . the proportions of anterior chamber cells of grade 3 or more and grade 4 were significantly higher in the eyes of the hla - b27 positive group than in the eyes of the hla - b27 negative group ( p=0.001 and 0.041 , respectively ) ( fig . 1 ) . in the analysis of the anterior chamber inflammation between the eyes of groups iia and iib , there were no statistically significant differences ( p>0.05 , table 2 , fig . 1 ) . a clear distinction of the visual outcome during the active inflammatory stage was observed between hla - b27 negative and positive groups ( table 2 ) . approximately four out of five hla - b27 negative au eyes ( 81.5% ) had bcva of 20/40 or better during the active inflammatory stage , whereas approximately two out of three hla - b27 positive au eyes ( 65.7% ) had bcva worse than 20/40 . in addition , about one out of three hla - b27 positive au eyes ( 32.8% ) had bcva of 20/200 or worse , whereas less than one out of ten hla - b27 negative au eyes ( 7.4% ) had bcva of 20/200 or worse ( p=0.011 ) . the proportion of eyes with bcva of 20/40 or better was significantly larger in group i than in group ii ( p<0.001 ) , and the proportion of eyes with bcva between 20/40 and 20/100 and the proportion of eyes with bcva between 20/100 and 20/400 were significantly larger in group ii than in group i ( p=0.031 and 0.030 , respectively ) . in the analysis of the worst bcva between the eyes of groups iia and iib , there were no significant differences in all ranges of visual acuity ( p>0.05 , table 2 ) . systemic or periocular steroids were required in significantly more patients in the hla - b27 positive group ( approximately 4 out of 5 patients ) than in the hla - b27 negative group ( approximately 1 out of 2 patients ) ( p=0.015 , table 3 ) . further analysis revealed that both oral and periocular steroids were used more frequently in hla - b27 positive patients than in hla - b27 negative patients ( p=0.032 and 0.021 , respectively ) . the presence of a systemic disease association made no significant difference in the medical treatment of au in the hla - b27 positive group ( p>0.05 , table 3 ) . the disease duration and course including the frequency of uveitis attack are summarized in table 4 . approximately 9 out of 10 study patients ( 92.6% of group i and 92.7% of group ii ) had limited ( 3 months or less ) au with a mean duration of about one month , and approximately 9 out of 10 study patients had repeated episodes of au ( 85.2% of group i and 90.9% of group ii ) . there were no significant differences in the proportions of limited / persistent , single / repeated , and chronic cases and in the duration of an episode between hla - b27 negative and positive au groups ( p>0.05 ) , but au occurred significantly more often in patients with the hla - b27 haplotype ( 2.07 attacks / year ) than in those without the hla - b27 haplotype ( 1.47 attacks / year ) ( p=0.007 ) . no significant differences in the disease duration and course were observed between hla - b27 positive patients with or without associated systemic disease ( p>0.05 , table 4 ) . the ocular complications and final visual outcomes in the four study groups are described in table 5 . the most common ocular complication observed in both hla - b27 negative and positive groups was increased intraocular pressure ( iop ) ( 40.0% and 27.3% , respectively ) . however , there were no significant differences in the proportions of eyes with cataract , cystoid macular edema , increased iop , secondary glaucoma , or posterior synechia between hla - b27 negative and positive au groups and between hla - b27 positive au groups with or without systemic disease ( p>0.05 ) . group i and ii ( and group iia and iib also ) showed similar good final visual outcomes ( table 5 ) . over 90% of eyes in each study group had final bcva of 20/40 or better , and 86.1% ( 31/36 ) , 82.8% ( 72/87 ) , 89.1% ( 41/46 ) , and 75.6% ( 31/41 ) of the eyes in groups i , ii , iia , and iib , respectively , had final bcva of 20/20 . there were no significant differences in the mean logmar ( logarithm of the minimum angle of resolution ) bcva between the eyes of groups i and ii and between the eyes of groups iia and iib ( light perception was assigned the logmar equivalent 2.70 ) ( p=0.687 and 0.161 , respectively ) 2 shows the kaplan - meier survival curves of eyes with a potential bcva of 20/40 and 20/32 in hla - b27 negative versus positive au patients , and there was no significant difference between hla - b27 negative and positive au patients ( p=0.8118 and 0.4286 , respectively ; by log rank test ) . there was only one legally blind eye ( bcva of 20/200 or worse ) with final bcva of light perception among all study eyes . this patient , who had ankylosing spondylitis and the hla - b27 haplotype , had his first au occurrence in the left eye when he was 24 yr old , and he had secondary chronic angle closure glaucoma after recurrent au episodes . glaucoma implant surgery and a penetrating keratoplasty were performed in his left eye , but his bcva eventually decreased to light perception . there was no obvious male or female predominance in group i ( male : female ratio , 1.08:1 ) , whereas there were approximately three times more male patients in group ii ( male : female ratio , 2.9:1 ) , and this difference between groups i and ii was statistically significant ( p=0.040 ) . among the two subgroups of the hla - b27 positive group , group iia ( male : female ratio , 8.7:1 ) had a significantly higher male : female ratio than did group iib ( male : female ratio , 1.4:1 ) ( p=0.007 ) . the age of onset of au was statistically similar between hla - b27 negative and positive au groups ( p=0.919 ) , and the age of onset of group iia was significantly younger than that of group iib ( p=0.015 ) . hla - b27 positive au showed more severe anterior chamber inflammation than its hla - b27 negative counterpart . the mean grade of anterior chamber cells and the rate of hypopyon formation were significantly higher in the eyes of group ii than in those of group i ( p=0.006 and 0.034 , respectively ) ; hypopyon occurred almost exclusively in hla - b27 positive eyes ( table 2 ) . the proportions of anterior chamber cells of grade 3 or more and grade 4 were significantly higher in the eyes of the hla - b27 positive group than in the eyes of the hla - b27 negative group ( p=0.001 and 0.041 , respectively ) ( fig . 1 ) . in the analysis of the anterior chamber inflammation between the eyes of groups iia and iib , there were no statistically significant differences ( p>0.05 , table 2 , fig . 1 ) . a clear distinction of the visual outcome during the active inflammatory stage was observed between hla - b27 negative and positive groups ( table 2 ) . approximately four out of five hla - b27 negative au eyes ( 81.5% ) had bcva of 20/40 or better during the active inflammatory stage , whereas approximately two out of three hla - b27 positive au eyes ( 65.7% ) had bcva worse than 20/40 . in addition , about one out of three hla - b27 positive au eyes ( 32.8% ) had bcva of 20/200 or worse , whereas less than one out of ten hla - b27 negative au eyes ( 7.4% ) had bcva of 20/200 or worse ( p=0.011 ) . the proportion of eyes with bcva of 20/40 or better was significantly larger in group i than in group ii ( p<0.001 ) , and the proportion of eyes with bcva between 20/40 and 20/100 and the proportion of eyes with bcva between 20/100 and 20/400 were significantly larger in group ii than in group i ( p=0.031 and 0.030 , respectively ) . in the analysis of the worst bcva between the eyes of groups iia and iib , there were no significant differences in all ranges of visual acuity ( p>0.05 , table 2 ) . systemic or periocular steroids were required in significantly more patients in the hla - b27 positive group ( approximately 4 out of 5 patients ) than in the hla - b27 negative group ( approximately 1 out of 2 patients ) ( p=0.015 , table 3 ) . further analysis revealed that both oral and periocular steroids were used more frequently in hla - b27 positive patients than in hla - b27 negative patients ( p=0.032 and 0.021 , respectively ) . the presence of a systemic disease association made no significant difference in the medical treatment of au in the hla - b27 positive group ( p>0.05 , table 3 ) . the disease duration and course including the frequency of uveitis attack are summarized in table 4 . approximately 9 out of 10 study patients ( 92.6% of group i and 92.7% of group ii ) had limited ( 3 months or less ) au with a mean duration of about one month , and approximately 9 out of 10 study patients had repeated episodes of au ( 85.2% of group i and 90.9% of group ii ) . there were no significant differences in the proportions of limited / persistent , single / repeated , and chronic cases and in the duration of an episode between hla - b27 negative and positive au groups ( p>0.05 ) , but au occurred significantly more often in patients with the hla - b27 haplotype ( 2.07 attacks / year ) than in those without the hla - b27 haplotype ( 1.47 attacks / year ) ( p=0.007 ) . no significant differences in the disease duration and course were observed between hla - b27 positive patients with or without associated systemic disease ( p>0.05 , table 4 ) . the ocular complications and final visual outcomes in the four study groups are described in table 5 . the most common ocular complication observed in both hla - b27 negative and positive groups however , there were no significant differences in the proportions of eyes with cataract , cystoid macular edema , increased iop , secondary glaucoma , or posterior synechia between hla - b27 negative and positive au groups and between hla - b27 positive au groups with or without systemic disease ( p>0.05 ) . group i and ii ( and group iia and iib also ) showed similar good final visual outcomes ( table 5 ) . over 90% of eyes in each study group had final bcva of 20/40 or better , and 86.1% ( 31/36 ) , 82.8% ( 72/87 ) , 89.1% ( 41/46 ) , and 75.6% ( 31/41 ) of the eyes in groups i , ii , iia , and iib , respectively , had final bcva of 20/20 . there were no significant differences in the mean logmar ( logarithm of the minimum angle of resolution ) bcva between the eyes of groups i and ii and between the eyes of groups iia and iib ( light perception was assigned the logmar equivalent 2.70 ) ( p=0.687 and 0.161 , respectively ) . 2 shows the kaplan - meier survival curves of eyes with a potential bcva of 20/40 and 20/32 in hla - b27 negative versus positive au patients , and there was no significant difference between hla - b27 negative and positive au patients ( p=0.8118 and 0.4286 , respectively ; by log rank test ) . there was only one legally blind eye ( bcva of 20/200 or worse ) with final bcva of light perception among all study eyes . this patient , who had ankylosing spondylitis and the hla - b27 haplotype , had his first au occurrence in the left eye when he was 24 yr old , and he had secondary chronic angle closure glaucoma after recurrent au episodes . glaucoma implant surgery and a penetrating keratoplasty were performed in his left eye , but his bcva eventually decreased to light perception . many studies have been reported on the clinical features and prognosis of hla - b27 positive au ( 7 - 14 , 17 , 18 ) . however , the ocular complications and visual outcomes of hla - b27 positive au compared to hla - b27 negative au continue to be controversial . as mentioned in an earlier report ( 2 ) , some of the conflicting findings may result from differences in the follow - up period and in the inclusion and diagnostic criteria used in various studies . evaluation of the effect of the hla - b27 haplotype on the ocular and visual prognosis of au in patients with hla - b27 positive au compared to the hla - b27 negative au group is complicated by the heterogeneity of the hla - b27 negative au group ( 2 ) . that is , inclusion of distinct uveitic entities , which are likely to be associated with ocular complications and visual prognosis independent of the hla - b27 antigen ( such as sarcoidosis and behcet 's disease ) , may make it difficult to evaluate the effect of the hla - b27 antigen on the complications and outcomes of au . in the present study , only idiopathic cases of hla - b27 negative au were included to minimize potential confounding factors , similarly to a previous report ( 9 ) . in addition , the effects of associated systemic disease on hla - b27 positive au were also evaluated by further dividing the hla - b27 positive au group into two subgroups : patients with a systemic disease association and those who had no evidence of systemic disease association . the clinical features of hla - b27 positive au in this study are generally similar to those in previously published reports from other western ( 7 - 12 , 14 , 17 ) and asian countries ( 13 , 18 ) . the common characteristics include an increased frequency among male patients and a significant association with hla - b27-related systemic disease as well as a significant cellular reaction in the anterior chamber with hypopyon requiring more aggressive treatment and with a higher frequency of recurrent attacks . in terms of the ocular complications and final visual outcomes , for which there have been conflicting results reported , the present study revealed no significant difference in the overall outcome between the hla - b27 negative and positive groups . a previous study with less favorable outcomes for hla - b27 positive au was conducted on patients seen between 1982 and 1993 ( 9 ) . therapeutic strategies for hla - b27 positive au have somewhat changed , and more intensive treatment is now given to hla - b27 positive au patients . this more aggressive treatment for hla - b27 positive au may explain the similar overall outcomes between hla - b27 negative and positive groups in the current study , which reviewed au patients treated between 1996 and 2006 . in terms of the effect of the systemic disease association on the hla - b27 positive au , our study showed no significant difference in the ocular complications and final visual outcomes between hla - b27 positive au patients with systemic disease ( group iia ) and those with no systemic disease ( group iib ) . this finding is in accord with the results of earlier reports ( 9 , 16 ) . one of the earlier reports revealed that the mean recurrence rate of inflammation was higher in hla - b27 positive patients with systemic disease ( 3.6 attacks ) than in those with no systemic disease ( 5.2 attacks ) ( 9 ) . however , another report showed that there was no significant difference in the mean frequency of attacks between hla - b27 positive au patients with systemic disease ( 0.8 attacks / year ) and those with no systemic disease ( 0.7 attacks / year ) ( 16 ) , and our study results were consistent with the latter one with the median frequency of attacks of 2.07 and 2.05 attacks / year in groups iia and iib , respectively . previous reports on the clinical characteristics of hla - b27 positive au in asian countries are scarce , and no comparative study on the clinical characteristics of hla - b27 positive and negative aus in korean populations has been reported in the international ophthalmic literature . in an earlier report ( 18 ) , chinese au patients with the hla - b27 haplotype had a generally good visual prognosis with 90.3% of the affected eyes having a visual acuity better than 20/40 , although 66.8% of patients had recurrent uveitis episodes and 31.4% and 14.3% of the affected eyes had posterior synechiae and cataract , respectively . in another report from thailand ( 13 ) , clinical features of hla - b27 positive and negative acute au were similar to those reported in western countries with no significant differences in the visual outcomes between hla - b27 positive and negative groups . in contrast to most prior western reports that noted a larger proportion of increased iop in the hla - b27 positive group than in the hla - b27 negative group ( 9 , 12 ) , however , the proportion of cases with increased iop was significantly higher in hla - b27 negative group than in hla - b27 positive group . in the current study , an increased iop ( > 21 mmhg ) and secondary glaucoma were more common in the hla - b27 negative patients ( 40.0% and 10.0% , respectively ) than in the hla - b27 positive patients ( 27.3% and 2.3% , respectively ) , although there were no statistically significant differences ( p=0.150 and 0.076 , respectively ) . the increased iop may be attributed to the au itself and/or the steroid treatment for au . in addition , the etiology of hla - b27 negative au in asia might include more cases of various viral infections ( often associated with increased iop ) as suggested in a previous study ( 13 ) . previous studies on the clinical characteristics and prognosis of hla - b27 positive and/or negative au in korean patients were published in a domestic ophthalmologic journal ( 19 , 20 ) . ( 19 ) recruited 32 au patients from 462 hla - b27 positive spondyloarthropathy patients ; the clinical characteristics of hla - b27 positive au were similar to this study except for the prominent unilateral involvement ( 31/32 , 96.8% ) and no occurrence of hypopyon ( 0/32 , 0% ) . however , the follow - up period for the 32 au patients was not mentioned and therefore these differences might be due to a shorter follow - up . however , patients with posterior uveitis were included ( 4/27 eyes ) making the group more heterogeneous . a comparative study between hla - b27 positive and negative au patients ( 5 ) showed more severe inflammation in the anterior chamber and more aggressive treatment in hla - b27 positive au compared to hla - b27 negative au , and the final visual outcomes were similar between the two groups . however , the number of subjects was relatively small ( 16 in hla - b27 positive group and 18 in hla - b27 negative group ) . the current study analyzed the largest data set to date available on korean patients with hla - b27 positive and negative au . in addition , we compared the subgroups of hla - b27 positive au with or without systemic disease . the significance of our results may partly be limited by referral bias as this study was based at a university hospital ( tertiary referral center ) with more complicated and severe cases . thus , the clinical features of both hla - b27 negative and positive aus would generally have a better prognosis than is described in this study , as pointed out in a previous report ( 2 ) . in addition , our results are also subject to selection bias because hla - b27 typing was conducted only for patients who agreed to perform the test after receiving information on the diagnostic , therapeutic , and prognostic significance of hla - b27 typing . in conclusion , the ocular and visual outcomes of hla - b27 positive au patients in korea are generally good and show no significant difference from those of hla - b27 negative au . the more aggressive treatment for significantly severer and recurrent anterior chamber inflammation in hla - b27 positive au is a feasible explanation for these findings . no significant differences in clinical characteristics , medical treatment , disease duration and course , and final ocular and visual outcomes were observed between the hla - b27 positive aus with or without associated systemic disease .

clinical features and prognosis of hla - b27 positive anterior uveitis ( au ) were assessed compared with hla - b27 negative au in a korean population , based on the medical records of au patients seen at a university hospital . twenty - seven hla - b27 negative , idiopathic au patients ( group i ) and 55 hla - b27 positive au patients ( group ii ) were studied . hla - b27 positive group was further divided into 29 with associated systemic disease ( seronegative spondyloarthropathy ) ( group iia ) and 26 without associated systemic disease ( group iib ) . significantly more severe anterior chamber inflammation in terms of anterior chamber cells ( p=0.006 ) and hypopyon formation ( p=0.034 ) was observed with higher frequency of au attacks ( p=0.007 ) in the hla - b27 positive group than in the hla - b27 negative group . systemic / periocular steroids were required in significantly more patients in the hla - b27 positive group than in the hla - b27 negative group ( p=0.015 ) . however , no significant differences were observed for final ocular and visual outcomes between these two groups . associated systemic disease made no significant difference in the clinical features and prognosis in the hla - b27 positive au patients . in conclusion , despite more severe inflammation and a higher recurrence rate , hla - b27 positive au shows similar good final ocular and visual outcomes compared to hla - b27 negative , idiopathic au in a korean population .

INTRODUCTION MATERIALS AND METHODS RESULTS Clinical features Ocular complications and final visual outcomes DISCUSSION

as the second most common au next to idiopathic hla - b27 negative au , hla - b27 positive au accounts for 18% to 32% of all au cases in western countries and 6% to 13% of all au cases in asia ( 2 ) . in a previous hospital - based study , au accounted for approximately 28.1% of all uveitis cases in korea ( 3 ) , and 35% to 47% of au patients were positive for hla - b27 in other studies ( 4 , 5 ) . hla - b27 positive au is distinguishable from its hla - b27 negative counterpart and is characterized by early onset , male preponderance , frequent association with systemic diseases , and a high tendency for recurrence ( 2 , 6 ) . there have been many comparative studies on the prognosis of hla - b27 positive and negative au with conflicting results . in addition , an equal ocular outcome was observed for both hla - b27 positive and negative groups in some studies ( 10 - 14 ) . this study was performed to assess the clinical features and prognosis of hla - b27 positive au compared with hla - b27 negative au in a korean population . in addition , the effects of associated systemic disease on hla - b27 positive au were also evaluated . the medical records of all patients with au seen at the uveitis service of a university hospital in korea , between january 1996 and december 2006 were reviewed throughout patients ' entire follow - up and those who were tested for the presence of the hla - b27 allele were included in this study . , fuchs ' heterochromic cyclitis or infectious uveitis ) and other systemic and/or ocular diseases that are likely to be associated with ocular complications and visual prognosis independent of the hla - b27 antigen ( e.g. patients were divided into two groups : those with a negative hla - b27 haplotype who presented with idiopathic au ( group i ) and those who were positive for hla - b27 ( group ii ) . group ii was further divided into two subgroups ; patients with a systemic disease association ( e.g. , spondyloarthropathy , inflammatory bowel disease ) ( group iia ) and those who had no evidence of systemic disease association ( group iib ) . eighty - two korean patients were included in the present study , and 27 patients ( 40 eyes ) tested hla - b27 negative and suffered from au of unknown etiology ( group i ) . fifty - five hla - b27 positive patients ( 88 eyes ) ( group ii ) were further divided into 29 ( 52.7% ) patients ( 47 eyes ) with systemic disease association ( group iia ) and 26 ( 47.3% ) patients ( 41 eyes ) with no systemic disease association ( group iib ) . among the two subgroups of the hla - b27 positive group , group iia ( male : female ratio , 8.7:1 ) had a significantly higher male : female ratio than did group iib ( male : female ratio , 1.4:1 ) ( p=0.007 ) . the age of onset of au was statistically similar between hla - b27 negative and positive au groups ( p=0.919 ) , and the age of onset of group iia was significantly younger than that of group iib ( p=0.015 ) . hla - b27 positive au showed more severe anterior chamber inflammation than its hla - b27 negative counterpart . the mean grade of anterior chamber cells and the rate of hypopyon formation were significantly higher in the eyes of group ii than in those of group i ( p=0.006 and 0.034 , respectively ) ; hypopyon occurred almost exclusively in hla - b27 positive eyes ( table 2 ) . the proportions of anterior chamber cells of grade 3 or more and grade 4 were significantly higher in the eyes of the hla - b27 positive group than in the eyes of the hla - b27 negative group ( p=0.001 and 0.041 , respectively ) ( fig . approximately four out of five hla - b27 negative au eyes ( 81.5% ) had bcva of 20/40 or better during the active inflammatory stage , whereas approximately two out of three hla - b27 positive au eyes ( 65.7% ) had bcva worse than 20/40 . in addition , about one out of three hla - b27 positive au eyes ( 32.8% ) had bcva of 20/200 or worse , whereas less than one out of ten hla - b27 negative au eyes ( 7.4% ) had bcva of 20/200 or worse ( p=0.011 ) . systemic or periocular steroids were required in significantly more patients in the hla - b27 positive group ( approximately 4 out of 5 patients ) than in the hla - b27 negative group ( approximately 1 out of 2 patients ) ( p=0.015 , table 3 ) . further analysis revealed that both oral and periocular steroids were used more frequently in hla - b27 positive patients than in hla - b27 negative patients ( p=0.032 and 0.021 , respectively ) . the presence of a systemic disease association made no significant difference in the medical treatment of au in the hla - b27 positive group ( p>0.05 , table 3 ) . approximately 9 out of 10 study patients ( 92.6% of group i and 92.7% of group ii ) had limited ( 3 months or less ) au with a mean duration of about one month , and approximately 9 out of 10 study patients had repeated episodes of au ( 85.2% of group i and 90.9% of group ii ) . there were no significant differences in the proportions of limited / persistent , single / repeated , and chronic cases and in the duration of an episode between hla - b27 negative and positive au groups ( p>0.05 ) , but au occurred significantly more often in patients with the hla - b27 haplotype ( 2.07 attacks / year ) than in those without the hla - b27 haplotype ( 1.47 attacks / year ) ( p=0.007 ) . no significant differences in the disease duration and course were observed between hla - b27 positive patients with or without associated systemic disease ( p>0.05 , table 4 ) . however , there were no significant differences in the proportions of eyes with cataract , cystoid macular edema , increased iop , secondary glaucoma , or posterior synechia between hla - b27 negative and positive au groups and between hla - b27 positive au groups with or without systemic disease ( p>0.05 ) . group i and ii ( and group iia and iib also ) showed similar good final visual outcomes ( table 5 ) . there were no significant differences in the mean logmar ( logarithm of the minimum angle of resolution ) bcva between the eyes of groups i and ii and between the eyes of groups iia and iib ( light perception was assigned the logmar equivalent 2.70 ) ( p=0.687 and 0.161 , respectively ) 2 shows the kaplan - meier survival curves of eyes with a potential bcva of 20/40 and 20/32 in hla - b27 negative versus positive au patients , and there was no significant difference between hla - b27 negative and positive au patients ( p=0.8118 and 0.4286 , respectively ; by log rank test ) . among the two subgroups of the hla - b27 positive group , group iia ( male : female ratio , 8.7:1 ) had a significantly higher male : female ratio than did group iib ( male : female ratio , 1.4:1 ) ( p=0.007 ) . the age of onset of au was statistically similar between hla - b27 negative and positive au groups ( p=0.919 ) , and the age of onset of group iia was significantly younger than that of group iib ( p=0.015 ) . hla - b27 positive au showed more severe anterior chamber inflammation than its hla - b27 negative counterpart . the mean grade of anterior chamber cells and the rate of hypopyon formation were significantly higher in the eyes of group ii than in those of group i ( p=0.006 and 0.034 , respectively ) ; hypopyon occurred almost exclusively in hla - b27 positive eyes ( table 2 ) . the proportions of anterior chamber cells of grade 3 or more and grade 4 were significantly higher in the eyes of the hla - b27 positive group than in the eyes of the hla - b27 negative group ( p=0.001 and 0.041 , respectively ) ( fig . a clear distinction of the visual outcome during the active inflammatory stage was observed between hla - b27 negative and positive groups ( table 2 ) . approximately four out of five hla - b27 negative au eyes ( 81.5% ) had bcva of 20/40 or better during the active inflammatory stage , whereas approximately two out of three hla - b27 positive au eyes ( 65.7% ) had bcva worse than 20/40 . in addition , about one out of three hla - b27 positive au eyes ( 32.8% ) had bcva of 20/200 or worse , whereas less than one out of ten hla - b27 negative au eyes ( 7.4% ) had bcva of 20/200 or worse ( p=0.011 ) . systemic or periocular steroids were required in significantly more patients in the hla - b27 positive group ( approximately 4 out of 5 patients ) than in the hla - b27 negative group ( approximately 1 out of 2 patients ) ( p=0.015 , table 3 ) . further analysis revealed that both oral and periocular steroids were used more frequently in hla - b27 positive patients than in hla - b27 negative patients ( p=0.032 and 0.021 , respectively ) . the presence of a systemic disease association made no significant difference in the medical treatment of au in the hla - b27 positive group ( p>0.05 , table 3 ) . approximately 9 out of 10 study patients ( 92.6% of group i and 92.7% of group ii ) had limited ( 3 months or less ) au with a mean duration of about one month , and approximately 9 out of 10 study patients had repeated episodes of au ( 85.2% of group i and 90.9% of group ii ) . there were no significant differences in the proportions of limited / persistent , single / repeated , and chronic cases and in the duration of an episode between hla - b27 negative and positive au groups ( p>0.05 ) , but au occurred significantly more often in patients with the hla - b27 haplotype ( 2.07 attacks / year ) than in those without the hla - b27 haplotype ( 1.47 attacks / year ) ( p=0.007 ) . no significant differences in the disease duration and course were observed between hla - b27 positive patients with or without associated systemic disease ( p>0.05 , table 4 ) . the most common ocular complication observed in both hla - b27 negative and positive groups however , there were no significant differences in the proportions of eyes with cataract , cystoid macular edema , increased iop , secondary glaucoma , or posterior synechia between hla - b27 negative and positive au groups and between hla - b27 positive au groups with or without systemic disease ( p>0.05 ) . group i and ii ( and group iia and iib also ) showed similar good final visual outcomes ( table 5 ) . there were no significant differences in the mean logmar ( logarithm of the minimum angle of resolution ) bcva between the eyes of groups i and ii and between the eyes of groups iia and iib ( light perception was assigned the logmar equivalent 2.70 ) ( p=0.687 and 0.161 , respectively ) . 2 shows the kaplan - meier survival curves of eyes with a potential bcva of 20/40 and 20/32 in hla - b27 negative versus positive au patients , and there was no significant difference between hla - b27 negative and positive au patients ( p=0.8118 and 0.4286 , respectively ; by log rank test ) . many studies have been reported on the clinical features and prognosis of hla - b27 positive au ( 7 - 14 , 17 , 18 ) . however , the ocular complications and visual outcomes of hla - b27 positive au compared to hla - b27 negative au continue to be controversial . evaluation of the effect of the hla - b27 haplotype on the ocular and visual prognosis of au in patients with hla - b27 positive au compared to the hla - b27 negative au group is complicated by the heterogeneity of the hla - b27 negative au group ( 2 ) . that is , inclusion of distinct uveitic entities , which are likely to be associated with ocular complications and visual prognosis independent of the hla - b27 antigen ( such as sarcoidosis and behcet 's disease ) , may make it difficult to evaluate the effect of the hla - b27 antigen on the complications and outcomes of au . in the present study , only idiopathic cases of hla - b27 negative au were included to minimize potential confounding factors , similarly to a previous report ( 9 ) . in addition , the effects of associated systemic disease on hla - b27 positive au were also evaluated by further dividing the hla - b27 positive au group into two subgroups : patients with a systemic disease association and those who had no evidence of systemic disease association . the clinical features of hla - b27 positive au in this study are generally similar to those in previously published reports from other western ( 7 - 12 , 14 , 17 ) and asian countries ( 13 , 18 ) . the common characteristics include an increased frequency among male patients and a significant association with hla - b27-related systemic disease as well as a significant cellular reaction in the anterior chamber with hypopyon requiring more aggressive treatment and with a higher frequency of recurrent attacks . in terms of the ocular complications and final visual outcomes , for which there have been conflicting results reported , the present study revealed no significant difference in the overall outcome between the hla - b27 negative and positive groups . a previous study with less favorable outcomes for hla - b27 positive au was conducted on patients seen between 1982 and 1993 ( 9 ) . therapeutic strategies for hla - b27 positive au have somewhat changed , and more intensive treatment is now given to hla - b27 positive au patients . this more aggressive treatment for hla - b27 positive au may explain the similar overall outcomes between hla - b27 negative and positive groups in the current study , which reviewed au patients treated between 1996 and 2006 . in terms of the effect of the systemic disease association on the hla - b27 positive au , our study showed no significant difference in the ocular complications and final visual outcomes between hla - b27 positive au patients with systemic disease ( group iia ) and those with no systemic disease ( group iib ) . one of the earlier reports revealed that the mean recurrence rate of inflammation was higher in hla - b27 positive patients with systemic disease ( 3.6 attacks ) than in those with no systemic disease ( 5.2 attacks ) ( 9 ) . however , another report showed that there was no significant difference in the mean frequency of attacks between hla - b27 positive au patients with systemic disease ( 0.8 attacks / year ) and those with no systemic disease ( 0.7 attacks / year ) ( 16 ) , and our study results were consistent with the latter one with the median frequency of attacks of 2.07 and 2.05 attacks / year in groups iia and iib , respectively . previous reports on the clinical characteristics of hla - b27 positive au in asian countries are scarce , and no comparative study on the clinical characteristics of hla - b27 positive and negative aus in korean populations has been reported in the international ophthalmic literature . in another report from thailand ( 13 ) , clinical features of hla - b27 positive and negative acute au were similar to those reported in western countries with no significant differences in the visual outcomes between hla - b27 positive and negative groups . in contrast to most prior western reports that noted a larger proportion of increased iop in the hla - b27 positive group than in the hla - b27 negative group ( 9 , 12 ) , however , the proportion of cases with increased iop was significantly higher in hla - b27 negative group than in hla - b27 positive group . in the current study , an increased iop ( > 21 mmhg ) and secondary glaucoma were more common in the hla - b27 negative patients ( 40.0% and 10.0% , respectively ) than in the hla - b27 positive patients ( 27.3% and 2.3% , respectively ) , although there were no statistically significant differences ( p=0.150 and 0.076 , respectively ) . in addition , the etiology of hla - b27 negative au in asia might include more cases of various viral infections ( often associated with increased iop ) as suggested in a previous study ( 13 ) . previous studies on the clinical characteristics and prognosis of hla - b27 positive and/or negative au in korean patients were published in a domestic ophthalmologic journal ( 19 , 20 ) . ( 19 ) recruited 32 au patients from 462 hla - b27 positive spondyloarthropathy patients ; the clinical characteristics of hla - b27 positive au were similar to this study except for the prominent unilateral involvement ( 31/32 , 96.8% ) and no occurrence of hypopyon ( 0/32 , 0% ) . a comparative study between hla - b27 positive and negative au patients ( 5 ) showed more severe inflammation in the anterior chamber and more aggressive treatment in hla - b27 positive au compared to hla - b27 negative au , and the final visual outcomes were similar between the two groups . however , the number of subjects was relatively small ( 16 in hla - b27 positive group and 18 in hla - b27 negative group ) . the current study analyzed the largest data set to date available on korean patients with hla - b27 positive and negative au . in addition , we compared the subgroups of hla - b27 positive au with or without systemic disease . thus , the clinical features of both hla - b27 negative and positive aus would generally have a better prognosis than is described in this study , as pointed out in a previous report ( 2 ) . in conclusion , the ocular and visual outcomes of hla - b27 positive au patients in korea are generally good and show no significant difference from those of hla - b27 negative au . the more aggressive treatment for significantly severer and recurrent anterior chamber inflammation in hla - b27 positive au is a feasible explanation for these findings . no significant differences in clinical characteristics , medical treatment , disease duration and course , and final ocular and visual outcomes were observed between the hla - b27 positive aus with or without associated systemic disease .

rheumatoid arthritis ( ra ) is a progressive , inflammatory disease which is characterised by inflammation of the joint synovium that could ultimately progress to joint destruction [ 1 , 2 ] . due to its chronic , immune - mediated course , long - term treatment with immune - modulatory drugs is generally required . this disabling condition imposes substantial economic burden through the decreased quality of life ( qol ) and loss of productivity . recent advances in biotechnology and pathogenesis of ra have led to the discovery of biological dmards . biological agents inhibit pro - inflammatory cytokines which are believed to have a crucial role in the inflammatory process within the synovial joint . tnf- inhibitors have proved their clinical efficacy and raised the previous goals of ra treatment [ 5 , 8 ] . clinicians nowadays aim to achieve low disease activity or preferably remission and not merely slowing the progression of the disease and controlling symptoms . the discovery of biological agents has led to a drastic shift in the therapeutic approach to ra , leading to a better qol . yet , these breakthrough drugs are associated with high procurement costs . this ultimately increases the financial burden ra imposes on society [ 4 , 5 ] . such a scenario has elicited the need to carry out pharmacoeconomical assessments in order to inform policy and decision makers of the cost - effectiveness of the biological dmards [ 5 , 8 ] . hence , this study was undertaken to determine the improved qol and the incremental cost - effectiveness ratio ( icer ) involved in treating maltese patients suffering from resistant ra , with tnf- inhibitors . data collection used to conduct this 6-month study was carried out at the rheumatology outpatient clinic at mater dei hospital . patients were eligible to participate in the study provided that they had been diagnosed with ra according to the 1987 acr classification criteria , failed to achieve a low disease activity despite dmard / s therapy ; and were switched onto a tnf- inhibitor ( etanercept , adalimumab or infliximab ) . patients were not eligible to participate if pregnant or planning to conceive suffering from tb or hep b ; they are diagnosed with juvenile chronic arthritis , ankylosing spondylitis , osteoarthritis , psoriatic arthritis , and/or any other rheumatological condition . following approval from the maltese research ethics committee board , patients identified for participation were briefed on the purpose of the study . a signed consent form was obtained from every patient who accepted to participate , out of their own voluntarily will . the disease - specific health assessment questionnaire ( haq ) was chosen as an outcome measure tool to assist in the evaluation of the functional improvement experienced by patients that were switched onto tnf- inhibitor therapy . the haq is a reliable tool used by various studies to assess daily activities , namely , dressing , grooming , eating , walking , hygiene , and so forth . each of these items is given a score from 0 ( without any difficulty ) to 3 ( unable to perform ) reflecting the patient 's ability in performing a particular activity . the generic sf-36 is a health status questionnaire which has become a predominant tool in assessing various medical interventions consisting of 8 domains and 2 summary scores . unlike the haq , the higher the score obtained in each of the sf-36 domains , the more it indicates a better health status in the respective domain . the das-28 was used as a clinical outcome measure tool in order to monitor disease activity with biological intervention . the das-28 generates a continuous scale ( 0 to 9.4 ) through the assessment of tender joints ( 028 ) , swollen joints ( 028 ) , erythrocyte sedimentation rate ( mm / hr ) , and the visual analogue scale ( 0100 ) . this prospective study , carried out between 2010 and 2011 , had a time phase of 6 months , during which patients were assessed 3 times through the sf-36 and haq . at phase 1 ( t0 months ) , patients were still being treated with conventional dmard therapy . failure to achieve the desired outcome and following discussions with the rheumatology consultant , patients were identified as suitable candidates for initiation of tnf- inhibitor therapy . subjects who accepted to participate in the study were interviewed by the investigator using the haq and sf-36 . raw data obtained for both the sf-36 and the haq were inputted in a microsoft excel database . the final haq score and summary scores for every sf-36 domain were calculated . at phase 2 ( t3 months after initiation of tnf- inhibitor therapy ) , participants were reinterviewed using the same questionnaires . at phase 3 ( t6 months ) , data from the sf-36 and haq were again collected following 6 months of tnf- inhibitor therapy . the sf-36 scores were converted into sf-6d scores using the algorithms provided by the university of sheffield . the excel program based on nonparametric bayesian preference weights was chosen since the non - parametric model has shown added benefits in the predictive capacity of the model over the parametric random effects model . the raw scores for every item within the sf-36 for all the 13 patients were inputted in the excel programme which generated utility values , anchored at 0 for dead and 1 indicative of full health . during both phases 1 and 3 , patients were examined by the rheumatology consultant and assigned a das-28 score as a measure of disease activity . in both phases , medical case notes of patients were reviewed and related data were collected , namely , das-28 scores , treatment regimens , adverse events , history of hospitalisation during the study period , and surgeries performed related to ra . data collected from the haq and sf-36 questionnaires were analyzed statistically using spss version 18.0 . the one - way anova test was used to compare the mean scores obtained during phase 1 , 2 , and 3 for both the haq and the sf-36 . scores obtained in the haq and sf-36 were also analysed using the post hoc tukey 's test where pairwise comparison between the mean rating scores of any two visits was carried out . the costs of tnf- inhibitors , dmards , and glucocorticoids involved in the study were obtained from the maltese government health pharmaceutical services ( ghps ) . treatment regimens for every patient were obtained by scrutiny of patients ' notes prior to initiation of tnf- inhibitor therapy ( treatment b ) and after 6 months of biological therapy ( treatment a ) . costs of treatment b included the costs of dmards and concomitant steroids for a period of 6 months prior to phase 1 . hospitalization and hip replacement surgery costs were included , assuming that the use of a tnf - inhibitor would have decreased the need of hospitalization and hip replacement surgery . currently , maltese patients , who have an inadequate response to multiple dmards and suffer from moderate to severe ra , are considered for biological agent therapy as recommended by the acr guidelines . prior to commencement of a tnf- inhibitor , patients participating in the study had been on the same nonbiological dmard / s for at least 6 months . the direct costs of treatment a included the cost of the biological agent being administered , any concomitant dmard , and steroids for a period of 6 months . the economic evaluation used in this study was a cost effectiveness analysis ( cea ) which combines the difference between the costs of treatments with improvement achieved through biological intervention . the difference in costs between treatments was established by subtracting the cost of treatment b from treatment a for every patient . the improvement in the haq was chosen as a unit of health benefit since the haq is a strong predictor of the functional disability observed in patients with ra through loss of productivity [ 5 , 16 ] . the icer per unit of improvement in haq was calculated using the following formula : ( 1)icer per unit improvement in haq = ( cost of treatment acost of treatment b ) (haq score on treatment b haq score on treatment a)1 . the das-28 score was also used as a unit of health benefit , as it highly reflects the disease activity and is thus useful in indicating the suppression of disease activity following a therapeutic intervention . the icer per unit of improvement in the das-28 through biological intervention was calculated using the following formula : ( 2)icer per unit improvement in das-28 = ( cost of treatment acost of treatment b ) (das-28 score on treatment b das-28 score on treatment a)1 . both the haq and the das-28 give an ordinal scoring system which is often criticized if used as a unit of health benefit to express the incremental icer . the sf-6d provides scores on a cardinal scale from 0 to 1 with equal grades and can therefore be used in assessing cost effectiveness of a health intervention . hence , the icer was expressed in terms of unit improvement in the sf-6d using the following formula : ( 3)icer per unit improvement in sf-6d = ( cost of treatment acost of treatment b ) (sf-6d utility index on treatment b sf-6d utility index treatment a)1 . comparison between resulting icers using the 3 different methodologies could not be made since the ( i ) haq gave scores ranging from 0 to 3 , ( ii ) the das-28 scores ranged from 0 to 9.4 , and ( iii ) the sf-6d ranged from 0 to 1 . this has prompted the researcher to devise a scoring scheme ranging from 0 to 10 in order to compare icers obtained over a 6-month period . the resulting icers from all 3 different methodologies were calculated and expressed in terms of this scoring scheme . one - way anova testing was conducted on the resulting icers in order to assess significance between outcome health benefits chosen . 13 patients were recruited within the study out of which 85% were females , with a mean age of 54 years . patients had been suffering from ra , prior to initiation of tnf- inhibitor therapy for a mean of 11 years ranging from 4 to 31 years . as shown in figure 1 , the mean haq scores achieved in phase 1 were the highest indicating the functional disability experienced despite conventional dmard therapy . progress achieved was sustained and continued to improve during the last 3 months of the study , yet to a lesser extent than in the previous 3 months . table 1 shows that all activities assessed within the haq have improved significantly ( p < 0.05 ) between phase 1 and 2 . the mean difference in the haq scores obtained between phase 2 and phase 3 was not statistically significant ( p > 0.05 ) . during the whole study , this result indicates that significant improvement is observed as early as 3 months after initiation of tnf- inhibitor therapy . table 1 shows the difference in mean scores obtained between phases for every activity assessed within the haq and their relative level of significance ( significance noted at p < 0.05 * ) . as noted from figure 3 , patients reported low functional status in the sf-36 whilst on dmard therapy . during phase 2 and 3 , all patients reported an improvement in the functionality for all domains within the sf-36 . the greatest improvement was observed in the physical and emotional role between phase 1 and 2 . every sf-36 domain improved significantly between phase 1 ( baseline ) and phase 2 since p values obtained were < 0.05 level of significance ( table 2 ) . as observed with the haq scores , p values between phase 2 and 3 for every sf-36 domain were not significant ( p > 0.05 ) . sf-36 domains improved significantly after 6 months of biological intervention between phase 1 and 3 ( p < 0.05 ) . lower p values were obtained for the er and bp when compared with other domains between phase 2 and 3 , thereby indicating continuous improvement during the latter part of the study . table 2 shows the difference in mean scores obtained between phases for every sf-36 domain and their relative level of significance ( significance noted at p < 0.05 * ) . during phase 1 , as observed in figure 2 , 85% of patients ( n = 11 ) scored higher than 5.1 indicating a high disease activity according to the eular criteria . in accordance with the eular criteria response , a decrease of 1.2 from the initial das-28 score has been found to indicate a significant change and is thus considered as a good response . after 6 months of biological intervention , the das-28 decreased significantly ( > 1.2 ) for all 13 patients . 31% of patients ( n = 4 ) scored < 3.2 in the das-28 which corresponds to low disease activity whilst 15.4% ( n = 2 ) scored < 2.6 which is compatible with disease remission . 54% of patients ( n = 7 ) scored values compatible with moderate disease activity ( > 3.2 5.1 ) . a slight improvement in the haq score despite biological intervention results in a high icer ( table 3 ) . patient 10 experienced a minimal improvement in the quality of life which gave an icer of 26,030 over a period of 6 months . the rest of subjects ( n = 12 ) experienced a much lower icer due to a higher improvement recorded in the quality of life . patient 13 had undergone a hip replacement surgery prior to phase 1 and hence gave a more favorable icer , due to a lower discrepancy between the costs of treatments . in order to improve by 10% in the quality of life as measured by the haq , an average addition of 1,977 is incurred over a period of 6 months . table 3 shows the icers per unit improvement and per 10% improvement in the haq following 6 months of tnf- inhibitor therapy . the least reduction in disease activity was again reported by patient 10 ( table 4 ) . the lowest resulting icer was incurred again by patient 13 . for every unit of improvement in the das-28 ( reduction in disease activity by 1 on a scale from 0 to 9.4 ) an increase of 2,200 the average reduction in disease activity ( das-28 ) was of 2.71 , which is considered as a significant good response ( > 1.2 ) . in order to achieve a reduction of 10% in the das-28 score ( i.e. , 0.94 ) table 4 shows the icers per unit improvement and per 10% improvement in the das-28 following 6 months of tnf- inhibitor therapy . an average patient , following 6 months of tnf- inhibitor , gained a 0.27 improvement in the utility health index , the sf-6d . theoretically , an icer of 23,164 is incurred for a patient to regain a perfect quality of life ( 1 ) from a state ( 0 ) equivalent to death ( table 5 ) . an improvement of 0.1 as assessed through the sf-6d would cost the payer an additional 2,316 . table 5 shows the icers per unit improvement and per 10% improvement in the sf-6d following 6 months of tnf- inhibitor therapy . no significant difference ( p > 0.05 ) was noted between the 3 different methodologies used to calculate the icer ( table 6 ) . the choice of the sf-6d yielded the higher icer when compared to resulting icers in terms of the haq and das-28 . table 6 shows the level of significance ( significance noted at p < 0.05 ) and standard deviations between the haq , das-28 , and the sf-6d . results obtained within this study highlight the evidence of the significant improvement gained in the qol caused by this disabling condition in the initial treatment phases . various studies have used the haq to shed light on the quality of life and physical disability endured by patients . at baseline , patients reported a score ranging between 1.3 and 2.65 . in a study by del moral et al . , an haq score > 1.5 was correlated with a significant number of working days absenteeism ( > 50% ) than those scoring < 0.5 . this implies that costs are further increased by the loss of productivity experienced by patients . during this study , indirect costs such as loss of work were not included since a high percentage of the subjects were housewives and thus it would be very difficult to quantify the work carried out or the lack of it . following 3 months of treatment with tnf- inhibitors , scores ranged from 0.2 to 1.5 , indicating a considerable significant improvement ( p < 0.05 ) . scores continued to decrease furthermore during the last 3 months of the study yet significance was not noted ( p > 0.05 ) . apart from the functional impairment assessed within the haq , which hinders patients from their daily activities , ra affects considerably the vitality , emotional wellbeing , and mental health . this has been confirmed by the low mean scores reported in the study for all domains within the sf-36 at phase 1 . despite multiple dmard therapy and concomitant glucocorticoids all domains improved significantly after 3 months of tnf- inhibitors ( p < 0.05 ) . in the latter half of the study , significance was not noted since scores achieved were similar to phase 2 apart from the emotional role which continued to improve ( p > 0.05 ) . this is in concordance with the findings from the haq which suggests that a plateau is reached after 3 months of biological intervention . such an observation highlights the need for long - term studies , in order to assess whether improvement gained is sustained and to what extent . findings from this study are in accordance with results obtained from the generic sf-36 questionnaire in a study conducted on a spanish population . findings from both hrqol questionnaires used indicate the clinical effectiveness of tnf- antagonists in achieving a better qol , as documented in various studies [ 20 , 21 ] . the das-28 score used a clinical marker to monitor the disease activity after intervention with tnf- inhibitors . at phase 1 , 85% of subjects were experiencing a high disease activity ( > 5.1 ) as devised by the eular criteria . baseline das-28 results further support the resulting disability and compromised quality of life reported in the hrqol questionnaires . following 6 months of biological intervention , prevalence of das-28 < 3.2 was 31% ( n = 4 ) which is compatible with low disease activity . all patients ( n = 13 ) experienced significant reduction in disease activity ( > 1.2 ) as defined by the acr / eular criteria . acr / eular remission ( < 2.6 ) was met by 15.4% ( n = 2 ) of subjects . abalos medina reported a higher prevalence of acr / eular remission , yet this must be interpreted in the light of the limited number of participants and length of the current study . recent therapeutic strategies in ra aim to slow down disease progression and reduce functional impairment . tnf- antagonists are the forerunners in achieving such a therapeutic goal as they have been proven to suppress disease activity . since the procurement of tnf- antagonists is associated with incredibly high costs when compared with conventional dmard therapy , economic evaluations are essential as benefit from biological treatment is evident , but financial constraints limit its availability . the haq was chosen to assess the improvement achieved through biological intervention , since a close correlation was reported between haq and health care costs [ 2426 ] . a unit of improvement in the haq ( any 1 unit difference between 0 and 3 ) was chosen as a health outcome benefit in order to assess the icer . the resulting icers per unit of haq varied between 1,201 and 26,030 with a mean of 6,588 . discrepancy between costs of treatment was offset by hip replacement surgery for patient 13 which resulted in a more favourable icer . such result is enlightening as it demonstrates that tnf- antagonist therapy is more likely to be cost effective in patients with severe functional disability , who may ultimately require surgical interventions . the least favourable icer was achieved by patient 10 who experienced the least improvement in the haq ( gain of 0.25 ) . in such a scenario , it is evident that the disease is resistant despite biological treatment and alternative treatments should be initiated in order to gain a better control of the disease and consequently qol . kobelt et al . described a similar situation where direct costs with a score > 2.6 was approximately twice the direct costs for patients achieving a score less than 0.6 . this highlights the need for effective treatment in order to achieve maximal improvement in qol and hence diminish the costs incurred . such measure of health benefit was chosen as it is free of any bias from both the researcher and the patient . subjects achieved an average significant reduction of 2.71 ( > 1.2 ) following 6 months of treatment . the highest icer in terms of das-28 was again reported by patient 10 which resulted due to a minor improvement reported in the das-28 . the resulting icers ranged from 897 to 4,368 with the lowest icer reported by patient 13 . the icer per unit of improvement in the sf-6d gave an average of 23,164 which is extremely higher than the icers per unit of improvement in the haq and das-28 . yet such a high cost resulted since it accounts for a transition between death and a perfect quality of life . all icer results were expressed in terms of the same scoring scheme to be able to compare results . for every 10% improvement in the sf-6d , a cost of 2,316 was incurred after 6 months of tnf- antagonist intervention . statistical significance was not noted ( p > 0.05 ) between results obtained per 10% improvement in the respective health outcome . this could be either due to the limited number of observations ( n = 13 ) or due to the large variation in costs between the 13 observations of each method as shown in the large standard deviations ( table 6 ) . similar studies should be undertaken to be able to calculate the cost effectiveness of such agents in terms of qaly so as to provide to the health payer , with comparative data of the cost effectiveness of this clinically effective treatment . biologic registers are of uttermost importance for such evaluations as they can provide long - term data on tnf- antagonists and facilitate the path to model building for the calculation of qaly . this data , which is currently unavailable in malta , could shed a more decisive light on the timely intervention of tnf- antagonists which could ultimately result in a clinically effective , yet cost - effective treatment . the economic burden of the biological agents and the use of tnf- antagonists in rheumatology is many times questioned by policy makers and financial settings within a national health service scheme . an improvement in quality of life is achieved within 6 months of therapy , as shown in this short - term study , and if this is sustained , the magnitude of the pharmacoeconomic impact increases substantially . this initial data could be useful to transmit the message to policy makers that for each individual patient , the potential of improving the quality of life and the pharmacoeconomic benefit is evident .

background . tnf- inhibitors have shown to be effective in reducing disease activity and improving the quality of life . due to the high costs associated with acquisition of this treatment , this study was undertaken to evaluate the icer of tnf- antagonists ( etanercept , adalimumab , and infliximab ) in improving the quality of life . methods . the haq and sf-36 were administered at phases 1 , 2 , and 3 , in order to assess the improvement in the qol . suppression of disease activity was assessed through the das-28 . results . statistically significant improvements ( p < 0.05 ) were noted for the sf-36 and haq after 3 months and for the das-28 after 6 months of tnf- inhibitor therapy . the mean icer per 10% improvement in the haq , das-28 , and sf-6d were 1976.5 , 2086.5 , and 2316.4 , respectively , following 6 months of tnf- intervention . most favorable icers were reported from a patient who had to undergo surgical intervention whilst on dmard therapy . conclusion . significant improvement was observed in patients ' quality of life , after a short timeframe of 6 months . such data is useful information in the light of convincing policy makers , in terms of providing access to the medications to individual patients on national health service schemes .

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

rheumatoid arthritis ( ra ) is a progressive , inflammatory disease which is characterised by inflammation of the joint synovium that could ultimately progress to joint destruction [ 1 , 2 ] . this disabling condition imposes substantial economic burden through the decreased quality of life ( qol ) and loss of productivity . recent advances in biotechnology and pathogenesis of ra have led to the discovery of biological dmards . biological agents inhibit pro - inflammatory cytokines which are believed to have a crucial role in the inflammatory process within the synovial joint . tnf- inhibitors have proved their clinical efficacy and raised the previous goals of ra treatment [ 5 , 8 ] . yet , these breakthrough drugs are associated with high procurement costs . this ultimately increases the financial burden ra imposes on society [ 4 , 5 ] . such a scenario has elicited the need to carry out pharmacoeconomical assessments in order to inform policy and decision makers of the cost - effectiveness of the biological dmards [ 5 , 8 ] . hence , this study was undertaken to determine the improved qol and the incremental cost - effectiveness ratio ( icer ) involved in treating maltese patients suffering from resistant ra , with tnf- inhibitors . data collection used to conduct this 6-month study was carried out at the rheumatology outpatient clinic at mater dei hospital . patients were eligible to participate in the study provided that they had been diagnosed with ra according to the 1987 acr classification criteria , failed to achieve a low disease activity despite dmard / s therapy ; and were switched onto a tnf- inhibitor ( etanercept , adalimumab or infliximab ) . a signed consent form was obtained from every patient who accepted to participate , out of their own voluntarily will . the disease - specific health assessment questionnaire ( haq ) was chosen as an outcome measure tool to assist in the evaluation of the functional improvement experienced by patients that were switched onto tnf- inhibitor therapy . the haq is a reliable tool used by various studies to assess daily activities , namely , dressing , grooming , eating , walking , hygiene , and so forth . unlike the haq , the higher the score obtained in each of the sf-36 domains , the more it indicates a better health status in the respective domain . the das-28 was used as a clinical outcome measure tool in order to monitor disease activity with biological intervention . the das-28 generates a continuous scale ( 0 to 9.4 ) through the assessment of tender joints ( 028 ) , swollen joints ( 028 ) , erythrocyte sedimentation rate ( mm / hr ) , and the visual analogue scale ( 0100 ) . this prospective study , carried out between 2010 and 2011 , had a time phase of 6 months , during which patients were assessed 3 times through the sf-36 and haq . failure to achieve the desired outcome and following discussions with the rheumatology consultant , patients were identified as suitable candidates for initiation of tnf- inhibitor therapy . subjects who accepted to participate in the study were interviewed by the investigator using the haq and sf-36 . raw data obtained for both the sf-36 and the haq were inputted in a microsoft excel database . at phase 2 ( t3 months after initiation of tnf- inhibitor therapy ) , participants were reinterviewed using the same questionnaires . at phase 3 ( t6 months ) , data from the sf-36 and haq were again collected following 6 months of tnf- inhibitor therapy . the sf-36 scores were converted into sf-6d scores using the algorithms provided by the university of sheffield . the excel program based on nonparametric bayesian preference weights was chosen since the non - parametric model has shown added benefits in the predictive capacity of the model over the parametric random effects model . the raw scores for every item within the sf-36 for all the 13 patients were inputted in the excel programme which generated utility values , anchored at 0 for dead and 1 indicative of full health . during both phases 1 and 3 , patients were examined by the rheumatology consultant and assigned a das-28 score as a measure of disease activity . in both phases , medical case notes of patients were reviewed and related data were collected , namely , das-28 scores , treatment regimens , adverse events , history of hospitalisation during the study period , and surgeries performed related to ra . data collected from the haq and sf-36 questionnaires were analyzed statistically using spss version 18.0 . the one - way anova test was used to compare the mean scores obtained during phase 1 , 2 , and 3 for both the haq and the sf-36 . scores obtained in the haq and sf-36 were also analysed using the post hoc tukey 's test where pairwise comparison between the mean rating scores of any two visits was carried out . the costs of tnf- inhibitors , dmards , and glucocorticoids involved in the study were obtained from the maltese government health pharmaceutical services ( ghps ) . treatment regimens for every patient were obtained by scrutiny of patients ' notes prior to initiation of tnf- inhibitor therapy ( treatment b ) and after 6 months of biological therapy ( treatment a ) . costs of treatment b included the costs of dmards and concomitant steroids for a period of 6 months prior to phase 1 . prior to commencement of a tnf- inhibitor , patients participating in the study had been on the same nonbiological dmard / s for at least 6 months . the direct costs of treatment a included the cost of the biological agent being administered , any concomitant dmard , and steroids for a period of 6 months . the economic evaluation used in this study was a cost effectiveness analysis ( cea ) which combines the difference between the costs of treatments with improvement achieved through biological intervention . the improvement in the haq was chosen as a unit of health benefit since the haq is a strong predictor of the functional disability observed in patients with ra through loss of productivity [ 5 , 16 ] . the icer per unit of improvement in haq was calculated using the following formula : ( 1)icer per unit improvement in haq = ( cost of treatment acost of treatment b ) (haq score on treatment b haq score on treatment a)1 . the das-28 score was also used as a unit of health benefit , as it highly reflects the disease activity and is thus useful in indicating the suppression of disease activity following a therapeutic intervention . the icer per unit of improvement in the das-28 through biological intervention was calculated using the following formula : ( 2)icer per unit improvement in das-28 = ( cost of treatment acost of treatment b ) (das-28 score on treatment b das-28 score on treatment a)1 . both the haq and the das-28 give an ordinal scoring system which is often criticized if used as a unit of health benefit to express the incremental icer . hence , the icer was expressed in terms of unit improvement in the sf-6d using the following formula : ( 3)icer per unit improvement in sf-6d = ( cost of treatment acost of treatment b ) (sf-6d utility index on treatment b sf-6d utility index treatment a)1 . comparison between resulting icers using the 3 different methodologies could not be made since the ( i ) haq gave scores ranging from 0 to 3 , ( ii ) the das-28 scores ranged from 0 to 9.4 , and ( iii ) the sf-6d ranged from 0 to 1 . this has prompted the researcher to devise a scoring scheme ranging from 0 to 10 in order to compare icers obtained over a 6-month period . the resulting icers from all 3 different methodologies were calculated and expressed in terms of this scoring scheme . one - way anova testing was conducted on the resulting icers in order to assess significance between outcome health benefits chosen . patients had been suffering from ra , prior to initiation of tnf- inhibitor therapy for a mean of 11 years ranging from 4 to 31 years . as shown in figure 1 , the mean haq scores achieved in phase 1 were the highest indicating the functional disability experienced despite conventional dmard therapy . progress achieved was sustained and continued to improve during the last 3 months of the study , yet to a lesser extent than in the previous 3 months . table 1 shows that all activities assessed within the haq have improved significantly ( p < 0.05 ) between phase 1 and 2 . the mean difference in the haq scores obtained between phase 2 and phase 3 was not statistically significant ( p > 0.05 ) . during the whole study , this result indicates that significant improvement is observed as early as 3 months after initiation of tnf- inhibitor therapy . table 1 shows the difference in mean scores obtained between phases for every activity assessed within the haq and their relative level of significance ( significance noted at p < 0.05 * ) . as noted from figure 3 , patients reported low functional status in the sf-36 whilst on dmard therapy . during phase 2 and 3 , all patients reported an improvement in the functionality for all domains within the sf-36 . the greatest improvement was observed in the physical and emotional role between phase 1 and 2 . every sf-36 domain improved significantly between phase 1 ( baseline ) and phase 2 since p values obtained were < 0.05 level of significance ( table 2 ) . as observed with the haq scores , p values between phase 2 and 3 for every sf-36 domain were not significant ( p > 0.05 ) . sf-36 domains improved significantly after 6 months of biological intervention between phase 1 and 3 ( p < 0.05 ) . lower p values were obtained for the er and bp when compared with other domains between phase 2 and 3 , thereby indicating continuous improvement during the latter part of the study . table 2 shows the difference in mean scores obtained between phases for every sf-36 domain and their relative level of significance ( significance noted at p < 0.05 * ) . during phase 1 , as observed in figure 2 , 85% of patients ( n = 11 ) scored higher than 5.1 indicating a high disease activity according to the eular criteria . after 6 months of biological intervention , the das-28 decreased significantly ( > 1.2 ) for all 13 patients . 31% of patients ( n = 4 ) scored < 3.2 in the das-28 which corresponds to low disease activity whilst 15.4% ( n = 2 ) scored < 2.6 which is compatible with disease remission . a slight improvement in the haq score despite biological intervention results in a high icer ( table 3 ) . patient 10 experienced a minimal improvement in the quality of life which gave an icer of 26,030 over a period of 6 months . the rest of subjects ( n = 12 ) experienced a much lower icer due to a higher improvement recorded in the quality of life . patient 13 had undergone a hip replacement surgery prior to phase 1 and hence gave a more favorable icer , due to a lower discrepancy between the costs of treatments . in order to improve by 10% in the quality of life as measured by the haq , an average addition of 1,977 is incurred over a period of 6 months . table 3 shows the icers per unit improvement and per 10% improvement in the haq following 6 months of tnf- inhibitor therapy . the least reduction in disease activity was again reported by patient 10 ( table 4 ) . for every unit of improvement in the das-28 ( reduction in disease activity by 1 on a scale from 0 to 9.4 ) an increase of 2,200 the average reduction in disease activity ( das-28 ) was of 2.71 , which is considered as a significant good response ( > 1.2 ) . in order to achieve a reduction of 10% in the das-28 score ( i.e. , 0.94 ) table 4 shows the icers per unit improvement and per 10% improvement in the das-28 following 6 months of tnf- inhibitor therapy . an average patient , following 6 months of tnf- inhibitor , gained a 0.27 improvement in the utility health index , the sf-6d . theoretically , an icer of 23,164 is incurred for a patient to regain a perfect quality of life ( 1 ) from a state ( 0 ) equivalent to death ( table 5 ) . an improvement of 0.1 as assessed through the sf-6d would cost the payer an additional 2,316 . table 5 shows the icers per unit improvement and per 10% improvement in the sf-6d following 6 months of tnf- inhibitor therapy . no significant difference ( p > 0.05 ) was noted between the 3 different methodologies used to calculate the icer ( table 6 ) . the choice of the sf-6d yielded the higher icer when compared to resulting icers in terms of the haq and das-28 . table 6 shows the level of significance ( significance noted at p < 0.05 ) and standard deviations between the haq , das-28 , and the sf-6d . results obtained within this study highlight the evidence of the significant improvement gained in the qol caused by this disabling condition in the initial treatment phases . various studies have used the haq to shed light on the quality of life and physical disability endured by patients . following 3 months of treatment with tnf- inhibitors , scores ranged from 0.2 to 1.5 , indicating a considerable significant improvement ( p < 0.05 ) . scores continued to decrease furthermore during the last 3 months of the study yet significance was not noted ( p > 0.05 ) . apart from the functional impairment assessed within the haq , which hinders patients from their daily activities , ra affects considerably the vitality , emotional wellbeing , and mental health . this has been confirmed by the low mean scores reported in the study for all domains within the sf-36 at phase 1 . despite multiple dmard therapy and concomitant glucocorticoids all domains improved significantly after 3 months of tnf- inhibitors ( p < 0.05 ) . in the latter half of the study , significance was not noted since scores achieved were similar to phase 2 apart from the emotional role which continued to improve ( p > 0.05 ) . this is in concordance with the findings from the haq which suggests that a plateau is reached after 3 months of biological intervention . such an observation highlights the need for long - term studies , in order to assess whether improvement gained is sustained and to what extent . findings from this study are in accordance with results obtained from the generic sf-36 questionnaire in a study conducted on a spanish population . findings from both hrqol questionnaires used indicate the clinical effectiveness of tnf- antagonists in achieving a better qol , as documented in various studies [ 20 , 21 ] . the das-28 score used a clinical marker to monitor the disease activity after intervention with tnf- inhibitors . at phase 1 , 85% of subjects were experiencing a high disease activity ( > 5.1 ) as devised by the eular criteria . baseline das-28 results further support the resulting disability and compromised quality of life reported in the hrqol questionnaires . following 6 months of biological intervention , prevalence of das-28 < 3.2 was 31% ( n = 4 ) which is compatible with low disease activity . abalos medina reported a higher prevalence of acr / eular remission , yet this must be interpreted in the light of the limited number of participants and length of the current study . tnf- antagonists are the forerunners in achieving such a therapeutic goal as they have been proven to suppress disease activity . since the procurement of tnf- antagonists is associated with incredibly high costs when compared with conventional dmard therapy , economic evaluations are essential as benefit from biological treatment is evident , but financial constraints limit its availability . the haq was chosen to assess the improvement achieved through biological intervention , since a close correlation was reported between haq and health care costs [ 2426 ] . a unit of improvement in the haq ( any 1 unit difference between 0 and 3 ) was chosen as a health outcome benefit in order to assess the icer . such result is enlightening as it demonstrates that tnf- antagonist therapy is more likely to be cost effective in patients with severe functional disability , who may ultimately require surgical interventions . the least favourable icer was achieved by patient 10 who experienced the least improvement in the haq ( gain of 0.25 ) . in such a scenario , it is evident that the disease is resistant despite biological treatment and alternative treatments should be initiated in order to gain a better control of the disease and consequently qol . this highlights the need for effective treatment in order to achieve maximal improvement in qol and hence diminish the costs incurred . subjects achieved an average significant reduction of 2.71 ( > 1.2 ) following 6 months of treatment . the highest icer in terms of das-28 was again reported by patient 10 which resulted due to a minor improvement reported in the das-28 . the icer per unit of improvement in the sf-6d gave an average of 23,164 which is extremely higher than the icers per unit of improvement in the haq and das-28 . yet such a high cost resulted since it accounts for a transition between death and a perfect quality of life . all icer results were expressed in terms of the same scoring scheme to be able to compare results . for every 10% improvement in the sf-6d , a cost of 2,316 was incurred after 6 months of tnf- antagonist intervention . statistical significance was not noted ( p > 0.05 ) between results obtained per 10% improvement in the respective health outcome . this could be either due to the limited number of observations ( n = 13 ) or due to the large variation in costs between the 13 observations of each method as shown in the large standard deviations ( table 6 ) . similar studies should be undertaken to be able to calculate the cost effectiveness of such agents in terms of qaly so as to provide to the health payer , with comparative data of the cost effectiveness of this clinically effective treatment . biologic registers are of uttermost importance for such evaluations as they can provide long - term data on tnf- antagonists and facilitate the path to model building for the calculation of qaly . this data , which is currently unavailable in malta , could shed a more decisive light on the timely intervention of tnf- antagonists which could ultimately result in a clinically effective , yet cost - effective treatment . the economic burden of the biological agents and the use of tnf- antagonists in rheumatology is many times questioned by policy makers and financial settings within a national health service scheme . an improvement in quality of life is achieved within 6 months of therapy , as shown in this short - term study , and if this is sustained , the magnitude of the pharmacoeconomic impact increases substantially . this initial data could be useful to transmit the message to policy makers that for each individual patient , the potential of improving the quality of life and the pharmacoeconomic benefit is evident .

electron transfer ( et ) is one of the simplest and most ubiquitous reactions in nature . it has been extensively investigated and since the ground - breaking work by marcus and successors is seemingly well - understood . however , some important details still remain unclear , in particular in the context of low solvent permittivity and whenever transient complexes play a role . furthermore , the questions of whether et proceeds as a contact or distant reaction or whether the loose or contact ion pair is initially formed remain a matter of debate . these questions are not easily answered , even using ultrafast spectroscopy , because the data analysis usually builds upon multiparametric models , which can give rise to ambiguous interpretations and quantification is difficult . in the case of photoinduced et , the picture is getting even more complicated if exciplexes are involved . exciplexes constitute important intermediates in many photochemical reactions in chemical or biological systems , and their contribution to et quenching is debated even in polar media . in general , exciplexes can be monitored by their emission . this effect is brought about by the equilibration of the exciplex with the corresponding spin - correlated radical ion pair ( rip ) , which can undergo singlet triplet transitions and subsequently recombines . figure 1 depicts a reaction scheme of the photoinduced et processes in a typical exciplex forming donor , the ordinate corresponds to free energy and the abscissa can be expressed as an effective reaction coordinate involving the distance between fluorophore ( f ) and quencher ( q ) and the marcus outer - sphere electron transfer reaction coordinate . the ordinate corresponds to free energy ; the abscissa is a projection of the two - dimensional reaction coordinate comprising the marcus outer - sphere electron transfer reaction coordinate ( i.e. , solvent polarization ) and the interparticle distance . the blue and red arrows indicate the ( radiative ) decay processes of either the locally excited state or the exciplex that are observed in the experiment . the presented scheme is a unification of two reaction schemes suggested by weller et al . in refs ( 28). photoexcitation of the fluorophore ( 1 ) opens up two different pathways ( numbers refer to figure 1 ) . full et may directly yield the rip ( 3 ) or the exciplex may be generated by partial et in a contact reaction ( 2a ) . if the charge transfer within the exciplex is complete , the term contact ion pair is more qualified . clearly , in the latter case , with vanishing coupling the charge transfer emission also disappears . note that often only one of these reaction pathways ( 2a or 3 ) is exclusively , and not seldomly arbitrarily , assumed . if the exciplex is the primary reaction product , the rip can be formed in a secondary step by the dissociation of the exciplex ( 2b ) , with suitable solvent permittivities and viscosities taken for granted . in any case , the initial et processes preserve the overall spin of the reactant pair , i.e. , the rip and the exciplex are formed in their singlet state . if the radical ion pair diffusively separates such that the exchange coupling becomes sufficiently weak , the electronic zeeman and hyperfine interactions can coherently convert the electronic singlet state into an electronic triplet state ( 4 ) . this process depends on the external magnetic field and , for the organic systems studied here , is less efficient if a magnetic field on the order of tens of millitesla is applied . . however , here we focus on relatively high fields . ) in the presence of such magnetic fields , the zeeman - splitting of the triplet energy levels causes the intersystem crossing probability to diminish , since near - degeneracy can only be attained between the singlet state and one of the triplet states , whereas all of the three triplet states are involved in the interconversion process at low external magnetic fields . note , that the singlet ( s ) and triplet ( t ) radical ion pairs can undergo charge recombination , yielding the ground state and the fluorophore in its triplet state , respectively . the singlet back electron transfer to the ground state is typically located in the marcus inverted region and , hence , is slow . in addition , only the singlet ion pair can re - form the exciplex ( 5 ) , and thus , its formation and emission ( 6 ) can be influenced by the external magnetic field . note that magnetohydrodynamic effects and susceptibility differences do not play a role at the low magnetic field used here . in summary , the following elementary processes give rise to a magnetic field effect ( mfe ) on the exciplex emission : photoexcitation ( 1 ) , exciplex formation and its dissociation into ions ( 2a , 2b ) , direct formation of the ions via distant et ( 3 ) , spin evolution accompanied by spin - selective charge recombination ( 4 ) , re - formation of the exciplex ( 5 ) , and exciplex emission ( 6 ) . note that all relevant species can be described in a unified picture as a function of interparticle distance and a solvent coordinate on a common free - energy hypersurface . this model was recently used to rationalize the exciplex emission using a mulliken hush - like approach . in this work we will heavily rely on magnetic field effects and spin chemical ideas , which are now well - understood and the subject of comprehensive reviews . in fact , the radical pair mechanism is now again in the spotlight of current research , since it probably is the common denominator of the ( adverse ) effects of magnetic fields on biological systems or the cryptochrome - based magnetic compass of several migratory animals . the possibility of monitoring the magnetosensitive exciplex emission opens up the possibility of detecting radical pair dynamics with a much higher sensitivity than usually attained by making use of absorption spectroscopic techniques . only recently could the sensitivity of the detection of time - resolved mfes based on transient absorption be improved considerably using cavity ring - down detection . yet , at the moment of writing , the time resolution so obtainable is lower than in the experiments presented herein . recent studies that rely on the exciplex fluorescence as a probe of magnetic field effects comprise such diverse topics as degenerate electron transfer , the recombination function , radio- and microwave magnetic field effects , or excited state reversibility . several attempts to detect the increased exciplex luminescence in a time - resolved manner have been realized . however , most of these studies were focused on intramolecular systems ( chain linked donor acceptor pairs ) . staerk and co - workers , for example , investigated single photon timing traces of intramolecularly linked pyrene a proof of concept study has employed time - resolved measurements of the mfe on freely diffusing ( intermolecular ) exciplexes , but no systematic studies have been undertaken so far . furthermore , time - resolved studies of the reaction products , i.e. , ions , have frequently been employed , but the time resolution of these studies was rarely sufficient to observe the evolution of the geminate mfe in real time . the effect of microwave excitation on the time - resolved exciplex emission of a polymethylene - linked donor acceptor system has also been studied . in most cases , however , a more quantitative interpretation is desirable , since it could give further insight into the details of the reaction mechanism and the role of the exciplex in the magnetic field effect . only a single attempt has been made so far to simulate the experimental data theoretically . in ref ( 59 ) the authors made use of a simple analytical model . a reaction scheme according to which the rip is formed initially and the exciplex recombination occurs irreversibly was used . approximating the singlet probability by linear functions unfortunately , no details about the mechanism as to the role of the exciplex could be extracted from the time - resolved data , since it was a priori assumed that the radical ion pair is the primary quenching product . in the literature dealing with magnetic field effects it is generally assumed that the radical ion pair is initially generated by distant electron transfer and it later irreversibly combines to give the exciplex . in contrast to that , many studies focusing predominantly on photochemical aspects can be found , suggesting that the exciplex is the initial quenching product even at fairly high permittivities . in this paper , we would like to address these two opposing paradigms , in which either the exciplex or the radical ion pair is the initial quenching product in the above - mentioned reaction scheme . we will show that , by using time - resolved exciplex emission , we are able to identify the nature of the initial reaction product without introducing biasing assumptions but rather by direct observation of the mfe . our simple idea relies on the fact that the exciplex dissociation is a comparably slow process . the ions resulting from the exciplex dissociation will be formed with delay with respect to those formed by direct electron transfer . as a consequence the mfe generated by the exciplex route will be delayed as well . in this way the time - resolved mfe allows discriminating both pathways ( 2 versus 3 ) and can effectively complement ultrafast observations . the paper is structured as follows : in section ii we commence by presenting photochemical properties of the donor acceptor system 9,10-dimethylanthracene / dma before we acquaint the reader with time - resolved mfe measurements and demonstrate how these measurements relate to steady - state results . thereafter , in section iii , three models will be discussed , two of which represent the current paradigm of irreversible exciplex formation ( termed models 1a and 1b , sections iii.i and iii.ii ) . in addition , we introduce model 2 ( section iii.iii ) , which includes the reaction pathway of direct exciplex formation ( 2a ) , exciplex dissociation into a loose ion pair ( 2b ) , and reencounter ( 5 ) . we continue by showing that only the latter model is suitable to explain the observed time - resolved mfes . we then use this model in section iv to extract pertinent parameters of the exciplex kinetics , the most important parameter being the probability that the initial photoinduced et reaction directly yields the exciplex . we show that even in comparably polar media the direct exciplex formation is found to be significant , and we close with a discussion and conclusions of these findings ( sections iv and v ) . we have studied the time - dependent mfe on the 9,10-dimethylanthracene ( f)/n , n - dimethylaniline ( dma , q ) system in propyl acetate ( pa , r = 6)/butyronitrile ( bn , r = 24 ) solvent mixtures . these mixtures allow for a systematic variation of the solvent permittivity , r , in a range from 6 to 24 ( at 295 k ) while the viscosity and , thus , the diffusion coefficients are kept constant . in addition , the refractive index ( nd = 1.3845 ) is amazingly invariant with the solvent composition , such that the pekar factor [ ( 1/n ) ( 1/r ) = 0.459 ] , which governs the outer - sphere electron transfer reorganization energy and , thus , the rate of et processes , changes by less than 6% for r in the range from 11 to 24 . at permittivities lower than r = 11 the mfe of the investigated system decays strongly . at the lowest permittivity considered in this study ( r = 10 ) , the deviation of the pekar factor amounts to only 8% . moreover , the zero zero energy is practically constant in the used solvent mixture . hence , the quenching and the back - et reactions can to a good approximation be considered solvent - independent , and the changes of the mfe are predominantly brought about by the modulation of the radical pair interaction , i.e. , the potential of mean force governing the diffusive excursion and the re - encounter probability of the rip . absorption and emission spectra of 9,10-dimethylanthracene in the absence ( bottom ) and presence ( top ) of 0.05 m n , n - dimethylaniline . the solvent is a mixture of propyl acetate and butyronitrile of relative dielectric constant r = 14 ( 43 wt % butyronitrile ) . the emissions of the exciplex and the locally excited fluorophore are shaded in red and blue , respectively . figure 2 depicts the absorption and emission spectra of 9,10-dimethylanthracene in the absence and presence of dma . the emission spectrum is decomposed into emission of the locally excited fluorophore and the exciplex emission . to separate the two contributions , the exciplex emission is modeled as a superposition of franck condon weighted vibronic transitions . the spectrum of the pure fluorophore being known , linear least - squares fitting provides access to the spectrum of the exciplex . when this donor acceptor system is immersed in a magnetic field , the exciplex emission increases . this is expected on the basis of the radical pair mechanism , since the singlet triplet degeneracy is lifted in the presence of an external magnetic field and the t states no longer efficiently participate in the singlet triplet conversion within the separated , yet spin - correlated , rip . the extent of the mfe , e , is quantified as the relative change of emission intensity by applying a saturating magnetic field of 150 mt . for the investigated system , the mfe goes through a maximum of approximately 11% for a dielectric constant of 19 . it strongly depends on the permittivity , since r balances the radical ion pair separation , which is necessary for efficient spin evolution and the ( re)encounter of the pair . note in this context that radical pair lifetimes on the order of nanoseconds are required for efficient singlet triplet mixing by the hyperfine interaction in organic radicals . in addition to the exciplex , the locally excited fluorophore shows a feeble mfe brought about by excited state reversibility . the time - resolved data were measured using a custom - built time - correlated single photon counting apparatus with an excitation wavelength of 395 or 374 nm and a long - pass filter in front of the detection to ensure the observation of pure exciplex emission . a detailed description of the experimental procedure and the apparatuses used is given in the first three pages of the supporting information . note that this technique employs very low light intensities and tiny fluorophore ( and hence rip ) concentrations , such that no ions accumulate and bulk , i.e. , bimolecular , processes are negligible next to the geminate processes . this is fundamentally different from the experimental conditions frequently employed in , for example , transient absorption experiments ( if not making use of the recently introduced cavity ring - down detection ) . the exciplex time traces rise with a time constant of approximately 1.8 ns , which is within experimental error independent of the magnetic field . the decay kinetics is in general rather complex and includes the dissociation into free ions and recombination giving rise to delayed emission . when an external magnetic field is applied , the delayed fluorescence intensity increases , which can be seen in figure 3 ( lower panel ) . we have adjusted the time profiles by matching the intensities during the initial rise up to and including the maximum and formed the difference i(t ) = i(t , b0 ) i(t , b=0 ) to obtain the time - resolved mfe , as also shown in figure 3 ( upper panel ) . using an ultrafast setup we have verified that no significant mfe is detectable within the matching interval . lower panel : exciplex emission decays of the 9,10-dimethylanthracene ( 10 m)/n , n - dimethylaniline ( 0.05 m ) system in pure butyronitrile at r = 24.3 in the presence and absence of an external magnetic field ( ca . 150 mt ) observed with a long - pass filter ( > 600 nm ) after excitation at 395 nm . in the presence of an external magnetic field , upper panel : time - resolved magnetic field effect i obtained by taking the difference of the experimental single photon timing traces recorded in the presence and absence of an additional external magnetic field ( gray ) and the fit to the experimental data ( black ) . there is an intimate relation of i and the total mfe observed in a steady - state experiment : subsuming all time - resolved intensities the steady - state intensities can be calculated . ( this holds true assuming that the time resolution of the experiment is sufficiently high to detect the entire decay kinetics , which is clearly fulfilled in the present case.)1 we have integrated the matched time traces for t < 400 ns to determine e . the integrated mfes are plotted in figure 4 and compared to the steady - state results . the two sets of data apparently coincide , indicating that the entire mfe indeed occurs within the accessible time window ( 400 ns or significantly less at higher permittivities ) , under low light intensity and low concentration conditions . this applies to solutions of low and comparably high dielectric constants in the same manner . the agreement of the values for the absolute magnetic field effect obtained from steady state and time - resolved data suggests that no bulk processes contribute to the mfes observed here . in particular , f - pairs , i.e. , reencountering ions in the bulk , and processes involving triplet states of the fluorophore are negligible . this is corroborated by the fact that the time span of the mfe is reduced with increasing permittivity , even though the significance of processes stemming from diffusively separated ions is expected to increase . solvent polarity dependence of the magnetic field effect of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex determined from steady - state ( filled circles ) and time - resolved ( open circles with error bars ) measurements . the dielectric constant is varied by altering the composition of the binary solvent mixture of propyl acetate and butyronitrile . within experimental error , the time - resolved mfe for different permittivities is shown in figure 5 together with simulations , to be discussed below . no mfe is detected within the first two nanoseconds of the experiment , which is expected in view of the slowness of the spin evolution . the maximum of the time - resolved mfe occurs in the range from 20 to 60 ns after excitation , with the larger values occurring at lower permittivities . in general , i peaks at times where the delayed fluorescence is substantial and thereafter the effect decays and reaches the noise level of the experiment within a time window of 400 ns ( or significantly earlier at higher permittivities ) . experimental time - dependent magnetic field effects at different dielectric constants ( as given in the figure ) for the system 9,10-dimethylanthracene / n , n - dimethylaniline in propyl acetate / butyronitrile mixtures . the differences of the single photon timing traces in the presence and absence of an external magnetic field ( 150 mt ) are shown in gray . the time traces have been amplitude - matched during the buildup of the exciplex ( t < 2 ns after excitation ) . the green dotted lines are simulations of the data using model 2 with i = 1 ( ions are generated first ) , whereas the red dashed lines have been obtained using model 2 with i = 0 ( exciplex is generated first ) . the solid blue lines are simulations of the experimental data using model 2 and fitting i ; no scaling factor was used . the left - hand side shows the normalized data , which emphasizes the differences in the temporal evolution for i = 0 and i = 1 . note that only by using model 2 and a variable i the shape and the amplitude of the experimental data can be reproduced without scaling . to model the time - resolved mfe data depicted in figure 5 we started off using the commonly applied procedures found in the literature . traditionally , the mfe on the exciplex has been modeled using2where ps(t , b0 ) is the probability that the rip born in the singlet state is in its singlet state at time t , and f(t ) is the recombination function , i.e. , the probability that the rip recombines at time t. e is the intrinsic exciplex lifetime . among these quantities , the only one depending on the external magnetic field , b0 , is ps(t , b0 ) von neumann equation in the limit of low viscosities and/or not too low permittivities . in this low - viscosity approximation , distant and spin - selective recombination reactions as well as the exchange interaction can be neglected in the first order on account of the fact that the development of the mfe is a slow process and that trajectories yielding a large mfe predominantely involve diffusively separated particles . for these pairs , the details of the recombination and the exchange interaction are negligible during the majority of their lifetime . model 1a assumes that the initial quenching product is the singlet radical ion pair ; i.e. , it describes the irreversible pathway ( 3 4 5 6 ) in figure 1 . despite its simplicity , the model has been very successfully used to describe the mfe on exciplexes and to model the dependence on solvent properties , degenerate electron exchange , and rip recombination . model 1a can easily be modified to account for initial exciplex formation by introducing a parameter i describing the probability that the initial charge separation process yields the loose rip ( path 3 in figure 1 ) , thereby giving rise to model 1b . the irreversible model introducing the possibility of initial exciplex formation will be referred to as model 1b . the exciplex probability can in this case be calculated from3 both models discussed so far rely on the assumption that the magnetic field effect only results from ions that irreversibly combine to the exciplex . when modeling experimental data using these models , the only adjustable parameter is the exciplex lifetime e . time - dependent magnetic field effect traces calculated with model 1b are shown in figure 6 . normalized simulated time traces using the irreversible model 1b at different values of the only ( artificially ) variable parameter e ( from 5 to 30 ns ) as opposed to a noise - free representation of the experimental data at e = 18 ns , which was obtained by fitting a combination of exponential functions to the experimental time - dependent magnetic field effect . the experimental data was measured using the system 9,10-dimethylanthracene ( 10 m)/n , n - dimethylaniline ( 0.05 m ) in a mixture of propyl acetate / butyronitrile at r = 20 ( 75 wt % butyronitrile ) . note that variations of i have predominantly the effect of changing the amplitude of the simulated trace , leaving its shape approximately unaltered . i in model 1b has thus the role of a mere scaling factor , while the only parameter influencing the shape is e . from figure 6 it becomes clear that the data , obtained for an experimentally determined exciplex lifetime of 18 ns , can not be described using this irreversible model . even when artificially varying e in the range from 5 to 30 ns as shown in figure 6 , the experimental data can not be reproduced . in all cases , the experimental mfe rises significantly more slowly than predicted by model 1b . e only increases the time span of the decay of the effect , but it does not lead to a significant shift in the peak position . there are little doubts about the validity of the low - viscosity approximation and ps , which , in the same manner , do not only apply to exciplexes but also to pulse radiolysis studies , etc . in fact , the applicability of the low - viscosity limit has been thoroughly tested and found valid for exciplex systems in ref ( 55 ) . we therefore propose that the discrepancy between model and experiments results from an incomplete description of the reaction scheme and that the exciplex dissociation must be taken into account . position of the maximum ( or center of gravity ) of the time - resolved magnetic field effect in time after excitation as a function of the exciplex lifetime . additionally , we observe that the center of gravity and/or the position of the maximum of i are linearly related to the lifetime of the exciplex , as is shown in figure 7 . as the lifetime of the exciplex relates to its initial decay , the observation of a linear relationship supports our proposition , since it suggests that the dissociation of the exciplex is the rate - limiting step for the mfe and needs to be taken into account for a proper description of the experimental data . we thus introduce model 2 , which takes the dissociation of the exciplex into account . equation 2 has to be extended by terms accounting for the initial quenching product and the exciplex kinetics . we propose evaluating the probability that the radical ion pair exists as exciplex , pe(t , b0 ) , from4where kd is the rate of exciplex dissociation and r(t , b0|ri ) dt denotes the probability that the rip formed at t = 0 at distance ri has recombined until t. ri is the formation distance of the loose ion pair by ( distant ) et , and re is the contact distance of donor and acceptor at which the transition of the loose ion pair to the exciplex ( or contact ion pair ) occurs , e.g. , by assuming a favorable ( relative and internal ) orientation , giving rise to the possibility of assuming a stacked arrangement with large electronic coupling . typically exciplex formation will eventually give rise to an interparticle distance smaller than re . re is also the interparticle distance to which the dissociation of the exciplex leads . the different terms on the right - hand side of eq 4 can be understood as follows : the first summand , e = 1 i , specifies the probability that the exciplex is formed initially ( path 2a in figure 1 ) , while the second summand gives the probability that the initially formed rip forms an exciplex until t. the third term gives the probability that the exciplex dissociates at time and is re - formed until t , and the last term describes the depopulation by dissociation or radiative / nonradiative decay . graphic visualization of the employed theoretical models for the irreversible case ( radical ion pair is generated first , models 1a and 1b , left and center ) and the reversible case ( model 2 , right ) . equation 4 is a volterra integral equation of the second kind with convolution kernel . the equation has been solved numerically by discretizing the time axes on an equidistant grid and substituting the integral by the well - known quadrature expression based on repeated application of simpson s rule . the singlet recombination probability r(t , b0|ri ) depends on the singlet probability ps(t , b0 ) , the mutual diffusion of the rip , and the recombination rate ka in figure 8 . as a consequence , the time evolution of the mfe depends on the parameters of the diffusive motion via r(t , b0|ri ) , the exciplex lifetime , and its dissociation quantum yield , diss = kd e , and parameters of the radical ion pair , in particular i and the association constant , ka= ka / kd , and the parameters governing the spin evolution ( hyperfine coupling constants ) . the singlet probability also depends on the rate of degenerate electron exchange of the donors , and this effect has been fully taken into account ( cf . it turns out that there are only two parameters , namely , i and ka , which have a strong effect on the shape and the magnitude of the time - resolved mfe ( cf . these parameters can be extracted from the experimental data by least - squares fitting . figure 5 shows simulations of the time - resolved mfes for several solvent permittivities using model 2 . the simulation reproduces the experimental findings with respect to both shape ( left panels ) and magnitude ( right panels in figure 5 ) . the exciplex lifetime , e , was determined from the initial decay of the time - correlated single photon counting traces of the exciplex , and the dissociation quantum yield , diss , was estimated from the dependence of e on permittivity assuming that for r = 6 no dissociation occurs and that the radiative and nonradiative rates of the exciplex are constant ( and/or small compared to the dissociation rate ) within the polarity range studied here . figure 5 shows fits to the experimental mfes for different solvent permittivities using model 2 . the simulations obtained in the limit of no initial exciplex formation ( i = 1 ) and no initial ion formation ( i = 0 ) are also shown for comparison . as can be seen from the figure , the mfe always rises steeply for i = 1 , with the maximum occurring too early in time , as has been discussed before . from the fit of model 2 to the experimental data , values for i and ka are obtained as a function of permittivity and are shown in figure 9 . the plot of i as a function of permittivity shows , first , that direct exciplex formation contributes at all permittivities and , second , that the probability of distant electron transfer quenching increases with increasing polarity of the solution . for the investigated permittivity range , i levels off at relative permittivities exceeding 20 . note , that the hypothesis of exciplexes contributing to the quenching in polar solution has been invoked several times to account for electron transfer quenching kinetics in polar solution , such as acetonitrile . in ref ( 19 ) , for instance , the authors already recognized the importance of the two parallel channels , i.e. , contact and loose ion pair formation were found to contribute simultaneously ( based on a kinetic scheme and the determination of rate constants and ion yields , i.e. , from indirect considerations ) . for 9,10-dicyanoanthracene / hexamethylbenzene in acetonitrile the efficiency of contact ion pair formation has been estimated to be around 0.7 . however , here we are limited to moderate r for which ample exciplex emission is detectable . the dependence of the association constant ka extracted from the least - squares fits ( cf . figure 9 ) indicates a weak exergonic association . using sutin s approach , the free energies of association from the ion pair to the exciplex can be estimated ( assuming a contact distance of 6.5 and a reaction zone of 0.8 width ) . potential wells in the range from 5.3 kbt ( low r ) to 3.2 kbt ( high r ) are found in this way . in one analysis run , we have also adjusted diss , the quantum yield of exciplex dissociation , in a least - squares sense . the resulting values are also shown in figure 9 and compared with the corresponding values estimated on the basis of e , as discussed above . the dependences are nearly identical , suggesting that , for the permittivities giving rise to large mfes , the exciplex lifetime is indeed mainly governed by separation into the ions . the delay of the maximum of the mfe with respect to the simplistic model 1a ( eq 2 ) results from the fact that , first , the exciplex is formed directly and , second , that its dissociation into the ions is slow , the rate being given by disse . the ions formed in this delayed manner will via the radical pair mechanism eventually give rise to a delayed mfe . this offers insight into the initial products of quenching , which is very difficult to obtain by different means . solvent dependence of the association constant ( ka , top panel ) , the dissociation quantum yield of the exciplex ( diss , center ) , and the fraction of excited fluorophore quencher pairs deactivating to solvent - separated ions ( i , bottom ) as opposed to the exciplex . the dissociation quantum yield has been evaluated from the exciplex lifetime under the assumption that for r = 6 the exciplex does not dissociate and that its radiative and nonradiative rate constants do not change with solvent permittivity in the range accessible by the used solvent mixture ( 6 r 25 ) . fitting the dissociation quantum yield diss did not give rise to significantly altered results for either ka or i . the reliability of the proposed method can be tested using isotopologues . substituting the protons in the fluorophore by deuterium has a huge impact on the singlet probability , ps(t , b0 ) , since the hyperfine coupling constants of the deuteriums amount only to 15% of those of the protons and , in addition , the spin quantum numbers of the nuclei differ ( i d = 1 , ih = /2 ) . time - resolved magnetic field effect of the exciplex of pyrene ( py - h10 ) ( bottom panel ) and perdeuterated pyrene ( py - d10 ) ( top panel ) with n , n - dimethylaniline in butyronitrile . for both fits the following identical set of parameters was used : i = 0.55 , ka = 4.6 m , diss 1 , e = 8 ns . only if the degenerate electron exchange of the n , n - dimethylaniline radical cation is taken into account does the model consistently , i.e. , with a common set of parameters , describe both the pyrene and the pyrene - d10 system . in figure 10 , the experimental time traces are compared for perdeuterated pyrene , py - d10 , and pyrene of natural isotope composition , py - h10 , with dma as quencher . no marked difference of the size of the mfe ( e , h = 8.3% and e , d = 8.4% ) as well as of its time dependence is observed . this is surprising at first view , since the singlet probabilities , calculated without taking degenerate electron exchange of the quencher dma into account , differ markedly ( cf . figure s3 in the supporting information ) and so do the calculated time curves . however , taking degenerate electron exchange into account , the differences in ps(t , b0 ) nearly disappear and the time - resolved mfe can indeed be modeled with one and the same set of parameters for py - h10 and py - d10 ( cf . figure 10 ) . for this system , too , a significant amount of exciplex is directly formed from the excited fluorophore quencher pair ( 1 i = 0.45 ) . furthermore , this result emphasizes that it is mandatory to explicitly take degenerate electron exchange into account . the effect is particularly pronounced if the hyperfine contribution of the exchanging radical ion exceeds that of the radical ion originating from the fluorophore . with the weak hyperfine field of the deuterated pyrene , the singlet triplet conversion is essentially governed by the dma and thus the mfe is more sensitive to its degenerate electron exchange . for the system 9,10-dimethylanthracene / dma of natural abundance note that model 2 introduced here reproduces the mfes with respect to both shape and absolute value . in particular , the time - integral can be used to model the steady - state mfe depicted in figure 4 . previously , we have modeled the steady - state data in terms of the recombination probability of a radical ion pair , i.e. , in terms of a model that does not refer to properties of the exciplex . the fact that this approach was successful despite neglecting essential details of the exciplex kinetics shows that the steady - state data are not suitable to discern these details . only the time - resolved mfe technique can reveal the details of the exciplex kinetics and yield a comprehensive description of the mfe . in this study we presented measurements of the time - resolved magnetic field effect on the exciplex kinetics in organic donor the integrated experimental data reproduces the steady - state data published earlier , demonstrating that bulk recombination of rips can be ruled out as a major contribution to the magnetic field effects observed here . by systematically varying the solvent permittivity while leaving most parameters influencing electron transfer rates and the solvent viscosity constant , we could show that the irreversible model ( model 1a or 1b ) , which is implicitly assumed in the commonly employed data treatment , is not able to reproduce the time - resolved experimental observations . introducing a new model , we demonstrated that it is mandatory to take the dissociation equilibrium of the exciplex into account . only then can a proper description of the shape and the magnitude of the time - resolved magnetic field effect be achieved . although the proposed model may seem to introduce many additional free parameters , only two parameters , namely , the association constant , ka , and the probability that the rip is formed initially , i , need to be fitted . all other parameters can be obtained experimentally from independent measurements or do not show a pronounced effect on the result . the exciplex lifetime in solvents of different permittivity was shown to provide a good estimate of the dissociation yield diss . the irreversible model commonly applied is contained in our model as the limiting case for i = 1 and diss = 0 . the reliability of the model established on the basis of the results of the measurements on the donor / acceptor pair 9,10-dimethylanthracene / dma was tested by applying the model to measurements on the classical model system py / dma . results obtained with py - h10 and its isotopologue py - d10 showed that ( 1 ) our model is well - applicable for other systems as well and that ( 2 ) for a proper description of the experimental data degenerate electron exchange needs to be taken into account . finally , addressing the question whether the exciplex or the radical ion pair is the initial quenching product , we could show that , even at relatively high permittivities , the modeling of our time - resolved mfe data suggests that direct exciplex formation contributes significantly . this has already been postulated in the literature , but has so far not been considered in the context of magnetic field effects . we showed that the irreversible description of the magnetic field effect ( i = 1 ) , which is commonly applied , fails to reproduce the time - resolved experimental findings and that it is mandatory to account for the exciplex kinetics . at low permittivities , electron transfer thus seems to be a contact reaction indicating that the exciplex is formed initially . at higher polarity , electron transfer becomes more distant , reflected in our model parameter i , which increases slightly with increasing solvent permittivity ( r ) . all in all , we have successfully demonstrated that time - resolved magnetic field effect studies have the potential to provide new insights into the reaction dynamics of exciplexes and radical ion pairs that are difficult to obtain by other methods .

we describe the experimental investigation of time - resolved magnetic field effects in exciplex - forming organic donor acceptor systems . in these systems , the photoexcited acceptor state is predominantly deactivated by bimolecular electron transfer reactions ( yielding radical ion pairs ) or by direct exciplex formation . the delayed fluorescence emitted by the exciplex is magnetosensitive if the reaction pathway involves loose radical ion pair states . this magnetic field effect results from the coherent interconversion between the electronic singlet and triplet radical ion pair states as described by the radical pair mechanism . by monitoring the changes in the exciplex luminescence intensity when applying external magnetic fields , details of the reaction mechanism can be elucidated . in this work we present results obtained with the fluorophore - quencher pair 9,10-dimethylanthracene / n , n - dimethylaniline ( dma ) in solvents of systematically varied permittivity . a simple theoretical model is introduced that allows discriminating the initial state of quenching , viz . , the loose ion pair and the exciplex , based on the time - resolved magnetic field effect . the approach is validated by applying it to the isotopologous fluorophore - quencher pairs pyrene / dma and pyrene - d10/dma . we detect that both the exciplex and the radical ion pair are formed during the initial quenching stage . upon increasing the solvent polarity , the relative importance of the distant electron transfer quenching increases . however , even in comparably polar media , the exciplex pathway remains remarkably significant . we discuss our results in relation to recent findings on the involvement of exciplexes in photoinduced electron transfer reactions .

Introduction Experimental Results Simulations Discussion Conclusions

this effect is brought about by the equilibration of the exciplex with the corresponding spin - correlated radical ion pair ( rip ) , which can undergo singlet triplet transitions and subsequently recombines . figure 1 depicts a reaction scheme of the photoinduced et processes in a typical exciplex forming donor , the ordinate corresponds to free energy and the abscissa can be expressed as an effective reaction coordinate involving the distance between fluorophore ( f ) and quencher ( q ) and the marcus outer - sphere electron transfer reaction coordinate . if the charge transfer within the exciplex is complete , the term contact ion pair is more qualified . if the exciplex is the primary reaction product , the rip can be formed in a secondary step by the dissociation of the exciplex ( 2b ) , with suitable solvent permittivities and viscosities taken for granted . if the radical ion pair diffusively separates such that the exchange coupling becomes sufficiently weak , the electronic zeeman and hyperfine interactions can coherently convert the electronic singlet state into an electronic triplet state ( 4 ) . in the presence of such magnetic fields , the zeeman - splitting of the triplet energy levels causes the intersystem crossing probability to diminish , since near - degeneracy can only be attained between the singlet state and one of the triplet states , whereas all of the three triplet states are involved in the interconversion process at low external magnetic fields . note , that the singlet ( s ) and triplet ( t ) radical ion pairs can undergo charge recombination , yielding the ground state and the fluorophore in its triplet state , respectively . in addition , only the singlet ion pair can re - form the exciplex ( 5 ) , and thus , its formation and emission ( 6 ) can be influenced by the external magnetic field . in summary , the following elementary processes give rise to a magnetic field effect ( mfe ) on the exciplex emission : photoexcitation ( 1 ) , exciplex formation and its dissociation into ions ( 2a , 2b ) , direct formation of the ions via distant et ( 3 ) , spin evolution accompanied by spin - selective charge recombination ( 4 ) , re - formation of the exciplex ( 5 ) , and exciplex emission ( 6 ) . in this work we will heavily rely on magnetic field effects and spin chemical ideas , which are now well - understood and the subject of comprehensive reviews . in fact , the radical pair mechanism is now again in the spotlight of current research , since it probably is the common denominator of the ( adverse ) effects of magnetic fields on biological systems or the cryptochrome - based magnetic compass of several migratory animals . only recently could the sensitivity of the detection of time - resolved mfes based on transient absorption be improved considerably using cavity ring - down detection . recent studies that rely on the exciplex fluorescence as a probe of magnetic field effects comprise such diverse topics as degenerate electron transfer , the recombination function , radio- and microwave magnetic field effects , or excited state reversibility . the effect of microwave excitation on the time - resolved exciplex emission of a polymethylene - linked donor acceptor system has also been studied . in most cases , however , a more quantitative interpretation is desirable , since it could give further insight into the details of the reaction mechanism and the role of the exciplex in the magnetic field effect . approximating the singlet probability by linear functions unfortunately , no details about the mechanism as to the role of the exciplex could be extracted from the time - resolved data , since it was a priori assumed that the radical ion pair is the primary quenching product . in the literature dealing with magnetic field effects it is generally assumed that the radical ion pair is initially generated by distant electron transfer and it later irreversibly combines to give the exciplex . in contrast to that , many studies focusing predominantly on photochemical aspects can be found , suggesting that the exciplex is the initial quenching product even at fairly high permittivities . in this paper , we would like to address these two opposing paradigms , in which either the exciplex or the radical ion pair is the initial quenching product in the above - mentioned reaction scheme . we will show that , by using time - resolved exciplex emission , we are able to identify the nature of the initial reaction product without introducing biasing assumptions but rather by direct observation of the mfe . in this way the time - resolved mfe allows discriminating both pathways ( 2 versus 3 ) and can effectively complement ultrafast observations . the paper is structured as follows : in section ii we commence by presenting photochemical properties of the donor acceptor system 9,10-dimethylanthracene / dma before we acquaint the reader with time - resolved mfe measurements and demonstrate how these measurements relate to steady - state results . in addition , we introduce model 2 ( section iii.iii ) , which includes the reaction pathway of direct exciplex formation ( 2a ) , exciplex dissociation into a loose ion pair ( 2b ) , and reencounter ( 5 ) . we show that even in comparably polar media the direct exciplex formation is found to be significant , and we close with a discussion and conclusions of these findings ( sections iv and v ) . we have studied the time - dependent mfe on the 9,10-dimethylanthracene ( f)/n , n - dimethylaniline ( dma , q ) system in propyl acetate ( pa , r = 6)/butyronitrile ( bn , r = 24 ) solvent mixtures . in addition , the refractive index ( nd = 1.3845 ) is amazingly invariant with the solvent composition , such that the pekar factor [ ( 1/n ) ( 1/r ) = 0.459 ] , which governs the outer - sphere electron transfer reorganization energy and , thus , the rate of et processes , changes by less than 6% for r in the range from 11 to 24 . hence , the quenching and the back - et reactions can to a good approximation be considered solvent - independent , and the changes of the mfe are predominantly brought about by the modulation of the radical pair interaction , i.e. absorption and emission spectra of 9,10-dimethylanthracene in the absence ( bottom ) and presence ( top ) of 0.05 m n , n - dimethylaniline . this is expected on the basis of the radical pair mechanism , since the singlet triplet degeneracy is lifted in the presence of an external magnetic field and the t states no longer efficiently participate in the singlet triplet conversion within the separated , yet spin - correlated , rip . it strongly depends on the permittivity , since r balances the radical ion pair separation , which is necessary for efficient spin evolution and the ( re)encounter of the pair . when an external magnetic field is applied , the delayed fluorescence intensity increases , which can be seen in figure 3 ( lower panel ) . we have adjusted the time profiles by matching the intensities during the initial rise up to and including the maximum and formed the difference i(t ) = i(t , b0 ) i(t , b=0 ) to obtain the time - resolved mfe , as also shown in figure 3 ( upper panel ) . lower panel : exciplex emission decays of the 9,10-dimethylanthracene ( 10 m)/n , n - dimethylaniline ( 0.05 m ) system in pure butyronitrile at r = 24.3 in the presence and absence of an external magnetic field ( ca . in the presence of an external magnetic field , upper panel : time - resolved magnetic field effect i obtained by taking the difference of the experimental single photon timing traces recorded in the presence and absence of an additional external magnetic field ( gray ) and the fit to the experimental data ( black ) . the agreement of the values for the absolute magnetic field effect obtained from steady state and time - resolved data suggests that no bulk processes contribute to the mfes observed here . solvent polarity dependence of the magnetic field effect of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex determined from steady - state ( filled circles ) and time - resolved ( open circles with error bars ) measurements . the maximum of the time - resolved mfe occurs in the range from 20 to 60 ns after excitation , with the larger values occurring at lower permittivities . experimental time - dependent magnetic field effects at different dielectric constants ( as given in the figure ) for the system 9,10-dimethylanthracene / n , n - dimethylaniline in propyl acetate / butyronitrile mixtures . traditionally , the mfe on the exciplex has been modeled using2where ps(t , b0 ) is the probability that the rip born in the singlet state is in its singlet state at time t , and f(t ) is the recombination function , i.e. among these quantities , the only one depending on the external magnetic field , b0 , is ps(t , b0 ) von neumann equation in the limit of low viscosities and/or not too low permittivities . for these pairs , the details of the recombination and the exchange interaction are negligible during the majority of their lifetime . the exciplex probability can in this case be calculated from3 both models discussed so far rely on the assumption that the magnetic field effect only results from ions that irreversibly combine to the exciplex . normalized simulated time traces using the irreversible model 1b at different values of the only ( artificially ) variable parameter e ( from 5 to 30 ns ) as opposed to a noise - free representation of the experimental data at e = 18 ns , which was obtained by fitting a combination of exponential functions to the experimental time - dependent magnetic field effect . the experimental data was measured using the system 9,10-dimethylanthracene ( 10 m)/n , n - dimethylaniline ( 0.05 m ) in a mixture of propyl acetate / butyronitrile at r = 20 ( 75 wt % butyronitrile ) . position of the maximum ( or center of gravity ) of the time - resolved magnetic field effect in time after excitation as a function of the exciplex lifetime . as the lifetime of the exciplex relates to its initial decay , the observation of a linear relationship supports our proposition , since it suggests that the dissociation of the exciplex is the rate - limiting step for the mfe and needs to be taken into account for a proper description of the experimental data . we propose evaluating the probability that the radical ion pair exists as exciplex , pe(t , b0 ) , from4where kd is the rate of exciplex dissociation and r(t , b0|ri ) dt denotes the probability that the rip formed at t = 0 at distance ri has recombined until t. ri is the formation distance of the loose ion pair by ( distant ) et , and re is the contact distance of donor and acceptor at which the transition of the loose ion pair to the exciplex ( or contact ion pair ) occurs , e.g. the different terms on the right - hand side of eq 4 can be understood as follows : the first summand , e = 1 i , specifies the probability that the exciplex is formed initially ( path 2a in figure 1 ) , while the second summand gives the probability that the initially formed rip forms an exciplex until t. the third term gives the probability that the exciplex dissociates at time and is re - formed until t , and the last term describes the depopulation by dissociation or radiative / nonradiative decay . as a consequence , the time evolution of the mfe depends on the parameters of the diffusive motion via r(t , b0|ri ) , the exciplex lifetime , and its dissociation quantum yield , diss = kd e , and parameters of the radical ion pair , in particular i and the association constant , ka= ka / kd , and the parameters governing the spin evolution ( hyperfine coupling constants ) . it turns out that there are only two parameters , namely , i and ka , which have a strong effect on the shape and the magnitude of the time - resolved mfe ( cf . the exciplex lifetime , e , was determined from the initial decay of the time - correlated single photon counting traces of the exciplex , and the dissociation quantum yield , diss , was estimated from the dependence of e on permittivity assuming that for r = 6 no dissociation occurs and that the radiative and nonradiative rates of the exciplex are constant ( and/or small compared to the dissociation rate ) within the polarity range studied here . the plot of i as a function of permittivity shows , first , that direct exciplex formation contributes at all permittivities and , second , that the probability of distant electron transfer quenching increases with increasing polarity of the solution . using sutin s approach , the free energies of association from the ion pair to the exciplex can be estimated ( assuming a contact distance of 6.5 and a reaction zone of 0.8 width ) . the delay of the maximum of the mfe with respect to the simplistic model 1a ( eq 2 ) results from the fact that , first , the exciplex is formed directly and , second , that its dissociation into the ions is slow , the rate being given by disse . solvent dependence of the association constant ( ka , top panel ) , the dissociation quantum yield of the exciplex ( diss , center ) , and the fraction of excited fluorophore quencher pairs deactivating to solvent - separated ions ( i , bottom ) as opposed to the exciplex . substituting the protons in the fluorophore by deuterium has a huge impact on the singlet probability , ps(t , b0 ) , since the hyperfine coupling constants of the deuteriums amount only to 15% of those of the protons and , in addition , the spin quantum numbers of the nuclei differ ( i d = 1 , ih = /2 ) . time - resolved magnetic field effect of the exciplex of pyrene ( py - h10 ) ( bottom panel ) and perdeuterated pyrene ( py - d10 ) ( top panel ) with n , n - dimethylaniline in butyronitrile . only if the degenerate electron exchange of the n , n - dimethylaniline radical cation is taken into account does the model consistently , i.e. however , taking degenerate electron exchange into account , the differences in ps(t , b0 ) nearly disappear and the time - resolved mfe can indeed be modeled with one and the same set of parameters for py - h10 and py - d10 ( cf . the effect is particularly pronounced if the hyperfine contribution of the exchanging radical ion exceeds that of the radical ion originating from the fluorophore . with the weak hyperfine field of the deuterated pyrene , the singlet triplet conversion is essentially governed by the dma and thus the mfe is more sensitive to its degenerate electron exchange . only the time - resolved mfe technique can reveal the details of the exciplex kinetics and yield a comprehensive description of the mfe . in this study we presented measurements of the time - resolved magnetic field effect on the exciplex kinetics in organic donor the integrated experimental data reproduces the steady - state data published earlier , demonstrating that bulk recombination of rips can be ruled out as a major contribution to the magnetic field effects observed here . by systematically varying the solvent permittivity while leaving most parameters influencing electron transfer rates and the solvent viscosity constant , we could show that the irreversible model ( model 1a or 1b ) , which is implicitly assumed in the commonly employed data treatment , is not able to reproduce the time - resolved experimental observations . only then can a proper description of the shape and the magnitude of the time - resolved magnetic field effect be achieved . the reliability of the model established on the basis of the results of the measurements on the donor / acceptor pair 9,10-dimethylanthracene / dma was tested by applying the model to measurements on the classical model system py / dma . results obtained with py - h10 and its isotopologue py - d10 showed that ( 1 ) our model is well - applicable for other systems as well and that ( 2 ) for a proper description of the experimental data degenerate electron exchange needs to be taken into account . finally , addressing the question whether the exciplex or the radical ion pair is the initial quenching product , we could show that , even at relatively high permittivities , the modeling of our time - resolved mfe data suggests that direct exciplex formation contributes significantly . we showed that the irreversible description of the magnetic field effect ( i = 1 ) , which is commonly applied , fails to reproduce the time - resolved experimental findings and that it is mandatory to account for the exciplex kinetics . all in all , we have successfully demonstrated that time - resolved magnetic field effect studies have the potential to provide new insights into the reaction dynamics of exciplexes and radical ion pairs that are difficult to obtain by other methods .

pancreatic ductal adenocarcinoma ( pdac ) , commonly known as pancreatic cancer ( pc ) , is the fourth leading cause of cancer deaths world - wide , with high age - standardized incidence rates occurring in north america and asia . pc is diagnosed in more than 337,000 individuals each year , accounts for 4% of all cancer deaths , and has the lowest five - year relative survival rate of all leading cancers , at 9% . prognosis is poor because diagnosis typically occurs at a late , incurable stage , and prevention and early detection methods are lacking . risk factors including age , tobacco , diabetes , pancreatitis , heavy alcohol use , family history , and hereditary conditions explain only a proportion of pcs . being overweight [ body mass index ( bmi)25 kg / m ] or obese ( bmi30 kg / m ) increases pc risk by 30% , has a population attributable fraction up to 16% , and influences pc survival . given the rise in the prevalence of obesity in north america and asia and the fact that obesity is a modifiable pc risk factor , an understanding of obesity s role in early pancreatic carcinogenesis is crucial for pc prevention and early detection . we contend that commonly - detected pc precursors may be attributed to obesity , and that proper diagnosis and treatment of precursors and underlying obesity offer potential to reduce pc burden . intraductal papillary mucinous neoplasms ( ipmns ) are macrocystic pc precursors ( precancers ) that comprise half of the ~150,000 pancreatic cysts detected incidentally in 3% of computed tomography ( ct ) scans and 20% of magnetic resonance imaging ( mri ) studies each year , making them more amenable to study than the microscopic pc precursor , pancreatic intraepithelial neoplasia ( panin ) . once detected , the only way to accurately determine ipmn severity/ pathology [ which spans from low - grade ( lg ) and moderate - grade ( mg ) to high - grade ( hg ) dysplasia & invasive carcinoma ] is surgical resection , which is associated with an operative mortality of 2%4% and morbidity of 40%50% . the guidelines recommend that those with ' high risk stigmata ' undergo resection as most harbor hg or invasive disease . high risk stigmata include : main pancreatic duct ( md ) involvement/ dilatation 10 mm , jaundice , or an enhanced solid component / nodule ) . worrisome features ( md dilation 59 mm , size 3 cm , thickened cyst walls , non - enhanced mural nodules , or pancreatitis ) are recommended for surveillance with an invasive endoscopic ultrasound - guided fine needle aspirate procedure despite poor sensitivity and complications . however , consensus guidelines incorrectly predict pathology in 30%70% of cases , causing under- and over - treatment . thus , rationale exists for identifying noninvasive markers to improve diagnostic accuracy for ipmns , especially those without high risk stigmata . increased glucose uptake and energy metabolism is prominent in pdacs and correlates with ipmn grade . very high bmi ( 35 kg / m ) was associated with a high prevalence of malignancy in side branch duct ( bd ) ipmns . bd - ipmns without high risk stigmata are challenging to manage , and if obesity is a marker of malignant bd - ipmns , this could aid in management . one major limitation of prior studies is that bmi was used to measure obesity . bmi is imprecise and can not differentiate between subcutaneous fat accumulation ( which represents the normal physiological buffer for excess energy intake ) and abdominal / visceral adiposity , a facilitator of carcinogenesis through metabolic disturbances , inflammation , and fat infiltration in the pancreas . abdominal / visceral fat area ( vfa ) is a risk factor for pancreatic fat infiltration in patients with pc and panins , and is associated with poor pc outcomes . routine abdominal ct scans are the gold - standard for investigating quantitative radiologic features of abdominal adiposity ( such as vfa ) , yet no published studies of these features exist for ipmn patients . we sought to determine if quantitative radiologic features of obesity extracted from abdominal ct scans can help to distinguish risk of malignant versus benign ipmns . the study population included a fixed cohort of 37 patients with ipmns whose pre - operative ct images had recently been evaluated as part of a different study . the cases had initially been identified using a prospectively maintained clinical database of individuals who underwent a pancreatic resection for an ipmn between 2006 and 2011 at moffitt cancer center and research institute ( moffitt ) and provided written consent for medical images and clinical data to be donated for research through protocols approved by the institutional review board ( irb ) of the university of south florida , including total cancer care . for all cases , demographic and clinical data ( presenting systems , age at diagnosis , past medical and surgical history , and information on known and suspected cancer risk factors such as smoking , family history , and body mass index calculated from pre - surgical height and weight ) was obtained from the electronic medical record and patient questionnaire . detailed imaging studies , surgical details , pathology results , lab values ( serum ca 19 - 9 ) , and treatment information was collected from the medical record and moffitt s cancer registry . board - certified pathologists with expertise in pdac and ipmn pathology ( kj , dc , bac ) previously histologically confirmed the diagnosis and degree of dysplasia using world health organization guidelines . the final diagnosis represented the most severe grade of dysplasia observed in the neoplastic epithelium . most of the ct scans from this series of patients were obtained on the siemens sensation ( 16 , 40 , or 64 ) using an abdominal or pancreatic ct angio ( cta ) protocol according to standard operating procedures described previously . archived non - enhanced ct images performed within the three months prior to surgery , were acquired from moffitt s ge centricity picture archiving and communication system ( pacs ) . the imaging team , led by our board - certified abdominal radiologists ( dj and jc ) , were blinded to the final pathology . contrast enhanced axial venous phase images were used and reviewed for high risk stigmata and worrisome features of the pancreatic lesions . non enhanced axial ct images were utilized and have previously shown to be adequate for visceral and subcutaneous fat measurements . measures of total abdominal fat ( taf ) area , vfa , and subcutaneous fat area ( sfa ) were obtained using the volume segmentation and thresholding tools in aw server version 2.0 software ( general electric , waukesha , wi , usa ) . the axial l2-l3 intervertebral disc level was used for analysis because adipose tissue at this level corresponds to whole body quantities and is well distinguished from skeletal muscle and other structures . ct attenuation thresholds to define adipose tissue were set between 249 and 49 hounsfield units . taf area on an l2l3 axial slice nearest the superior endplate of l3 was calculated by counting the volume of voxels that meet fat attenuation thresholds divided by slice thickness , which allowed standardization of measurements despite potentially different ct scan protocols . the vfa to sfa ratio ( v / s ) was calculated with v / s>0.4 cm defined as viscerally obese . manual tracing of the visceral fascial plane allowed the radiologist to exclude any fat density regions within bowel or fatty lesions within organs . for select variables , descriptive statistics were calculated using frequencies and percents for categorical variables and means and standard deviations ( sd ) for continuous variables . the distributions of covariates were compared across groups using the wilcoxon two sample two - sided exact test for continuous variables and fisher s exact tests for categorical variables . spearman correlations were calculated to evaluate the relationship between bmi and quantitative radiologic obesity measures . estimates of sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) were calculated for key variables . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , north carolina ) . the study population included a fixed cohort of 37 patients with ipmns whose pre - operative ct images had recently been evaluated as part of a different study . the cases had initially been identified using a prospectively maintained clinical database of individuals who underwent a pancreatic resection for an ipmn between 2006 and 2011 at moffitt cancer center and research institute ( moffitt ) and provided written consent for medical images and clinical data to be donated for research through protocols approved by the institutional review board ( irb ) of the university of south florida , including total cancer care . for all cases , demographic and clinical data ( presenting systems , age at diagnosis , past medical and surgical history , and information on known and suspected cancer risk factors such as smoking , family history , and body mass index calculated from pre - surgical height and weight ) was obtained from the electronic medical record and patient questionnaire . detailed imaging studies , surgical details , pathology results , lab values ( serum ca 19 - 9 ) , and treatment information was collected from the medical record and moffitt s cancer registry . board - certified pathologists with expertise in pdac and ipmn pathology ( kj , dc , bac ) previously histologically confirmed the diagnosis and degree of dysplasia using world health organization guidelines . the final diagnosis represented the most severe grade of dysplasia observed in the neoplastic epithelium . most of the ct scans from this series of patients were obtained on the siemens sensation ( 16 , 40 , or 64 ) using an abdominal or pancreatic ct angio ( cta ) protocol according to standard operating procedures described previously . archived non - enhanced ct images performed within the three months prior to surgery , were acquired from moffitt s ge centricity picture archiving and communication system ( pacs ) . the imaging team , led by our board - certified abdominal radiologists ( dj and jc ) , were blinded to the final pathology . contrast enhanced axial venous phase images were used and reviewed for high risk stigmata and worrisome features of the pancreatic lesions . non enhanced axial ct images were utilized and have previously shown to be adequate for visceral and subcutaneous fat measurements . measures of total abdominal fat ( taf ) area , vfa , and subcutaneous fat area ( sfa ) were obtained using the volume segmentation and thresholding tools in aw server version 2.0 software ( general electric , waukesha , wi , usa ) . the axial l2-l3 intervertebral disc level was used for analysis because adipose tissue at this level corresponds to whole body quantities and is well distinguished from skeletal muscle and other structures . ct attenuation thresholds to define adipose tissue were set between 249 and 49 hounsfield units . taf area on an l2l3 axial slice nearest the superior endplate of l3 was calculated by counting the volume of voxels that meet fat attenuation thresholds divided by slice thickness , which allowed standardization of measurements despite potentially different ct scan protocols . the vfa to sfa ratio ( v / s ) was calculated with v / s>0.4 cm defined as viscerally obese . manual tracing of the visceral fascial plane allowed the radiologist to exclude any fat density regions within bowel or fatty lesions within organs . for select variables , descriptive statistics were calculated using frequencies and percents for categorical variables and means and standard deviations ( sd ) for continuous variables . the distributions of covariates were compared across groups using the wilcoxon two sample two - sided exact test for continuous variables and fisher s exact tests for categorical variables . spearman correlations were calculated to evaluate the relationship between bmi and quantitative radiologic obesity measures . estimates of sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) were calculated for key variables . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , north carolina ) . radiologic measures of obesity were successfully calculated for 34 of the 37 cases ; three cases did not have available scans in our pacs including the axial l2l3 intervertebral disc level views for adiposity measurement . clinical , epidemiologic , and imaging characteristics of the 34 cases ( 17 benign ; 17 malignant ) investigated in this analysis are in table 1 and are in line with published data on other ipmn cohorts . seventy - six percent with malignant pathology had md involvement on ct versus 24% with benign pathology ( p=0.005 ) . mean lesion size was higher in the malignant compared to the benign group ( 3.4 versus 1.9 cm),p=0.003 . malignant ipmns , particularly those deemed to be invasive , were predominately located in the pancreatic head . the majority of cases ( 82% ) with malignant pathology had one or more high risk stigmata ( md involvement / dilatation>10 mm , obstructive jaundice with a cystic lesion in the pancreatic head , or an enhanced solid component within the cyst ) , versus 18% of those with benign pathology ( p<0.001 ) . md dilation 59 mm , cyst size>3 cm , thickened enhanced cyst walls , non - enhanced mural nodules , or acute pancreatitis ) was not associated with malignancy ( p=0.708 ) in this cohort . bmi was higher in malignant ( 28.9 kg / m , 95% ci : 26.331.4 kg / m ) versus benign cases ( 25.8 kg / m , 95% ci : 23.328.3 kg / m ) , withp=0.045 . mean bmi was similar in males and females , at 27.8 and 27.0 kg / m , respectively . characteristics of ipmn cases in the study cohort ( n=34 ) mean vfa was higher in patients with malignant ( 199 cm ) versus benign ( 120 cm ) ipmns , but did not reach statistical significance with the wilcoxon two - sample exact test ( p=0.092 ) ( table 2 ) . mean v / s was substantially higher in malignant versus benign ipmns , with values of 1.25 cm and 0.69 cm , respectively ( p=0.013 ) . we found no statistically significant differences between taf and sfa in the malignant and benign groups . quantitative radiologic measures of obesity , by ipmn pathology males had a higher mean vfa value ( 202.4 cm ) than females ( 133.6 cm ) and a higher mean v / s value ( 1.25 cm ) than females ( 0.80 cm ) . stratified analyses revealed that among both males and females , mean bmi , taf , and vfa values were higher for patients with malignant compared to benign ipmns , though results were not statistically significant ( p>0.05 ) for either gender ( table 3 ) . among females , while no correlation existed between bmi and v / s ( r=0.16,p=0.35 ) , significant positive correlations were found between bmi and vfa ( r=0.68,p<0.0001 ) and between bmi and sfa ( r=0.71,p<0.0001 ) . gender - specific differences in bmi and quantitative radiologic measures of obesity , by ipmn pathology of clinical importance , figure 1 displays ct scans from two ipmn patients who did not present with high risk stigmata on imaging . both have similar bmis but vastly different vfa and v / s values , with case 1 having higher vfa and v / s and a worrisome feature ( cyst size>3 cm ) and high - grade pathology and case 2 having lower vfa and v / s and low - grade pathology at resection . these data suggest that visceral fat may be added as another risk factor to potentially aid in directing management towards a necessary surgery to remove what turned out to be a high - grade lesion ( case 1 ) and avoided an unnecessary surgery for a low - grade lesion ( case 2 ) . the accuracy , sensitivity , specificity , ppv , and npv of v / s in predicting malignant ipmn pathology were 74% , 71% , 76% , 75% and 72% , respectively . axial post contrast cts ( a and b ) and quantitative segmentation ( c and d ) for two representative side bd ipmn cases with main pancreatic ducts normal in caliber . case 1 has a well - demarcated homogenous hypodense 4.8 cm cystic lesion in the pancreatic neck ( yellow arrow ) . case 2 has a poorly defined 1.3 cm hypoenhancing pancreatic neck lesion ( yellow arrow ) . c and d : ( 1 ) axial ct image through l2l3 intervertebral disc level . abdominal wall and paraspinal muscle area were segmented and thresholds set to voxels with hounsfield units ( hu ) 29 to 150 . visceral fat , intra - abdominal organs , and vasculature were subtracted . although skeletal muscle indices can be obtained in a complementary manner to visceral fat measurements , these were not directly analyzed in this study . ( 3 ) total abdominal fat with hu thresholds applied to include fat density voxels with hu 249 to 49 ( green ) . ( radiologic measures of obesity were successfully calculated for 34 of the 37 cases ; three cases did not have available scans in our pacs including the axial l2l3 intervertebral disc level views for adiposity measurement . clinical , epidemiologic , and imaging characteristics of the 34 cases ( 17 benign ; 17 malignant ) investigated in this analysis are in table 1 and are in line with published data on other ipmn cohorts . seventy - six percent with malignant pathology had md involvement on ct versus 24% with benign pathology ( p=0.005 ) . mean lesion size was higher in the malignant compared to the benign group ( 3.4 versus 1.9 cm),p=0.003 . malignant ipmns , particularly those deemed to be invasive , were predominately located in the pancreatic head . the majority of cases ( 82% ) with malignant pathology had one or more high risk stigmata ( md involvement / dilatation>10 mm , obstructive jaundice with a cystic lesion in the pancreatic head , or an enhanced solid component within the cyst ) , versus 18% of those with benign pathology ( p<0.001 ) . md dilation 59 mm , cyst size>3 cm , thickened enhanced cyst walls , non - enhanced mural nodules , or acute pancreatitis ) was not associated with malignancy ( p=0.708 ) in this cohort . bmi was higher in malignant ( 28.9 kg / m , 95% ci : 26.331.4 kg / m ) versus benign cases ( 25.8 kg / m , 95% ci : 23.328.3 kg / m ) , withp=0.045 . mean bmi was similar in males and females , at 27.8 and 27.0 kg / m , respectively . mean vfa was higher in patients with malignant ( 199 cm ) versus benign ( 120 cm ) ipmns , but did not reach statistical significance with the wilcoxon two - sample exact test ( p=0.092 ) ( table 2 ) . mean v / s was substantially higher in malignant versus benign ipmns , with values of 1.25 cm and 0.69 cm , respectively ( p=0.013 ) . we found no statistically significant differences between taf and sfa in the malignant and benign groups . quantitative radiologic measures of obesity , by ipmn pathology males had a higher mean vfa value ( 202.4 cm ) than females ( 133.6 cm ) and a higher mean v / s value ( 1.25 cm ) than females ( 0.80 cm ) . stratified analyses revealed that among both males and females , mean bmi , taf , and vfa values were higher for patients with malignant compared to benign ipmns , though results were not statistically significant ( p>0.05 ) for either gender ( table 3 ) . among females , while no correlation existed between bmi and v / s ( r=0.16,p=0.35 ) , significant positive correlations were found between bmi and vfa ( r=0.68,p<0.0001 ) and between bmi and sfa ( r=0.71,p<0.0001 ) . gender - specific differences in bmi and quantitative radiologic measures of obesity , by ipmn pathology of clinical importance , figure 1 displays ct scans from two ipmn patients who did not present with high risk stigmata on imaging . both have similar bmis but vastly different vfa and v / s values , with case 1 having higher vfa and v / s and a worrisome feature ( cyst size>3 cm ) and high - grade pathology and case 2 having lower vfa and v / s and low - grade pathology at resection . these data suggest that visceral fat may be added as another risk factor to potentially aid in directing management towards a necessary surgery to remove what turned out to be a high - grade lesion ( case 1 ) and avoided an unnecessary surgery for a low - grade lesion ( case 2 ) . the accuracy , sensitivity , specificity , ppv , and npv of v / s in predicting malignant ipmn pathology were 74% , 71% , 76% , 75% and 72% , respectively . axial post contrast cts ( a and b ) and quantitative segmentation ( c and d ) for two representative side bd ipmn cases with main pancreatic ducts normal in caliber . case 1 has a well - demarcated homogenous hypodense 4.8 cm cystic lesion in the pancreatic neck ( yellow arrow ) . case 2 has a poorly defined 1.3 cm hypoenhancing pancreatic neck lesion ( yellow arrow ) . c and d : ( 1 ) axial ct image through l2l3 intervertebral disc level . abdominal wall and paraspinal muscle area were segmented and thresholds set to voxels with hounsfield units ( hu ) 29 to 150 . although skeletal muscle indices can be obtained in a complementary manner to visceral fat measurements , these were not directly analyzed in this study . ( 3 ) total abdominal fat with hu thresholds applied to include fat density voxels with hu 249 to 49 ( green ) . ( 4 ) manual segmentation of visceral fat regions ( green ) . this pilot project represents the first to study objectively quantitative radiologic measures of obesity as diagnostic markers of ipmn pathology . in addition to observing higher pre - operative bmi values in patients confirmed to have malignant ipmns , vfa and v / s values were also observed in the malignant ipmn group compared to those with benign ipmns . we also observed that males with ipmns had higher vfa and v / s values than female cases , in line with the observation that visceral fat is more common in males , and showed that women with benign ipmns had a significantly lower v / s ratio ( 0.5 cm ) than those with malignant ipmns ( 1.4 cm ) , women with benign ipmns in our cohort appeared to have a higher sfa than other cohort members , consistent with data suggesting that subcutaneous fat may not be a marker of malignancy . previous authors have suggested that in an asymptomatic adult cohort , men have significantly higher v / s ratios . however , no standardized gender - based v / s values are currently available which suggests further research is needed to define visceral obesity in each gender . our small cohort , however , had relatively more females in the benign pathology group and more males in the malignant pathology group , so firm conclusions can not be drawn based on these preliminary findings . despite this , findings suggest that being overweight or obese , particularly in the intra - abdominal area , may be a prognostic marker for malignant potential of ipmns . given that abdominal / visceral adiposity has been shown to influence carcinogenesis and that bmi is an imprecise proxy for abdominal adiposity , biologically - driven radiologic measures of visceral fat may have greater clinical utility than bmi in predicting ipmn pathology . further research with a larger sample size is clearly needed to distinguish the relationship between radiologic measures of visceral fat , gender , and malignancy . few studies have reported on quantitative radiologic measures of obesity in patients with pdac . in a study of 9 pdac cases and matched controls , no significant differences in sfa , vfa , tfa , or v pre - operative visceral fat was shown to be a prognostic indicator in patients with pdac , with increased visceral fat being associated with worse survival in patients with lymph node metastases . elevated visceral fat defined by the v / s has also been shown to predict recurrence among locally advanced rectal cancer patients . collectively , these and other studies provide plausibility for our observation that vfa and v / s may be associated with more advanced ipmn pathology . although limitations of this pilot study include its small size and retrospective design , characteristics of this cohort are representative of other ipmn cohorts , suggesting potential generalizability . furthermore , with a larger sample size , multivariable modeling and receiver characteristic curve analyses will be helpful to determine the utility of gender - specific radiologic measures of abdominal obesity in discriminating malignant from benign ipmns , independent of and in combination with novel molecular and radiologic markers , standard clinical and radiologic features encompassed by consensus guidelines , and bmi . in summary , use of quantitative radiologic measures of abdominal obesity could provide a noninvasive , rapid , low cost , and repeatable way of investigating features that may potentially aid in personalizing care for patients with pancreatic cancer precursors . given that a reduction in abdominal adiposity by lifestyle , diet , and/or pharmacologic intervention would be impactful and could translate into a decreased burden of pc , obesity , and other diseases , further studies in this area are warranted .

objective : intra - abdominal fat is a risk factor for pancreatic cancer ( pc ) , but little is known about its contribution to pc precursors known as intraductal papillary mucinous neoplasms ( ipmns ) . our goal was to evaluate quantitative radiologic measures of abdominal / visceral obesity as possible diagnostic markers of ipmn severity / pathology.methods : in a cohort of 34 surgically - resected , pathologically - confirmed ipmns ( 17 benign ; 17 malignant ) with preoperative abdominal computed tomography ( ct ) images , we calculated body mass index ( bmi ) and four radiologic measures of obesity : total abdominal fat ( taf ) area , visceral fat area ( vfa ) , subcutaneous fat area ( sfa ) , and visceral to subcutaneous fat ratio ( v / s ) . measures were compared between groups using wilcoxon two - sample exact tests and other metrics.results:mean bmi for individuals with malignant ipmns ( 28.9 kg / m2 ) was higher than mean bmi for those with benign ipmns ( 25.8 kg / m2 ) ( p=0.045 ) . mean vfa was higher for patients with malignant ipmns ( 199.3 cm2 ) compared to benign ipmns ( 120.4 cm2),p=0.092 . v / s was significantly higher ( p=0.013 ) for patients with malignant versus benign ipmns ( 1.25vs . 0.69 cm2 ) , especially among females . the accuracy , sensitivity , specificity , and positive and negative predictive value of v / s in predicting malignant ipmn pathology were 74% , 71% , 76% , 75% , and 72% , respectively . conclusions : preliminary findings suggest measures of visceral fat from routine medical images may help predict ipmn pathology , acting as potential noninvasive diagnostic adjuncts for management and targets for intervention that may be more biologically - relevant than bmi . further investigation of gender - specific associations in larger , prospective ipmn cohorts is warranted to validate and expand upon these observations .

Introduction Materials and methods Study population and data Histopathologic analysis CT imaging, acquisition, and abdominal obesity assessment Statistical analysis Results Study population characteristics Analysis of quantitative radiologic measures of obesity Discussion Conflicts of interest statement

pancreatic ductal adenocarcinoma ( pdac ) , commonly known as pancreatic cancer ( pc ) , is the fourth leading cause of cancer deaths world - wide , with high age - standardized incidence rates occurring in north america and asia . being overweight [ body mass index ( bmi)25 kg / m ] or obese ( bmi30 kg / m ) increases pc risk by 30% , has a population attributable fraction up to 16% , and influences pc survival . given the rise in the prevalence of obesity in north america and asia and the fact that obesity is a modifiable pc risk factor , an understanding of obesity s role in early pancreatic carcinogenesis is crucial for pc prevention and early detection . we contend that commonly - detected pc precursors may be attributed to obesity , and that proper diagnosis and treatment of precursors and underlying obesity offer potential to reduce pc burden . intraductal papillary mucinous neoplasms ( ipmns ) are macrocystic pc precursors ( precancers ) that comprise half of the ~150,000 pancreatic cysts detected incidentally in 3% of computed tomography ( ct ) scans and 20% of magnetic resonance imaging ( mri ) studies each year , making them more amenable to study than the microscopic pc precursor , pancreatic intraepithelial neoplasia ( panin ) . the guidelines recommend that those with ' high risk stigmata ' undergo resection as most harbor hg or invasive disease . very high bmi ( 35 kg / m ) was associated with a high prevalence of malignancy in side branch duct ( bd ) ipmns . bd - ipmns without high risk stigmata are challenging to manage , and if obesity is a marker of malignant bd - ipmns , this could aid in management . bmi is imprecise and can not differentiate between subcutaneous fat accumulation ( which represents the normal physiological buffer for excess energy intake ) and abdominal / visceral adiposity , a facilitator of carcinogenesis through metabolic disturbances , inflammation , and fat infiltration in the pancreas . abdominal / visceral fat area ( vfa ) is a risk factor for pancreatic fat infiltration in patients with pc and panins , and is associated with poor pc outcomes . routine abdominal ct scans are the gold - standard for investigating quantitative radiologic features of abdominal adiposity ( such as vfa ) , yet no published studies of these features exist for ipmn patients . we sought to determine if quantitative radiologic features of obesity extracted from abdominal ct scans can help to distinguish risk of malignant versus benign ipmns . the study population included a fixed cohort of 37 patients with ipmns whose pre - operative ct images had recently been evaluated as part of a different study . the cases had initially been identified using a prospectively maintained clinical database of individuals who underwent a pancreatic resection for an ipmn between 2006 and 2011 at moffitt cancer center and research institute ( moffitt ) and provided written consent for medical images and clinical data to be donated for research through protocols approved by the institutional review board ( irb ) of the university of south florida , including total cancer care . for all cases , demographic and clinical data ( presenting systems , age at diagnosis , past medical and surgical history , and information on known and suspected cancer risk factors such as smoking , family history , and body mass index calculated from pre - surgical height and weight ) was obtained from the electronic medical record and patient questionnaire . detailed imaging studies , surgical details , pathology results , lab values ( serum ca 19 - 9 ) , and treatment information was collected from the medical record and moffitt s cancer registry . board - certified pathologists with expertise in pdac and ipmn pathology ( kj , dc , bac ) previously histologically confirmed the diagnosis and degree of dysplasia using world health organization guidelines . the imaging team , led by our board - certified abdominal radiologists ( dj and jc ) , were blinded to the final pathology . measures of total abdominal fat ( taf ) area , vfa , and subcutaneous fat area ( sfa ) were obtained using the volume segmentation and thresholding tools in aw server version 2.0 software ( general electric , waukesha , wi , usa ) . the vfa to sfa ratio ( v / s ) was calculated with v / s>0.4 cm defined as viscerally obese . the distributions of covariates were compared across groups using the wilcoxon two sample two - sided exact test for continuous variables and fisher s exact tests for categorical variables . spearman correlations were calculated to evaluate the relationship between bmi and quantitative radiologic obesity measures . estimates of sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) were calculated for key variables . the study population included a fixed cohort of 37 patients with ipmns whose pre - operative ct images had recently been evaluated as part of a different study . the cases had initially been identified using a prospectively maintained clinical database of individuals who underwent a pancreatic resection for an ipmn between 2006 and 2011 at moffitt cancer center and research institute ( moffitt ) and provided written consent for medical images and clinical data to be donated for research through protocols approved by the institutional review board ( irb ) of the university of south florida , including total cancer care . for all cases , demographic and clinical data ( presenting systems , age at diagnosis , past medical and surgical history , and information on known and suspected cancer risk factors such as smoking , family history , and body mass index calculated from pre - surgical height and weight ) was obtained from the electronic medical record and patient questionnaire . detailed imaging studies , surgical details , pathology results , lab values ( serum ca 19 - 9 ) , and treatment information was collected from the medical record and moffitt s cancer registry . non enhanced axial ct images were utilized and have previously shown to be adequate for visceral and subcutaneous fat measurements . measures of total abdominal fat ( taf ) area , vfa , and subcutaneous fat area ( sfa ) were obtained using the volume segmentation and thresholding tools in aw server version 2.0 software ( general electric , waukesha , wi , usa ) . the vfa to sfa ratio ( v / s ) was calculated with v / s>0.4 cm defined as viscerally obese . for select variables , descriptive statistics were calculated using frequencies and percents for categorical variables and means and standard deviations ( sd ) for continuous variables . the distributions of covariates were compared across groups using the wilcoxon two sample two - sided exact test for continuous variables and fisher s exact tests for categorical variables . spearman correlations were calculated to evaluate the relationship between bmi and quantitative radiologic obesity measures . estimates of sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) were calculated for key variables . radiologic measures of obesity were successfully calculated for 34 of the 37 cases ; three cases did not have available scans in our pacs including the axial l2l3 intervertebral disc level views for adiposity measurement . clinical , epidemiologic , and imaging characteristics of the 34 cases ( 17 benign ; 17 malignant ) investigated in this analysis are in table 1 and are in line with published data on other ipmn cohorts . seventy - six percent with malignant pathology had md involvement on ct versus 24% with benign pathology ( p=0.005 ) . mean lesion size was higher in the malignant compared to the benign group ( 3.4 versus 1.9 cm),p=0.003 . the majority of cases ( 82% ) with malignant pathology had one or more high risk stigmata ( md involvement / dilatation>10 mm , obstructive jaundice with a cystic lesion in the pancreatic head , or an enhanced solid component within the cyst ) , versus 18% of those with benign pathology ( p<0.001 ) . md dilation 59 mm , cyst size>3 cm , thickened enhanced cyst walls , non - enhanced mural nodules , or acute pancreatitis ) was not associated with malignancy ( p=0.708 ) in this cohort . bmi was higher in malignant ( 28.9 kg / m , 95% ci : 26.331.4 kg / m ) versus benign cases ( 25.8 kg / m , 95% ci : 23.328.3 kg / m ) , withp=0.045 . mean bmi was similar in males and females , at 27.8 and 27.0 kg / m , respectively . characteristics of ipmn cases in the study cohort ( n=34 ) mean vfa was higher in patients with malignant ( 199 cm ) versus benign ( 120 cm ) ipmns , but did not reach statistical significance with the wilcoxon two - sample exact test ( p=0.092 ) ( table 2 ) . mean v / s was substantially higher in malignant versus benign ipmns , with values of 1.25 cm and 0.69 cm , respectively ( p=0.013 ) . quantitative radiologic measures of obesity , by ipmn pathology males had a higher mean vfa value ( 202.4 cm ) than females ( 133.6 cm ) and a higher mean v / s value ( 1.25 cm ) than females ( 0.80 cm ) . stratified analyses revealed that among both males and females , mean bmi , taf , and vfa values were higher for patients with malignant compared to benign ipmns , though results were not statistically significant ( p>0.05 ) for either gender ( table 3 ) . among females , while no correlation existed between bmi and v / s ( r=0.16,p=0.35 ) , significant positive correlations were found between bmi and vfa ( r=0.68,p<0.0001 ) and between bmi and sfa ( r=0.71,p<0.0001 ) . gender - specific differences in bmi and quantitative radiologic measures of obesity , by ipmn pathology of clinical importance , figure 1 displays ct scans from two ipmn patients who did not present with high risk stigmata on imaging . both have similar bmis but vastly different vfa and v / s values , with case 1 having higher vfa and v / s and a worrisome feature ( cyst size>3 cm ) and high - grade pathology and case 2 having lower vfa and v / s and low - grade pathology at resection . these data suggest that visceral fat may be added as another risk factor to potentially aid in directing management towards a necessary surgery to remove what turned out to be a high - grade lesion ( case 1 ) and avoided an unnecessary surgery for a low - grade lesion ( case 2 ) . the accuracy , sensitivity , specificity , ppv , and npv of v / s in predicting malignant ipmn pathology were 74% , 71% , 76% , 75% and 72% , respectively . axial post contrast cts ( a and b ) and quantitative segmentation ( c and d ) for two representative side bd ipmn cases with main pancreatic ducts normal in caliber . visceral fat , intra - abdominal organs , and vasculature were subtracted . although skeletal muscle indices can be obtained in a complementary manner to visceral fat measurements , these were not directly analyzed in this study . ( 3 ) total abdominal fat with hu thresholds applied to include fat density voxels with hu 249 to 49 ( green ) . ( radiologic measures of obesity were successfully calculated for 34 of the 37 cases ; three cases did not have available scans in our pacs including the axial l2l3 intervertebral disc level views for adiposity measurement . clinical , epidemiologic , and imaging characteristics of the 34 cases ( 17 benign ; 17 malignant ) investigated in this analysis are in table 1 and are in line with published data on other ipmn cohorts . seventy - six percent with malignant pathology had md involvement on ct versus 24% with benign pathology ( p=0.005 ) . mean lesion size was higher in the malignant compared to the benign group ( 3.4 versus 1.9 cm),p=0.003 . malignant ipmns , particularly those deemed to be invasive , were predominately located in the pancreatic head . the majority of cases ( 82% ) with malignant pathology had one or more high risk stigmata ( md involvement / dilatation>10 mm , obstructive jaundice with a cystic lesion in the pancreatic head , or an enhanced solid component within the cyst ) , versus 18% of those with benign pathology ( p<0.001 ) . md dilation 59 mm , cyst size>3 cm , thickened enhanced cyst walls , non - enhanced mural nodules , or acute pancreatitis ) was not associated with malignancy ( p=0.708 ) in this cohort . bmi was higher in malignant ( 28.9 kg / m , 95% ci : 26.331.4 kg / m ) versus benign cases ( 25.8 kg / m , 95% ci : 23.328.3 kg / m ) , withp=0.045 . mean bmi was similar in males and females , at 27.8 and 27.0 kg / m , respectively . mean vfa was higher in patients with malignant ( 199 cm ) versus benign ( 120 cm ) ipmns , but did not reach statistical significance with the wilcoxon two - sample exact test ( p=0.092 ) ( table 2 ) . mean v / s was substantially higher in malignant versus benign ipmns , with values of 1.25 cm and 0.69 cm , respectively ( p=0.013 ) . quantitative radiologic measures of obesity , by ipmn pathology males had a higher mean vfa value ( 202.4 cm ) than females ( 133.6 cm ) and a higher mean v / s value ( 1.25 cm ) than females ( 0.80 cm ) . stratified analyses revealed that among both males and females , mean bmi , taf , and vfa values were higher for patients with malignant compared to benign ipmns , though results were not statistically significant ( p>0.05 ) for either gender ( table 3 ) . among females , while no correlation existed between bmi and v / s ( r=0.16,p=0.35 ) , significant positive correlations were found between bmi and vfa ( r=0.68,p<0.0001 ) and between bmi and sfa ( r=0.71,p<0.0001 ) . gender - specific differences in bmi and quantitative radiologic measures of obesity , by ipmn pathology of clinical importance , figure 1 displays ct scans from two ipmn patients who did not present with high risk stigmata on imaging . both have similar bmis but vastly different vfa and v / s values , with case 1 having higher vfa and v / s and a worrisome feature ( cyst size>3 cm ) and high - grade pathology and case 2 having lower vfa and v / s and low - grade pathology at resection . these data suggest that visceral fat may be added as another risk factor to potentially aid in directing management towards a necessary surgery to remove what turned out to be a high - grade lesion ( case 1 ) and avoided an unnecessary surgery for a low - grade lesion ( case 2 ) . the accuracy , sensitivity , specificity , ppv , and npv of v / s in predicting malignant ipmn pathology were 74% , 71% , 76% , 75% and 72% , respectively . axial post contrast cts ( a and b ) and quantitative segmentation ( c and d ) for two representative side bd ipmn cases with main pancreatic ducts normal in caliber . although skeletal muscle indices can be obtained in a complementary manner to visceral fat measurements , these were not directly analyzed in this study . ( 3 ) total abdominal fat with hu thresholds applied to include fat density voxels with hu 249 to 49 ( green ) . ( 4 ) manual segmentation of visceral fat regions ( green ) . this pilot project represents the first to study objectively quantitative radiologic measures of obesity as diagnostic markers of ipmn pathology . in addition to observing higher pre - operative bmi values in patients confirmed to have malignant ipmns , vfa and v / s values were also observed in the malignant ipmn group compared to those with benign ipmns . we also observed that males with ipmns had higher vfa and v / s values than female cases , in line with the observation that visceral fat is more common in males , and showed that women with benign ipmns had a significantly lower v / s ratio ( 0.5 cm ) than those with malignant ipmns ( 1.4 cm ) , women with benign ipmns in our cohort appeared to have a higher sfa than other cohort members , consistent with data suggesting that subcutaneous fat may not be a marker of malignancy . previous authors have suggested that in an asymptomatic adult cohort , men have significantly higher v / s ratios . however , no standardized gender - based v / s values are currently available which suggests further research is needed to define visceral obesity in each gender . despite this , findings suggest that being overweight or obese , particularly in the intra - abdominal area , may be a prognostic marker for malignant potential of ipmns . given that abdominal / visceral adiposity has been shown to influence carcinogenesis and that bmi is an imprecise proxy for abdominal adiposity , biologically - driven radiologic measures of visceral fat may have greater clinical utility than bmi in predicting ipmn pathology . further research with a larger sample size is clearly needed to distinguish the relationship between radiologic measures of visceral fat , gender , and malignancy . few studies have reported on quantitative radiologic measures of obesity in patients with pdac . in a study of 9 pdac cases and matched controls , no significant differences in sfa , vfa , tfa , or v pre - operative visceral fat was shown to be a prognostic indicator in patients with pdac , with increased visceral fat being associated with worse survival in patients with lymph node metastases . elevated visceral fat defined by the v / s has also been shown to predict recurrence among locally advanced rectal cancer patients . collectively , these and other studies provide plausibility for our observation that vfa and v / s may be associated with more advanced ipmn pathology . furthermore , with a larger sample size , multivariable modeling and receiver characteristic curve analyses will be helpful to determine the utility of gender - specific radiologic measures of abdominal obesity in discriminating malignant from benign ipmns , independent of and in combination with novel molecular and radiologic markers , standard clinical and radiologic features encompassed by consensus guidelines , and bmi . in summary , use of quantitative radiologic measures of abdominal obesity could provide a noninvasive , rapid , low cost , and repeatable way of investigating features that may potentially aid in personalizing care for patients with pancreatic cancer precursors . given that a reduction in abdominal adiposity by lifestyle , diet , and/or pharmacologic intervention would be impactful and could translate into a decreased burden of pc , obesity , and other diseases , further studies in this area are warranted .