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acute exacerbations of copd are characterized by a change in cough , sputum production , and respiratory symptoms from baseline that cause the patient to seek medical attention and involve increasing the dose and/or frequency of existing bronchodilator therapy . systemic corticosteroids are also recommended in patients with more advanced copd that may require hospitalization . exacerbations may be mild , necessitating only outpatient therapy such as antibiotics and/or systemic steroids but often may be severe enough to require hospitalization and admission to the intensive care unit . therefore , prevention of exacerbations is one of the most important goals of chronic obstructive pulmonary disease ( copd ) management ( global initiative for chronic obstructive lung disease 2005 ) because of the impact on patients daily activities and health - related quality of life ( hrqol ) , with these effects lasting for several weeks or months ( haughney and gruffydd - jones 2004 ) . frequent exacerbations also lead to a poorer recovery in terms of improvement in health status ( haughney and gruffydd - jones 2004 ) . furthermore , the frequency of acute exacerbation has a significant impact on the accelerated decline of lung function ( donaldson et al 2002 ) and also on healthcare costs ( haughney and gruffydd - jones 2004 ) . consequently , strategies for preventing copd exacerbations may have an important effect on the natural course , cost of management , and mortality in copd ( martinez and anzueto 2005 ) . in recent years , several large - scale clinical trials in copd have analyzed the exacerbation - preventing aspects of different treatments ( niewoehner 2004 ; solr 2005 ) . these include inhaled bronchodilators , inhaled corticosteroids ( icss ) , and a limited number of antimicrobial strategies . however , most antimicrobial strategies have been used therapeutically in the form of antibiotic treatment during an established exacerbation , apart from vaccinations against influenza and pneumococci , which can protect from these infections which are particularly dangerous for copd patients ( cazzola et al 2005 ) . long - acting 2-agonists ( labas ) have both bronchodilator and non - bronchodilator properties , whereas icss are potent anti - inflammatory agents ( cazzola and dahl 2004 ) . it is , therefore , obvious that labas and icss treat different aspects of copd ( cazzola and dahl 2004 ) . labas are central in the symptomatic control of copd ( american thoracic society / european respiratory society 2005 ) , whereas conflicting results have been reported with icss ( barnes 2000 ; calverley 2000 ) . this may reflect the fact that the inflammation in copd is poorly suppressed by corticosteroids , with small reduction in inflammatory cells , cytokines , or proteases in induced sputum even with oral corticosteroids ( barnes 2004 ) , although there are some subgroups of patients ( eg , those with eosinophilia ) who respond very well ( chanez et al 1997 ) . nonetheless , findings from several clinical studies have shown that treatment with icss improve expiratory airflow and reduce symptoms and the rate of exacerbations associated with copd ( alsaeedi et al 2002 ) , and , moreover , reduce all - cause mortality in copd ( sin et al 2005 ) . icss in relatively high doses ( eg , budesonide 800 g / day or fluticasone propionate 1 mg / day ) reduce exacerbations by 20%30% and improve the health status of copd patients by a similar amount compared with placebo ( man and sin 2005 ) . in particular , a meta - analysis of six trials , which evaluated the long - term effects ( 6 months ) of icss in stable copd and provided information about total exacerbations , demonstrated that icss reduce exacerbation rates in copd by nearly one third relative to placebo ( relative risk [ rr ] , 0.70 ; 95% ci , 0.580.84 ) ( alsaeedi et al 2002 ) . in the isolde ( inhaled steroid in obstructive lung disease in europe ) study , the major benefit of therapy was seen in those whose lung function was less than 50% of predicted ; however , the number of patients having at least one exacerbation per year was reduced with icss irrespective of baseline lung function ( jones et al 2003 ) . a convincing demonstration of the effectiveness of these agents in copd patients has come from the cope study ( an investigation of copd in the department of pulmonology , enschede , the netherlands ) ( van der valk et al 2002 ) . this study showed that the discontinuation of therapy with icss was associated with a more rapid onset and a higher risk of recurrence of exacerbations , and with a significant deterioration in aspects of hrqol , in the majority of patients . however , 40% of subjects in the cope study experienced no untoward effect from the withdrawal of icss . this finding indicates that there is an urgent need to identify which subgroups of copd patients respond well to prolonged icss therapy . in any case , a recent article that has reviewed the diverse approaches to data analysis used in the randomized trials of icss in the treatment of copd and their meta - analysis , has concluded that the reports that icss reduce copd exacerbation rates are the result of improper statistical analysis techniques ( suissa 2006 ) . the only two studies that used the correct statistical approach found insignificant effects with these drugs . it is not a surprise , therefore , that the evaluation of a cohort of newly treated copd patients formed from the administrative databases of saskatchewan health documented that icss do not seem to be beneficial in preventing the risk of a first exacerbation of copd ( de melo et al 2004 ) . interestingly , a recent paper has documented that immortal time bias can not account for the risk reduction associated with icss exposure in observational studies ( kiri et al 2005 ) . this finding is strengthened by the observed relationship between increased regularity of icss prescriptions and reductions in event rates . although labas have been shown to reduce exacerbation rates of copd , the magnitude of the effect appears modest at best . published trials reveal that salmeterol is the only laba with a significant effect on the rate of exacerbations . in a 1-year study comparing salmeterol with placebo , salmeterol significantly reduced the mean number of exacerbations per patient by 20% and the mean number of exacerbations requiring oral corticosteroids per patient by 29% ( calverley et al 2003a ) . recently , it has been documented that the addition of salmeterol to existing treatment in patients with a history of copd exacerbations and poor reversibility of airflow obstruction reduces exacerbations in patients who comply with treatment ( stockley et al 2006 ) . on the contrary , formoterol , in twice daily doses , failed to show such an effect in two other trials ( calverley et al 2003b ; szafranski et al 2003 ) , although a subsequent study has documented that it can influence exacerbation surrogates , for example bad days salmeterol has been shown in several model systems to reduce inflammatory cell activation , to reduce formation of edema and to enhance edema clearance , and to mitigate epithelial damage induced by bacteria ( rennard 2004 ) . whether these effects have any clinical importance remains to be determined , but they may account for its ability to reduce frequency of exacerbations of copd . formoterol may have similar non - bronchodilator effects ; however , studies are needed to document these effects . combining two therapies that possess different modes of action could be expected to have a greater benefit in the management of copd . there is an exciting possibility that the potential benefit in combining icss and labas might be due to a synergistic interaction . however , the basic molecular mechanism of such an interaction has still to be fully identified . recent in vitro and in vivo evidence suggest a mechanistic interaction at the molecular level between icss and labas . corticosteroids have been shown to up - regulate the 2-adrenoceptor in the human airways , which in turn may provide more receptors for 2-agonists to activate ( mak et al 1995 ; baraniuk et al 1997 ) . at the same time , labas may potentiate the molecular mechanism of corticosteroid actions by increasing the nuclear translocation of glucocorticoid receptors ( grs ) and thus causing an additive or sometimes synergistic suppression of inflammatory mediator release ( eickelberg et al 1999 ; korn et al 2001 ; pang and knox 2000 , 2001 ) . similar interactions between icss and labas in vivo are a likely explanation for the effects seen in the study of barnes and coworkers ( 2006 ) documenting that the combination of inhaled salmeterol and fluticasone propionate significantly reduced the absolute numbers of biopsy ( cd45 ) leukocytes , cd8 cells , and cd4 cells together with decreases in cells expressing genes for the proinflammatory mediators ifn- and tnf-. in the complementary airway compartment sampled by induced sputum , combination treatment significantly reduced sputum differential neutrophils and total eosinophils . furthermore , in vitro studies suggest that the addition of a laba to an ics may counter some of the potential negative effects of the corticosteroid ( tse et al 2003 ) or alternatively , some of those elicited by laba ( seeto et al 2003 ) . in addition to the complementary interaction of labas and icss at the molecular level , the delivery of these medications together in a single device may also help simplify treatment regimens and improve patient adherence ( cazzola and dahl 2004 ) . it is widely recognized that adherence to treatment of chronic obstructive airways diseases declines as the regime becomes more complicated , either by increasing the number of medications and/or the number of daily doses ( coutts et al 1992 ; chapman et al 2000 ) . as a result , improved compliance would be expected to occur through the use of a single combination inhaler simply because of the reduction in the number of medications and actuations required with such a regime . in particular , it would be expected that the use of combination therapy should improve compliance with ics therapy as its use is linked with laba therapy , which patients are more likely to take because of the greater symptomatic benefit . because of the above effects , it is not unexpected that long - term use of icss with labas may be associated with a reduction in copd exacerbations ( cazzola and dahl 2004 ) . however , the capacity of both icss and labas to reduce the total number of bacteria adhering to the respiratory mucosa in a concentration - dependent manner without altering the bacterial tropism for mucosa , and to preserve ciliated cells , is important in this context ( dowling et al 1999 ) . icss and labas , when administered together at low concentrations , exhibited a synergistic effect with respect to the preservation of ciliated cells , showing a trend toward reduced damage and a significant preservation of the number of ciliated cells compared with either agent alone at the same concentrations . this result may have clinical significance as it is thought that ciliated cells are the most sensitive to damage by bacterial infection ( tsang et al 1994 ) . it is well - known that airway colonization and chronic infection contribute to progressive pulmonary damage in copd patients via the action of proinflammatory substances in what is known as the vicious circle theory ( wilson 1991 ) . recently , the synergistic effects of salmeterol and fluticasone in human rhinovirus - induced proinflammatory cytokine production have also been documented ( edwards et al 2006 ) . rhinoviruses are implicated in many acute exacerbations of copd , perhaps by inducing proinflammatory cytokines ( seemungal et al 2001 ) . combining two therapies that possess different modes of action could be expected to have a greater benefit in the management of copd . there is an exciting possibility that the potential benefit in combining icss and labas might be due to a synergistic interaction . however , the basic molecular mechanism of such an interaction has still to be fully identified . recent in vitro and in vivo evidence suggest a mechanistic interaction at the molecular level between icss and labas . corticosteroids have been shown to up - regulate the 2-adrenoceptor in the human airways , which in turn may provide more receptors for 2-agonists to activate ( mak et al 1995 ; baraniuk et al 1997 ) . at the same time , labas may potentiate the molecular mechanism of corticosteroid actions by increasing the nuclear translocation of glucocorticoid receptors ( grs ) and thus causing an additive or sometimes synergistic suppression of inflammatory mediator release ( eickelberg et al 1999 ; korn et al 2001 ; pang and knox 2000 , 2001 ) . similar interactions between icss and labas in vivo are a likely explanation for the effects seen in the study of barnes and coworkers ( 2006 ) documenting that the combination of inhaled salmeterol and fluticasone propionate significantly reduced the absolute numbers of biopsy ( cd45 ) leukocytes , cd8 cells , and cd4 cells together with decreases in cells expressing genes for the proinflammatory mediators ifn- and tnf-. in the complementary airway compartment sampled by induced sputum , combination treatment significantly reduced sputum differential neutrophils and total eosinophils . furthermore , in vitro studies suggest that the addition of a laba to an ics may counter some of the potential negative effects of the corticosteroid ( tse et al 2003 ) or alternatively , some of those elicited by laba ( seeto et al 2003 ) . in addition to the complementary interaction of labas and icss at the molecular level , the delivery of these medications together in a single device may also help simplify treatment regimens and improve patient adherence ( cazzola and dahl 2004 ) . it is widely recognized that adherence to treatment of chronic obstructive airways diseases declines as the regime becomes more complicated , either by increasing the number of medications and/or the number of daily doses ( coutts et al 1992 ; chapman et al 2000 ) . as a result , improved compliance would be expected to occur through the use of a single combination inhaler simply because of the reduction in the number of medications and actuations required with such a regime . in particular , it would be expected that the use of combination therapy should improve compliance with ics therapy as its use is linked with laba therapy , which patients are more likely to take because of the greater symptomatic benefit . because of the above effects , it is not unexpected that long - term use of icss with labas may be associated with a reduction in copd exacerbations ( cazzola and dahl 2004 ) . however , the capacity of both icss and labas to reduce the total number of bacteria adhering to the respiratory mucosa in a concentration - dependent manner without altering the bacterial tropism for mucosa , and to preserve ciliated cells , is important in this context ( dowling et al 1999 ) . icss and labas , when administered together at low concentrations , exhibited a synergistic effect with respect to the preservation of ciliated cells , showing a trend toward reduced damage and a significant preservation of the number of ciliated cells compared with either agent alone at the same concentrations . this result may have clinical significance as it is thought that ciliated cells are the most sensitive to damage by bacterial infection ( tsang et al 1994 ) . it is well - known that airway colonization and chronic infection contribute to progressive pulmonary damage in copd patients via the action of proinflammatory substances in what is known as the vicious circle theory ( wilson 1991 ) . recently , the synergistic effects of salmeterol and fluticasone in human rhinovirus - induced proinflammatory cytokine production have also been documented ( edwards et al 2006 ) . rhinoviruses are implicated in many acute exacerbations of copd , perhaps by inducing proinflammatory cytokines ( seemungal et al 2001 ) . the efficacy of combination therapy with icss and labas delivered via a single inhaler to copd patients has repeatedly been documented ( table 1 ) . in particular , the impact on acute exacerbations of long - term treatments with combined therapy is shown in figures 1 and 2 , and table 2 . it is very important to also mention that the definition of exacerbation and the patient population enrolled in these studies are not uniform ( table 3 ) , and these facts have to be taken in consideration when analyzing their results . in a multicenter , randomized , placebo - controlled study , hanania et al ( 2003 ) compared the efficacy and safety of fluticasone propionate ( 250 g ) and salmeterol ( 50 g ) , when administered twice daily together in a single device for 6 months , with that of placebo and the individual agents alone in patients with copd . this study was unable to observe significant differences among treatment groups in the number of exacerbations or the time to first exacerbation . however , this study was of short duration and was designed and powered to evaluate the treatment effect on fev1 , which was the primary measure of efficacy , rather than to evaluate the rates of exacerbations . in a study with a similar design but evaluating the higher dose formulation of this combination therapy , fluticasone propionate ( 500 g ) with salmeterol ( 50 g ) twice daily given for 6 months ( mahler et al 2002 ) , there were no statistically significant differences between treatment groups in time to exacerbation . this study also demonstrated no difference in the number of participants who exacerbated once ( placebo : 8.8% ; salmeterol : 5.6% ; fluticasone : 10.1% ; fluticase / salmeterol combination [ fsc ] : 8.5% ) , but no p values were presented . also this study was of short duration and was designed and powered to evaluate the treatment effect on fev1 , which was the primary measure of efficacy , rather than to evaluate the rates of exacerbations . the tristan study ( calverley et al 2003a ) , a randomized controlled trial over 1 year of combination treatment with salmeterol ( 50 g ) and fluticasone ( 500 g ) administered together in a single device twice daily versus each of the components alone and versus placebo , enrolled patients with a baseline fev1 before bronchodilation that was 25%70% of that predicted . in this study , the rate of exacerbations requiring medical intervention , defined as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids , or both , fell by 25% in the combination group and by 20% and 19% in the salmeterol and fluticasone groups , respectively , compared with placebo . in particular , there was a significant difference in the mean exacerbation rate per participant per year in favor of the combination therapy when compared with placebo ( fsc : 0.97 vs placebo : 1.30 , p<0.0001 ) , but , interestingly , differences among the three active treatment arms were small and not statistically significant . the treatment effect was more pronounced in patients with severe disease ( ie , a baseline fev1 < 50% of predicted ) , who showed a 30% reduction with combination therapy compared with placebo , compared with a 10% reduction in patients who had a baseline fev1 greater than 50% of predicted . however , this beneficial effect was also seen for exacerbations treated with oral corticosteroids , which have conventionally been thought to be more severe episodes . the combination therapy was associated with an approximate 39% reduction in the number of this type of exacerbations compared with placebo ( 0.46 vs 0.76 exacerbations per year , p<0.0001 ) but , again , the differences between fsc and salmeterol or fluticasone were not significant . it is worth mentioning that the tristan was longer , but still based on fev1 . hence , the ability to observe exacerbation differences may have been helped by study duration . in a study using a design similar to that of the tristan study but including patients with an fev1 below 50% predicted , szafranski et al ( 2003 ) compared the effects of two inhalations twice daily of either budesonide / formoterol combination ( bfc ) 160/4.5 g ( delivered dose ) in a single inhaler , budesonide 200 g ( metered dose ) , formoterol 4.5 g , or placebo for 12 months . data from this study demonstrate a significant reduction in the number of severe exacerbations by 24% in the bfc group vs placebo , 23% vs formoterol , and 11% vs budesonide . this reflects a mean reduction in the mean annual exacerbation rates of 0.42 ( 95% ci 0.140.70 ) , 1.841.42 exacerbations per year when combination therapy was used . compared with placebo , both bfc and budesonide significantly reduced the number of oral steroid courses used in association with exacerbations ( 31% and 29% , respectively , compared with 3% for formoterol vs placebo ) . in addition , bfc significantly reduced the number of oral steroid courses compared with formoterol ( 28% ) . mild exacerbations were also significantly reduced on bfc when compared with placebo ( 62% ) and budesonide ( 35% ) , but not with formoterol ( 15% ) . in another study investigating bfc , calverley et al ( 2003b ) studied patients who initially received formoterol alone ( 9 g delivered dose twice daily ) and a short course of oral prednisolone ( 30 mg once daily ) for 2 weeks before randomization . the patients were subsequently randomized to twice daily inhaled bfc 320/9 g ( delivered dose ) , budesonide 400 g ( metered dose ) , formoterol 9 g , or placebo for 12 months , bfc prolonged time to first exacerbation requiring medical intervention , defined as a need for antibiotics and/or oral corticosteroids and/or hospitalization due to respiratory symptoms , compared with all other treatments . hazard rate analysis showed that the risk of having an exacerbation while being treated with bfc was reduced by 22.7% , 29.5% , and 28.5% vs budesonide , formoterol , and placebo , respectively . the exacerbation rate with bfc was reduced compared with placebo ( 23.6% ) and formoterol ( 25.5% ) but not with budesonide alone ( 13.6% ) . furthermore , bfc prolonged the time to first course of oral corticosteroids after randomization ; risk reductions were 32.7% and 33.8% vs budesonide and formoterol , respectively , and 42.3% vs placebo . bfc also reduced the rate of oral corticosteroid courses by 28.2% , 30.5% , and 44.7% vs budesonide , formoterol , and placebo , respectively ; budesonide alone reduced the number of oral corticosteroid courses compared with placebo but formoterol did not . data of three large studies ( calverley et al 2003a , 2003b ; szafranski et al 2003 ) that have examined the effects of a combination of laba and ics on the risk of developing exacerbation in copd have shown that the patients receiving combination therapy were less likely to report an exacerbation than those receiving the laba alone . nevertheless , a recent cochrane review ( nannini et al 2004 ) concluded that there were conflicting results when the different combination therapies were compared with the single components alone . in particular , the results of the studies showed that bfc and fsc were effective and reduced the frequency of exacerbations compared with dummy medication to a level of three quarters of the previous rates , with one exacerbation prevented every 24 years in the participants in the clinical trials . when the combination treatment was compared with one of the component drugs given as single treatments , bfc was better than its component laba at preventing the frequency of exacerbations , but fsc did not show a significant advantage over laba . recently , two different reviews ( halpin 2005 ; cazzola 2006 ) , which have evaluated the applicability and clinical relevance of number needed to treat ( nnt ) analysis for determining the effectiveness of combination therapies against copd exacerbations , have calculated that for every 100 patients treated with bfc for 1 year , between 42 and 47 exacerbations requiring medical intervention would be prevented vs laba therapy alone , whereas for every 100 patients treated with fsc for 1 year , only 7 exacerbations would be prevented vs laba alone ( table 2 ) . it should be noted , however , that the nnt value relating to fsc should be interpreted with caution as there was no significant difference between fsc and salmeterol alone ( p=0.345 for treatment ratios for combination vs salmeterol therapy ) . this means that the hypothetical 95% ci would include infinity and that an nnt of infinity should be more appropriately applied to the difference between the two treatments , which indicates no true net clinical benefit of fsc over salmeterol alone . in any case , in a recent study investigating the effect withdrawal of fluticasone from patients with severe copd ( fev1 < 50% predicted ) using fsc ( cosmic study ) for 3 months , wouters et al ( 2005 ) demonstrated an acute and sustained decrease in fev1 , increase in symptoms , and an increase in mild exacerbations in patients treated with salmeterol alone compared with those who continued treatment with fsc over the one year of the study . the conflicting findings between the results of the different studies may be accounted for by dissimilar study designs . in particular , definitions of exacerbation that have been used in the examined studies have been rather different ( table 3 ) . for example , in mahler s study ( mahler et al 2002 ) , exacerbation was defined by treatment ( mild = increased use of salbutamol ; moderate = use of either oral antibiotics and/or corticosteroids ; severe = hospitalization ) . in the szafranski s study ( szafranski et al 2003 ) , the definition of mild exacerbation was a day with 4 inhalations of reliever medication above the mean run - in use , whereas that of severe exacerbation was a requirement for oral steroids and/or antibiotics and/or hospitalization due to respiratory symptoms . in the tristan study ( calverley et al 2003a ) , exacerbations were defined a priori as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids , or both . pauwels et al ( 2004 ) correctly highlighted that considerable heterogeneity in the aetiology and manifestation of copd exacerbations makes identification and quantification of defining symptoms extremely difficult . however , with the emergence of a number of drugs designed or developed specifically to treat copd and associated exacerbations , the absence of a uniform definition is a significant issue . in fact , the choice of definition can significantly affect study outcomes , with varying criteria likely to result in different levels of demonstrated treatment success . furthermore , emerging data confirm that exacerbation is a term not easily understood by patients , who prefer to use simpler words such as crisis it is also interesting to stress that there was some difference in the patient populations enrolled in the studies described above . patients enrolled in the tristan study ( calverley et al 2003a ) had to have suffered from at least one episode of acute copd symptom exacerbation per year in the previous 3 years , and at least one exacerbation in the year immediately before trial entry that required treatment with oral corticosteroids , antibiotics , or both . in the szafranski study ( szafranski et al 2003 ) , patients had to have suffered from at least one severe ( use of oral corticosteroids and/or antibiotics and/or hospitalization due to respiratory symptoms ) copd exacerbation within 212 months before the first clinic visit . moreover , the mean pre - trial fev1 was 45% predicted in the tristan study ( calverley et al 2003a ) and 36% predicted in the szafranski s study ( szafranski et al 2003 ) . moreover , the mean total exacerbation rate per patient per year under placebo was 1.30 in the tristan study ( calverley et al 2003a ) and 1.9 in the szafranski study ( szafranski et al 2003 ) . this differences indicate that the szafranski s study ( szafranski et al 2003 ) probably enrolled patients with more severe disease or , at least , with a higher possibility of suffering from acute exacerbation . in fact , also in the calverley study ( calverley et al 2003b ) , the mean pre - trial fev1 was 36% predicted and the mean total exacerbation rate per patient per year under placebo was 1.80 . as correctly highlighted by calverley et al ( 2003b ) , the more severe disease in the patients enrolled in bfc studies is the likely explanation of the greater number of episodes that they observed compared with other studies , a difference that increased the power of the study to detect an effect of treatment . it is also important to highlight that the authors of the three studies did not report the number of exacerbations in the year before the enrolment in the studies . this is a real lack of information because , as recently documented by donaldson et al ( 2003 ) , annual exacerbation frequency remains constant over time . as rabe ( 2003 ) stated in his editorial comment on the calverley paper ( calverley et al 2003b ) , the data of the combination therapy trials in copd seem to indicate that patients experiencing more severe exacerbations benefit the most from combination therapy , whereas , if the definition of a ( mild ) exacerbation for clinical trials also includes aggravation of symptoms , such as in the paper by szafranski et al ( 2003 ) , the relative effect of a long - acting bronchodilator might be more pronounced . this clearly highlights the importance of definitions of exacerbations for clinical trials and calls for studies comparing the effect of maximal bronchodilation with , for example , the combination of labas with long - acting anticholinergics ( lamas ) in copd with mild and severe exacerbations as an outcome . in effect , some studies are documenting the clinical value of combining a laba with a lama in copd ( cazzola et al 2004 ; van noord et al 2005 , 2006 ) , although the information of the impact of such a type of combination on exacerbations is still lacking . borrowing jones opinion ( jones 2004 ) , we believe that there are a number of possible mechanisms to account for the apparent additive effect of inhaled combination therapy with icss and labas in preventing exacerbations and to explain the discrepancies observed between the studies . it is likely that : ( 1 ) the individual drugs were not at the top of their dose response curve for this outcome ; ( 2 ) they have different , and additive , mechanisms of action , which can also be linked to the specific compound ; or ( 3 ) the different agents were preventing different types of exacerbation . jones ( 2004 ) has highlighted that an operational definition based upon a worsening of symptoms and the treatment required ( moderate exacerbations are those that can be treated with oral corticosteroids and/or antibiotics , severe exacerbations are those requiring hospital admission ) does not identify the type of exacerbation . this emphasizes again the importance for the need of a uniform definition of copd exacerbation for clinical trials in order to reach more solid conclusions . whatever the case may be , it must be emphasized that a treatment regimen with administration of an ics and laba in a single inhaler prolongs the time to a first copd exacerbation , compared with treatment with the single components in patients with an fev1 < 50% predicted ( calverley et al 2003b ) . intriguingly , this finding has been observed with bfc ( calverley et al 2003b ) , but not with fsc ( calverley et al 2003a ) . the prolonged time to first exacerbation may delay the deterioration of the disease and help maintain health status and patient well - being . it is an important outcome because it indicates that following aggressive treatment of an acute exacerbation with an oral corticosteroid , therapy with an ics / laba combination may better control the disease than treatment with bronchodilator alone . in particular , as highlighted by calverley ( 2004 ) , combining treatments is certainly something to be considered in patients who have reported exacerbations on a regular basis but have not yet received ics or laba therapy in combination , although there are concerns about the increased risk of side - effects and cost of using laba / ics therapy in copd . data of three large studies ( calverley et al 2003a , 2003b ; szafranski et al 2003 ) that have examined the effects of a combination of laba and ics on the risk of developing exacerbation in copd have shown that the patients receiving combination therapy were less likely to report an exacerbation than those receiving the laba alone . nevertheless , a recent cochrane review ( nannini et al 2004 ) concluded that there were conflicting results when the different combination therapies were compared with the single components alone . in particular , the results of the studies showed that bfc and fsc were effective and reduced the frequency of exacerbations compared with dummy medication to a level of three quarters of the previous rates , with one exacerbation prevented every 24 years in the participants in the clinical trials . when the combination treatment was compared with one of the component drugs given as single treatments , bfc was better than its component laba at preventing the frequency of exacerbations , but recently , two different reviews ( halpin 2005 ; cazzola 2006 ) , which have evaluated the applicability and clinical relevance of number needed to treat ( nnt ) analysis for determining the effectiveness of combination therapies against copd exacerbations , have calculated that for every 100 patients treated with bfc for 1 year , between 42 and 47 exacerbations requiring medical intervention would be prevented vs laba therapy alone , whereas for every 100 patients treated with fsc for 1 year , only 7 exacerbations would be prevented vs laba alone ( table 2 ) . it should be noted , however , that the nnt value relating to fsc should be interpreted with caution as there was no significant difference between fsc and salmeterol alone ( p=0.345 for treatment ratios for combination vs salmeterol therapy ) . this means that the hypothetical 95% ci would include infinity and that an nnt of infinity should be more appropriately applied to the difference between the two treatments , which indicates no true net clinical benefit of fsc over salmeterol alone . in any case , in a recent study investigating the effect withdrawal of fluticasone from patients with severe copd ( fev1 < 50% predicted ) using fsc ( cosmic study ) for 3 months , wouters et al ( 2005 ) demonstrated an acute and sustained decrease in fev1 , increase in symptoms , and an increase in mild exacerbations in patients treated with salmeterol alone compared with those who continued treatment with fsc over the one year of the study . the conflicting findings between the results of the different studies may be accounted for by dissimilar study designs . in particular , definitions of exacerbation that have been used in the examined studies have been rather different ( table 3 ) . for example , in mahler s study ( mahler et al 2002 ) , exacerbation was defined by treatment ( mild = increased use of salbutamol ; moderate = use of either oral antibiotics and/or corticosteroids ; severe = hospitalization ) . in the szafranski s study ( szafranski et al 2003 ) , the definition of mild exacerbation was a day with 4 inhalations of reliever medication above the mean run - in use , whereas that of severe exacerbation was a requirement for oral steroids and/or antibiotics and/or hospitalization due to respiratory symptoms . in the tristan study ( calverley et al 2003a ) , exacerbations were defined a priori as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids , or both . pauwels et al ( 2004 ) correctly highlighted that considerable heterogeneity in the aetiology and manifestation of copd exacerbations makes identification and quantification of defining symptoms extremely difficult . however , with the emergence of a number of drugs designed or developed specifically to treat copd and associated exacerbations , the absence of a uniform definition is a significant issue . in fact , the choice of definition can significantly affect study outcomes , with varying criteria likely to result in different levels of demonstrated treatment success . furthermore , emerging data confirm that exacerbation is a term not easily understood by patients , who prefer to use simpler words such as crisis it is also interesting to stress that there was some difference in the patient populations enrolled in the studies described above . patients enrolled in the tristan study ( calverley et al 2003a ) had to have suffered from at least one episode of acute copd symptom exacerbation per year in the previous 3 years , and at least one exacerbation in the year immediately before trial entry that required treatment with oral corticosteroids , antibiotics , or both . in the szafranski study ( szafranski et al 2003 ) , patients had to have suffered from at least one severe ( use of oral corticosteroids and/or antibiotics and/or hospitalization due to respiratory symptoms ) copd exacerbation within 212 months before the first clinic visit . moreover , the mean pre - trial fev1 was 45% predicted in the tristan study ( calverley et al 2003a ) and 36% predicted in the szafranski s study ( szafranski et al 2003 ) . moreover , the mean total exacerbation rate per patient per year under placebo was 1.30 in the tristan study ( calverley et al 2003a ) and 1.9 in the szafranski study ( szafranski et al 2003 ) . this differences indicate that the szafranski s study ( szafranski et al 2003 ) probably enrolled patients with more severe disease or , at least , with a higher possibility of suffering from acute exacerbation . in fact , also in the calverley study ( calverley et al 2003b ) , the mean pre - trial fev1 was 36% predicted and the mean total exacerbation rate per patient per year under placebo was 1.80 . as correctly highlighted by calverley et al ( 2003b ) , the more severe disease in the patients enrolled in bfc studies is the likely explanation of the greater number of episodes that they observed compared with other studies , a difference that increased the power of the study to detect an effect of treatment . it is also important to highlight that the authors of the three studies did not report the number of exacerbations in the year before the enrolment in the studies . this is a real lack of information because , as recently documented by donaldson et al ( 2003 ) , annual exacerbation frequency remains constant over time . as rabe ( 2003 ) stated in his editorial comment on the calverley paper ( calverley et al 2003b ) , the data of the combination therapy trials in copd seem to indicate that patients experiencing more severe exacerbations benefit the most from combination therapy , whereas , if the definition of a ( mild ) exacerbation for clinical trials also includes aggravation of symptoms , such as in the paper by szafranski et al ( 2003 ) , the relative effect of a long - acting bronchodilator might be more pronounced . this clearly highlights the importance of definitions of exacerbations for clinical trials and calls for studies comparing the effect of maximal bronchodilation with , for example , the combination of labas with long - acting anticholinergics ( lamas ) in copd with mild and severe exacerbations as an outcome . in effect , some studies are documenting the clinical value of combining a laba with a lama in copd ( cazzola et al 2004 ; van noord et al 2005 , 2006 ) , although the information of the impact of such a type of combination on exacerbations is still lacking . borrowing jones opinion ( jones 2004 ) , we believe that there are a number of possible mechanisms to account for the apparent additive effect of inhaled combination therapy with icss and labas in preventing exacerbations and to explain the discrepancies observed between the studies . it is likely that : ( 1 ) the individual drugs were not at the top of their dose response curve for this outcome ; ( 2 ) they have different , and additive , mechanisms of action , which can also be linked to the specific compound ; or ( 3 ) the different agents were preventing different types of exacerbation . jones ( 2004 ) has highlighted that an operational definition based upon a worsening of symptoms and the treatment required ( moderate exacerbations are those that can be treated with oral corticosteroids and/or antibiotics , severe exacerbations are those requiring hospital admission ) does not identify the type of exacerbation . this emphasizes again the importance for the need of a uniform definition of copd exacerbation for clinical trials in order to reach more solid conclusions . whatever the case may be , it must be emphasized that a treatment regimen with administration of an ics and laba in a single inhaler prolongs the time to a first copd exacerbation , compared with treatment with the single components in patients with an fev1 < 50% predicted ( calverley et al 2003b ) . intriguingly , this finding has been observed with bfc ( calverley et al 2003b ) , but not with fsc ( calverley et al 2003a ) . the prolonged time to first exacerbation may delay the deterioration of the disease and help maintain health status and patient well - being . it is an important outcome because it indicates that following aggressive treatment of an acute exacerbation with an oral corticosteroid , therapy with an ics / laba combination may better control the disease than treatment with bronchodilator alone . in particular , as highlighted by calverley ( 2004 ) , combining treatments is certainly something to be considered in patients who have reported exacerbations on a regular basis but have not yet received ics or laba therapy in combination , although there are concerns about the increased risk of side - effects and cost of using laba / ics therapy in copd .

acute exacerbations of copd can complicate the course of the disease in patients with severe airway obstruction . reduction of exacerbations is an important clinical outcome in evaluating new therapies in copd . combination therapies with long - acting -agonists and inhaled corticosteroids have now been approved for use . three 1-year randomized clinical trials , which studied the effect of combining a long - acting 2-agonist with an inhaled corticosteroid in copd , documented that exacerbation frequency was lower with therapy than placebo . combination therapy had a similar effect to its monocomponents in the trial evaluating salmeterol / fluticasone combination . however , when patients with more severe copd were studied using a combination of budesonide and formoterol , a clear improvement was seen in the overall exacerbation rates compared with the use of a long - acting 2-agonist alone .

reverse shoulder prosthesis is an excellent surgical option for patients with certain shoulder pathologies , of which rotator cuff arthropathy is one . one area where we are gaining a greater understanding is that of managing acromial pathology . recent studies are showing that not all acromial pathology is a contraindication to reverse shoulder arthroplasty ; in both pre- and post - operative patients , small lateral fractures of the acromion can be treated non - operatively or fixed surgically with a tension band technique . overall , results comparable to that of reverse arthroplasty can be achieved with either of these treatment options . however , fractures at the base of the acromion or of the scapular spine present a different problem . in these types of fractures , a larger length of deltoid muscle is detached , and therefore defunctioned . in reverse shoulder arthroplasty , in order to achieve a stable shoulder , the deltoid must be tensioned a great deal . this in turn causes a deforming force and distraction at the fracture site , making for a poor healing potential . also , as the reverse shoulder prosthesis relies on the deltoid muscle to move the arm , if the deltoid is detensioned or improperly tensioned , this will lead to poor functional capacity . we present a case of treatment of a fracture at the base of the acromion using a 90/90 plating construct that healed in a good position . a 71-year - old right - handed woman presented with a 2-year history of right shoulder pain and dysfunction , which began abruptly when lifting a heavy load . at the initial evaluation , her range of motion was limited , particularly in active abduction as she achieved only 45 degrees in this plane . a cuff arthropathy ( hamada grade 3 ) was diagnosed and the decision was taken to proceed with reverse shoulder arthroplasty . to complete the pre - operative assessment , a ct scan was performed which confirmed there was no acromial pathology involved . a reverse shoulder prosthesis and a biceps tenodesis was carried out ( delta xtend , depuy , warsaw , in , usa ) through a deltopecoral approach [ figure 1 ] . the patient 's right arm was immobilized in a sling and the range of motion exercises were started 6 weeks after arthroplasty [ figure 1a ] . five months after surgery , she sustained a blow to the right shoulder but sought medical attention 1 month later , at her scheduled follow - up visit . radiographs showed a displaced fracture at the base of the acromion [ figure 1 ] . this showed that both the humeral and glenoid components were well fixed and the only pathology was the fracture . antero - posterior radiographs showing the patient 's right shoulder immediately after reverse shoulder arthroplasty ( a ) and after fracture , 5 months later ( b ) . the post - operative image shows no pre - existing acromial pathology she was taken to the operating room and her previous incision was extended into a sabre incision to better expose the acromion . two small fragment ( 3.5 mm ) locking plates ( synthes , west chester , pa , usa ) were used in a 90/90 configuration ; one was a fragment specific clavicular plate and the other a reconstruction plate . the fragment specific clavicular plate was applied on the superior edge of the scapular spine in a compression mode . a second reconsrtuction plate was applied from the posterior acromion to the posterior cortex of the scapular spine , in the infraspinatus fossa . fixation was solid , but in order to protect the construct , the patient was placed in an abduction brace for 6 weeks . eighteen months after fracture fixation , the patient was satisfied with the clinical result [ figure 2 ] . she was capable of 125 degrees of abduction , 160 degrees of forward flexion , 85 degrees of external rotation in adduction , and 60 degrees of internal rotation . her quickdash score was 29.5 ( compared to 82.5 pre - op ) and constant score was 69 on the affected side compared to 85 on the left side , for a good functional outcome . a medline search using the key words [ acromion fracture ] , [ scapula fracture ] , [ reverse arthroplasty ] was performed . we then sought to classify and separate the cases by location ; there were 30 cases of fractures at the base of the acromion , and scapular spine . when our case is added , we have a total of 31 cases . results are summarized in table 1 . in brief , of the 30 more proximal cases , 21 were treated non - operatively in a sling , 7 were treated with open reduction and open fixation and one with revision of the prosthesis . of those treated non - operatively , and for whom results are published , fourteen had a non - union , four had a malunion and in two cases , it was unclear whether union was achieved . for patients receiving fixation of the fracture , there was one non - union , two repeat fixations , and one patient required removal of the fixation . the patient who had a revision of the prosthesis had a malunion of the acromion . results of the literature review . in the fracture location column , the fracture description provided by the authors is presented . although recent studies have shown that good outcomes may be achieved with non - surgical management of lateral acromial fractures , the same does not hold true for basal acromial fractures . the likely reason for this is that with fractures at the base of the acromion , a large length of the deltoid is defunctioned and the deltoid muscle is essential to the functioning of the reverse shoulder prosthesis . previously described techniques , such as tension band fixation achieved poor functional results for fractures at the base of the acromion . we postulate that this is because the fixation method is insufficient to withstand the forces generated by the deltoid muscle a tension band can neutralize forces parallel to the axis of the band but in the case of the deltoid , force vectors are generated in different directions . it also allows for compression along the fracture site and resists motion in all directions . good screw purchase can be achieved by angling the screws either toward the scapular spine or the coracoid . the locking option enables good fixation and improves cut - out strength in osteopenic or osteoporotic bone . in addition to the stable fixation achieved with this technique , we further recommend immobilization of the affected shoulder using a sling and an abduction pillow to detension the deltoid , thus at least partially removing the distractive forces at the fracture site . also essential to achieving a good functional result we thus also recommend a program of range of motion exercises followed by strengthening exercises to maximize functional capabilities . based on results from previous studies , fractures of the tip were those of the most lateral or anterior portion of the acromion . fractures of the body of the acromion are those medial to the tip of the acromion and lateral to the beginning of the scapular base . the scapular base is the lateral border of the scapular spine , which is smooth and round . in our nomenclature , fractures at the scapular base are termed fractures at the base of the acromion as functionally , this zone connects the acromion to the rest of the scapula and this term avoids confusion with any more medially occurring fractures . coronal ( a ) and axial ( b ) representations of the proposed classification scheme for acromial fractures associated with revere arthroplasty we propose the above mentioned classification system as it provides a nomenclature for acromial fractures that is descriptive and is based on the anatomy and functionality of the scapula . as the results from the review of the literature indicate , the more medial the fracture , the worse the prognosis with non - operative treatment . thus , this classification system may be used to predict outcomes and determine treatment offered . it differs from other classification systems ( crosby ) in that it further subclassifies more medial fractures , which are the more ominous fractures . as more research is carried out in this field

fractures of the acromion and scapula are known to occur after reverse shoulder arthroplasty . we present a case of a fracture at the base of the acromion 5 months after arthroplasty treated successfully with dual plating of the acromion . eighteen months after fracture fixation , the patient had 160 degrees of active forward flexion , a quickdash of 29.5 , a constant score of 69 and she was satisfied with the result . a concomitant review of the literature produced , in addition to our patient , 56 cases . these were used to produce a classification system , based on bony and functional anatomy as follows . tip fractures are of the most lateral or anterior portion of the acromion , those of the body of the acromion are medial to the tip but lateral to the beginning of the scapular base . fractures at the scapular base are termed fractures of the base of the acromion and those more medial to that , fractures of the scapular spine . the functional results of these case series demonstrated poorer functional outcomes for more medial fractures . as future research in this domain increases , clarity on the nomenclature of these fractures will allow for prognostication and treatment based on fracture location as well as comparison between studies .

besides its classical role in calcium and bone homeostasis , vitamin d is considered a potent immunomodulator that can affect the pathogenesis of several autoimmune diseases . our aim is to evaluate the effect of vitamin d correction to a patient with new onset graves disease ( gd ) with an underlying vitamin d deficiency . we describe the effect of vitamin d3 on untreated graves disease with vitamin d deficiency . a healthy saudi woman in her 40s sought consultation with a three - month history of palpitation . she denied any history of heat intolerance , weight loss , menstrual irregularity or sweating . . physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit . the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5 vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7 tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution . the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( prescribed by an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month . follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 . vitamin d deficiency may exacerbate the onset and/or development of gd and correction of the deficiency may be able to reverse it . however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd . more recently , vitamin d has been shown to be a modulator in both innate and adaptive immunity.1 there is a well - established link between vitamin d deficiency and various autoimmune diseases , including type 1 diabetes mellitus ( t1 dm ) , systemic lupus erythematosus ( sle ) , rheumatoid arthritis ( ra ) , inflammatory bowel disease ( ibd ) , and multiple sclerosis ( ms ) . furthermore , it has been found that the supplementation of vitamin d can prevent the onset and/or development of different kinds of autoimmune disorders in human beings and animal models.2 in addition , it has been shown that the prevalence of vitamin d deficiency is common in patients with graves disease ( gd),3 and is associated with higher thyroid volume.4 in our case report , we evaluated the effect of vitamin d correction to a patient with new onset gd with an underlying vitamin d deficiency . a healthy saudi woman in her 40s sought consultation with a 3 months history of palpitation . she denied any history of heat intolerance , weight loss , menstrual irregularity , diarrhea , or sweating . there was no personal or family history of thyroid disease and no specific medication history . physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit . the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5 vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7 anti - thyroid antibodies showed a tg , 17.1 iu / ml ; tpo , 0.19 iu / ml with a positive tsh receptor antibody . tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution ( fig . the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( took it from an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month . the serial thyroid function tests , vitamin d levels , and titer autoantibodies are summarized in table 1 . follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 . written informed consent was obtained from the patient for the publication of this case and accompanying images . it has become apparent that multiple factors contribute to the etiology of gd , including genetic and environmental factors . these activated t cells in turn increase the secretion of thyroid - specific autoantibodies from b cells . the prevalence of vitamin d deficiency was reported to be common in patients with gd.3 whether vitamin d deficiency has a causal relationship with gd remains a controversial issue . misharin et al.5 observed that vitamin d deficiency was found to modulate graves hyperthyroidism induced in balb / c mice by thyrotropin receptor immunization . in this study , balb / c mice on a vitamin d deficient diet were more likely to develop persistent hyperthyroidism than other mice receiving adequate vitamin d supply . in another study , combination treatment with methimazole and vitamin d3 ( 1,25 ( oh)2d ) in patients with gd has more rapid euthyroidism achievement compared with patients receiving methimazole alone.6 in addition , vitamin d supplementation has been shown to inhibit inflammatory responses in human thyroid and t cells.7 interestingly , vitamin d deficiency is found to be associated with higher thyroid volume in patients with newly onset gd.4 it has been recently discovered that vitamin d - receptor gene and vitamin d - binding protein gene polymorphisms are associated with gd.8,9 our present case supports the current literature and strongly suggests that vitamin d deficiency may exacerbate the onset and/or development of gd and correction of which may be able to reverse it . however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd .

objectivebesides its classical role in calcium and bone homeostasis , vitamin d is considered a potent immunomodulator that can affect the pathogenesis of several autoimmune diseases . our aim is to evaluate the effect of vitamin d correction to a patient with new onset graves disease ( gd ) with an underlying vitamin d deficiency.methodwe describe the effect of vitamin d3 on untreated graves disease with vitamin d deficiency.resultsa healthy saudi woman in her 40s sought consultation with a three - month history of palpitation . she denied any history of heat intolerance , weight loss , menstrual irregularity or sweating . she has a history of chronic muscle aches and pains . physical examination revealed a mild diffusely enlarged and non - tender thyroid gland with no bruit . she had no signs of graves ophthalmopathy . in laboratory examinations , the initial thyroid function test , which was done in an outside hospital , revealed a tsh , 0.01 miu / l ; ft4 , 22.5 pmol / l and ft3 , 6.5 pmol / l . vitamin d 25-oh level was done in our hospital and showed a result of 26.0 nmol / l with a tsh , 0.013 miu / l ; ft4 , 16.7 pmol / l ; and ft3 , 3.8 pmol / l . tsh receptor antibody was positive . tc-99 m thyroid scintigraphy demonstrated an enlarged thyroid gland with increased radiotracer trapping and heterogeneous distribution . the patient was given only oral cholecalciferol 4000 iu per day since november 2012 ( prescribed by an outside hospital ) then from may 2013 onwards she was given 50,000 iu per month . follow - up laboratory exams revealed improved vitamin d levels as well as tsh and ft4 . she eventually improved both clinically and biochemically with a satisfactory outcome.conclusionvitamin d deficiency may exacerbate the onset and/or development of gd and correction of the deficiency may be able to reverse it . however , further prospective clinical studies will be needed to define the role of vitamin d treatment in gd .

nigella sativa l. ( ns ) is a vegetal specie of the ranunculaceae family , commonly known as black cumin seed , neguilla or ajemuz , that is widely cultivated in the mediterranean region . its seeds have played an important role over the years in ancient islamic system of herbal medicine and in spain , where they have been traditionally used in folk medicine . ns seeds have shown several therapeutic effects such as prevention of cancer , antihypertensive effect , anti - inflammatory , analgesic , and antihistaminic actions . the volatile oil from this plant presents a relaxant action on different smooth muscles and tracheal muscles of guinea pigs . there is evidence of anticonvulsant and antioxidant effects against pentylenetetrazol - induced kindling in mice . . likewise , aqueous extract of this plant suppresses penicillin - induced epileptic activity in rats . this anticonvulsant effect is a consequence of selectively altering the monoamine level in different brain regions . in recent works , histopathological changes of neurodegeneration in the frontal cortex and brain stem in neurons after exposition to toluene have been observed [ 15 , 16 ] . the administration of ns extract and thymoquinone ( major component from ns volatile oil ) causes morphologic improvement over apoptosis and indicates that ns therapy is useful as a potential treatment of neurodegeneration prevention . ns seeds composition includes nutritional components such as carbohydrates ( glucose , xylose , rhamnose , and arabinose ) , vitamins as thiamine , riboflavin , pyridoxine , niacin and folic acid , mineral elements , and proteins . the ns seeds are also a source of calcium , iron and potassium , alkaloids ( nigellidine , nigellimine , and nigellicine ) , 36%38% fixed oil and 0.4%2.5% essential oil . the fixed oil is mainly composed of unsaturated and essential fatty acids ( linoleic acid , followed by oleic acid ) whereas the volatile oil has been shown to contain 18.424% thymoquinone and 46% monoterpenes such as p - cymene and pinene [ 5 , 20 ] . thimoquinone , as indicated above , is thought to be the main active component of ns seeds and suppresses itself epileptic seizures in rats , while a monodesmosidic triterpene saponin , -hederin , has also been isolated from the extract of ns seeds and proved to exert antitumoral activity . three flavonoid glycosides and triterpene saponins were also identified from nigella sativa , together with four phospholipid classes : phosphatidylcholine phosphatidylethanolamine , phosphatidylserine , and phosphatitdylinositol [ 24 , 25 ] . in previous studies we demonstrated that aqueous and methanolic extracts of ns seeds exert a potent sedative and depressive effect on cns and induce analgesia . the effect on cns resulted in a significant reduction of spontaneous motility , a decrease in normal body temperature and significant analgesic action against hot - plate and pressure tests . body temperature reduction can be interpreted as an index of alteration of various central neurotransmitters ; anxiety and sedation are mainly mediated by the gaba - a receptor in the cns . since depressant action was confirmed specially for the methanolic extract , we decided to study whether the addition of this methanolic dry extract in cortical neurons culture could exert any influence on the secretion of the excitatory amino acids aspartate ( asp ) and glutamate ( glu ) , and the inhibitory amino acids gaba and glycine ( gly ) , as well as the presence of these amino acid neurotransmitters in the extract . we also considered its effect on the amino acids secretion when stimulated by a depolarizing agent and its effect on cultured neurons viability . the viability of the cultured neurons after exposition to ns extract concentrations 2.5 , 25 , and 250 g / ml during 15 and 60 minutes are shown in figure 1 . results are expressed as a percentage with respect to control value ( 100% viability ) . the amino acids gaba , gly , glu , and asp were measured in ns methanolic extract by hplc . the results are expressed as pmoles / mg of ns extract ( table 1 ) . the results showed gly as the most abundant , followed by gaba , glu , and asp , respectively . the amino acids content in the extract was subtracted from the total amino acid content measured in the cellular medium after neuronal stimulation with ns extract so we can state that the final amino acids content in cell culture is a direct consequence of cell release . the release of the four amino acids after stimulation with the chosen nontoxic concentrations of ns extract ( 2.5 , 25 , and 250 g / ml ) showed a diminished secretion response that was statistically significant ( p < .05 for gly and glu ; p < .01 for gaba ; p < .001 for asp ) after 15 minutes of incubation with respect to control ( neuronal cells stimulated with normal locke medium during the same period of time and considered as 100% secretion ) ( figures 2 , 3 , 4 , and 5 ) . gly and asp release was reduced in a dose dependent manner ; glu and gaba showed a tendency to retrieve control values , although their secretion was lower than control . this fall of aminoacids release is greater for higher extract concentration except for glu and gaba , which showed a tendency to recuperation to control values at the same time ( figure 2 ) . the hplc analysis revealed the same behavior for all the amino acids , with the exception of gaba , after treatment with ns extract during 60 minutes . the increased presence of this amino acid was statistically significant for 25 and 250 g / ml ns extract . in order to know the response to a depolarizing agent , cortical neurons were stimulated with ns extract at the indicated concentrations , during 15 and 60 minutes previous to depolarization with 60 mm kcl ( figures 6 and 7 ) . the neurons treated with ns extract during 15 minutes and subsequently stimulated with kcl showed a dose - dependent decrease in amino acids secretion with respect to control value ( neuronal cells stimulated with locke medium ) , which was considered as 100% . the observed behaviour was more relevant for glu and asp at 25 and 250 g / ml than for gaba and gly under the same conditions ( figure 6 ) . measurement of secretion mediated by kcl during 60 minutes revealed an inhibition of the liberation of these neurotransmitters . in this case , only gaba and glu were released in a dose - dependent manner ( figure 7 ) . the aim of the study was to determine the effects of ns methanolic extract on the release of neurotransmitter amino acids by measuring their concentrations in the culture media using hplc precolumn derivatization technique . three concentrations of ns extract ( 2.5 , 25 , and 250 g / ml ) and two time points ( 15 and 60 min ) for the determination of the effects were used . this is a preliminary study which shows that exposure of the cultured neurons have a modulatory effect on the release and contents of these aminoacids . the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide ( mtt ) assay was employed to estimate the cells viability when neurons were treated with ns extract . the three concentrations of dry methanolic extract used in our study did not affect cellular respiratory capacity at any of the two periods of time considered . these results allowed us to consider the adequate approach to the study of amino acid secretion in the conditions selected and to confirm the innocuous characteristics of the chosen extract concentrations . our previous in vitro findings support the hypothesis that the sedative and depressive effects of nigella sativa ( ns ) observed in vivo , could be based on changes of inhibitory / excitatory amino acids levels . several authors attribute the sedative effects of different plant extracts ( valeriana officinalis l. , scutellaria lateriflora l. ) [ 27 , 28 ] to its endogenous gaba concentration , although they also hypothesize that there exist other components of the vegetal extracts with benzodiazepine - like effects that may account for their in vivo effects . the aim of our research was to determine whether these substances that are present in the ns extract could mediate the specific effects previously observed in vivo through the secretion of asp , glu , gaba , and gly . first of all , the presence and content of the four amino acids in the extract was analyzed by hplc , showing a major presence of inhibitory amino acids ( gly and gaba ) ( table 1 ) . then , the cell amino acids release was assayed after addition of several concentrations of ns extract to cultured neurons during two different periods of time ( 15 and 60 minutes ) . the aim of this approach was to observe whether neuronal secretion could be modified depending on the exposition time . high secretion of gaba was observed after 60 minutes ' contact with ns extract at 25 and 250 g / ml . this time is close to the one that allowed the maximum effect in our in vivo study , close to 40 minutes . under these conditions , similar results have been observed when high gaba concentrations are present in an aqueous extract of valeriana officinalis root which induced in vitro liberation of [ h]gaba in rat synaptosomes by reversal of the gaba carrier and inhibition of its reuptake . both plants are widely prescribed as sedative / anxiolytic ones . as the gaba presence in the methanolic extract of ns was confirmed with our results , it is possible to assume that a high level of gaba in the medium during cellular incubation and a longer period of exposition at this one could exert the same effect above described . recent findings suggest that ns protects from induced generalized epilepsy in rats by selectively altering the monoamine level in different brain regions . this study pointed that ns possibly facilitates the inhibitory activity of the gabaergic system through a competitive agonist action in the benzodiazepine ( bzd ) site of the gaba receptor as well as the involvement of dopaminergic and noradrenergic system . the confirmed presence of gaba in the methanolic extract could be directly related to its conduct over the gaba receptor and this could explain the potent sedative and depressive effect on cns as previously reported . moreover , the important presence of gly in the extract could be also related to its inhibitory action . the binding of both neurotransmitters to their receptors on the neuronal membrane induces hyperpolarization that could be responsible for a significantly lower secretion of amino acids with respect to control values . in addition to this , our results showed a decrease in neuronal excitatory activity derived from a diminished asp and glu secretion , specially the second one . our results show the behavior of the cortical neurons and confirm what we expected from the previous study performed in vivo . likewise the methanolic extract composition is able to mediate in the neuronal amino acids release . in this case , the ns extract had an influence on neuronal transmission because it modifies the neurotransmitter amino acids release . depolarization of neuronal cells by application of high k+ concentration ( 60 mm kcl ) induced a diminished amino acid release in both periods of time assayed . this diminished release was similar to nondepolarizing secretion except for gaba which also diminished its secretion . although multiple mechanisms of neurotransmitter release evoked by elevated extracellular k+ may be involved [ 29 , 30 ] , we consider that the effect of ns extract over neuronal cells produces a drop in the transmission and is responsible for of the inhibitory effect indicated above . a possible action of ns extract over l - type calcium channels or an opening on potassium channels had been suggested by others authors [ 3135 ] , and it could contribute to the relaxant activities of this plant . furthermore , its possible effect over these channels could persist even after the extract was eliminated from the medium before depolarization . with respect to gaba secretion , the only amino acid which differs in its behavior with respect to nondepolarizing liberation , the diminished secretion may derivate from the membrane transporter - reversal for gaba above suggested . in our study , we have considered the possibility that the gaba carrier had been previously affected by the presence of ns extract during 15 or 60 minutes , inhibiting gaba reuptake and favouring its liberation from neurons . other authors have concluded that extracellular k+ ( 50 mm kcl ) provokes gaba release by reversal transporter of gaba . in our case , we observed a diminished liberation that could be justified by the previous loss of cellular gaba content as the neurons had been treated with the ns extract . in conclusion , this study suggests a sedative effect of ns methanolic extract by modification of neurotransmitter amino acids release , because the ns extract may induce an important release of gaba and gly in the cultured neurons medium and therefore , exert an increase in the agonist action over their receptors . the results explain the sedative and depressive effects observed in vivo by an increase in inhibitory amino acids at the synaptic terminals . even more , this effect is complemented with a possible decrease of excitatory transmission , as it has been demonstrated in vitro and could contribute to inhibitory response . minimum essential eagle 's medium ( emem ) was obtained from bio - whittaker , and foetal bovine serum ( fbs ) and horse serum ( hs ) were procured from sera - lab ( sussex , england ) . standards of glutamate , aspartate , gaba and glycine were purchased from sigma ( st . louis , mo , usa ) . reagents and solvents for hplc were triethylamine from sigma ( st . louis , mo , usa ) , acetic acid and methanol ultra gradient grade from merck ( darmstadt , germany ) . distilled water used for the preparation of buffers and standards was deionized with milli - q purification system . syringe filters millex - gv were obtained from millipore ( milford , ma , usa ) . membrane filters ( 0.45 m pore size ) from tecknochroma ( barcelona , spain ) were used for filtration of the mobile phase and samples . cell proliferation kit ii ( xtt ) , colorimetric determination , was purchased from roche diagnostics gmbh ( mannheim , germany ) . , usa ) coupled with a photodiode array detector shimadzu spd-10a ( izasa , madrid , spain ) was used for the amino acids isolation and quantification . the analytical system consisted of a waters ods spherisorb 150 4.6 mm i.d . ; 5 m packed column ( teknokroma , barcelona , spain ) as stationary phase , preceded by a guard column spherisorb rp-18 , 5 m , 4 mm 4 mm . a microplate fluorescence reader fl600-biotek spectrofluorimeter was used for the quantification of cell viability at 492 nm . seeds of nigella sativa l. were supplied by the medicinal and aromatic plants research institute of egypt ( el cairo , egypt ) . herbarium samples were authenticated by a taxonomist and a voucher specimen was deposited in the herbarium of the faculty of pharmacy , universidad complutense de madrid , with voucher number maf 161043 . the methanolic extract of the plant was prepared according to ( science and technology program for development ) cyted protocol for vegetal species from countries that are included in this program . brain neurons were obtained from foetal rat brains of 19 days of gestation as previously described with minor modifications . isolated neurons were suspended in eagle 's minimum essential medium ( emem ) containing 0.3 g / l glutamine , 0.6% glucose , 5% phosphate buffered saline ( pbs ) , 5% horse serum ( hs ) , 100 u / ml penicillin and 100 g / ml streptomycin . cells , at a density of 1 10 cells / well , were placed on plastic petri dishes of 24 wells , treated with 10 g / ml poly - l - lysine to aid attachment . the plates were incubated in a humidified incubator in an atmosphere of 5% co2/95% air at 37c . after 72 hours , nonneuronal cells ( contaminating glial cells ) were mitotically inhibited by exposure to cytosine arabinoside . the incubation medium was replaced by fresh medium to which cytosine arabinoside was added to a final concentration of 10 m . after 3 days , this medium was replaced by fresh medium and experiments were carried out using cultures ranging from 1015 days . cell purity was checked by both cells staining with cresyl violet to identify neurons and with the specific antiglial fibrillary acidic protein ( gfap ) antibody to identify glial cells . cortical neurons , after 7 days in culture , were detached from the culture plates with trypsin solution ( 0.25% trypsin and 0.02% edta in dulbecco 's buffered saline without calcium and magnesium ) and then the cells were fixed ( during 30 min ) with 2% p - formaldehyde . after two washes with 1 ml of pbs , the cells were treated ( during 1 h ) with anti - gfap antirabbit ( at dilution of 1/500 ) . subsequently , the cells were washed with pbs and treated with antirabbit conjugated igg fitc , at 1/100 , for 30 min . the glial cells in the cultures were 8.3 3.6% of the total ( neural + glial cells ) . this assay is used as an index of cell survival or cellular respiratory capacity - based on method of mossmann and improved by weislow et al . and roehm et al . . the tetrazolium assay is based on the mitochondrial dehydrogenases ( md ) activity and their inactivation after cell death . in live cells , mtt is reduced to a highly water - soluble orange colored product , formazan dye . neuronal cells were seeded in 96-multiwell plates at a density of 36 10 cells / well ( in 200 l medium ) and kept in the incubator until 8090% confluence . after this , the medium was removed and the cells were washed twice with pbs and the ns extract previously dissolved in pbs was added to the wells and incubated for 15 minutes or 1 hour . after each one of the treatment periods ( 15 or 60 min ) , the medium was removed and incubated with mtt solution ( final concentration 0.3 mg / ml ) , according to the kit specifications . after 2 h incubation at 37c in a humidified atmosphere , orange dye solution was spectrophotometrically quantified using an elisa plate reader at 492 nm . the amount of orange formazan formed , as monitored by the absorbance , directly correlates to the number of living cells . hplc analysis of amino acids was performed by a previously described method with minor modifications and with the equipment and conditions that were previously developed for the amino acids determination . prior to hplc amino acid secretion analysis cells were washed twice , at 10 min intervals , with 1 ml of locke medium . after removing the medium , cells were stimulated for 15 min or 1 hour at 37c with 250 l fresh locke medium ( control cells ) or with 250 l of locke medium containing the dry methanolic extract of ns seeds at different concentrations ( 2.5 , 25 and 250 g / ml ) . after stimulation , the wells medium ( supernatant s1 ) was taken for amino acid valuation and cells were then stimulated with 250 l of 60 mm kcl for 15 min . after this , the cells secretion ( supernatant s2 ) was removed for their valuation and cells were lysated with 250 l of distilled water for total intracellular amino acids content determination . the different supernatants obtained , s1 and s2 , as well as the cell lysated , were lyophilized for their dansylation . the content of the four amino acids present in the methanolic extract was also determined by hplc analysis under the same conditions and these results were subtracted from the total amino acids content in cellular medium in presence of ns extract ( s1 ) in order to obtain cellular secretion value itself . the results were calculated as the amino acid release into the incubation medium with respect to the total amino acid content . these results were expressed as a percentage of secretion with respect to control experiments that were considered as 100% . data are presented as means sem of four separated experiments from different cell cultures , each one performed in triplicate with different batches of neuronal cells .

nigella sativa l. ( ns ) has been used for medicinal purposes since ancient times . this study aimed to investigate the cytotoxicity of ns dry methanolic extract on cultured cortical neurons and its influence on neurotransmitter release , as well as the presence of excitatory ( glutamate and aspartate ) and inhibitory amino acids ( gamma - aminobutyric acid gaba and glycine ) in ns extract . cultured rat cortical neurons were exposed to different times and concentrations of ns dry methanolic extract and cell viability was then determined by a quantitative colorimetric method . ns did not induce any toxicity . the secretion of different amino acids was studied in primary cultured cortical neurons by high - performance liquid chromatography ( hplc ) using a derivation before injection with dansyl chloride . ns modulated amino acid release in cultured neurons ; gaba was significantly increased whereas secretion of glutamate , aspartate , and glycine were decreased . the in vitro findings support the hypothesis that the sedative and depressive effects of ns observed in vivo could be based on changes of inhibitory / excitatory amino acids levels .

pheochromocytomas are rare catecholamine producing tumors arising from chromaffine cells in the sympatho adrenal system . its prevalence is estimated at 0.1% to 0.6% . they secrete various catecholamines , predominantly norepinephrine , and epinephrine to small extent . these catecholamines are responsible for the manifestations with sustained or paroxysmal symptoms . diagnosis is established by measuring metanephrines in the urine or blood . localization of the tumor is done using computed tomography ( ct ) or magnetic resonance imaging ( mri ) scans . thrombosis of the inferior vena cava ( ivc ) has comparable etiological factors to lower limb deep venous thrombosis . hypercoagulability related to hematological or neoplastic abnormalities , venous stasis secondary to extraluminal pressure from tumors or inflammatory processes , and vessel injury due to trauma have all been implicated as primary mechanism in the pathophysiology of ivc thrombosis . however , its association with pheochromocytoma in indian subjects has not been reported till date . a 48-year - old man was admitted to our hospital with complaints of headache , sweating , anxiety , dizziness , nausea and vomiting . the patient was 164 cm tall and weighed 57 kg . on physical examination , there were no caf au lait spots or neurofibromas . hematological analysis confirmed normocytic anemia with hemoglobin 11.3 gm / dl , a raised erythrocyte sedimentation rate ( esr ) ( 130 mm fall in the first hour ) , while the total and differential leukocyte counts were normal . biochemical parameters such as liver and kidney functions , and serum electrolytes , calcium , phosphorous , alkaline phosphatase and d - dimer were within normal limits . the endocrinological evaluation revealed increased urine catecholamines and urinary vanillyl mandelic acid ( vma ) [ table 1 ] . baseline biochemical parameters of the patient abdominal ct revealed a well defined , heterogenous mass lesion of size 7.6 5.3 4.8 cms with attenuation score of 35 hu at the upper pole of right kidney without any calcifications [ figure 1 ] . there was no involvement of renal vein , hepatic veins and veins of lower limbs demonstrated by doppler ultrasound . magnetic resonance imaging ( mri ) revealed intraluminal thrombus extending proximally up to the confluence of hepatic veins immediately inferior to the right atrium without distal extension to femoral veins bilaterally [ figure 2 ] . an ivc venogram via the right jugular vein demonstrated multiple filling defects indicating occlusion of the ivc inferior to the right atrium [ figure 3 ] . there was simultaneous enlargement of distal part of ivc . computed tomography of the abdomen- showing a well defined , heterogeneously enhancing mass lesion of size 7.6 5.3 4.8 cm at the upper pole of right kidney without any calcifications . the left adrenal gland appeared to be normal t2-weighted axial magnetic resonance imaging demonstrating the mass ( predominantly high signal ) between the inferior vena cava and right kidney ( black arrow ) compressing the overlying inferior vena cava ( white arrow ) ivc venogram showing multiple filling defects indicating occlusion of the inferior vena cava inferior to the right atrium . there is distal enlargement of inferior vena cava a diagnosis of ivc thrombosis with pheochromocytoma was established , and surgical treatment was planned . alpha receptor blocking therapy with prazosin was instituted , followed by blocker , after testing for adequacy of blockade . the patient was treated conservatively with subcutaneous low molecular weight heparin followed by oral warfarin . after 2 weeks , hypertension was well controlled and the remaining symptoms disappeared . with adequate blood pressure control , biopsy of the specimen revealed a typical organoid or zellballen pattern with no cytoplasmatic inclusion , pleomorphism , cytological alterations or necrosis ; and , the mitotic index was low [ figure 4 ] . during the postoperative period , the patient 's blood pressure remained normal . a 24-hour urine specimen collected for metanephrine and vma , revealed levels within normal limits . at present , the patient is asymptomatic , requires no medications , and is employed as an engineer . mri imaging demonstrated resolution of the thrombosis and return of patency of the ivc at 4 months [ figure 5 ] . the typical growth pattern of nests of tumor cells ( zellballen ) surrounded by a discontinuous layer of sustentacular cells and fibrovascular stroma in the biopsy specimen of the patient in the study . blood vessels surrounding tumor nests are composed of round to oval cells t2-weighted axial magnetic resonance imaging comparable in position and image acquisition to figure 2 demonstrating complete resolution of inferior vena cava thrombosis ( white arrow ) after 4-months of oral anticoagulation therapy two aspects render our case unusual : 1 ) the coexistence of pheochromocytoma with ivc thrombosis 2 ) though there are case reports citing the association between malignant pheochromocytoma and ivc thrombus , to our sincere belief ; this is the first such report citing this uncommon association from india . although the lifetime incidence of venous thrombosis is 0.1% , it still remains a rare condition especially in patients below 30 years of age . predisposing factors include alterations in blood flow [ stasis ] , injury to the vascular endothelium and abnormalities in the constitution of blood hypercoagulability ( virchow 's triad ) . endothelial damage is invariably an acquired phenomenon , whereas hypercoagulability may result from both congenital and acquired risk factors ( especially in the peri - operative period ) . the classical presentation of ivc thrombus varies according to the level of the thrombosis with up to 50% of patients presenting with bilateral lower extremity swelling and dilatation of superficial abdominal vessels . whilst some patients remain asymptomatic , lower back pain , nephrotic syndrome , hepatic engorgement , cardiac failure and pulmonary embolus have also been described . tsuji et al . reported a series of 10 patients where 40% were pyrexic at presentation , with an associated elevation in d - dimer levels and inflammatory markers ( white cell count , c - reactive protein ) . our patient had no lower limb , liver or kidney involvement , and this might be ascribed to the partial occlusion of ivc . we could not explain normal d - dimer levels in the backdrop of such a large thrombus in our patient . ct scan with contrast enhanced images and mri scan are used to localize adrenal pheochromocytoma . meta - iodobenzylguanidine ( mibg ) and positron emission tomography ( pet ) scanning ( gallium- dota - toc / noc and dopa - pet perform better than fdg- pet ) are largely reserved for extraadrenal paraganglioma , or very large tumors to rule out metastasis . heterogeneity , high hounsfield density on ct ( > hu ) , marked enhancement with intravenous contrast and delayed contrast washout ( < 60 % at 10 minutes ) , high signal intensity on t2 weighted mri , cystic and hemorrhagic changes point to pheochromocytoma , adrenocortical carcinoma or metastasis . however , pheochromocytoma with lipid degeneration can result in low attenuation scores ( < 10 hu ) and > 60% washout at delayed ct scanning . benign adrenal incidentalomas are characterized by size < 5 cm , sharp margins , smooth contours , lack of demonstrable growth on serial examinations , attenuation scores < 10 hu , and > 60% washout at delayed ct scanning . in our patient , ct scan revealed nonhomogenous mass of hu 35 without any calcification . histologically , pheochromocytomas are capsulated and are composed of round or polygonal epithelioid / chief cells arranged in characteristic compact cell nests ( zellballen ) or trabecular patterns . the chief cells have centrally located nuclei with finely clumped chromatin , and a moderate amount of eosinophilic , granular cytoplasm . tumors of higher grade are characterized by a progressive loss in the relationship between chief cells and sustentacular cells , and a decrease in the number of sustentacular cells . in our patient , typical zellballen pattern was found . presence of markers like chromogranin a ( cga ) , neuron specific enloase , synaptophsyin serve as additional tools to confirm the neuroendocrine nature of the chief cells . the only reliable clue to the presence of a malignant pheochromocytoma is local invasion or distant metastases , which may occur as long as 20 years after resection . benign on pathologic examination , long term follow - up is indicated in all patients to confirm that impression . other markers for malignancy are absent or weak expression of inhibin / activin- beta b subunit , and presence of succinate dehydrogenase b ( sdh b ) subunit is seen . in absence of any invasion , we considered the mass in our patient to be benign . the simultaneous occurrence of pheochromocytoma and ivc thrombosis is reported sporadically . ivc thrombosis in this case could be because of : 1 ) local compression leading to alteration in blood flow and stasis 2 ) sustained hypertension leading to vascular endothelial injury and hypercoagulability , 3 ) association of pheochromocytoma with systemic lupus erythematous and behcet 's disease might explain the triggering of an autoimmune phenomenon leading to a hypercoagulable state , and 4 ) an underlying anatomic abnormality or coagulation disorder . it also could be a chance association between these 2 conditions . in our case , recent advances in the utilization of ultrasound , ct and mri imaging as well as endovascular procedures have resulted in an increase in detection rates of ivc anomalies , as well as an increase in the incidental discovery of such abnormalities during unrelated investigations , therapeutic endovascular or surgical procedures . contrast venography remains the standard for diagnosis of ivc thrombosis with a low false - positive rate , and the advantage of access for immediate treatment if required . however , it is an invasive procedure associated with a 2%-10% incidence of post - procedural deep venous thrombosis ( dvt ) . duplex ultrasound scanning has become an accurate non - invasive method of diagnosing ivc thrombosis and is often the first - line investigative modality . however , duplex usg is operator dependant and can be limited by body habitus or the presence of bowel gas and may occasionally fail to identify any ivc anomaly . ct imaging is a rapid non - invasive method which can accurately diagnose and assess the extent of thrombus as well as delineate any associated abdominal or pelvic abnormality . mri imaging is now replacing ct as the optimal investigative tool avoiding radiation and giving more accurate delineation of thrombus as well as any ivc anomaly . mri is also used to follow - up patients to determine morphological changes in the thrombus following therapy . management of patients with coexisting pheochromocytoma and ivc thrombosis needs operative resection of the adrenal mass and medical / interventional management of ivc thrombosis . the goals of operation include 1 ) removal of the tumor with postoperative normotension , and 2 ) ivc luminal restoration and anticoagulation . minimally invasive techniques are being increasingly used for resection of adrenal tumors and to treat renal artery lesions . our patient was subjected to laparoscopic adrenalectomy after adequate preoperative blood pressure control by blockers , followed by blockers . treatment options in the case of ivc thrombus without anatomical variance include anticoagulation , mechanical thrombectomy , systemic thrombolytic therapy , transcatheter regional thrombolysis , pulse - spray pharmacomechanical thrombolysis and angioplasty . there is no specific literature describing the ideal duration of anticoagulation in these instances ; however , case evidence identifies a trend toward treatment for a minimum of one year with the interplay of hypercoagulability disorders needing to be factored into any decision . surgical reconstruction of the ivc and bypass of an aberrant section are both recognized modalities reserved for the most severe cases and are associated with morbidity and mortality risk . endovascular stent placement in combination with angioplasty is recommended in the cases of residual stenosis and chronic ivc occlusion . in the case of ivc thrombus associated with an aberrant ivc , with no other predisposing factors , treatment involves anti - coagulation . the duration of this treatment is widely debated with no extensive literature to provide an evidence based approach . dean et al . took a view , which is quite similar to that of ours , that a caval anomaly is a permanent risk factor for venous stasis and thrombosis and that anticoagulant treatment should be lifelong . since our patient had no anatomic abnormality or any other predisposing factors , we decided to give the treatment for 4 months only and stopped it then after documenting radiologic luminal restoration . though cases of renal artery stenosis , renal artery aneurysm and inferior vena cava thrombosis have been described , we found the uncommon association with ivc thrombosis in an indian patient . ct or preferably mri imaging are required to delineate ivc anatomy and ascertain proximal extent of the thrombus . although invasive therapeutic modalities exist , long - term and commonly life - long anticoagulation is often required . pheochromocytoma does not seem to have any effect on the outcome of the coexisting ivc thrombosis .

pheochromocytomas have been described in association with vascular abnormalities like renal artery stenosis . a 48-year - old man was admitted to our hospital with the complaints of headache , sweating , anxiety , dizziness , nausea , vomiting and hypertension . for last several days , he was having a dull aching abdominal pain . abdominal computed tomography ( ct ) revealed the presence of a left adrenal pheochromocytoma . an inferior vena cava ( ivc ) venogram via the right jugular vein demonstrated occlusion of the ivc inferior to the right atrium . surgical removal of pheochromocytoma was done , followed by anticoagulant treatment for ivc thrombosis , initially with subcutaneous low molecular weight heparin , and then with oral warfarin , resulting in restoration of patency . to the best of our knowledge , the occurrence of pheochromocytoma in ivc thrombosis has not been reported so far from india . possible mechanisms of such an involvement are discussed .

in recent years , core training has been widely studied since it has been considered a pivotal issue in health , rehabilitation and sports performance ( hibbs et al . , 2008 ) . however , the definition of the core varies with the interpretation of the literature ( hibbs and thompson , 2008 ) . anatomically , the core region has been described as the area bounded by the abdominal muscles in the front , by paraspinal and gluteal muscles in the back , by diaphragm on the top and by pelvic floor and girdle musculature at the bottom ( richardson et al . , 1999 ) . the core represents the connection between lower and upper limbs and should be considered as a functional unit in which different muscles interact , even if not located in the thoraco - lumbar region ( such as shoulders and pelvic muscles ) . however , literature concerning core training sometimes fails to distinguish between concepts of core stability and core strength . faries and greenwood ( in hibbs and thompson , 2008 ) formulated the following clear definitions : core stability refers to the ability to stabilize the spine as a result of muscle activity , while core strength refers to the ability of muscles contractions to produce and transfer force as a result of muscle activity . since strength and motor control are complementary qualities , the core training programmes can target mainly , but not exclusively , at muscle strengthening and/or motor control of core musculature . motor control training seems to require low intensity stabilization exercises focused on efficient integration of low threshold recruitment of local and global muscle systems . conversely , core strength training seems to require high intensity and overload training of the global muscle system . vezina and hubley - kozey ( 2000 ) suggested that core stability programmes should include muscle activation below 25% of maximum voluntary contraction ( mvc ) , while core strength training should include activation higher than 60% of mvc to result in strength benefits . the available evidence suggests that to adequately train the core muscles in athletes , strength and conditioning specialists should focus on implementing multi - joint full body exercises , rather than core - specific exercises ( martuscello et al . , 2013 ) . exercises involving the full body linkage such as plank exercises , have been advocated to the capacity of transmitting force through the body linkage ( schoenfeld et al . , 2014 ) . training with labile systems has been documented to offer unique opportunities for linkage training challenges ( mcgill et al . , 2015 ) . several studies examined core muscle activation during the execution of various exercises on stable and unstable surfaces ( for a review see : behm et al . , 2010 ) . the use of unstable surfaces contacting the subject s feet or hands is becoming popular in strength training . instability can be obtained through the use of many devices and techniques including , but not limited to , unstable platforms such as bosu or swiss balls . more recently , suspension training systems have been added to the list of instability training devices . in suspension training , lower or upper limbs many core directed exercises are designed with such a device , creating a wide variety of challenges . these exercises consist of multi - planar and multijoint movements , and are executed with complex techniques . it is important to quantify the muscle contraction intensity since it is a key factor in establishing training effects induced by this sort of exercises . although considerable research has examined more traditional means of instability training ( behm and drinkwater , 2010 ) , little previous research has evaluated the effects of suspension training on muscle activation . in particular , some studies focused on core - directed exercises ( atkins , 2014 ; byrne et al . , 2014 ; czaprowski et al . , 2014 ; mok et al . , 2014 ; snarr and esco , 2014 ) , whereas others investigated the effect of the application of suspension system on core muscle activity in push exercises ( calatayud et al . , 2014 further investigation of these exercise approaches is needed to understand their influence on muscle activation and joint load levels . the primary purpose of this study therefore , was to examine differences in core muscle activation across four full - body linkage exercises using a suspension training system . these exercises were chosen from a spectrum of whole body linkage exercises focused on the anterior core musculature executed in instable conditions , including a roll - out , bodysaw , pike , and knee - up . although the selected exercises were mainly focused on anterior slings , we wanted to provide a comprehensive view of core muscle activation by monitoring rectus abdominis , internal and external oblique , and paraspinal muscles . it was hypothesized that significant differences would be found in core muscles among exercises . the second aim of the study was to determine which of these exercises would reach the threshold of 60% of mvc , expected to be high enough to increase muscle strength . it was hypothesized that the four exercises would elicit muscle activity in excess of 60% of mvc in the rectus abdominis , i.e. the muscle on which the main focus was put considering the selected exercises . seventeen healthy participants were recruited ( age 27.32.4 years , body height 1725 cm , body mass 69.29.3 kg ) . all participants were physically active , declaring three practice sessions per week of resistance training . inclusion criteria for study participation were as follows : no past or present neurological or musculoskeletal trunk or limb pathology , no cardiorespiratory disease , no history of abdominal , shoulder or back surgery , and no psychological problems . participants were instructed to refrain from performing strenuous physical activity in the 24 hours preceding all experimental sessions . the study was previously approved by the research ethics committee of the department of medical sciences , university of turin . the surface electromyographic ( emg ) signals were obtained from six trunk muscles with concentric bipolar electrodes ( code , spes medica , battipaglia , italy ) . before the placement of the electrodes , the skin was slightly abraded with adhesive paste and cleaned with water in accordance to seniam recommendation for skin preparation ( hermens et al . , 2000 ) . the electrodes were placed according to the instructions described in previous methodological works ( beretta piccoli et al . , 2014 ; boccia and rainoldi , 2014 ) lower rectus abdominis : on the lower part of the rectus abdominis , 3 cm lateral to the midline ; upper rectus abdominis : on the upper part of the rectus abdominis , 3 cm lateral to the midline ; external oblique : 14 cm lateral to the umbilicus , above the anterior superior iliac spine ( asis ) ; internal oblique : 2 cm lower with respect to the most prominent point of the asis , just medial and superior to the inguinal ligament ; lower erector spinae : 2 cm lateral to the l5-s1 ; upper erector spinae : 6 cm lateral to the l1-l2 . the electrodes were placed only on the left ( randomly chosen ) side of the body ; the reference electrode was positioned on the wrist . the signal of a biaxial electrogoniometer ( sg 150 , biometrics ltd , gwent , uk ) positioned at the level of the shoulders ( for the roll - out and bodysaw ) or the hips ( for the pike and knee - tuck ) , depending on which joint was more involved during the exercise , was used as a trigger to highlight exercise repetitions . the emg signals were synchronized with the electrogoniometer signal , amplified ( emg - usb , ot bioelettronica , torino , italy ) , sampled at 2048 hz , bandpass filtered ( 3-db bandwidth , 10 - 450 hz , 12 db / oct slope on each side ) , and converted to digital data by a 12-bit a / d converter . samples were visualized during acquisition and then stored in a personal computer using ot biolab software ( version 1.8 , ot bioelettronica , torino , italy ) for further analysis . the participants recruited were instructed with regard to the correct technique of suspension exercise and the mvc procedure during the first experimental session conducted one week before the measurement session . the participants were asked to refrain from physical activity 24 hours before the measurements . during the measurement session , participants performed 4 exercises with the use of suspension straps ( trx suspension trainer ; fitness anywhere lcc , san francisco , ca , usa ) in random order . the exercises were selected based on a previous study ( behm and drinkwater , 2010 ) that indicated them as important in developing core strength . at the beginning of the measurement session , three mvc exercises were performed twice for 5 s , with 2 min rest between them . the following standardized exercises ( ng et al . , 2002 ) were used to activate maximally the trunk muscles ( figure 1 ) : upper rectus abdominis ( ura ) and lower rectus abdominis ( lra ) : body supine with hips and knees flexed 90 , with feet locked . participants flexed the trunk ( i.e. crunch execution ) against resistance at the level of the shoulders;external oblique ( eo ) and internal oblique ( io ) : side - lying with the hip at the edge of the bench and feet locked by a second operator . participants performed side - bend exercise against resistance at the level of the shoulder;lower erector spinae ( les ) and upper erector spinae ( ues ) : prone position with asis at the edge of the bench and feet locked by a second operator . upper rectus abdominis ( ura ) and lower rectus abdominis ( lra ) : body supine with hips and knees flexed 90 , with feet locked . participants flexed the trunk ( i.e. crunch execution ) against resistance at the level of the shoulders ; external oblique ( eo ) and internal oblique ( io ) : side - lying with the hip at the edge of the bench and feet locked by a second operator . participants performed side - bend exercise against resistance at the level of the shoulder ; lower erector spinae ( les ) and upper erector spinae ( ues ) : prone position with asis at the edge of the bench and feet locked by a second operator . standardized exercises used to maximally activate trunk muscles : lower rectus abdominis and upper rectus abdominis ( left ) ; internal oblique and external oblique ( middle ) ; lower erector spinae and upper erector spinae ( right ) . participants were required to achieve a range of motion with the correct technique execution and to maintain a neutral position of the spine and pelvis in each exercise . a certified strength and conditioning coach monitored the exercise performance to ensure that the exercise was properly executed considering its technique . each exercise was repeated three times and lasted 6 s. a metronome set at 30 beats per minute was used to ensure proper timing ( with 4 beats for each repetition ) : 2 s from the initial position to the final position ( concentric phase ) ; 2 s of maintenance ( isometric phase ) ; and 2 s returning to the starting position ( eccentric phase ) . the exercises were performed with 3 min of rest in - between to allow complete recovery . the random order of the exercises allowed to mitigate the effects of cumulative fatigue on emg estimates . the following exercises were used ( figure 2 ) : roll - out : participants assumed an inclined standing position while placing each hand on the strap handles , with elbows and wrists placed below the shoulders , arms perpendicular to the floor and shoulders flexed approximately 45 ; they then performed a shoulder flexion moving the hands forward;bodysaw : participants assumed a prone position , they placed elbows below the shoulders , both forearms touching the floor , while placing each foot on the strap handle ; participants then flexed the shoulders and extended the elbows pushing the body backwards;pike : participants assumed a push - up position with the feet in strap handles , then they flexed hips to approximately 90 , while keeping the knees fully extended;knee - tuck : participants assumed a push - up position while placing each foot in the strap handle , then they flexed both hips and knees to approximately 90 , bringing the knees forward . roll - out : participants assumed an inclined standing position while placing each hand on the strap handles , with elbows and wrists placed below the shoulders , arms perpendicular to the floor and shoulders flexed approximately 45 ; they then performed a shoulder flexion moving the hands forward ; bodysaw : participants assumed a prone position , they placed elbows below the shoulders , both forearms touching the floor , while placing each foot on the strap handle ; participants then flexed the shoulders and extended the elbows pushing the body backwards ; pike : participants assumed a push - up position with the feet in strap handles , then they flexed hips to approximately 90 , while keeping the knees fully extended ; knee - tuck : participants assumed a push - up position while placing each foot in the strap handle , then they flexed both hips and knees to approximately 90 , bringing the knees forward . initial and final positions of each exercise : 1 ) roll - out ; 2 ) bodysaw ; 3 ) pike ; 4 ) knee - tuck . the average rectified value ( arv ) of emg signals was computed off - line with numerical algorithms using non - overlapping signal epochs of 0.5 s ( hibbs et al . , 2011 ) . the mean value of arv over the two repetitions was calculated for each muscle and normalized with respect to the maximum arv obtained during the correspondent mvc . the normality assumption of the data was evaluated with the shapiro - wilk test ; homoscedasticity and autocorrelation of the variables were assessed using the breusch - pagan and durbin - watson tests . the differences between exercises ( pike bodysaw knee - tuck roll - out ) and between muscles ( lra ura eo io les ues ) were compared with the 2-way analysis of variance ( anova ) . for the purpose of this report statistical analyses were conducted using the r statistical package ( version 3.0.3 , r core team , foundation for statistical computing , vienna , austria ) . all participants managed to complete each exercise trial and thus , were included in the data analysis . figure 3 shows the box plots of the activation values ( % of mvc ) of each muscle during the four exercises . muscle activation ( median , ir ) figure 3each box plot shows the muscle activation ( as percentage of maximum voluntary contraction ) during exercise . table 1muscle activation ( median , ir ) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction . results of the two - way anova after tukey multiple comparisons are reported as symbols ; p < 0.01.lower rectus abdominisupper rectus abdominisexternal obliqueinternal obliquelower erector spinaeupper erector spinaepike57 ( 36 ) 41 ( 48 ) 55 ( 21)23 ( 20)12 ( 7)9 ( 4)bodysaw100 ( 42 ) 57 ( 52)59 ( 33)32 ( 20)4 ( 3)8 ( 6)knee - tuck54 ( 50 ) 44 ( 41 ) 42 ( 7 ) 18 ( 26)8 ( 5)6 ( 5)roll - out140 ( 89 ) 67 ( 78 ) 71 ( 44 ) 40 ( 31)9 ( 5)11 ( 6)indicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the pikeindicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the bodysawindicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the knee - tuckindicates statistically significant difference betweenthe indicated exercise ( explained in row ) with respect to the roll - out each box plot shows the muscle activation ( as percentage of maximum voluntary contraction ) during exercise . muscle activation ( median , ir ) expressed as percentage values of electromyographic amplitude normalized to maximum voluntary contraction . results of the two - way anova after tukey multiple comparisons are reported as symbols ; p < 0.01 . indicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the pike indicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the bodysaw indicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the knee - tuck indicates statistically significant difference between the indicated exercise ( explained in row ) with respect to the roll - out the normalized lra activity was 140% ( ir , 89% ) of mvc during the roll - out , 100% ( ir , 42% ) of mvc during the bodysaw , 57% ( ir , 36% ) of mvc during the pike and 54% ( ir , 50% ) of mvc during the knee - tuck . the normalized lra values were significantly higher ( p < 0.01 ) during the roll - out and bodysaw compared to the pike and knee - tuck . the roll - out exercise showed significantly greater activation ( p < 0.01 ) than the bodysaw . the normalized ura activity was 67% ( ir , 78% ) of mvc during the roll - out , 57% ( ir , 52% ) of mvc during the bodysaw , 41% ( ir , 48% ) of mvc during the pike and 44% ( ir , 41% ) of mvc during the knee - tuck . the normalized ura values were significantly higher ( p < 0.01 ) during the roll - out compared to the pike and knee - tuck . the normalized eo activity was 71% ( ir , 44% ) of mvc during the roll - out , 59% ( ir , 33% ) of mvc during the bodysaw , 55% ( ir , 21% ) of mvc during the pike and 42% ( ir , 7% ) of mvc during the knee - tuck . the normalized eo values were significantly higher ( p < 0.01 ) during the roll - out compared to the knee - tuck . the normalized io activity was 40% ( ir , 31% ) of mvc during the roll - out , 32% ( ir , 20% ) of mvc during the bodysaw , 23% ( ir , 20% ) of mvc during the pike and 18% ( ir , 26% ) of mvc during the knee - tuck . during all exercises the normalized io values were not significantly higher ( p < 0.01 ) . the normalized les activity was 9% ( ir , 5% ) of mvc during the roll - out , 4% ( ir , 3% ) of mvc during the bodysaw , 12% ( ir , 7% ) of mvc during the pike and 8% ( ir , 5% ) of mvc during the knee - tuck . during all exercises the normalized les values were not significantly higher ( p < 0.01 ) . the normalized ues activity was 11% ( ir , 6% ) of mvc during the roll - out , 8% ( ir , 6% ) of mvc during the bodysaw , 9% ( ir , 4% ) of mvc during the pike and 6% ( ir , 5% ) of mvc during the knee - tuck . during all exercises the normalized ues values were not significantly higher ( p < 0.01 ) . table 2 shows the estimate ( difference of means ) at 95% of the confidence interval after tukey multiple comparisons ; in this case only exercise factor was considered . estimate at 95% of the confidence interval after tukey multiple comparisons with the exercise factor considered . the estimate shows the difference of means ( % of maximum voluntary contraction ) . indicates the statistical significance of the adjusted p - value . the roll - out exercise showed significantly ( p < 0.01 ) higher activation compared to the bodysaw ( 16% , ci 8 - 23% ) , pike ( 26% , ci 18 - 33% ) and knee - tuck ( 29% , ci 21 - 37% ) . pike and knee - tuck exercises showed significantly higher activation compared to the bodysaw of 10% ( 28% ) and 13% ( 6 - 21% ) . suspension training has become increasingly popular as a training tool . despite this popularity , relatively little research exists on the effects of such training on muscle activation magnitude . the first objective of the study was to investigate the activation differences of four exercises ( roll - out , bodysaw , pike and knee - tuck ) to better characterize suspension training . our findings indicate that suspension exercises could be an effective strategy to reach high to very high activation of abdominal muscles such as the rectus abdominis and external oblique . to facilitate comparisons between exercises and previous studies , we categorized muscle activation into four levels according to previous studies , with < 21% as low , 2140% as moderate , 4160% as high , and > 60% as very high ( escamilla et al . , 2010 ) . exercises used in the present study provide a range of medium to high intensity exercises through which participants or athletes can progress during a training or rehabilitation programme ( blanchard and glasgow , 2014 ) ( figure 2 ) . roll - out exercise was the most challenging for core musculature , followed by bodysaw , pike and knee - tuck exercises ( table 2 ) . the roll - out showed the highest activation of rectus abdominis and oblique muscles compared to other exercises . although lra showed much greater activation in roll - out and bodysaw compared to pike and knee - tuck exercises , the other muscles showed smaller differences . these findings could suggest that in the exercises characterized by shoulder flexion ( such as roll - out and bodysaw ) , the increased requirement of core stability was reflected more by the lower rectus abdominis . according to vezina and hubley - kozey ( 2000 ) , the exercises that generate muscle activity greater than 60% of mvc might be more conducive to developing muscular strength . the rectus abdominis ( both parts ) and eo reached activation higher than 60% of mvc ( or very close to that threshold , 55% ) in the roll - out and bodysaw ; consequently these exercises can be considered suitable for strength training of these muscles . although in the knee - tuck and pike , the rectus abdominis and eo did not reach the threshold of 60% , they presented high activation levels ( 41 - 60% mvc ) . while strengthening of the core is important , an activation level below 60% might be beneficial in increasing muscle endurance within the core . since the core muscles are primarily composed of type i fibres ( haggmark and thorstensson , 1979 ) , muscular endurance should also be a major concern when designing strength and conditioning programmes ( vezina and hubley - kozey , 2000 ) . due to large demand for muscle activation , all the proposed exercises might be appropriate for extremely fit individuals in the latter stages of a progressive abdominal strengthening or rehabilitation programme . this is an expected result as all exercises focused on anterior abdominal wall muscles . this finding confirms that in the herein selected whole - body linkage exercises , the activation of core muscles can be mainly focused on abdominal muscles while keeping the paraspinal muscles involved with low intensity . although no direct comparison can be made between the selected suspension exercises compared to previously reported similar exercises , it is possible to highlight the following differences . we can compare only the activation of the rectus abdominis , since for oblique muscles we used a different normalization exercise than the other three studies . plank exercises are frequently included in spine stabilization programmes as a means of improving motor control for spine stabilization . when plank exercises are performed on stable or unstable support surfaces , the reported activation level of the rectus abdominis and eo ranges from low to moderate ( garcia - vaquero et al . , 2012 ) . when executed in suspension condition , rectus abdominis muscles also showed moderate activation ( byrne and bishop , 2014 ) . only when the planks were performed with a similar technique ( instability on lower limb and shoulder flexion ) was the activation similar to that reported here , which was very high for the rectus abdominis ( mcgill and andersen , 2015 ) . therefore , we can assume that our exercises were more challenging than an isometric plank in a stable condition . in the roll - out , we found very high activation of lra ( 140% ) and ura ( 67% ) . these levels were higher than previously reported values obtained during the execution of the roll - out with the swiss - ball ( about 50 - 60% for rectus abdominis ) ( escamilla and lewis , 2010 ; marshall and desai , 2010 ) and similar to the values reported with the use of the power wheel , being very high for ura ( 76% ) and lra ( 81% ) ( escamilla et al . , 2006 ) . in the pike , we found high activation of lra ( 57% ) and ura ( 41% ) . the values reported for the pike executed with the swiss ball ( escamilla and lewis , 2010 ) and power wheel ( escamilla and babb , 2006 ) were similar for ura ( swiss ball 47% ; power wheel 41% ) and lra ( swiss ball 55% ; power wheel 53% ) . in the knee - tuck , we observed high activation of lra ( 54% ) and ura ( 44% ) . otherwise , the values reported for the knee - tuck executed with the swiss ball ( escamilla and lewis , 2010 ) and power wheel ( escamilla and babb , 2006 ) were lower for both ura ( swiss ball 32% ; power wheel 41% ) and lra ( swiss ball 35% ; power wheel 45% ) . our findings suggest that the two parts of the rectus abdominis can be activated differently according to the needs of the motor task ( kibler et al . , 2006 ) . this finding could be explained by the possibility to ( voluntary or involuntary ) modulate the activation ratio between rectus abdominis parts in order to achieve the best control of the core region . this could be justified by the metameric innervation of rectus abdominis muscles ( duchateau et al . , 1988 ) , although this issue is still controversial ( monfort - panego et al . , 2009 ) . however , lra muscles were generally more active than ura because of confounding methodological factors . mvcs of the lra and ura in fact were estimated by a standardized exercise to activate maximally the trunk muscles : it could be argued that the same exercise fully activated ura whereas it failed to fully activate lra . hence , the emg amplitude recorded during mvc was not the maximum achievable . consequently , throughout experimental exercises , lra seemed relatively more active than ura because its reference value of mvc was underestimated . , arv estimates of emg signals exceeded the mvc reference values ( arv higher than 100% ) . this inconsistency might be due to incomplete activation during mvc ( as in the case of the lower rectus abdominis ) and other confounding factors related to emg technique ( relative shift of muscle belly with respect to electrodes occurring in dynamic tasks and different activation between isometric and dynamic tasks , among others ) . as widely reported , variability of muscular activation between participants was high . this suggests that performing these exercises , some individuals might produce more or less activation than the average activity indicated here . although 17 individuals participated in this research , the differences in their fitness level and exercise experience could have affected the performance of the exercises and the resulting activation levels . crosstalk between muscles was minimized by using an innovative detection system based on concentric - ring electrodes which had been reported as having higher spatial selectivity compared to the traditional detection systems and reducing the problem of crosstalk from nearby muscles ( farina and cescon , 2001 ) . findings from this study , based on electromyographic analysis , showed that roll - out exercise was the most challenging . moreover , roll - out and bodysaw exercises executed in suspension activated the rectus abdominis and external oblique muscles at intensities higher than , or very close to , 60% of the maximum voluntary contraction . based on these findings , we can assume that roll - out and bodysaw exercises can be used to adequately strengthen the antero - lateral , superficial aspect of the core region , and thus they can be considered core strength exercises . these findings appear to have particular relevance for well - trained individuals given the high demand imposed by these exercises .

abstracta quantitative observational laboratory study was conducted to characterize and classify core training exercises executed in a suspension modality on the base of muscle activation . in a prospective single - group repeated measures design , seventeen active male participants performed four suspension exercises typically associated with core training ( roll - out , bodysaw , pike and knee - tuck ) . surface electromyographic signals were recorded from lower and upper parts of rectus abdominis , external oblique , internal oblique , lower and upper parts of erector spinae muscles using concentric bipolar electrodes . the average rectified values of electromyographic signals were normalized with respect to individual maximum voluntary isometric contraction of each muscle . roll - out exercise showed the highest activation of rectus abdominis and oblique muscles compared to the other exercises . the rectus abdominis and external oblique reached an activation higher than 60% of the maximal voluntary contraction ( or very close to that threshold , 55% ) in roll - out and bodysaw exercises . findings from this study allow the selection of suspension core training exercises on the basis of quantitative information about the activation of muscles of interest . roll - out and bodysaw exercises can be considered as suitable for strength training of rectus abdominis and external oblique muscles .

acute mountain sickness ( ams ) occurs during exposure to high altitude ( ha ) and is a clinical syndrome characterised by headache , insomnia , malaise , and gastrointestinal symptoms . it is common , developing in 1030% at 25003000 meters and in up to 60% of those ascending to around 4500 meters . it causes significant morbidity and is a challenging clinical condition in remote environments . a biochemical marker of ams , particularly one available as a point - of - care test ( poc ) , could have widespread clinical utility . the pathophysiology of ams is not clearly understood but involves alterations in fluid balance , endothelial function , vascular permeability , inflammation , and oxidative stress . the renal response to ha is an important factor in acclimatization , and ha exposure leads to renal arteriole constriction and relative hypoxia [ 3 , 4 ] . despite the relative renal hypoxia , ngal ( neutrophil gelatinase - associated lipocalin ) is a 25 kda peptide , part of the lipocalin family of small soluble proteins . it is produced in a number of human tissues , notably the distal nephron but also in the lung ngal rises rapidly in the nephron in response to a renal insult and an ngal 150 ng / ml following acute kidney injury ( aki ) is predictive of acute renal failure ( arf ) well before creatinine has risen . ngal is also an acute - phase protein , has a role in inflammation [ 8 , 9 ] , and is upregulated in the lung during inflammation [ 5 , 10 , 11 ] . ngal is also known to rise in conditions associated with oxidative stress [ 12 , 13 ] , and oxidative stress has been implicated in ams [ 14 , 15 ] . we therefore hypothesised that ngal would increase at ha secondary to these various stimuli and that the magnitude of any increase might relate to the presence of ams . we therefore studied a combined cohort of trekkers from 2 expeditions to ha . in order to clarify the relative contribution of ams , hypoxia or exercise to ngal levels , we also studied a cohort pre- and postexercise at near sea level , and a further cohort exposed to acute normobaric hypoxia . the potential role of inflammation in stimulating ngal was assessed by the measurement of highly sensitive c - reactive protein ( hscrp ) in a subset of participants . all study protocols were approved by the ministry of defence research ethics committee , whitehall , uk , and satisfied the requirements of the declaration of helsinki . in all studies informed , thirty - two subjects participating in a defence medical services ( dms ) trekking expedition ( trek 1 ) in the khumbu region of nepal were studied . blood samples were taken from the antecubital fossa at 3 study altitudes : on day 2 at 3400 m , day 6 at 4270 m , and day 10 at 5150 m ( following ascent to everest base camp at 5364 m ) . all samples in this study were collected immediately following a day trekking ( posttrek ) to the study altitude . twenty subjects from a further dms expedition ( trek 2 ) to nepal were also studied . blood samples were again taken at 3 study altitudes : on day 2 ( 3400 m ) , day 6 ( 4270 m ) , and day 10 ( 5150 m ) ( following ascent to kala patthar ( kp ) , 5643 m ) . samples in this study were again collected immediately following a day trekking ( posttrek ) . additional samples were taken at rest in kathmandu ( kat ) at 1300 m and at rest the next morning at the 3 study altitudes . as serving members of the military , all subjects were able to fulfil the fitness criteria of their relevant service . this broadly includes an age - adjusted ability to run 1.5 miles in under approximately 11 minutes and to perform an age - adjusted number of sits - ups and push - ups within two blocks of 2 minutes . fourteen subjects underwent a 3-hour exposure to normobaric hypoxia ( fio2 11.6% , equivalent to 4800 m altitude ) in a hypoxic chamber . this exposure included a 5-minute step test ( step height of 25 cm , 1 complete step every 2 seconds ) at 95 minutes . a group of 22 subjects had ngal assayed at rest and after exercise at sl in the uk following ascent from sea level to 1085 m over 6 hours ( an equivalent gain in altitude and duration of exercise similar to that experienced on a trekking day in nepal ) . two subjects from trek 2 were part of the sl exercise group , but data collection occurred several months apart . ngal was analysed in the field on a biosite triage point of care monitor ( alere ltd , stockport , uk ) using a triage ngal test kit . the triage ngal test is a point - of - care , fluorescence - based immunoassay used which gives a rapid ( 15 minutes ) quantitative measurement of ngal in a range from 60 to 1,300 ng / ml . oxygen saturation ( digitally on warm hands at rest ) was measured using a nellcor np-20 pulse oximeter ( covidian , ma , usa ) during trek 1 + 2 and in the hypoxic chamber study at the same time as blood samples were taken . during trek 1 + 2 , twice - daily ams scores were assessed using the lake louise score ( lls ) questionnaire . the lls allocates a score of 0 to 3 ( symptom not present to severe ) for symptoms of ams ( headache , gastrointestinal symptoms , fatigue / weakness , dizzy / light - headedness , and difficulty sleeping ) . a score of 3 or more in the presence of headache is consistent with ams , a score of 6 or more with severe ams . the commercially available , highly sensitive , immunoturbidimetric assay ( roche diagnostics ) was used to measure crp in trek 2 at the same time points as ngal . mg / l and a between - run coefficient of variation between 2.5 and 5.7% . for statistical calculations , the software package spss 14.0 was used . for subjects with a ngal below the limit of detection of the assay ( 60 ng / ml ) , a value of 60 ng / ml was assigned for the purposes of statistical analysis . all data were tested for gaussian distribution using the kolmogorov - smirnov test and shapiro wilks statistic . for the analysis of dependent variables that were normally distributed , changes were tested by student 's paired t - test . for independent variables that were normally distributed , a within - subjects anova was performed to investigate any serial changes in ngal with ascent at rest and post - trek . a two - way mixed anova with either resting or after trek ngal at each study altitude as the within - subjects factor and the presence of ams ( according to the ll score at multiple altitudes ) as the between - subjects factor if the mauchly sphericity test was significant , then p values were expressed after multiplication by the greenhouse - geisser epsilon . correlation analyses for normally distributed data were performed by calculating the pearson coefficient of correlation . a p value < 0.05 ( two - sided ) was considered significant . as the ascent profile and route were closely matched in trek 1 and trek 2 , data were combined and analysed as a whole . taking medication ( acetazolamide , dexamethasone ) had no apparent effect on ngal values , and therefore these subjects ( n = 11 ) were not excluded from the analysis . demographic data for the field study ( trek 1 + 2 ) , the controls , and the hypoxic chamber study are shown in table 1 . in the 22 subjects ascending to 1085 m in the uk , there was no significant ( p = 0.084 ) rise in ngal following exercise : resting sl ngal was 64 11 ( ng / ml , mean sd , range 60104 ) and postexercise ngal was 71 14 ( ng / ml , mean sd , range 60100 ) . of the 52 subjects , spo2 ( % , mean sem ) dropped from 97 2 at kat ( 1300 m ) to 84 5 and 79 7 at 4270 and 5150 m , respectively ( p < 0.001 ) . there was a moderate inverse correlation between ngal and spo2 at 5150 m ( r = 0.477 , p = 0.001 ) ( figure 1 ) with a weaker inverse correlation between ngal and spo2 at 4270 m ( r = 0.340 , p = 0.019 ) . within the subjects , anova demonstrated a significant change in ngal with ascent both at rest ( p = 0.007 ) and after trek ( p = 0.001 ) ( figure 2 ) . spo2 ( % , mean sem ) dropped from 99 0.4 at baseline to a nadir of 79 5 ( p < 0.001 ) . despite an equivalent drop in spo2 to that seen in trek 1 + 2 , ngal ( ng / ml , mean sd , range ) showed no change between baseline and 180 minutes : 63 26 ( 2980 ) versus 67 25 ( 2784 ) , p = 0.538 . in trek 2 , serum creatinine { mol / l , mean sem , ( range ) , ( p value versus baseline at kat ) } was 78 2 ( 6395 ) at baseline ; at 3400 , 4270 and 5150 m it was 87 3 ( 72120 ) ( p = 0.001 ) ; 84 2 ( 72104 ) ( p < 0.001 ) ; and 94 5 ( 76142 ) ( p < 0.001 ) . according to their ll scores at the highest study altitude ( 5150 m ) , there were 23 subjects with no ams , 16 subjects with mild ams , and 7 subjects with severe ams . there was a significant difference between ngal depending on the presence or absence of ams at 5150 m ( figure 3 ) with higher values in those with ams and severe ams . a two - way mixed anova revealed a significant change ( p = 0.003 ) in resting ngal with ascent and an interaction with ams at 4270 m ( p = 0.017 ) and 4910 m ( p = 0.002 for change in ngal , p = 0.027 for interaction with ams ) . hscrp was ( mean sem , range ) : 1.6 0.4 ( 0.337.53 ) at baseline ; at 3400 m , 4270 m , and 5150 m post - trek : 7 2.9 ( 0.7847.93 ) ( p = 0.002 versus baseline ) ; 25.7 8.1 ( 0.58104 ) ( p < 0.001 versus baseline ) ; 9 3.2 ( 0.5644.62 ) ( p = 0.003 versus baseline ) . at 3400 m , 4270 m , and 5150 m at rest : 6.2 2.9 ( 1.8725.1 ) ( p = 0.001 versus baseline ) ; 21.6 5.7 ( 0.4983.9 ) ( p < 0.001 versus baseline ) ; and 5.8 2.1 ( 0.5426.59 ) ( p = 0.012 versus baseline ) . ngal at 5150 m , after trek was moderately correlated with hscrp at 5100 m after exercise ( rho 0.526 , p = 0.036 ) . this is the first report to describe an association between ngal and both the presence and severity of ams at ha . the significant novel findings are that ngal rises in response to sustained hypobaric hypoxia but not acute normobaric hypoxia or near sl exercise and that this rise is related to ams at 5150 m. the rise in ngal following trekking ( by day 2 at 3400 m ) was to the levels normally associated with the subsequent development of arf ( > 150 ng / ml ) , but this did not occur . although creatinine rose significantly with altitude , the rise was very modest , and we suspect that a combination of factors other than a simple renal insult is responsible for the increase in ngal at ha . our data suggest an inverse correlation between spo2 and ngal at 5150 m ( and to a lesser extent at 4270 m ) . although no such correlation was found at 3400 m , this may still suggest that prolonged renal hypoxia could be a significant drive to ngal release . in addition to renal hypoxia , we suspect that other factors may also contribute to the rise in ngal at ha . the significantly greater ngal in those with severe or mild ams versus those without at 5150 indeed , ngal is an acute - phase protein with a role in inflammation [ 8 , 10 , 11 ] . exercise stimulates an immune response , and hypoxia is also known to cause a response in immune and endothelial cells with inflammatory markers such as hscrp increasing with ha [ 1922 ] . consistent with this , we saw a significantly higher hscrp at all altitudes compared to baseline . limited data have suggested hscrp may be associated with ams but we did not demonstrate any evidence to support this . there was a weak correlation between hscrp and ngal at 5150 m but this can not explain the rise in ngal as a whole . ngal also rises with oxidative stress [ 9 , 12 ] which is increased by exercise , and ha - induced oxidative stress has been implicated in ams . as such , it is interesting to note that we found a higher ngal following trekking and in those with ams at the highest altitude . in an attempt to clarify the relative influence of exercise and hypoxia on ngal , we measured ngal before and after exercise of a similar duration ( 6 hrs ) and similar incremental altitude ( 1085 m ) as that experienced daily in nepal and also in a hypoxic chamber . in neither scenario did ngal rise . this may reflect inadequate duration or severity of stimulus but may also reflect that the ngal response is not due to exercise or hypoxia alone but is multifactorial involving hypoxia , oxidative stress , an inflammatory response , and other , as yet unidentified , stimuli . we also acknowledge limitations such as a lack of serum markers of oxidative stress and a lack of resting ngal data in trek 1 . in addition , we did not measure ngal at sl before departure to nepal , although the ngal recorded as a baseline at kat ( 1300 m ) ( 68 ng / ml ) was no different to those recorded at sl in the uk ( 63 and 64 ng / ml ) . we also acknowledge the fact that although we measured creatinine in trek 2 , we did not continue to monitor it after the cessation of trekking . as a consequence of creatinine rising more slowly in response to a renal insult than ngal , in conclusion , there are several interesting and novel findings that are worthy of further exploration . ngal rises in response to prolonged hypobaric hypoxia ; marked increases in ngal may occur without concomitant arf and the degree of ngal rise at ha is associated with the presence or absence of ams . the fact that ngal does not appear to rise secondary to acute normobaric hypoxia or exercise in isolation suggests that the rise at ha and relation with ams may have common pathways , perhaps related to prolonged hypoxia and an inflammatory response . with the huge and increasing popularity of recreational sports undertaken at both moderate and high altitude assessment of ngal takes a matter of minutes using poc testing , and its use in identifying ams requires further evaluation .

acute mountain sickness ( ams ) is a common clinical challenge at high altitude ( ha ) . a point - of - care biochemical marker for ams could have widespread utility . neutrophil gelatinase - associated lipocalin ( ngal ) rises in response to renal injury , inflammation and oxidative stress . we investigated whether ngal rises with ha and if this rise was related to ams , hypoxia or exercise . ngal was assayed in a cohort ( n = 22 ) undertaking 6 hours exercise at near sea - level ( sl ) ; a cohort ( n = 14 ) during 3 hours of normobaric hypoxia ( fio2 11.6% ) and on two trekking expeditions ( n = 52 ) to over 5000 m. ngal did not change with exercise at sl or following normobaric hypoxia . during the trekking expeditions ngal levels ( ng / ml , mean sd , range ) rose significantly ( p < 0.001 ) from 68 14 ( 60102 ) at 1300 m to 183 107 ( 65519 ) ; 143 66 ( 60315 ) and 150 71 ( 60357 ) at 3400 m , 4270 m and 5150 m respectively . at 5150 m there was a significant difference in ngal between those with severe ams ( n = 7 ) , mild ams ( n = 16 ) or no ams ( n = 23 ) : 201 34 versus 171 19 versus 124 12 respectively ( p = 0.009 for severe versus no ams ; p = 0.026 for mild versus no ams ) . in summary , ngal rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of ams .

from may 1 through july 30 , 2007 , a total of 29 cases of locally acquired cyclospora infection were reported in british columbia ( figure 1 ; table 1 ) . an initial investigation was conducted around the 6 laboratory - confirmed case - patients reported in the last 2 weeks of may and the first week of june ( phase 1 ) . no common exposure was reported , and case reports subsided . during the last week of june , case reports resumed , and phase 2 of the investigation was initiated . a total of 19 confirmed and 4 probable cases were identified with symptom onsets during june 28july 20 , 2008 . average time from symptom onset to positive laboratory result was 17 days ( range 631 days ) . confirmed and probable cases of cyclosporiasis ( n = 29 ) , by date of onset , british columbia , canada , may august 2007 . * laboratory - confirmed cases were reported to public health by medical diagnostic laboratories and specimens forwarded for confirmation to the public health reference laboratory . oocysts included shape ( spherical ) , size ( 810 m in diameter ) , oocyst wall ( well - defined ) , internal contents with refractile globules , autofluorescence , and modified acid - fast or safranin staining ( 1 ) . during phase 2 , a total of 17 confirmed case - patients were interviewed with hypothesis - generating questionnaires about items eaten in the 2 weeks before symptom onset . the instrument included questions about restaurant history with meal details ; grocery stores frequented ; and yes / no questions about > 70 fruits and vegetables , 8 herbs , and 16 mixed foods ( e.g. , salsa , pesto ) previously implicated in outbreaks of foodborne disease . frequently reported foods were compared with population controls from canadian ( waterloo , ontario ) and american ( oregon ; us foodborne diseases active surveillance network [ foodnet ] ) published food consumption surveys ( 35 ) . although such measurements may be limited by the timing of questionnaire administration and the recall period considered , they can be useful comparators during the hypothesis - generating stages of an investigation . by the end of phase 2 , strawberries , cilantro , and sweet basil garlic and red peppers also were commonly eaten by case - patients ; however , population comparisons were unavailable . eighty - eight percent of case - patients reported having eaten romaine lettuce ; 85% of controls in the waterloo survey ( 4,5 ) had eaten lettuce of any type , and romaine lettuce consumption was much less commonly reported in the foodnet survey ( 3 ) ( table 2 ) . a formal case control study was considered premature in the early stages of phase 2 because no strong hypothesis emerged from early interviews and comparisons to population controls . we further explored the plausibility of various hypotheses through a combination of methods described below that allowed room for additional hypotheses to emerge or existing hypotheses to strengthen as cases accrued . * case - patients were 17 persons with laboratory - confirmed cases interviewed during phase 2 ( june 24july 21 , 2007 ) . us foodnet , foodborne diseases active surveillance network ; na , not applicable . detailed questionnaires asked whether foods were eaten in a restaurant or were store - bought and about type of packaging and method of preparation ( because c. cayetanensis is heat - sensitive ) ( 6 ) . we reinterviewed early case - patients using the second questionnaire and interviewed later case - patients using both questionnaires . in phase 1 , garlic eaten at restaurants by all 4 persons with confirmed infections were traced back to different suppliers ; only 1 case - patient ate raw garlic in a restaurant . three case - patients also reported eating cooked garlic at home ; cooking would have inactivated the pathogen . early and proactive collaboration with cfia involved a general assessment of the country of origin and distribution patterns for frequently eaten foods . according to cfia records , romaine lettuce and red peppers sold during the exposure period were not imported from a known cyclospora - endemic country and were widely distributed in canada and the united states . comm . ) . because interviews , population control comparisons , and product distribution limited suspected foods to strawberries , cilantro , and basil , we began preliminary traceback of all 3 suspected items . environmental health officers and regional cfia staff interviewed grocery store owners , restaurant managers , and distributors to trace produce to its supplier . local strawberries eaten by case - patients from 3 small markets were traced back to 2 local farms in geographically separate regions of british columbia . cilantro eaten by case - patients was traced to 2 suppliers ; both supplied home - grown rather than imported produce . of 14 case - patients with confirmed basil exposures , 4 ( 57% ) ate only organic basil supplied by distributor a. additionally , 4 ( 29% ) reported multiple basil exposures , including exposure to organic basil from distributor a ( figure 2 ) . in british columbia , organic basil enjoys a smaller market share than the conventional product . traceback of basil eaten by persons with confirmed cyclosporiasis ( n = 14 ) , british columbia , canada , may august 2007 . in phase 2 , 12 ( 71% ) of 17 case - patients reported shopping at grocery c. records of any grocery store purchases for the households of 8 consenting case - patients were obtained through grocery c s savings card program ; other case - patients were not cardholders . all purchase histories were requested for 1 month before symptom onset to account for the typical incubation period plus product shelf life . records from 3 ( 38% ) case - patients showed purchases of the same organic basil supplied by distributor a. two case - patients had bought organic basil on the same day at the same location . of the remaining 5 case - patients who recalled purchasing organic basil but whose consumer card records did not confirm it , 2 had not used their cards for large portions of the incubation period . we collected supplier information for organic basil during a visit to the distribution warehouse and local farm site of distributor a. the remaining 2 ( 14% ) case - patients with basil exposure previously unlinked to distributor a were confirmed through trace - forward from distributor a. the first had eaten organic basil at a smaller market supplied by distributor a under another trade name . the second had eaten conventional basil from a grocery store supplied by distributor a. distributor a confirmed using organic basil to supplement conventional basil shipments when supply was low . late summer outbreaks of cyclosporiasis in british columbia are unusual ; distributor a confirmed that imported product was used throughout the summer in 2007 because of a poor local growing season . all case - patients in phase 2 who recalled basil exposure ( 82% ) could have been exposed to organic basil from distributor a. once this common vehicle was identified , cfia conducted a full traceback of organic basil by using formal documentation including invoices , shipment numbers , and airway bills . the suspected imported basil was no longer available for testing . using distributor a invoices , we identified a specific shipment of organic basil imported from 1 of 2 mexican supplier farms , and cfia notified mexican authorities . the mexican farm was located in a region previously linked to cyclosporiasis outbreaks ( r. cardinal , cfia , pers . detailed interviews , modified traceback of several suspected items , and information about product distribution and market share led to organic basil as a primary hypothesis . food regulators could pinpoint a specific shipment and trace it to its origin because consumer cards provided the exact purchase dates for basil that case - patients could not recall . overall , the approach used in this investigation increased the work load typically requested of team members during foodborne outbreaks . however , this combination of investigative methods successfully identified a single vehicle during a community cyclosporiasis outbreak where a common menu was not available .

investigations of community outbreaks of cyclosporiasis are challenged by case - patients poor recall of exposure resulting from lags in detection and the stealthy nature of food vehicles . we combined multiple techniques , including early consultation with food regulators , traceback of suspected items , and grocery store loyalty card records , to identify a single vehicle for a cyclosporiasis outbreak in british columbia , canada , in 2007 .

brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products and can lead to a systemic disease with any organ involvement . in this report , we describe a case of brucellosis - induced avascular necrosis of the hip . brucellosis was diagnosed through serological tests , and avascular necrosis of the femoral head was confirmed by pelvic mri . the patient was treated with a combination of antimicrobial treatments and referred to the orthopedic service for total hip arthroplasty . brucellosis may present with unusual manifestations and should be always taken into consideration , particularly in endemic areas . brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products of infected animals ( 1 ) . it is a serious public health problem particularly in endemic areas , and is accompanied by . the disease is endemic in iran and has a high prevalence in lorestan province , central iran ( 3 ) . brucellosis is a systemic disease that may involve any organ in the body ( 4 ) . osteoarticular involvements including arthritis , spondylitis , osteomyelitis , tendonitis , and bursitis occur frequently and are reported in 3085% of patients with brucellosis ( 5 ) . the involvement of the large peripheral joints is usually manifested as monoarthritis ( 1 ) . although the hip joints involvement may happen in brucellosis , brucellosis - induced avascular necrosis of the hip , we describe a case of brucellosis that complicated by avascular necrosis of the femoral head . the patient was a 50-year - old rural farmer and rancher who presented with groin pain for six months before admission . he reported no history of trauma , underlying diseases , and steroids and alcohol use . over the previous six months , he had experienced symptoms including .fever , night sweats , weakness , fatigue , anorexia , and weight loss of 10 kg . the patient was visited by a local therapist ; in addition there was a delay in timely referral due to his addiction to opium . the groin pain was his most severe complaint on admission so that he could not bear his weight on the right leg . the physical examinations on admission showed stable vital signs , there was no organomegaly , and the right hip was in flexion and external rotation position so that any active and passive motion increased the pain . the results of laboratory study were as follows : complete blood count ( cbc ) : nl , liver function test ( lft ) : nl , rheamatic factor ( rf ) : neg , antinuclear antibody ( ana ) : neg , blood culture : neg , elevated erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) : positive , and standard agglutination test for brucellosis : positive ( table 1 ) . pelvic radiography and magnetic resonance imaging ( mri ) were administered showing avascular necrosis of right femoral head ( fig . 1 , 2 ) . the patient was subsequently treated with standard antibrucellosis regimen : streptomycine 1gr / d i m for three weeks , doxycycline 100mg / bid po , and rifampin 600mg / d po . .despite the improvement in the patient s general condition and laboratory evidence showing infection control , the patient was suffered from right groin pain and limitation of motion in the hip joint . although osteoarticular involvements , especially arthritis in the large peripheral joints , are among the most common manifestations of brucellosis , no joint destruction has been reported in many studies ( 5 ) . despite few reports on the permanent joint complications of brucellosis - induced peripheral joint arthritis , hip joints pyogenic infection has a poor prognosis , particularly if treatment is delayed ( 6 ) . delays in diagnosis and treatment of brucellosis - induced hip arthritis could lead to complications including dislocation and avascular necrosis of the femoral head ( 4 ) . although aspiration and examination of the joint fluid were not performed due to lack of synovial effusion on admission , considering the positive serological tests for brucellosis and concurrent avascular necrosis of the femoral head on mri , it seems that the delay in diagnosis and treatment of brucellosis in this patient had led to avascular necrosis of the femoral head . finally , the following questions arise : was avascular necrosis caused by increased intra - articular pressure related to the infection or due to direct involvement of the femoral head by the organism ? since brucellosis is a systemic infection with a broad clinical spectrum ranging from asymptomatic forms to deaths in severe cases , this disease should be taken into considerations when dealing with any patient with various and nonspecific symptoms in endemic areas with extension of the clinical signs of brucellosis . moreover , early diagnosis and long - term treatment and follow - up are of great importance in brucellosis .

backgroundbrucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products and can lead to a systemic disease with any organ involvement . in this report , we describe a case of brucellosis - induced avascular necrosis of the hip . brucellosis was diagnosed through serological tests , and avascular necrosis of the femoral head was confirmed by pelvic mri . the patient was treated with a combination of antimicrobial treatments and referred to the orthopedic service for total hip arthroplasty . brucellosis may present with unusual manifestations and should be always taken into consideration , particularly in endemic areas .

, more than 9000 articles related to sids were published and over 100 sids explanations appeared in medical hypotheses . sids occurs when an infant dies suddenly , unexpected by history , and without a cause found at forensic autopsy or thorough death - scene investigation . in the past four decades several multifactor models three inter - related causal spheres of influence model in which any two of these three could cause sids : ( 1 ) subclinical tissue damage ( 2 ) deficiency in postnatal development of reflexes and responses , and ( 3 ) environmental factors . filiano and kinney also proposed a triple risk model but required three risk factors , similar to emery 's , to act simultaneously and described them as ( 1 ) vulnerable infant ; ( 2 ) critical development period ; and ( 3 ) environmental stressors . these and other models do not , however , address the mathematical character of sids . this paper addresses each characteristic factor of sids that must be explained : the gender distribution ; the age distribution ; the effect of prone and supine sleep positions ; the seasonal variation ; risk factors of anemic apnea , respiratory infection , and neurological prematurity , and links them together with other causes of respiratory deaths as a proposed unifying theory . this paper uses data on infant live births and deaths as reported by the u.s . the sids data are given by international classification of diseases ( icd ) for 19791998 as 9icd and for 19992005 as 10icd . although technically a sids diagnosis requires an autopsy without causal findings , not all these sids were autopsied and the percentage of sids without autopsy in the us has decreased monotonically from 1979 to the present day . however , cdc lists sids for all autopsied and nonautopsied cases without distinction . in the case of an interracial parentage consequently the cause of death and race of sids will have measurement error involved which will increase the chi - square 1 d.f . test statistics ( 1 ) of comparisons between predicted and observed sids numbers and races , above those for tabulated p - values that assume the variance is from sampling error only . therefore although p - values are presented they may give incorrect implications for rejection of hypotheses if they fall < p = .05 . we therefore rely upon the overall consistency of age and gender data to support our mathematical construct of sids . a characteristic male fraction is associated with sids . because of difficulty in detecting congenital anomalies and other subtle causes of death in < 28-day neonates , postneonatal sids the cdc reports there were 62,933 male and 40,952 female postneonatal sids during 1979 to 2005 for a male fraction of 0.606 . figure 1 shows the male fraction of us postneonatal sids of all races over this period fluctuating slightly about the mean value of 0.606 as the sids rate decreased markedly from the discovery that the prone sleep position was a major sids risk factor which was followed by a back - to - sleep campaign in 1992 . naeye et al . first hypothesized that the male excess in infant mortality could be x - linked . mage and donner [ 810 ] showed that this excessive male fraction could be related to an unknown x - linked gene locus with a dominant allele ( a ) protective against sids , perhaps by providing enzymatic activity to allow anaerobic oxidation to take place in respiratory control neurons of the brainstem during transient periods of cerebral anoxia . the corresponding recessive allele ( a ) , with frequency q , would not provide this protection and could allow critical cerebral neurons to die of anoxia , and sids to occur when the dominant allele a is not present . for xy males and xx females the recessive allele frequency ( q ) can be determined from ( 1a ) and ( 1b ) , as the ratios of susceptible infants ( 1a)fmmm = q2fbq mb , ( 1b)q = fm / fbmm / mb , where fm and mm are postneonatal female and male sids and fb and mb are female and male live birth rates , respectively , so q represents the female postneonatal sids rate divided by the male postneonatal sids rate per 1000 live births . the global average male fraction of 0.612 for autopsied postneonatal sids is higher than the 0.606 us male fraction for total autopsied and nonautopsied sids , perhaps due to false positive sids that have a lower male fraction . with a 5% average male excess live birth rate , when stratified by race , we obtain from the cdc us 19792005 total postneonatal sids and birth data there is a white male fraction of 0.622 , black male fraction of 0.570 , and other races combined = 0.594 . using ( 1b ) , q white = 0.639 , q black = 0.779 , and for other races combined q = 0.724 . such variation in allele fraction between races along with the establishment of hardy - weinberg equilibrium is expected for each racial grouping from genetic drift over a long period of time . by necessity we accept here the cdc racial designations and neglect the presence of interracial infants . in our genetic analysis we assume that all sids infants have only the recessive allele ( a ) and require their probability of genetic susceptibility pg to equal 1 . to support this genetic mechanism , we note in table 1 that other causes of respiratory deaths in infancy have a statistically similar male fraction to 0.606 for postneonatal us sids when all races are combined . not all of these cdc reported cases were autopsied , and false positive sids occur from infanticide by gentle suffocation that is virtually indistinguishable from sids at autopsy , so statistical testing assuming no autopsy error may not be cause for rejection at p = 0.05 . rds , also known as hyaline membrane disease , had a male fraction of 0.610 . bronchopulmonary dysplasia had a male fraction of 0.613 . with the discovery that the prone position is a risk factor , there is a trend to parse postneonatal sids into true sids and subcategories . two such alternatives to sids are accidental suffocation and strangulation in bed and unknown ill - defined unspecified - causes which had male fractions of 0.593 and 0.597 , respectively . . suffocations by inhalation of food or other foreign object ( siffo ) in infancy had a male fraction of 0.600 very close to 0.606 ( p = 0.52 ) for all postneonatal sids . the risk factors for infant inhalation of food or other object are morsel size , rounded shape , and slippery surface , like a grape . however , types of infant food , and mode and manner of preparation are identical for males and females , so these risk factors are independent of gender . we hold that all this tabulated male fraction similarity of order 0.61 is strong evidence of a common x - linked recessive susceptibility to the same terminal mechanism of cerebral anoxia . furthermore , the virtually identical male fraction of 0.6053 compared to 0.6057 for sids occurs for these same siffo icd codes combined for all children ages 1 to 14 years in the us from 1979 to 2005 , with 2,324 male and 1,515 female ( p = 0.98 ) . when broken into ages 14 , 59 , and 1014 years none of these groups are rejected . the implications of this consistent male fraction from infancy through adolescence is emphasized in the later discussion section . the siffo + igc data for the next cdc age group of 1519 years with a higher male fraction is not shown here because higher teenage male alcohol consumption is a new positive bias factor ( 496 male , 277 female : male fraction = 0.642 ) . the male fractions in 19791998 of all us infant deaths by all icd 9 chapters and for 19992005 in their icd 10 equivalents are shown in table 2 . these data show the well - known male excess in virtually all icd classes of infant death , with only the neoplasms showing no male or female excess as expected from a purely random initiation process as the 5% us male live birth excess corresponds to a male fraction of 105/205 = 0.5122 . ( 1 ) the differing male fractions for most of these disease classes are essentially similar between the two periods 19791998 and 19992005 . this suggests that there is something physiological involved that provides the apparent characteristic excess male risk for each such class of cause of death . for example , certain conditions arising in the perinatal period with some 350 000 deaths covered by icd9 and 100 000 covered by icd10 have male fractions of 0.566 and 0.567 , respectively . ( 2 ) the approximately 0.61 male fractions of table 1 for respiratory causes shown are found as expected for the congenital anomalies of the respiratory system ( 0.602 icd9 and 0.579 icd10 ) and diseases of the respiratory system ( 0.587 icd9 and 0.581 icd10 ) . what is surprising is that the male fraction of mortality from diseases of the digestive system in infancy is similar to that from respiratory causes , 0.588 in 9icd and 0.601 in 10icd . in the uk from 1979 to 2006 other diseases of the digestive system ( not ulcer - appendicitis - hernia - obstruction - chronic liver disease - cirrhosis ) were 458 male and 329 female for a male fraction of 0.581 similar to the us data . we speculate that a linkage between the mechanism for the similar male fraction from digestive disease as sids may be from digestive causes such as malabsorption of iron and glucose in celiac disease and insufficient vascularization that would limit uptake and transport of glucose , respectively . this could lead to hypoglycemia that is a known risk factor for sids and sudden death [ 15 , 16 ] . in the older infant , the resistance to hypoxia is much less than for the neonate , reflecting the diminished stores of glycogen and therefore limited substrate for anaerobic metabolism . an enzyme , such as glucose-6-phosphate dehydrogenase ( g6pd ) could play a role as its x - linked gene locus is at xq28 and it has a great multiplicity of alleles that are associated in their deficiency with nonspherocytic hemolytic anemia , and anemia is a likely risk factor for sids . g6pd catalyzes initiation of glucose oxidation via the hexose - monophosphate pathway that may be a critical requirement for neuronal survival during cerebral anoxia . there could be more complicated x - linked processes such as requiring two ( or more ) independent x - linked alleles with probabilities q1 and q2 , with probability of simultaneous presence ( q = q1q2 ) that would equal the qvalues listed above for a single x - linked allele . alternatively , a gene locus such as g6pd could have many recessive alleles ( q1 , q2 , q3 , ) that are nonprotective of sids that could sum up to the q values listed above for the same risk of sids ( q = q1 + q2 + q3 + ) . we have chosen a single - gene x - linkage process for simplicity of discussion , and note that any genome - wide association study required to test our model can test for all possibilities . the age distribution of sids is unique : any viable hypothesis for the cause of sids must account for its characteristic age distribution . . raring first noted that the unique and characteristic age distribution of sids appeared to follow a 2-parameter lognormal model . mage reviewed the sids age literature and in a meta - analysis of 15 global sids age data sets obtained the distribution of some 20 000 ages of sids shown in figure 2 . in construction of figure 2 , 1-month is < 28 days of life . age data in weeks of life were divided by 4.33 to convert to months and the althoff data from cologne reported as age within midmonth intervals ( e.g. , 1.52.5 month ) were plotted to estimate the corresponding integer month intervals ( e.g. , 1 - 2 months and 2 - 3 months ) for pooling with the other monthly sids data . the total of 19,949 sids includes 194 sids deaths that are predicted to occur after 1-year in these 15 cohorts by use of an exponential fit to monthly data intervals 5 to 12 that was then extrapolated and summed from 13 to 41 months . these data in figure 2 were fit by a 4-parameter lognormal distribution , also known as the johnson sb distribution , shown as ( 2 ) . here dp(m ) is the probability of sids occurring between ages m and m + dm in months , median = 3.1 months and standard deviation = 0.6617 , as fit by maximum likelihood ( 2)dp(m)dm=(22)1[(m+0.31)1 + ( 41.2m)1 ] exp [ log e2([(m+.31)(41.2)]/[(41.2m)(+.31)])/22 ] . equation ( 2 ) can be interpreted as a sum of products of three age dependent terms , denoted as pn , pi , and pa . let pn = 1/(m + 0.31 ) represent a risk factor of neurological prematurity leading to delays in development of respiratory reflexes and responses , that decreases with increasing age . neurological prematurity is a risk factor that is maximal at birth and decreases as the infant physically matures . kinney has found that an important subset of sids appears to have a deficiency in serotonin receptors that is hypothesized as a causal factor of those sids . let pi = 1/(41.2 m ) represent an infection risk factor that increases with increasing age . secondary sids , that have findings of low - grade respiratory infection at autopsy that of itself is insufficient to cause death . risk of such infection increases with age as infants lose passively acquired maternal immunoglobulin ( igg ) and they have increased exposure to pathogens as they have more contacts both within and without their immediate family . us dhhs linked birth and death certificate data for 19952004 show , in table 3 , that the rate of sids increases monotonically with live birth order ( lbo ) . it has been suggested that older school - age siblings may be an important respiratory infection vector . we assume here that the infant lives with two parents , all older siblings survived to the time of sids death , and no adoption of the sids infant or older siblings took place . for lbo 6 we assume only 5 siblings have contact with the infant . let the probability of a family member not carrying a respiratory infection communicable to the infant at any time = p. for infants with family size = 2 parents + ( lbo 1 ) siblings the probability of not having an infection vector present is equal p. the probability of an exposure to at least one carrier is then 1 p. by least squares analysis we found p = 0.9 with a scaling factor of 2.5 , so we model the rate of sids per 1000 = 2.5 ( 1 . infant anemia has not been considered directly as a risk factor for sids per se , because accurate hemoglobin [ hb ] levels can not be determined after death due to rapid hb breakdown resulting in the mottled and reddened areas known as livor mortis . a study in mice shows how hb is already significantly decreased in the first postmortem hour . because the exact time of sids during sleep is not known it would be impossible to correct for the variable amount of hb lost between the instant of sids death and autopsy . there is , however , indirect evidence suggesting a relationship between anemia and sids : the peak incidence of sids coincides with the nadir [ of hb ] in the physiological anemia of infancy . anemia does contribute to apnea and apparent life - threatening events ( altes ) from causing longer cyanotic breath - holding spells [ 2932 ] that are risk factors for sids , leading to the apnea hypothesis . [ 3 , 32 ] . therefore anemia is treated by us as a risk factor for sids . let pa = exp [ log e ( [ ( m + 0.31)/( + 0.31)]/[(41.2 m)/(41.2 )])/(2 ) ] , as found in the johnson sb model as ( 2 ) , represent an anemia - cum - apnea risk factor rising from 0 at birth , reaching a peak at the median ( = 3.1 months ) and decreasing to zero at 41.2 months . anemia in infancy may be defined relatively as any value for the hemoglobin [ hb ] less than two standard deviations ( < 2 ) below the mean for age , or absolutely as less than a fixed value , such as 13.5 g / dl which is the 2 level below mean cord blood hb and mean hb at 1 week . infant physiological anemia is a risk factor that is virtually zero at birth due to placental transfusion during labor and at birth hb concentration in the blood can reach + 2 of 23.7 g / dl . we propose that this high at birth hb phenomenon accounts for the relative protection from sids during the first week of life . in the following weeks , total hb decreases rapidly as fetal hemoglobin ( hbf ) is removed faster than it can be replaced by adult hemoglobin ( hba ) . a nadir in total hb occurs at or about 2 months of age for a term infant that corresponds to the 63rd day mode of the sids sb age distribution [ 18 , 21 ] . table 4 shows the 2 hb g / dl level ( lowest 2.5% of all infants ) . by definition , approximately 25 in 1000 term infants have a hb value below the 2 value shown , and preterm infants will fall under this value with a higher frequency , perhaps related to their increased risk of sids . of those 25 in 1000 , the one with the lowest hb would be at the highest risk of apnea and therefore sids . if physiological anemia is considered as a hb deficit from a fixed level of 13.5 g / dl , that could , combined with apnea , cause transient hypoxia and inability to meet neuronal oxygen demand in the brainstem of sids susceptible infants . if so , it could correspond , as shown in table 4 , to the rise - and - fall factor pa modeled from the johnsonsbdistribution as ( 2 ) fit to figure 2 . the presence of respiratory infection as a risk factor fits the characteristic of sids of a seasonal dependency , maximizing in the winter and minimizing in the summer , that has been associated with wide seasonal temperature changes . mage showed that in hawaii , a semitropical us state with only narrow seasonal change in mild temperatures , that 384 sids varied seasonally with calendar day ( t ) between 1979 and 2002 as a cosine function shown as ( 3 ) where the maximum sids rate is predicted to occur on january 30th ( t = 30 ) : ( 3)mortality on day t=0.810 + 0.241 [ 1+cosine2(t30)365.25],0<t<365.25 . this equation may be interpreted to show that in hawaii , 23% of sids have a seasonal infection component ( 0.241/1.051 ) and that 77% of sids occur at a constant rate due to other physiological factors such as anemia and neurological prematurity that have no known seasonality . the winter flux of infection vector would come via visitors from the us mainland and japan , so the authors expect that the proportion of seasonal infections and sids may be larger in temperate zones of the us with colder winter temperatures than found in hawaii . there has been a tendency for the winter peak to be reduced since the start of the back - to - sleep campaign that may be due to the lessening of the hypoxia caused by low - grade seasonal respiratory infection when sleeping supine [ 38 , 39 ] . the pre-1992 lognormal form of the age distribution of sids [ 40 , 41 ] remained the same during the change of preferred sleep position from prone to supine . pollack found the age distributions of us sids between 1989 and 1999 were virtually unchanged in the two cohorts . he reported that the stability of this distribution is remarkable when one considers the large decline in sids incidenceas shown in figure 1 . malloy and freeman also found little change in age distribution for us sids between 1992 and 1999 ( p = 0.025 ) . the derivation and its explanation for this consistency is aided by a venn diagram shown as figure 3 . let a prone sleeping infant be susceptible to sids in both the pg pa pn and pg pa pi areas of figure 3 even if missing the pi or pn risk factors , respectively . note that the sum of pg pa pn + pg pa pi represents two overlapping areas on the venn diagram because the central segment ( pg pa pn pi ) is counted twice . let the probability of a prone sleeping child = pp and that of a supine sleeping child = ps . for simplicity we include the side sleeping position with the prone , and we require pp + ps = 1 . then the probability of dying of sids at age m while prone ( ppsids ) is written as , ( 4)ppsids = pp pg pa ( [ pn+pi]pnpi ) . one can then write the probability of supine sids ( pssids ) as , ( 5)pssids = ps pg pa pn pi . combining ( 4 ) and ( 5 ) we get the total probability of sids ( psids ) as ( 6)psids = pp pg pa ( pn+pi)+(pspp ) pg pa pn pi . we then note that the sum of pn + pi has a similar mathematical form as pn pi as follows : ( 7a)pn+pi=1(m + 0.31)+1(41.2m)=[(41.2m)+(m+0.31)][(m+0.31 ) ( 41.2m ) ] , ( 7b)pn+pi=41.5[(m+0.31)(41.2m ) ] , ( 7c)pn pi=1[(m+0.31)(41.2m ) ] . thus the mathematical form for the age distribution of both supine sids and prone sids can be represented by the same relationship of c pa/[(m + 0.31 ) ( 41.2 m ) ] , where c is a constant , which implies that , in terms of relative probability at different values of m , ( 7b ) and ( 7c ) are the same . this is consistent with the report that there were similar frequencies of pathological findings in both supine and prone sids confirming that the mode and cause of sids death is apparently the same for both sleep positions . this derivation shows how the venn diagram and johnson sbage distribution predict that supine and prone sids have the same age distribution , with lower rates for the supine sids . this corresponds to the supine requirement to have all 4 risk factors ( pg pa pn pi ) as opposed to only 3 risk factors ( pa pg pi or pa pg pn ) that can allow a prone sids to happen more readily . factors that make the prone sleep position a risk factor for sids are rebreathing of exhaled breath with reduced oxygen and increased carbon dioxide and the finding that presence of a fan in the infants sleep environment , that disperses exhaled breath , decreases the sids rate . other hypotheses than the x - linkage hypothesis of naeye et al . for the male excess in sids and other causes of infant respiratory mortality have appeared in the literature [ 4547 ] . the mechanism behind the excess perimortality rate in male infants is not known . a genetic factor leading to reduced tolerance analyzed amniotic fluid and showed the male fetus developed pulmonary surfactants slower than the female fetus and suggested that this deficit at birth may cause the male excess in infant respiratory distress syndrome ( rds ) that matches that of sids . this is not likely because the measured deficit should decrease with maturity as the infant ages , but cdc reports that the male fraction of rds between 28 and 364 days [ 0.617 ] is greater than the male fraction [ 0.604 ] on the first day of life when the deficit is maximal . patterson et al . found in their sids cases that males had a larger deficiency in serotonin receptors in the brainstem than females and suggested that this may be related to the male excess in sids . as for the male surfactant deficit cited above , a greater male serotonin - receptor deficit at birth should decrease with infant maturity , but the 0.606 male fraction of sids between 28 and 364 days is also greater than the 0.548 male fraction for 06 days ( which may partially be related to false positive sids from undiscovered infanticide or subtle congenital anomalies ) . l'hoir et al . found in their study in the netherlands that male infants were placed to sleep in the prone position more often than females , and were more likely to turn prone from a side sleeping position than females , and suggested that this may be related to the male sids excess . however , as shown in figure 1 , the sids male fraction remained essentially the same as the recommended sleep position in the us changed from prone ( pre-1992 ) to supine ( post-1992 ) , even though the sids rate dropped by a factor of three from 1979 to 2005 . furthermore , any other hypothesized cause for sids that suggests that the sids male excess in mortality is related to a male underdevelopment relative to the female can not explain the fact that virtually exactly the same male fraction of 0.605 occurs for siffo between 1 and 14 years as the 0.600 in the first year of life shown in table 1 . the risk factors for siffo in children are independent of gender because food in the us is not chosen or prepared differently for males and females . types of food that are most often recovered from the upper airway at infant autopsy are raw carrot and apple , round and slippery items such as hotdog pieces without skin removed , candy , nuts , and grapes [ 4951 ] . foreign objects swallowed by children over 1 year of age are often balloons and small coins such as pennies . although the rates of siffo decrease with age , as dentition and swallowing control develop , and the types of food items eaten by children change as they go from infancy to 14 years of age ( e.g. , chewing gum is often inhaled ) , the male excess remains the same up to 14 years . as opposed to sids that predominantly occurs during sleep , siffo predominantly occurs while the infant is awake or being fed , and immediate first aid is attempted that is successful in approximately 99% of all cases [ 52 , 53 ] . yet , assuming equal siffo risks for males and females , more males than females can not be resuscitated in exactly the same proportion as dying in sids . virtually all other risk factors posited for sids are either independent of gender ( e.g. , parental smoking or autosomal genetic conditions ) or are inoperative for siffo between 1 and 14 years of age except the possible x - linkage . an obvious potential cause of an infant male excess for any icd class may be due to an androgen excess in the male . we discussed this previously and showed that during the first year of life , the sids male fraction remains relatively constant while the male serum testosterone is slightly higher than the female 's at birth , peaks for three months during the first six months to aid testicular descent , and falls back towards zero for the second six months of life , so an androgen interaction is not a likely factor . as in any epidemiology study , there is always a finite probability that these data are the result of happenstance and coincidence , known as sampling error , and that next year 's data may be cause for rejection of the developments presented . we have shown how all characteristic properties of sids , its gender , age , and seasonal distributions , along with the observed risk factors of apnea , respiratory infection , and neurological prematurity , can be tied to each other mathematically . these relationships presented here explain how the supine position reduces the rate of sids and why it does not change the gender distribution or the form of the age distribution from those of sids occurring predominantly in the prone position . because all sids risk factors except the hypothesized x - linkage are independent of gender , we propose that equal numbers of males and females , per equal numbers of live births , are at risk of having potentially fatal risk factors that we previously defined here as pa , pi , and pn . approximately 2/3 of all males and 4/9 of all females have a genetic risk factor pg that is necessary to cause sids but not sufficient by itself infants with the protective allele and the three other risk factors ( see figure 3 ) may be among the cohort of those presenting with apparent life - threatening episodes ( altes ) that do not then or later progress to sids . it is proposed that sids may occur for those genetically susceptible infants when repeated transient coincidences of factors reduce the oxygen supply ( apnea , anemia , rebreathing exhaled breath , etc . ) during a period of increased oxygen demand ( low grade respiratory infection raising body temperature ) . if the infant has a residual neurological prematurity , auto resuscitation by the gasp reflex may be delayed causing acute cerebral anoxia that may cause some respiratory - drive neurons in the brainstem to die ( emery 's subclinical tissue damage ) . when a sufficient number of such neurons die , the next sleep with identical risk factors causing anoxia may reduce the number of functioning neurons below a minimum critical requirement so auto resuscitation is impossible . the protected infant with an x - linked dominant allele ( a ) could switch over from aerobic oxidation to anaerobic oxidation to keep those critical neurons alive during the same transient anoxic conditions so that autoresuscitation could occur . in summary , the quadruple risk model presented here , with factors developed from pre-1994 gender data and from pre-1992 sids age data , predicts the age and gender distributions for post-1992 data as shown . the factors determining the age distribution mesh with the medical literature 's findings of the risk factors for sids . should the genetically susceptible infant pass through infancy unscathed , the genetic susceptibility to cerebral anoxia can still penetrate in childhood if anoxic circumstances arise as shown by the identical us postneonatal sids male fraction of 0.606 occurring in us children aged 1 to 14-years suffocating from inhalation of food or other foreign objects . so , in the absence of any other plausible explanation in the medical literature for the same siffo male excess from birth to 14 years of age as sids , a common x - linkage remains as the only possibility . furthermore there was a 45% excess adult male completion rate of suicide attempts by coal - gas inhalation in paris between 1949 and 1962 ( completions of 58% male versus 40% female ) . in conclusion , although modern thought is now that sids is a composite of independent and different causes of death , they all appear to have the same male fraction . we reason that all those different causes of death lead to the same cerebral anoxia that may result in respiratory failure from the absence of an x - linked dominant allele that supports anaerobic oxidation in respiratory control neurons of the brainstem . proof of this unifying mechanism must await genetic testing to identify , if correct , the unknown recessive x - linked allele that is exclusively present in all these icd codes with the statistically similar male excess of sids .

the sudden infant death syndrome ( sids ) has four distinctive characteristics that must be explained by any theory proposed for it . ( 1 ) a characteristic male fraction of approximately 0.61 for all postneonatal sids in the us ; ( 2 ) a distinctive lognormal - type age distribution arising from zero at birth , mode at about 2 months , median at about 3 months , and an exponential decrease with age going towards zero beyond one year ; ( 3 ) a marked decrease in sids rate from the discovery that changing the recommended infant sleep position from prone to supine reduced the rate of sids , but it did not change the form of the age or gender distributions cited above ; ( 4 ) a seasonal variation , maximal in winter and minimal in summer , that implies subsets of sids displaying evidence of seasonal low - grade respiratory infection and nonseasonal neurological prematurity . a quadruple - risk model is presented that fits these conditions but requires confirmatory testing by finding a dominant x - linked allele protective against cerebral anoxia that is missing in sids .

a 56-year - old man was referred for biliopancreatic diversion with a duodenal switch ( bpd - ds ) for intractable complications associated with morbid obesity . he had been unable to lose weight with orlistat 120 mg three times a day for three months . the patient had diabetes mellitus for twenty years associated with hypertension , " mal perforant " and hepatic steatosis . he had all features of the metabolic syndrome and was treated for sleep apnea with a nocturnal continuous positive airway pressure device ( c - pap ) . his body mass index ( bmi ) at the time of the surgery was 48.7 kg / m , weighting 157.7 kg . the patient 's blood tests showed normal electrolytes and a creatinine of 110 mol / l . the glycated hemoglobin was 7.1% ( normal range 4.46.5% ) with fasting glucose values ranging from 7 to 11 mmol / l with ldl - cholesterol and hdl - cholesterol levels of 2.63 and 0.99 mmol / l respectively . rest and exercise electrocardiograms were normal as well as a cardiac dobutamine stress echocardiography . a pseudonormalized pattern of left ventricular filling was present on a standard echocardiogram and a 24-hour holter was normal except for a slight decrease in heart rate variability . his medications before surgery consisted of metformin 850 mg tid , rosiglitazone 8 mg daily , irbesartan 300 mg daily , diltiazem 120 mg daily , orlistat 120 mg tid and furosemide 20 mg every other day . he was on insulin humalog tid ( 4246 u ) with insulin nph 90 u at bed time , for a total of > 200 u of insulin daily . he underwent a modified scoparino 's biliopancreatic diversion with a duodenal switch [ 1 - 5 ] . the patient had an hemodynamically stable pulmonary emboli on the day 12 after surgery and was anticoagulated . anthropometric measurements before and eleven months after the surgery are presented in the table 1 and figure 1 and metabolic improvements and echocardiographic findings are depicted in tables 2 and 3 respectively . eleven months after the surgery , the medication consisted of metformin 850 mg tid , irbesartan 150 mg daily , 50 000 u of vitamin d2 , ferrous sulfate 300 mg daily , 25 000 u of vitamin a , calcium 500 mg and one tablet of vitamins and mineral supplements daily ( centrum forte ) . anthropometric variables before and after biliopancreatic bypass surgery anthropometric variables before and after biliopancreatic diversion surgery anthropometric measurements were assessed using a bioimpedance balance ( tanita tbf 350 ) metabolic profile before and after biliopancreatic diversion surgery blood pressure and echocardiographic variables before and after biliopancreatic diversion surgery bp : blood pressure ef : ejection fraction from obesity is associated with an increased risk of coronary artery disease ( cad ) and mortality [ 6 - 8 ] . morbidity and mortality rates rise proportionally to the degree of obesity in men and women and the impact of excess body fat is more significant in younger subjects than older ones [ 9 - 12 ] . in a 10-year follow - up , men and women with a bmi 35.0 kg / mhad a relative increased risk of developing diabetes of ~23 and ~17 fold respectively compared to a control group with a bmi between 18.5 and 24.9 kg / m . independently of the bmi , the relative risk of developing diabetes mellitus increases with weight gain as shown in the nurse 's health study . moreover , in that study , women who voluntary lost more than 5.0 kg reduced their risk of diabetes by 50% . weight loss also lowers blood pressure . in a 3-year follow - up of non - morbidly obese patients with a mean bmi of 31 kg / m , patients who maintained a 4.5 kg weight loss had a relative risk of hypertension of 0.35 or , a reduction of 0.45 mmhg in systolic blood pressure and 0.35 mmhg in diastolic pressure per kg of weight lost . in addition to higher cardiac output in obese patients , left ventricular volume and filling pressures are higher than normal . this usually results in the development of left ventricular strain , which leads to hypertrophy , often of the eccentric type . left ventricular diastolic function is thus frequently impaired . weight loss has a beneficial impact on the functional and the structural cardiac status . in a study of obese patients with a mean bmi of 32.7 kg / m , weight loss of 8 kg over a period of 25 weeks was associated with a significant decrease in left ventricular mass . weight loss lowers oxygen consumption at any given work rate , decreases cardiac output and blood pressure while left ventricular filling pressures decreased as left ventricular stroke volume diminishes . in a study of obese patients with a bmi > 40 kg / min whom surgical weight loss with vertical gastric banding ( vgb ) induced a decrease in body weight of 20% at six months , left ventricular wall thickness , particularly the septal and posterior walls , decreased . the national institutes of health ( nih ) suggested that surgical therapy be proposed to those patients with a bmi level > 40 kg / mor > 35 kg / mwith serious medical conditions including hypertension and obstructive sleep apnea . surgical intervention , when indicated , brings significant improvement such as a decrease in excess weight and comorbidities ; these include hypertension , diabetes , dyslipidemia [ 3,14,25 - 28 ] and sleep - related disorders . one can expect a mean reduction of 60 to 75% of excess body weight with biliopancreatic diversion which can persist for 4 to 8 years after surgery [ 3 - 5,30,31 ] . the duodenal switch operation , introduced by hess in 1988 , variant of the biliopancreatic diversion of scopinaro , helps in preserving normal eating habits , and the majority of patients undergoing the procedure will have normalization of glucose levels , triglyceride levels and blood pressure early weeks after the surgery [ 3 - 5 ] . indeed , in a review of 440 obese patients ( mean weight of 183 kg ) who underwent biliopancreatic diversion with duodenal switch , all of the 36 type 2 diabetic patients discontinued their medication over a 7-year follow - up period . operative mortality is between 0.5% and 2% and early complications include pulmonary embolus ( 0.5% ) and anastomotic leaks ( 12.5% ) . late complications presents in the form of anemia , anastomotic ulcerations , bone demineralization , neurological complications and protein malabsorption ; all of which can be addressed with appropriate supplements . moreover , significant nutritional and metabolic complications may be less frequent than previously thought . eleven months after the surgery , our patient had lost 40% of his body weight , and body fat mass was reduced by 41 % ( figure 1 ) . the medication was greatly lightened while the patient maintained fasting glucose values ranging from 5 to 7 mmol / l and a blood pressure less than 130/80 mmhg ( table 2 ) . indeed , rosiglitazone was discontinued , the insulin that averaged > 200 u daily was no more necessary , diltiazem was discontinued and the dose of irbesartan was halved . the 25% decreased in waist circumference is probably clinically significant albeit waist circumference may be less reliable in patients with a bmi > 35 kg / m . finally , sleep apnea syndrome improved as the patient was no longer on c - pap . left ventricle hypertrophy is recognized as a strong independent risk factor for cardiovascular morbidity and death and changes in cardiac structure following surgical weight loss have been observed . in the present case , the left ventricle mass index decreased by 15% and the thickness of the septal and posterior walls of the left ventricle were reduced ( table 3 ) . moreover , using doppler mitral flow velocity with the e / a ratio , we demonstrated that left ventricular diastolic dysfunction actually improved . before the operation , the e / a ratio was higher than 1 with a significant decrease during the valsalva maneuver which corresponds to a pseudonormal ventricular filling or grade 2 filling pattern . after the operation , the e / a ratio was smaller than 1 indicating a delayed relaxation of grade 1 filling pattern , representing an improvement in the diastolic function ( table 4 ) . mitral doppler at rest and during the valsalva maneuver before and after biliopancreatic diversion surgery e wave : early transmitral filling velocity a wave : transmitral atrial filling velocity e / a : early / atrial transmitral filling velocity this case report emphasizes the improvement in cardiovascular parameters , including the diastolic function ( demonstrated by a pseudonormal ventricular filling or grade 2 filling pattern ) and sleep apnea syndrome , following weight loss - induced by the biliopancreatic diversion with duodenal switch . pp envisioned the paper and pw , pp prepared the initial draft of the article . bpd - ds : biliopancreatic diversion with a duodenal switch c - pap : continuous positive airway pressure device vgb : vertical gastric banding nih : national institutes of health e / a ratio : early / atrial transmitral filling velocity the authors want to express their gratitude to suzie laroche , louise marois and claudette fortin for the quality of their intervention and the work on this particular population .

backgroundsevere obesity is associated with important morbidity and increased mortality . the successes of lifestyle modifications and drug therapy have been partial and mostly unsustained in reducing obesity and its comorbidities . bariatric surgery , particularly biliopancreatic diversion with duodenal switch reduces efficiently excess body weight and improves metabolic and cardiovascular functions.case presentationa 56-year - old man with severe clinical obesity underwent a biliopancreatic diversion with a duodenal switch after unsuccessful treatment with weight loss pharmacotherapy . he had diabetes , hypertension and sleep apnea syndrome and was on three medications for hypertension and two hypoglycemic agents in addition to > 200 insulin units daily . eleven months after the surgery , he had lost 40% of his body weight . the lipid profile showed great improvement and the hypertension and diabetes were more easily controlled with no more insulin needed . the pseudonormalized pattern of left ventricular diastolic function improved and ventricular walls showed decreased thickness.conclusionbiliopancreatic diversion may bring metabolic and cardiovascular benefits in severely obese patients from a cardiovascular perspective .

the immunosuppressive receptor pd-1 and its ligand pd - l1 have been identified by dr . tasuku honjo and his colleagues at the kyoto university as factors that induce programmed cell death . since the mechanism mediated by pd-1 and its ligand was shown to be important for immune suppression and tolerance , it has also been reported to be involved in pathogenetic mechanisms of various diseases . pd-1 and pd - l1 are single - pass transmembrane molecules expressed on the cell surface , which belong to the b7 family . their expression is induced by immune - activating stimuli and is understood as a negative feedback mechanism to suppress excessive immune reactions and let immune responses cease . when the receptor pd-1 is bound by its ligand , src homology 2-domain - containing tyrosine phosphatase 2 ( shp-2 ) and shp-1 are recruited to immunoreceptor tyrosine - based inhibitory motif and immunoreceptor tyrosine - based switch motif in the intracellular region of pd-1 and suppress antigen receptor signaling mediators . this process results in reduced production of cytokines , such as interferon ( ifn)- and interleukin ( il)-2 , reduced cell proliferation and suppressed immune cell activation . in addition to the close involvement in homeostasis of the living body as summarized above , recent studies have shown that immunosuppressive factors , pd-1 and pd - l1 , are also involved in immunosuppression in cases of tumors and chronic infections . while generally not highly expressed in normal tissues , pd - l1 is expressed at a high rate in tumor tissues in melanoma , lung cancer , colorectal cancer and ovarian cancer , and has been demonstrated to be involved in immune evasion by tumors . in patients with renal cell cancer and gastric cancer , pd - l1 has been shown to be an important prognostic determinant ; also , the disease progression is faster , and the mortality is higher in patients with tumors expressing pd - l1 than in patients without detectable pd - l1 expression [ 51 , 53 ] . in addition , pd - l1 expression has also been reported for other tumors including breast cancer , pancreatic cancer and bladder cancer , and pd-1 expression in tumor - infiltrating lymphocytes has been confirmed in many types of tumors including melanoma , lung cancer and intrahepatic bile duct cancer . these findings have revealed the importance of the pd-1/pd - l1 pathway as an immunosuppression mechanism in a variety of tumors . based on the series of studies on pd-1/pd - l1 in tumor diseases , therapeutic means targeting this pathway are being developed . specifically , these new therapeutics are biopharmaceuticals based on anti - pd-1 antibodies , anti - pd - l1 antibodies or recombinant proteins that inhibit the pd-1/pd - l1 pathway . these biopharmaceuticals have been reported to show good anti - tumor effects regardless of the tumor type and are attracting growing attention as a new , promising class of anti - tumor therapy . fully humanized anti - pd-1 and anti - pd - l1 antibodies have already been produced and are actively tested as therapeutic agents in clinical studies ( trials ) , with good anti - tumor effects continuing to be reported in patients with melanoma , lung cancer , renal cell cancer and some other types of cancer . in melanoma , which has a very poor prognosis , clinical trials conducted in japan and the united states have reported not only the suppressed growth of cancer cells , but also complete remission in some cases . on september , 2014 , the anti - pd-1 antibody was finally released by ono pharmaceutical as a new anti - tumor therapeutic agent , and it has become a pioneer for innovative immune checkpoint inhibitors ( ono pharmaceutical : https://www.opdivo.jp/contents/action/ ) . this immune checkpoint - targeted immunotherapy was selected as breakthrough of the year 2013 by the journal science as a global revolutionary technology . currently , merck , roche and other major pharmaceutical companies around the world are accelerating their efforts to develop similar antibody - based drugs ; this class of therapeutics is gaining so much momentum and attention that the conventional concept of anti - tumor therapy is being overturned . the causative factors of bovine leukemia can be divided into viral and non - viral . non - viral bovine leukemia is sporadic and can be subdivided into calf type , thymic type and cutaneous type involving unknown causes . on the other hand , enzootic bovine leukemia , which is caused by blv , accounts for the vast majority of cases of bovine leukemia , and its prevalence continues to increase . blv infections are latent in the aleukemic ( al ) state , but can emerge as persistent lymphocystosis ( pl ) with non - malignant polyclonal expansion of cd5 b - cells that predominantly harbor blv provirus and rarely as malignant b - cell lymphoma in various lymph nodes after long periods of latency . the progression of enzootic bovine leukemia is accompanied by marked suppression of cell - mediated immunity [ 8 , 9 , 16 ] , and as the pathogenetic mechanism remains unknown , there are no effective vaccines or therapeutic methods available , meaning that affected animals eventually die . bovine leukemia was designated as a communicable disease obligated to notify under the act on domestic animal infectious diseases control when it was revised in 1997 . in 2015 , 2,896 cases of bovine leukemia were reported ( of which the largest number of 494 cases occurred in hokkaido ) , representing the disease reported in a greater number than any other bovine disease required to monitor by the act ( http://www.maff.go.jp/j/syouan/douei/kansi_densen/kansi_densen.html ) . this number is 29.3-fold the number of cases reported in 1998 ( 96 affected animals ) , indicating that the increase has not yet been halted . requests for urgent measures against this disease have been voiced very frequently by people practicing veterinary medicine and animal husbandry . however , a large - scale survey conducted by the national institute of animal health using specimens collected from 2009 to 2011 showed an approximate blv - positive rate of 35% , demonstrating the difficulty of implementing any project to select and replace infected cattle . comparative analyses of blv - infected cattle by clinical condition have suggested that animals with a high viral load and persistent lymphocytosis are at a high risk of disease onset , often serving as infection sources , have a high risk of vertical transmission and have increased levels of cd4cd25foxp3 treg cells , showing increased susceptibility to opportunistic infections due to transforming growth factor - beta produced by treg cells , which reduces the production of interferon - gamma and tumor necrosis factor - alpha by cd4 t cells and impairs cellular immunity mediated mainly by the cytotoxic activity of nk cells [ 28 , 48 ] . moreover , the proliferative ability of lymphocytes in response to blv was also significantly reduced in cattle with persistent lymphocytosis , and lymphocytes were found to produce reduced levels of anti - viral cytokines , such as ifn- , il-2 and il-12 [ 13 , 18 ] . therefore , we analyzed the expression of pd-1 and its ligand , pd - l1 . results showed that the pd-1 expression in cd4 and cd8 cells and the pd - l1 expression in virus - infected b cells increase as the disease progresses . furthermore , pd - l1 expression negatively correlated with the ifn- expression level , an indicator of immunosuppression , while positively correlating with leukocyte count , virus titer and provirus level . antibodies to bovine pd-1 and pd - l1 were established , and the results from pd-1 and pd - l1 binding inhibition assays confirmed that they activated anti - viral immunity and that the increase in ifn- production positively correlated with the pd-1 expression rate on cd4 t cells . and also , recombinant bovine pd - l2 ( pd - l2-ig ) significantly enhanced ifn- production from virus antigens - stimulated pbmcs derived from blv - infected cattle . interestingly , the pd - l2-ig - induced ifn- production was further enhanced by treatment with anti - bovine pd-1 antibody . these results indicated that the pd-1/pd - l pathway constitutes a part of the immunosuppressive mechanism in bovine leukemia ( table 2table 2.change of immune inhibitory molecules in the cause of bovine leukemia virus infectionreceptor / liganduninfectedinfected(disease status)referencesalpllymphomapd-1/pd - l1///// lag-3/mhcclass ii////n.d [ 17 , 41]tim-3/gal-9///// ctla-4/cd80 , cd86/n.d/n.d/n.d/n.dn.d n.d : not demonstrated . ) we also analyzed immunosuppressive receptors other than the pd-1/pd - l1 pathway , such as lymphocyte - activation gene 3 ( lag-3 ) [ 17 , 41 ] , t - cell immunoglobulin and mucin domain - containing protein 3 ( tim-3 ) , and cytotoxic t - lymphocyte antigen 4 ( ctla-4 ; cd152 ) expressed in antigen - specific lymphocytes and found that expression levels of lag-3 , tim-3 , ctla-4 and their ligands on lymphocytes increased as the disease progressed and anti - viral immunity was activated in binding inhibition assays , as was the case for pd-1 ( table 2 ) . currently , clinical studies of these drugs are being carried out at the hokkaido university and other institutions . although there are many diseases in cattle involving immune abnormalities ( impairments ) , the mechanisms underlying these diseases remain unknown . our previous analyses have shown that immunosuppressive factors , such as pd - l1 , are also involved in immune suppression seen in diseases other than bovine leukemia , namely johne s disease and bovine anaplasmosis . furthermore , recent evidence suggests that immunosuppressive factors , such as pd-1 , are involved in the reduced immune function in chronic infectious diseases , such as mastitis , bovine mycoplasmosis and bovine tuberculosis ( manuscript in preparation ) . our laboratory has been working on the development of a blv vaccine for many years . it is based on a vaccine antigen that was found to be promising in in vitro studies . however , although we tried various procedures , the vaccine did not prevent infection or even disease onset despite the fact that effector cells were present in vivo ( data not shown ) . results from the present analysis suggested that the virus s immune evasion mechanism for lymphocyte exhaustion might be related to the ineffectiveness of the vaccine . future measures against chronic infectious diseases will require the development of a new , pre - emptive control method that targets this formidable immune evasion mechanism . to achieve this goal , results from more detailed analyses of immune exhaustion in other chronic infections are awaited . for use in humans , several immune checkpoint - targeting biopharmaceuticals have been successively developed , including those described above , and they are being actively tested in clinical trials . in the future , it is anticipated that they will be applied to veterinary medicine and animal husbandry , including diseases in cattle .

recently , dysfunction of antigen - specific t cells is well documented as t - cell exhaustion and has been defined by the loss of effector functions during chronic infections and cancer in human . the exhausted t cells are characterized phenotypically by the surface expression of immunoinhibitory receptors , such as programmed death 1 ( pd-1 ) , lymphocyte activation gene 3 ( lag-3 ) , t - cell immunoglobulin and mucin domain - containing protein 3 ( tim-3 ) and cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) . however , there is still a fundamental lack of knowledge about the immunoinhibitory receptors in the fields of veterinary medicine . in particular , very little is known about mechanism of t cell dysfunction in chronic infection in cattle . recent our studies have revealed that immunoinhibitory molecules including pd-1/ programmed death - ligand 1 ( pd - l1 ) play critical roles in immune exhaustion and disease progression in case of bovine leukemia virus ( blv ) infection , johne s disease and bovine anaplasmosis . this review includes some recent data from us .

all bacterial isolates related to the outbreaks ( 1 per patient ) were obtained from clinical samples . the strains were identified phenotypically by rapid i d 32 strep ( biomrieux , marcy letoile , france ) , which yielded profile 22025001100 ( leuconostoc spp . ( biolog , hayward , ca , usa ) ( 98% , t = 0.708 ) . the results were confirmed by 16s rdna sequence analysis , by a previously reported method ( 9 ) , and the analysis of 1,4201,500 bp showed 99% probability that the species were lm , when compared with genbank database sequences . antimicrobial drug susceptibility was determined by microdilution , with dademicroscan system ( baxter health care , west sacramento , ca , usa ) , and mics were confirmed by e - test ( ab biodisk , solna , sweden ) . for interpretation of antimicrobial drug susceptibility , clinical and laboratory standards insitute criteria ( 10 ) for leuconostoc spp . or when appropriate streptococcus spp . the antimicrobial drug susceptibility profiles were almost identical for all genotypes and showed susceptibility to penicillin and gentamicin ( mics of 0.25 mg / l and < 2 mg / l , respectively ) and to levofloxacin , tetracycline , quinupristin - dalfopristin , linezolid , daptomycin , erythromycin , clindamycin , and chloramphenicol . a pulsed - field gel electrophoresis ( pfge ) technique was used to assess the possibility of a clonal relationship among the 48 lm strains . genomic dna was extracted , restricted with apai , and electrophoresed with chef - driii apparatus ( bio - rad laboratories , richmond , ca , usa ) . no differences in the band profile were observed among the 42 strains of the first outbreak ( genotype 1 ) . analysis of the 6 strains isolated in the 2006 outbreak showed different dna band patterns from those corresponding to genotype 1 ( figure 2 ) . of the 6 isolates , 4 shared the same genotype , designated genotype 2 , whereas the remaining 2 isolates showed 2 new genotypes ( genotypes 3 and 4 ) . one lm strain , isolated from the parenteral nutrition catheter of a patient involved in the 2006 outbreak ( genotype 2 ) , was identical to those isolated from blood of the same patient ( figure 2 ) and from 3 other patients involved in the 2006 outbreak ( data not shown ) . mw , molecular weight marker at indicated sizes ; lines 1 to 9 , representative lm isolates from the first outbreak ( genotype 1 ) ; lines 10 , 11 , lm isolates obtained from parenteral nutrition catheter and blood from the same patient ( genotype 2 ) and identical to those from 3 different patients infected in the second outbreak ( data not shown ) ; lines 12 , 13 , lm isolates from 2 different patients involved in the second outbreak ( genotypes 3 and 4 ) most of the 42 patients infected with lm genotype 1 in the first outbreak displayed severe underlying diseases ( table 1 ) ; 9 of the patients died , and 3 of the deaths ( 7.1% ) were directly related to the leuconostoc infection . the bacterial isolates were isolated from blood ( 52.1% ) , catheter ( 21.8% ) , or both ( 26.1% ) . to assess risk factors related to acquisition of lm strains , we performed a case control study . control - patients ( n = 61 ) were randomly selected among remaining patients with another nosocomial infection caused by a non leuconostoc spp . microorganism isolated from a catheter , blood , or both , who were admitted to the same department and at the same time as the patients defined as case - patients . sd , standard deviation ; or , odds ratio ; ci , confidence interval ; ns , variable did not meet criterion for remaining in the multivariate model . predictor variables with p<0.10 in univariate analysis were included in the multivariate model to enable simultaneous adjustment . any process that modifies the gastrointestinal barrier ( inflammation , atresia , resection , obstruction ) . nosocomial infection criteria were those previously established by the centers for disease control and prevention ( atlanta , ga , usa ) ( 12 ) . a multiple logistic regression model was developed to identify potential independent factors associated with acquisition of lm strains . predictor variables with p<0.10 in univariate analysis were included in the multivariate model to enable adjustment . statistical analyses were conducted with spss 14.0 software ( spss inc . , chicago , il , usa ) . according to the multivariate analysis , previous infections ( 38.2% were bacteremias ) ( odds ratio [ or ] = 4.2 ) and parenteral nutrition ( or = 27.8 ) all case - patients received parenteral nutrition , with the exception of 2 , although they received enteral nutrition . parenteral nutrition is a putative source of the infection because all parenteral and enteral nutrition bags are prepared in the central hospital pharmacy and then distributed to the different medical units in the hospital . this possibility was further supported by 1 finding : pfge analysis of isolates obtained from a parenteral nutrition catheter connected to a patient during the second outbreak yielded the same genotype as the isolates obtained from blood from the same patient ( figure 2 ) and from another 3 physically separated , infected patients . the physical distance between these patients as well as the impossibility of retrograde displacement of the bacterial isolate from patient s blood makes it unlikely that the lm strain was acquired by contamination from the blood and indicates parenteral nutrition as the main source of lm transmission in the hospital outbreak . microbiologic controls of parenteral nutrition were reinforced during the second outbreak , and as stated , only 6 cases were detected . moreover , during the second outbreak , microbiologic analysis of environmental samples as well as samples from the digestive tract , skin , and throat of all patients involved did not yield any leuconostoc strains . parenteral nutrition controls performed in the hospital pharmacy department are now routinely assayed for lm isolation . since the last lm outbreak in november 2006 , that 42 lm isolates from the first outbreak shared the same genotype and 4 of 6 isolates in the second outbreak also shared the same ( another ) genotype rules out the possibility of endogenous infections among patients and suggests a common source for each outbreak . the occurrence of cases in patients in areas that were physically separated rules out the possibility of indirect patient - to - patient spread through the hands of healthcare workers or contaminated hospital equipment ( different departments do not share healthcare workers and equipment ) . enteral and parenteral nutrition has previously been described ( 13,14 ) as a risk factor associated with leuconostoc - infections , although no microbiologic evidence was provided in any of the studies . with regard to previous infections in the multiple logistic regression model , this may be related to the alteration of the immune system caused by the microorganism that caused the previous infections . two previous reports have described hospital transmission of leuconostoc spp ( 7,15 ) ; both outbreaks affected a small number of patients , and no epidemiologic studies were conducted to clarify the genetic relationship among the bacterial strains involved or the source of the nosocomial infection . although up to 88 cases of leuconostoc infection have been reported in the scientific literature in the past 25 years , these cases may not be comparable to those reported here , the largest nosocomial outbreak caused by leuconostoc spp . worldwide . this outbreak highlights the importance of lm as an emerging hospital pathogen in patients with underlying diseases and in whom parenteral nutrition may be the source of the initial infection and its spread . every infection with lm could be a yet undetected outbreak and should result in an investigation that focuses on parenteral nutrition or products manufactured in a centralized hospital pharmacy .

from july 2003 through october 2004 , 42 patients became infected by strains of leuconostoc mesenteroides subsp . mesenteroides ( genotype 1 ) in different departments of juan canalejo hospital in northwest spain . during 2006 , 6 inpatients , also in different departments of the hospital , became infected ( genotypes 24 ) . parenteral nutrition was the likely source .

the work of korean medical radiological technologists is divided into three areas : diagnosis , treatment , and nuclear medicine1 . the department of diagnostic radiology ( department of radiology ) is in charge of general x - ray examinations , magnetic resonance imaging , ultrasonography , and computerized tomography scans for conducting precise examinations of the inside of the body . the department of radiation oncology deals with cancer treatment , using high energy radiation to kill cancer cells ; the department of nuclear medicine uses radioactive isotopes with cutting - edge technologies for diagnosis and treatment of certain cancers2 . to perform these responsibilities , radiological technologists are constantly required to perform physical work , such as lifting adult patients who can not move adequately by themselves , pushing or pulling to move heavy equipment , and continuous work with a computer keyboard or mouse3 , 4 . during these activities , radiological technologists are sometimes at risk for musculoskeletal disorders5 . the acts of moving patients who can not move well by themselves and pushing and pulling heavy equipment , are known to be major causes of lower back pain , while the repetitive movements performed when utilizing computers for functions , such as use of the picture archiving communication system ( pacs ) and the order communication system ( ocs ) , are causes of upper extremity musculoskeletal disorders6 . in these ways , the exposure of radiological technologists to the risks of work - related musculoskeletal disorders can be said to be increasing . however , because awareness of occupational safety for radiological technologists has been concentrated on exposure to radiation and on the necessary defense against radiation exposure , the issue of safety in relation to work - related musculoskeletal disorders has tended to be neglected . because the significance of work - related musculoskeletal disorders has generally come to be recognized , a law was established in korea in 2005 that aims to prevention and control work - related musculoskeletal disorders . based on this legislation , physical therapists have worked in different industrial fields to analyze work movements and organize educational programs to prevent work - related musculoskeletal disorders . thus , the working area of physical therapists has been expanded to include this area7 . however , data that can be utilized by physical therapists in analyzing work - related musculoskeletal disorders is rarely reported , because ergonomic assessment is conducted only in certain types of jobs where there is high labor intensity . in general , hospitals are special workplaces in which the concept of medical personnel is stronger than the concept of workers , and the concept of medical practice is stronger than the concept of labor or work . however , since hospital workers are also workers who conduct physical activities using their bodies , they are also exposed to the risk of work - related musculoskeletal disorders . in particular , radiological technologists are exposed to higher levels of the risks relating to musculoskeletal disorders because they are forced to use inconvenient working postures due to the nature of their work , such as manually handling adult patients , frequently exceeding 60 kg in weight , without any generalized mechanical assistance . in these situations , they are required to adjust their own postures to accommodate the positions and weights of their patients8 . hospital workers are repeatedly exposed to the risk of work - related musculoskeletal disorders , and frequently exacerbate the problem because they do not recognize , or are indifferent to the problem . to prevent and effectively control musculoskeletal disorders , the potential disorders should first be accurately diagnosed and classified , and then their scales should be evaluated6 . the aim of this study was to analyze , through ergonomic analyses , those motions most used by radiological technologists that can cause musculoskeletal disorders so that the results can be utilized as the basis for diagnosis , classification and prevention of occupational musculoskeletal disorders in radiological technologists . in this study , designed to assess the ergonomic risks of radiological technologists , a field survey was conducted on august 1 and august 2 , 2013 , at a hospital located in gangwon - do , republic of korea . this was a university hospital in which all three areas of radiological technologists work were performed . the purpose , method , and research process of the study were explained to the general manager of the department of radiology , and the field survey was conducted after getting his consent . the study s objectives and procedure were explained to the study subjects , and their consent was obtained . the study was approved by the ethics committee of the kangwon national university hospital institutional review board . the work processes performed by the radiological technologists were then videotaped from the left side , right side , and front so that the movements of their joints were clearly visible . based on this data , the parts of the videotapes judged to demonstrate working postures that would impose the heaviest burdens on the technologists bodies were each captured and analyzed three times . the captured working postures were assessed and analyzed using the following ergonomic assessment methods : the rapid entire body assessment ( reba ) , the rapid upper limb assessment ( rula ) , the national institute for occupational safety and health lifting equation ( nle ) , and the strain index ( si ) . three professors of industrial safety conducted the analysis to preserve the objectivity of the study . the professors assessed the scores through discussion and analyzed the different categories of working posture . reba was developed for the purpose of analyzing the degree of exposure of the body to physical burden and the risk factors in the service industries , including nursing , cleaning , and other jobs with dynamic and unpredictable work postures . different factors , including repetition , static work , working postures , and duration of working hours , among others , are evaluated in relation to two groups , group a ( trunk , neck , leg ) and group b ( humerus , shoulder , forearm , wrist ) , using recorded video and direct observation . reba categorizes the danger levels into 5 stages using a grading system with a 15-point scale and suggests appropriate safety measures9 . rula enables convenient and swift assessment of the workload imposed by working postures by focusing on an upper limb analysis , whether in assembly work , service work , meat processing , dental clinic work , or video display terminal tasks , especially those involving the shoulders , wrists , neck , and other upper body parts . it divides the body parts into group a ( upper arm , lower arm , wrist ) and group b ( neck , trunk , legs ) and assesses them in terms of the number of movements , the static muscle work , force , and working postures as the workload parameters . rula categorizes the danger levels into 4 stages using a grading system with a 7-point scale and suggests appropriate safety measures10 . the ovako working posture analysing system ( owas ) is an assessment technique developed by the ovako steel company to define and assess workers working postures in relation to their suitability for manual handling of heavy objects in the course of their work . working postures are considered in relation to four items : the waist , the upper extremities , the lower extremities , and the weight of the object handled . the effects of these factors on the musculoskeletal system are divided into four levels , and the results are recorded11 . the si is a method for evaluating jobs to determine whether they expose workers to increased risk of developing musculoskeletal disorders of the distal upper extremity ( due)12 . the due is defined as the elbow , forearm , wrist , and hand . musculoskeletal disorders of the due include specific diagnoses ( e.g. , epicondylitis , peritendinitis , tendon entrapment at the wrist or finger , and carpal tunnel syndrome ) and less specific symptomatic conditions related to the muscle - tendon units of the due . the si uses six task variables to describe hand exertions : intensity of exertion , duration of exertion , exertions per minute , hand / wrist posture , speed of work ( how fast ) , and duration per day . among these factors an si higher than 5 indicates the occurrence of potential danger to the upper extremity . an si below 3 indicates safety , while an si above 7 is considered very dangerous . this study was conducted to analyze the working postures of radiological technologists and to utilize the results for the prevention and treatment of their musculoskeletal disorders . accordingly , their postures when moving patients , when pushing or pulling an apparatus , when conducting ultrasonography , and when handling a mouse for mri were analyzed . individual postures were assessed and scored according to the waist , neck , leg , shoulder , elbow , wrist , wrist twisting , load , handling , and activity , and the assessment ratings were analyzed . moving a patient was divided into a starting point and an ending point , and the postures at each point were assessed . at the starting point , in both the case when the radiological technologist was positioned on the right side of the patient and the case when the radiological technologist was positioned on the left side of the patient , the assessment rating was shown to be improve immediately therefore , the work of moving patients was shown to impose great burdens on the musculoskeletal system . burdens on the waist and shoulder were shown to be high , and of all the assessments , the burden on the waist was shown to be the highest ( table 1table 1.working postures for patient liftingworking posturerisk factorrebaowasrula(rt)rebaowasrula(lt)patient lifting starting pointwaist546546neck233233leg252141shoulder ( upper arm)414414elbow ( forearm)2323wrist2222wrist twisting1201load / force233233handle33activity2121as13481248ariiiiiipatient lifting ending pointwaist546445neck233233leg252252shoulder ( upper arm)414414elbow ( forearm)2323wrist2223wrist twisting1212load / force223233handle33activity2121as13481248ariiiiiireva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately ) . reva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately in assessing the posture during mouse handling for mri , the right hand and left hand were assessed separately . the assessment ratings for both the left and right hands were commonly shown to be improve soon ( reba ) or improve immediately ( rula ) . the mouse work required for mri was shown to impose large burdens on the neck , the shoulder , and the wrist ( table 2table 2.mouse handling work postureworking posturesrisk factorright handleft handrebarularebarulamouse handlingwaist3434neck3535leg1111shoulder ( upper arm)4444elbow ( forearm)2323wrist2334wrist twisting1212load / force0000handle11activity2121as108108arsisireva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately ; s , improve soon . ) . the sis indicated that continuously handling a mouse with the right hand placed the hand in a little in danger and that the level of risk for the left hand was uncertain . in the case of the left hand , work done with severe flexion of the wrist and without any support was shown to be particularly problematic ( table 3table 3.mouse handling work posture strain indexworking posturehand sideintensity ofexertionduration ofexertion ( % of cycle)effortsper minutehand / wristposturespeed of workduration of taskper day ( hours)si scoremouse handlingright3.00.51.51.51.51.05.06 a little dangerousleft3.00.51.02.01.01.03.00 uncertain ) . ultrasonography work postures were assessed after dividing them into three levels , from cases in which the distance between the radiology technologist and patient was the shortest to cases in which the distance was the longest . the assessment ratings for reba were shown to be should be improved and improve soon and that for rula was shown to be improve immediately . the results showed that when the distance between the radiology technologist and the patient was longer , the burden on the neck and shoulder was greater ( table 4table 4.ultrasonography work postureworking posturesrisk factorposture 1posture 2posture 3rebarularebarularebarulaposture 1 ( close to the patient)waist212133neck242436leg313531shoulder ( upper arm)333133posture 2 ( middle)elbow ( forearm)121223wrist122223wrist twisting121212load / force000000posture 3 ( patients with distant)handle1 - 1 - 1-activity111111as6898108arshould be improved4 . ireva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately ; s , improve soon . ) . the apparatus pushing work posture assessed was the radiological technologist s posture when pushing a heavy portable radiation apparatus to move it . the reba showed an assessment rating of should be improved and the rula showed an assessment rating of improve immediately . the load / force burdens were shown to be high ( table 5table 5.apparatus pushing working postureworking posturerisk factorrebarulapushing apparatuswaist22neck11leg22shoulder ( upper arm)22elbow ( forearm)22wrist11wrist twisting01load / force23handle0-activity11assessment score78assessment ratingshould be improvedimprove immediately ) . reva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately ; s , improve soon . reva , rapid entire body assessment ; owas , ovako working posture analysing system ; rula , rapid upper limb assessment ; as , assessment score ; ar , assessment rating ; i , improve immediately ; s , improve soon . this study was conducted to analyze the working postures of radiological technologists so that the results can be utilized for the prevention and treatment of musculoskeletal disorders . in this study , burdens on radiological technologists waists were shown to be high when they were moving patients for a ct scan . this result is supported by studies indicating that the lower back pain of radiological technologists is closely related to the frequency with which they assist in moving patients13 . the table for a ct scanner is just wide enough for a patient to lie on and is positioned at a height that is close to the hip joints of the radiological technologists . therefore , when moving patients , they use force after bending substantially at their waists and stretching their arms out far . as the trunk is bent forward , the load on the lumbar region increases markedly14 . in addition , since the movements are made in the limited space of the ct scan room and the height of the table is fixed , the radiological technologist can not take up a position that would distribute his or her weight to the lower extremities . therefore , these movements can be understood to impose large burdens on the waist . as a measure to prevent the musculoskeletal disorders that can occur when moving patients , making the height of the table that the patient lies on adjustable so that the radiological technologists can also use their lower extremities when moving heavy patients and thereby distribute the force to their lower extremities although there are diverse causes of lower back pain , manual transporting work , such as pushing , pulling , transporting , loading , and unloading , is an important cause15 . workers who are to engage in handling heavy objects should be adequately educated and trained in correct methods for lifting heavy objects , methods for lowering , methods for moving , methods for loading , working postures , exercises to prevent lower back pain , and other working methods before they are assigned to work in order to prevent lower back pain16 . this education and training should also be provided to radiological technologists to prevent lower back pain in them . in addition , habituation to the use of movement assistance systems , such as the easy - trans used when moving from sitting postures on a wheelchair or chair to a bed or when moving from a bed to a wheelchair , can also be a good way to reduce the burden on the waist and lower back . the assessment of postures during mouse handling for an mri scan showed that large burdens were imposed on the neck , shoulder , and wrist . repetitive keyboard entries , continuous movements in fixed postures ( static posture ) , inappropriate working postures , and prolonged periods of work cause pain in the neck , shoulder , elbow , wrist , and fingers , together with health hazards , such as numbness and tingling . among hospital workers , radiological technologists show a high incidence of vdt syndrome , thereby supporting this study result . therefore , to prevent not only vdt syndrome2 but also musculoskeletal disorders , a mouse pad with a wrist rest should be used for the right hand , and a support should be prepared for the left arm . in the case of ultrasonography working postures , larger burdens on the leg and neck were found when the patient s examination sites were located further away and the radiological technologist s upper extremities and arms were stretched further out . while maintaining a constant pressure , the radiological technologist is required to stretch his upper extremities out far while watching the computer monitor of the ultrasonography equipment . due to this position , when the patient s examination sites are located further away , the neck and trunk must be twisted further , thereby increasing the burden on the neck . therefore , the location of both the ultrasonography equipment and the patient should be arranged appropriately , and the computer monitor for the ultrasonography equipment should be situated at an appropriate level . the assessment of working postures when pushing a portable radiation apparatus showed that burdens on the musculoskeletal system increased as the weight of the apparatus being pushed by the radiological technologist increased . therefore , measures to reduce the weight of portable radiation apparatuses should be sought . among the radiological technologists in the department of diagnostic radiology the largest number of them reported pain in the lower extremity , followed by pain in the shoulder , hand , wrist , waist , neck , and elbow in order of precedence , and a higher ratio of females reported symptoms compared with males17 . in a study conducted by lee jin et al . , symptoms appeared most frequently in the shoulder , followed by the waist , neck , and leg / foot in order of precedence , and a slightly higher degree of fatigue was shown . the present study also showed that risks to the shoulder and waist were high , while the risks to the wrist and leg appeared to be highest , as was shown in the study of lee13 . although some differences were shown between the present study and a study conducted by kim , the differences are considered attributable to the fact that the study conducted by kim was limited to radiological technologists . radiological technologists are stressed due to the high tension of their work , and the muscles , blood vessels , and nerve tissues in their necks , shoulders , arms , hands , waists , legs , and knees are damaged or affected by their hard physical work , such as standing for most of their working hours , repetitive movements including waist flexion , and helping patients to move , together with static motions4 . although the musculoskeletal disorders of radiological technologists occur in various regions of their bodies , they occur most frequently in the shoulder and the lumbar region . however , as the issue of occupational safety of radiological technologists has been focused on possible exposure to radiation and defense against radiation exposure , the issue of safety in relation to work - related musculoskeletal disorders has been neglected , and education and interest regarding the prevention of musculoskeletal disorders has been insufficient . therefore , hospitals need to be educated regarding the concept of musculoskeletal disorders , and this needs to followed by education regarding prevention through use of correct working postures , exercise methods for radiological technologists .

[ purpose ] the aim of this study was to analyze , through ergonomic analyses , those motions most used by radiological technologists that can cause musculoskeletal disorders . [ subjects and methods ] the subjects were 7 radiological technologists with work experience in hospitals for more than 5 years . for the analysis of working postures , we simulated the work posture of radiological technologists when moving patients , when pushing or pulling an apparatus , when conducting ultrasonography , and when handling a mouse for mri were analyzed . [ results ] in this study , the burdens on the radiological technologists waists were shown to be high when they were moving patients for a ct scan . during mouse handling for an mri scan , large burdens were imposed on the neck . in the case of ultrasonography working postures , larger burdens on the leg and neck were found when the patient s examination sites were located further away . the assessment of working postures when pushing a portable radiation apparatus showed that burdens on the musculoskeletal system increased as the weight of the apparatus increased . [ conclusion ] the musculoskeletal disorders of radiological technologists occur in various regions of their bodies but occur most frequently in the shoulder and the lumbar region . therefore , hospitals need to be educated regarding the concept of musculoskeletal disorders .

blood is an invaluable , life - sustaining fluid . without a sufficient amount of blood , the cells of the human body could not receive adequate oxygen and nutrients they need to survive . large volume of blood could be lost as a result of numerously varying serious conditions such as road traffic accidents , obstetric and gynecological hemorrhages , surgery , trauma , chemotherapy , and long - term therapies as well as anemia of medical or hematologic conditions or cancer . because of these blood transfusion is considered as an integral and essential element of a health care system . besides , blood transfusion is one part of complex medical and surgical interventions which improves the life expectancy and life quality in patients with a variety of acute and chronic conditions . therefore , blood transfusion is now considered as an indispensable component of medical management of many diseases . blood donation is philanthropic deed in which the blood of a healthy person had been drawn voluntarily for the purpose of transfusion . the donated blood can be life - saving for individuals who have lost large amounts of blood because of serious accidents , as well as for individuals who have become severely anemic or have very low platelet counts and certain hematological disorders such as leukemia . besides , children being treated for cancer , premature infants , and children having heart surgery need blood and platelet transfusions to survive . world health organization ( who ) recommends countries to focus on young people to achieve 100% nonremunerated voluntary blood donation by 2020 . it also recommends that all countries should be self - sufficient in all blood products and that all blood donation should be voluntary , anonymous , and nonremunerated . according to its 2011 report , 107 million blood donations are collected globally ; approximately half of these are collected in the high - income countries , home to 15% of the world 's population . blood donation rate in high - income , middle - income , and low - income countries was 39.2 , 12.5 , and 4.0 donations per 1000 population , respectively . in low - income countries , up to 65% of blood transfusions are given to children under five years of age , whereas , in high - income countries , the most frequently transfused patient group is over 65 years of age , accounting for up to 76% of all transfusions . compared to the 2004 report , 7.70 million blood donations incensement was noticed from voluntary unpaid donors in 2011 . however , majority of countries still collect more than 50% of their blood supply from replacement or paid donors . about 234 million major operations are performed worldwide every year ; 63 million people undergo surgery for traumatic injuries , 31 million for treating cancers , and another 10 million for pregnancy - related complications . for all of these procedures , moreover , the demand of blood for patient management has been growing dramatically due to the sophistication and advancement of clinical medicine . however , the demand and supply have not yet balanced ; the demand is escalating . despite recommendations that all blood donations should be voluntary and nonremunerated , replacement and paid donors are common throughout sub - saharan african countries . surprisingly , 38 african countries collected fewer than 10 donations per 1000 people . there have been gross inadequacy and inequity in access to blood safety in who african region [ 7 , 8 ] . concurrently , in sub - saharan african countries , the need for blood transfusions is high because of maternal morbidity , malnutrition , and a heavy burden of infectious diseases such as malaria . in ethiopia , the national requirement for blood in ethiopia is between 80,000 and 120,000 units per year , but only 43% is collected . the percentage of blood collected from vbd and the average annual blood collection rate are extremely low . out of the 44 who african countries that reported the percentage of voluntary nonremunerated blood donation ( vnrbd ) , only 22% of blood is being donated by vbd in ethiopia ; the country is classified among countries that have least number of vbd ( group c , countries with < 50% vbd ) . adult population are potential source of great interest not only for the blood they could supply but also because of the information on the subject giving blood which could promote the spread of healthy lifestyles and acquisition of greater awareness about one 's own health and contribute to the development of a mature , responsible , and civic attitude . voluntary , nonremunerated blood donations are the cornerstone of a safe adequate supply of blood and blood components [ 12 , 13 ] . thus , the objective of this research was to assess knowledge , attitude , and practice towards blood donation among adult population in gondar town , northwest ethiopia . community based cross - sectional study was conducted in gondar town , northwest ethiopia , from february to may 2015 . the source populations were all adults who were residing in study area at least for 6 months and who were available during data collection period . those adults who were critically ill and had mental problems were excluded from the study . the independent variables were sociodemographic variables like sex , age , educational status , marital status , religion , and self - perceived health status . single population proportion formula , [ n = ( z/2)p(1 p)/d ] , was used to calculate the sample size . due to the lack of published information showing the knowledge , attitude , and practice of blood donation in this particular study area , we took 50% to get the maximum sample size by considering 95% confidence interval , marginal error ( d ) of 5% , and design effect of 2 . then , the final sample size was determined to be 768 . in the first stage of the sampling , three administrative areas ( subcities ) , lideta , maraki , and gebriel , were selected by using simple random sampling technique from the total 12 subcities . in the second stage of sampling , sanita ketena from lideta subcity , ketena two from maraki subcity , and , systematic sampling technique was employed to select households from each of the ketenas / kebeles . the numbers of households sampled from the selected ketenas and kebeles were determined using proportionate - to - population size . there were a total of 4603 households in three selected kebeles / ketenas : 1800 in sanita ketena of lideta subcity , 1960 in ketena two of maraki subcity , and 843 in kebele 14 of gebriel subcity . the interval ( k ) value was calculated for each selected kebele / ketena by dividing the total households in each selected kebele / ketena to the corresponding proportional sample size calculated for each ketena / kebele . . then other households were selected at every kth interval . whenever more than one eligible adult was found in the same selected household , only one of them was chosen using the lottery method for interview . in the case no eligible candidate was identified in a selected household or the selected household is closed even after revisit , the sampling process continued to the next household in the clockwise direction until getting an eligible person . knowledge about blood donation was assessed using 13 general questions which are deemed to be known by general population like place of blood donation , importance of blood donation , and eligibility for blood donation . each response was scored as 1 for correct response and 0 for incorrect response . knowledge scores for individuals were calculated and summed up to give the total knowledge score . participants who correctly responded to more than 50% of knowledge assessing questions were considered as having adequate knowledge about blood donation , whereas those who scored < 50% were considered as having inadequate knowledge about blood donation . similarly , 14 attitudes related questions were asked , and the responses of each question were scored as 1 for correct response and 0 for incorrect response . the attitude scoring ranges from 14 ( largest ) to 0 ( smallest ) . attitude scores for individuals were calculated and summed up to give the total attitude score . participants who correctly responded to more than 50% of attitude assessing questions were considered as having good attitude towards blood donation , whereas those who scored 50% were considered as having poor attitude towards blood donation . the practice was assessed by asking about history of previous donation and the frequency of donation . the practice was scored from largest ( the number of times a donor donated previously ) to smallest 0 ( never donated before ) . the data were entered using epi info version 3.5.1 and then cleaned and analyzed using spss version 20 software package . data cleaning was carried out by running frequency of each categorical variable and cross tabulation of different categorical variables . descriptive results were summarized as percentage , means , and standard deviations and presented in table . the association of the independent variable with the categorical outcome variable was measured by calculating odds ratio with p value and 95% confidence interval using bivariate and multivariate logistic regression . all independent variables with p value less than 0.2 were included in the multivariate models to identify factors associated with knowledge , attitude , and practice towards blood donation . besides , the relationships between knowledge , attitudes , and practice scores were examined using bivariate correlation analysis . the research was conducted after ethical approval letter was given from research and ethical committee of school of biomedical and laboratory science , university of gondar . in addition , after explaining the importance of study , permission letter was taken from each of the kebeles / ketenas administrators , and an informed consent was obtained from each study participant . from a total of 768 participants , 430 ( 56% ) were male and 338 ( 44% ) were female . more than half of the participants ( n = 402 ( 52.3% ) ) were in the age range of 2025 years . about 354 ( 46.1% ) and 189 ( 24.6% ) of the study participants had attained or have been attaining secondary and higher education , respectively ( table 1 ) . from the total study participants , 436 ( 56.8% ) had adequate knowledge towards blood donation . majority ( n = 704 , 91.8% ) of the study participants heard the idea of blood donation previously . about 678 ( 88.3% ) study participants thought that the importance of blood donation is to save life , while 24 ( 3.1% ) of them believed that it is to get health assurance ( table 2 ) . more than three - fourths , 630 ( 82% ) , of the respondents had good attitude towards blood donation . nearly all , 741 ( 96.5% ) , of the participants thought that blood donation is important ( table 3 ) . less than one - quarter , 141 ( 18.4% ) , of the respondents had an experience of blood donation , while the rest of the participants , 627 ( 81.6% ) , never donated blood before . of those who donated before , 86 ( 61% ) were voluntary donors , while the rest 39% of them were replacement donors . the major reasons mentioned for not donating blood among nondonors were perception of not being fitted to donate blood ( 21.2% ) , lack of information on where , when , and how to donate blood ( 17% ) , fear of being anemic after blood donation ( 12.6% ) , and fear of health risk after donation ( 12.3% ) ( table 4 ) . in bivariate logistic regression , age , occupation , marital status , educational status , and self - perceived health status were significantly associated with adequate knowledge about blood donation , while , in multivariate logistic regression controlling confounders , secondary educational status ( aor = 2.28 ; 95% ci : 1.51 , 3.44 ) and higher educational status ( aor = 2.88 ; 95% ci : 2.01 , 4.12 ) were significantly associated with adequate knowledge towards blood donation ( table 5 ) . in bivariate logistic regression marital status , religion , and self - perceiver health status were significantly associated with attitude of the participants , while in multivariate logistic regression religion was the factor which was significantly associated with attitude towards blood donation ( table 6 ) . in bivariate logistic regression analysis , age , sex , religion , marital status , and self - perceived health status were statistically associated with blood donation practice of the respondents , while in multivariate logistic regression analysis , participant 's age , sex , religion , and self - perceived health status were found to be significantly associated with practice of blood donation ( table 7 ) . in addition , we had tried to assess the correlation between knowledge , attitude , and practice scores of the study participants . knowledge and attitude scores of the participants achieved significant but weak positive correlation ( r = 0.238 ; p = 0.01 ) . similarly , knowledge and practice scores of the participants had shown statistically significant positive correlation , even though it is weak ( r = 0.26 ; p = 0.01 ) . moreover , the attitude and practice scores of the participants had fair positive correlation ( r = 0.31 ; p = 0.01 ) . in this study , an attempt has been made to assess the level and factors associated with knowledge , attitude , and practice of adults on blood donation . from the total study participants , 436 ( 56.8% ) had adequate knowledge regarding blood donation . the result is higher than a study done in jordan aimed at investigating knowledge and attitude of blood donors and barrier concerning blood donation among 500 blood donors which reported that 28.6% of them had adequate knowledge . the possible reason for this discrepancy might be due to the difference in the sample size . in this study , more than three - fourths ( 88.3% ) of the participants knew that the importance of blood donation is to save life . the result was higher than a study conducted in democratic republic of congo among 416 participants to assess the knowledge , attitude , and practice of the general population showing that only 183 ( 44.1% ) of them responded that the importance of blood donation is to save life . the difference might be due to variation in sample size and also variation in age of the study participants . in our study , only adult age group , 2040 years old , were included , whereas in the study of democratic republic of congo participants were in the age range of 1865 years . in the current study , multivariate logistic regression showed that educational status was the only variable that significantly associated with the knowledge of participants . participants who attended or had been attending secondary education ( aor = 2.9 ; 95% ci : 1.51 ; 3.44 ) and higher education ( aor = 2.9 ; 95% ci : 2.01 ; 4.12 ) were more likely to have adequate knowledge towards blood donation . thus , as the level of education increases , participants ' knowledge towards blood donation also increases . majority 630 ( 82% ) of the study participants have good attitude towards blood donation . about 282 ( 36.7% ) of them had a perception that blood donation causes anemia . this result is in line with a study conducted in mekelle city in which 370 ( 45.9% ) of the study participants believed that blood donation causes anemia . in this study , religion was the only variable significantly associated with the attitude of the participants using multivariate logistic regression . those participants who were catholic and jewish ( aor = 0.16 ; 95% ci : 0.05 , 0.51 ) were less likely to have good attitude towards blood donation . being catholic and jewish reduces the attitude towards blood donation by 84% compared to being orthodox christian by religion . this needs further in - depth behavioral study to explore the reason why being catholic and/or jewish by religion reduces blood donation perception . in the current study , less than one - fourth , 141 ( 18.4% ) , of the study participants had the experience of blood donation . the result is in agreement with studies done in trinidad and tobago ( 18.8% ) and north central nigeria ( 22.6% ) . on the contrary , it is lower than studies conducted in saudi arabia ( 58.2% ) , iran ( 26% ) , and southern brazil ( 32% ) . in saudi arabia , iran , and southern brazil studies , the study participants were in the age of range of 1850 , in the age range of 1865 , and above 20 years of age , respectively . however , in our study , the study 's population were within the age range of 2040 years . probably , individuals with age above 40 become socially responsible , and they do have increasing tendency to donate blood as supported by zago et al . . among donors , the rate of previous blood donation is higher than study done in saudi arabia ( 26.4% ) . the result is in contrast to studies done in north central nigeria and saudi arabia in which replacement donors were more frequent than voluntary donors . more than half , 432 ( 68.9% ) , of nondonors stated wrong perception like fear of being anemic , fear of weight loss , fear of health problem , perception of not being fit , and lack of information on where , when , and how to donate blood as a major reason for not donating . this result is consistent with study conducted in trinidad and tobago . in current study , multivariate logistic regression showed that participant 's age , sex , religion , and self - perceived health status were significantly associated with the practice of participants . those participants in age ranges of 3135 years ( aor = 2.61 ; 95% ci : 1.6 ; 4.86 ) and 3640 years ( aor = 3.8 ; 95% ci : 2.0 ; 7.31 ) were more likely to donate blood as compared to participants in age range of 2025 years . the possible reason for this might be due to the fact that participants at age range of 3040 years are in late adulthood stage so that they are assumed to be socially proactive and donate blood . this had also been supported by study done in southern brazil which revealed that individuals in the age range of 3049 had higher tendency to be loyal blood donors . in this study , the result is in line with a study conducted in togo which showed that majority ( 61% ) of blood donors were male . males were two times more likely to donate blood compared to females ( aor = 1.7 ; 95% ci : 1.14 ; 2.54 ) . this is in agreement with the study done in yazd , iran , which reported that significantly higher proportions of men were donors compared to women . the possible reason for this difference with regard to donation practice between women and men might be related with knowledge difference . culturally , the society is male dominated ; and there is disparity in access to education between women and men in ethiopia . moreover , our data showed that significant difference was observed in knowledge score between women and men : men had higher score than women ( = 22.4 ; p value = 0.049 ) . in this study , study participants with excellent self - perceived health status were two times more likely to donate blood as compared with those with good self - perceived health status ( aor = 2.23 ; 95% ci : 1.4 ; 3.62 ) . people who feel they are healthy are more confident and suitable for donating blood . in this study , knowledge and attitude scores of the participants had shown significant positive correlation even if the correlation is weak ( r = 0.238 ; p = 0.01 ) . likewise , knowledge and practice scores of the participants had shown positive but weak correlation ( r = 0.26 ; p = 0.01 ) . this result is in line with a study which was conducted in the city of yazd to assess the level of knowledge , attitude , and practice regarding blood donation . meanwhile attitude and practice of the participants have fair ( r = 0.31 ; p = 0.01 ) positive correlation . thus , as the attitude of the participants increases the level of practice also increases . in the current study , the major reasons mentioned by nondonors for not donating blood were perception of not being fitted to donate blood , lack of information on where , when , and how to donate blood , fear of being anemic after blood donation , and fear of health risk after donation . even though the extent of the problem varies with race , sociocultural values , and socioeconomic status of the population on which the studies focused , the blood donation barriers reported by nondonors in our study are nearly in agreement with other studies [ 2426 ] . this problem needs massive public health advocacy about the importance and related risk of blood donation to ensure steady supply and availability of safe blood for transfusion . in general , the study revealed that the proportion of adults who had adequate level of knowledge about blood donation and good attitude towards blood donation is high . however , the level of blood donation practice was low ; and perception like not being fitted to donate blood , fear of being anemic after blood donation , fear of health risk after donation , and lack of information on where , when , and how to donate blood were the major reason for not donating blood . regarding factors affecting attitude towards blood donation , religion was the only variable which remained to be significantly associated with attitude . besides age , sex , religion , and self - perceived health status were statistically significant variables that affect blood donation practice . the limitations of this study are similar to most of studies done on knowledge , attitudes and practices . one of the inherent limitations of such type of studies is that responses might be influenced by socially desirable traits and there might be the possibility of both interviewer and recall bias . the other limitation of this study is that the result can not be inferred to other populations in the country because in multicultural countries knowledge , attitude , and practices regarding blood donation might be greatly influenced by tradition and sociodemographic factors of the population in different parts of the country . recommendations . national blood bank agency , district blood banks , who , and other organizations working on assuring safe and adequate blood supply should design strategies and tailored programs that promote blood donation practice . besides , large scale in - depth behavioral studies need to be conducted to explore the distal and proximal societal factors that affect the communities ' perception towards blood donation and blood donation practice .

background . though world health organization recommends 100% voluntary blood donation , the percentage of blood collected from voluntary blood donors and the average annual blood collection rate are extremely low in ethiopia . the role of adults is crucial to meet the demand of safe blood . thus , this study aimed to assess knowledge , attitude , and practice of adult population towards blood donation in gondar town , northwest ethiopia . method . a community based cross - sectional study was conducted among 768 adults . multistage sampling technique together with simple random and systematic random sampling technique was employed . bivariate and multivariate logistic regression analysis and bivariate correlation analysis were done . result . about 436 ( 56.8% ) , 630 ( 82% ) , and 141 ( 18.4% ) study participants had adequate knowledge , good attitude , and experience of blood donation , respectively . secondary and higher educational statuses were significantly associated with adequate knowledge towards blood donation . participants who were protestant by religion were more likely to have good attitude towards blood donation . age , self - perceived health status , and religion were significantly associated with blood donation practice . conclusion . knowledge and attitude towards blood donation are high . however , the level of practice is low . district and national blood banks and transfusion agency should design strategies that promote and motivate the communities to donate blood .

the uptake of large amounts of lipids from plasma lipoproteins via endocytosis by macrophages in the artery wall to form lipid - engorged foam cells is an important initiating event in the development of atherosclerosis , a major cause of heart disease . the main lipoprotein implicated is low - density lipoprotein ( ldl ) , but oxidative modification of the particles , a process which can occur within the vessel wall , is required before foam cell formation is induced . the myosin superfamily is a group of actin - associated motor proteins that use atp hydrolysis to generate force or directional movement along actin filaments [ 2 , 3 ] . many new classes of myosin have been identified since the discovery , over seventy years ago , of myosin ii ( conventional myosin ) , which produces contractile force in muscle cells ; to date , 35 classes of myosin proteins have been characterised in mammalian cells . it is now known that these various classes of myosin motor are involved in a diverse range of intracellular processes , ranging from cell migration and division to cell anchorage and transport of cargo proteins along actin filaments . myosin vi ( myo6 ) , a member of the unconventional myosin classes , is an unusual member of the myosin superfamily in that its movement along actin filaments is towards the pointed ( ) end of an actin filament , the opposite direction to myosins of other classes . for this reason , myo6 is ideally suited for a role in endocytosis , which involves vesicle formation and/or transport away from the plasma membrane . evidence suggests that it may play a part in organization and/or anchorage of endocytic machinery , in the provision of force required for invagination and/or vesicle fission , and during transport of vesicles through the cell cortex [ 68 ] . attachment to vesicles is achieved through direct interaction with adaptor proteins ( aps ) , including disabled 2 ( dab2 ) ( which binds directly to receptors or indirectly via adaptor protein-2 ( ap2 ) ) and gaip interacting protein c terminus 1 ( gipc1 ) [ 79 ] . myo6 can be alternatively spliced in two regions of its cargo binding domain , giving rise to either a small insert ( 9aa ) ( si ) and/or a large insert ( up to 32aa ) ( li ) . thus , four variants are possible : myo6(+si , + li ) , myo6(si , + li ) , myo6(+si , li ) , and myo6 without inserts ( myo6(i ) ) . these variants are differentially expressed and appear to have distinct cellular targets and functions [ 6 , 7 ] . although the inserts contain no functional motifs or binding sites , they may affect the structure and therefore the affinity of myo6 for its binding partners [ 9 , 11 ] . endocytosis may occur by phagocytosis , the uptake of large particles , or by pinocytosis , the internalization of fluid and small particles [ 10 , 12 ] . phagocytosis is a regulated and selective process requiring receptor - mediated target recognition , but pinocytosis can be nonselective ( macropinocytosis ) or can be receptor - mediated . clathrin - mediated endocytosis ( cme ) is the most studied form of receptor - mediated pinocytosis , but other forms include caveolin - mediated endocytosis , as well as clathrin - and - caveolin - independent endocytosis [ 10 , 12 ] . most evidence supporting a role for myo6 in endocytosis relates to cme : it has been shown to colocalise with clathrin , dab2 , and ap-2 in polarised cells [ 1315 ] and with uncoated vesicles and gipc1 in unpolarised cells [ 13 , 16 ] . in addition to cme , myo6 has been found localized to sites of macropinocytosis in fibroblasts , and holt et al . have reported that loss of myo6 function in bone marrow - derived murine dendritic cells causes enhancement of macropinocytotic uptake , but its exact function in this process remains unclear [ 7 , 18 ] . given the importance of endocytosis in the uptake of lipoproteins by macrophages , it is possible that myo6 may have a role in foam cell formation and thus play a part in atherogenesis . little is known , however , about the expression and function of myo6 and its binding partners in macrophages . expression of myo6 , dab2 , and ap-2 has been detected in murine macrophages and expression of gipc in human peripheral blood monocytes , but , except for one report which found myo6 mrna in macrophages derived from the human monocyte cell line , thp-1 , no information is available on expression of these proteins in human macrophages . moreover , as far as we are aware , splice variant expression , subcellular localisation , and protein - protein interactions of myo6 have not been studied previously in any macrophage type . the aims of this study were to investigate the expression and function of myo6 and related proteins in human macrophages and to test the hypothesis that myo6 plays a role in the endocytosis of ldl and/or oxldl by these cells . expression of myo6 ( including splice variants ) , dab2 , ap-22 ( one of the large subunits of the ap-2 heterotetrameric complex ) , and gipc1 was demonstrated in both thp-1 macrophages and primary human monocyte - derived macrophages ( hmdm ) , and their subcellular location and interactions within the cells were investigated . to study the possible role of these proteins in macrophage endocytosis , thp-1 macrophages were used to evaluate the effects of ldl and oxldl on the expression of mrna and protein for myo6 , dab2 , ap-2 , and gipc1 , as well as the effects of inhibition of myo6 and dab2 expression by small interfering rna ( sirna ) on the uptake of the lipoproteins by the cells . the thp-1 cell line was established 30 years ago , and since then it has been used very extensively to study the role of monocyte / macrophages in cardiovascular disease . a recent comprehensive review of the value of this model concluded that , in defined conditions , thp-1 cells resemble hmdm and mimic the changes seen in atherosclerosis and that , provided results are interpreted cautiously , they are suitable for the study of the mechanisms by which these cells affect vascular function . the present work shows that the expression and subcellular location of myo6 and related proteins are generally similar in hmdm and thp-1 macrophages , but because of the practical difficulties in obtaining large numbers of hmdm , it was not possible to use primary cells in all of our experimental approaches . fetal bovine serum and penicillin / streptomycin were obtained from gibco ( paisley , uk ) , and dmem and l - glutamine were from paa uk ( yeovil , uk ) . rpmi 1640 medium , -mercaptoethanol , 4-phorbol 12 myristate 13-acetate ( pma ) , oil red o , ethidium bromide , and fluorescein - isothiocyanate- ( fitc- ) conjugated goat anti - mouse igg were supplied by sigma aldrich ( poole , uk ) . 4,6-diamidino-2-phenylindole ( dapi ) 1,1-dioctadecyl-3,3,33-tetramethylindo - carbocyanine perchlorate ( dii ) , rhodamine conjugated phalloidin , alexa488 conjugated goat anti - rabbit- , and alexa555 donkey anti - rabbit igg were purchased from invitrogen molecular probes ( paisley , uk ) . myo6 ( h-215 ) and dab2 ( h-110 ) rabbit polyclonal igg and normal donkey serum were from santa cruz biotechnology inc . other antibodies including mouse polyclonal anti - ap-22 and rabbit polyclonal anti--microglobulin igg ( abcam , cambridge , uk ) , mouse polyclonal anti - gipc1 igg ( abnova , heidelberg , germany ) , rabbit polyclonal anti--actin ( cell signaling technology , hitchin , uk ) , and horse radish peroxidase - conjugated goat anti - mouse and goat anti - rabbit igg ( thermo fisher scientific , cramlington , uk ) were from various suppliers as indicated , and normal goat serum was from dako uk ltd ( ely , uk ) . thp-1 monocytes were cultured in suspension in rpmi-1640 media , supplemented with 10% heat inactivated ( 56c , 30 min ) fetal bovine serum ( fbs ) , 1% penicillin / streptomycin , and 0.1% -mercaptoethanol ( culture medium ) . for differentiation into macrophages , monocytes were incubated with 200 ng / ml phorbol 12-myristate 13-acetate ( pma ) ( 200 ng / ml ) for 72 h. prior to experimental treatment , the medium containing pma and nonadherent or dead cells was removed , the cells were washed twice , and fresh culture medium was added . cells were incubated with or without treatment for up to 5 days , with media changes every 48 h. for hmdm preparation , primary human monocytes were isolated from the blood of healthy adult volunteers with ethical approval from the east london research ethics committee . blood was collected into tubes containing 15% edta ( v : v ) and processed immediately by the addition of pbs ( 1 : 1 , v : v ) , layering over 15 ml lymphoprep ( axis healthcare ltd , borehamwood , herts , uk ) and centrifugation at 800 g ( 30 min , 20c ) . the mononuclear cell layer was collected , mixed with an equal volume of ice - cold pbs containing 0.4% ( v / w ) trisodium citrate , and centrifuged at 800 g ( 5 min , 4c ) . after removal of the supernatant , the cell pellet was re - suspended in 0.2% ( w : v ) nacl for 30 sec at 4c to lyse any remaining red blood cells and 1.6% ( w : v ) , nacl was added , and the tubes were centrifuged as previously mentioned . resuspension in pbs containing 0.4% ( w : v ) trisodium citrate and centrifugation were then repeated 6 to remove contaminating platelets . the final pellet was resuspended in rpmi-1640 medium containing 5% fbs and 1% penicillin / streptomycin , and the cells were incubated at 37c in 5% co2 for 7 days to allow differentiation into macrophages . examination by light microscopy indicated that the cells showed a macrophage phenotype after 6 days ; cells were used on the 7th day after washing with pbs ( 4 ) to remove any remaining nonadherent cells . cos-7 adherent cells were cultured in dmem maintenance media supplemented with 10% fbs , 1% penicillin / streptomycin , and 1% l - glutamine . total rna was extracted from hmdm , thp-1 macrophages , and cos-7 cells using an extraction kit ( sigma - aldrich , poole , uk ) and rnase - free dnase ( qiagen , crawley , uk ) as recommended by the manufacturers . for reverse transcription , rna ( 1 g ) was used to generate cdna using omniscript reverse transcriptase ( qiagen ) and oligo-(dt ) primers according to the manufacturers ' instructions . for expression studies in untreated cells , pcr was used to amplify gene products for myo6 , the small and large insert regions of myo6 , dab2 , ap-22 , gipc1 , and in geneamp pcr system 9700 ( applied biosystems ) using the primers shown in table 1 and the following protocol : denaturing at 95c ( 5 min ) , followed by 35 cycles consisting of 30 sec at 94c , 90 sec at the appropriate specific annealing temperatures and extension at 72c for 60 sec , with a final extension at 72c for 10 min . pcr products were visualised on 1.2% or 3% agarose gels containing 0.01% ethidium bromide ( v : v ) . gels were viewed and photographed under uv light using the chemidoc xrs scanner ( bio - rad , hemel hempstead , uk ) . to determine the effects of ldl or oxldl on mrna expression , thp-1 macrophages were incubated with the lipoproteins ( 50 g protein / ml ) for 8 or 24 h , and total rna was then extracted and reverse transcribed as described previously . the abundance of mrna transcripts for myo6 , dab2 , ap-22 , gipc1 , -actin , 2-macroglobulin , and ribosomal protein l13a was assessed by quantitative pcr ( qpcr ) in an opticon 2 lightcycler system ( mj research , waltham , massachusetts , usa ) using the primers shown in table 1 employing the following protocol : denaturing at 94c ( 2 min ) , followed by 37 cycles of amplification consisting of denaturation at 94c ( 15 sec ) , incubation at an appropriate , specific annealing temperature ( 1 min ) , followed by extension at 72c ( 1 min ) . threshold cycle values were determined using opticon monitor 3 software and quantified using the standard curve for each gene . normalisation factors ( nf ) were calculated from the geometric means of the three most stably expressed reference genes ( -actin , 2-macroglobulin , and ribosomal l13a protein ) as determined using genorm software or , for sirna studies , to the reference gene 2-microglobulin run on the same qpcr plate with its own standard curve . in the latter case , annealing temperatures ( table 1 ) were adjusted to a temperature suitable for both the genes of interest and 2-microglobulin . for analysis of protein expression by western blotting , cells were washed twice with pbs and treated with ripa lysis buffer ( 150 mm nacl , 50 mm tris ( ph 8.0 ) , 1% triton x-100 , 0.5% deoxycholate ( doc ) , 0.1% sds ) containing protease inhibitor cocktail ( 10 l / ml ) ( 5 min , 4c ) . lysates were agitated for 30 min at 4c , centrifuged at 93 g ( 10 min at 4c ) to remove cell debris , and then denatured and reduced in nupage lds sample buffer ( 2 : 1 , v : v ) containing 10% -mercaptoethanol solution at 70c for 10 min . proteins ( 1030 g ) were separated by sds - polyacrylamide electrophoresis ( 8% or 10% resolving gel with 5% stacking gel or precast mini - protean tgx biorad graded gels ( 415% ) ( biorad ) ) and transferred to polyvinylidene fluoride ( pdvf ) membranes . blocking was carried out in 5% ( w : v ) nonfat milk powder at room temperature for 1 h , and membranes were then incubated with polyclonal rabbit anti - myo6 ( 1 : 200 ) , anti - dab2 ( 1 : 400 ) , anti--actin ( 1 : 1,000 ) or anti--microglobulin ( 1 : 700 ) , or mouse anti - gipc1 ( 1 : 500 ) igg overnight at 4c with agitation . after washing in tbst ( 4 15 min ) , horseradish peroxidase - conjugated goat anti - rabbit or anti - mouse secondary antibodies ( 2-microglobulin , dab2 , myo6 , 1 : 8,000 ; -actin , gipc1 , 1 : 10,000 ) were added and incubations were continued for 1 h at room temperature with agitation . finally membranes were washed in tbst ( 4 15 min ) and bands were visualized using enhanced chemiluminescence ( ge healthcare , little chalfont , uk ) . band densities were digitized from film to 12 bit images using a chemidoc xrs ( bio - rad ) and quantified as mean grey values measured over identical areas using quantity one software ( bio - rad ) . cls fluorescence microscopy was conducted using either a leica sp5 or a zeiss lsm510 confocal microscope . for all experiments , thp - macrophages or hmdm were incubated in 24-well plates containing coverslips , which were removed for mounting at the end of the procedure . coverslips were washed twice in 1 pbs , fixed with 4% formalin ( v : v ) for 15 min , and followed by cell permeabilisation in 0.25% triton x-100 for 5 min prior to blocking ( 1 h , 3% ( w : v ) bsa ) with normal goat ( ngs ) or donkey ( nds ) serum ( 1% w : v ) . igg polyclonal rabbit anti - myo6 , -gipc1 ( 1 : 100 , v : v ) , -dab2 ( 1 : 200 , v : v ) , or polyclonal mouse anti - ap-22 ( 1 : 100 , v : v ) ( diluted in 0.1% ( w : v ) bsa and 1% ngs / nds in pbs ) was then added . after 1 h , cells were washed ( 0.5% bsa in pbs , 5 min 4 and 15 min 1 ) and incubated with igg polyclonal secondary antibodies conjugated to a specific fluorochrome as follows : goat anti - rabbit antibody conjugated to alexa fluor 488 ( 1 : 1,000 , v : v ) , donkey anti - rabbit antibody conjugated to alexa fluor 555 ( 1 : 1,000 , v : v ) , or goat anti - mouse antibody conjugated to fluorescein isothiocyanate ( fitc ) ( 1 : 25 , v : v ) dilutions in 0.1% bsa and 1% ngs in pbs ( w : v ) . where double immunofluorescence staining was used , compatible pairs of primary or secondary antibodies were incubated with cells simultaneously . cells were then washed in pbs ( 5 min 3 , then 15 min 1 ) . to visualise cell nuclei , cells were incubated ( 3 min ) with 300 nm 4,6-diamidino-2-phenylindole ( dapi ) , a nucleic acid stain . the structure of f - actin was preserved prior to visualisation , with minimal loss of free myosin , by incubating cells ( 45 sec ) with buffer containing nacl , 137 mm ; kci , 5 mm ; na2hpo4 , 1.1 mm ; kh2po4 , 0.4 mm ; nahco3 , 4 mm ; glucose , 5.5 mm ; mgcl2 , 2 mm ; egta , 2 mm ; pipes , 5 mm ; ph 6.0 - 6.1 [ 25 , 26 ] supplemented with 0.32 m sucrose , 0.1% triton x-100 , and 1 g / ml phalloidin . cells were immediately incubated with 165 nm phalloidin conjugated to rhodamine , as a fluorescent marker ( 20 min ) . areas of fluorescence signal were detected by application of a threshold and the mean fluorescence intensity quantified using volocity ( 5.4 ) 3d image analysis software ( perkinelmer , waltham , ma , usa ) . g / ml ) was isolated from human plasma obtained from the national blood service ( north london , uk ) , using sequential density gradient ultracentrifugation . final preparations of native ldl ( nldl ) were dialysed against 0.9% nacl ( w / v ) containing 10 m edta ( 5l 4 ) . for ldl used in oxidation studies , human plasma was used within 1 month of purchase and ldl within 2 weeks of isolation . ldl was oxidised by incubation with cuso4 ( 5 m ) for 6 h , oxidation was terminated by the addition of edta ( 1% , 50 l / ml v / v ) , and the extent of oxidation was assessed by measuring the concentration of malondialdehyde ( mda ) ( nmol / mg ldl protein ) using the thiobarbituric acid reactive substances ( tbars ) assay . cuso4 was removed by dialysis at 4c against 0.9% nacl ( w / v ) containing 10 m edta ( 5l 4 ) . for labelling of nldl and oxldl with the fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylindo - carbocyanine perchlorate ( dii ) , lipoprotein deficient serum ( lpds ) , prepared from human plasma by sequential density gradient ultracentrifugation , was added to the ldl preparations ( 1 mg ldl protein/2 ml lpds ) followed by dii ( 300 g dii / mg ldl protein ) . to prevent oxidation , edta was added to the preparations at a final concentration of 10m , and the mixture was incubated for 8 h at 37c with shaking . the dii labelled lipoproteins ( dii - nldl / oxldl ) were then isolated by ultracentrifugation at d 1.063 g / ml ( 96,919 g , 15 h , 4c ) and dialysed as described previously before use . small interfering rna ( sirna ) sequences designed to target myo6 ( combination of two sequences ) , dab2 , ap-22 ( table 2 ) , or gipc1 ( sequence not released by the manufacturer ) were used to inhibit the expression of their cognate genes . thp-1 macrophages ( 6 10 well ) were transfected with sirna using hiperfect transfection reagent ( qiagen , crawley , uk ) . 5 nm ( ap-22 , gipc1 , dab2 ) or 40 nm ( myo6 ) sirna was added to 100 l ripa-1640 medium , then 20 l hiperfect was added , and the mixture was incubated for 10 min at room temperature to allow sirna - hiperfect complexes to form prior to their addition ( 100 l ) to cells . in all experiments , a nonsilencing scrambled sirna ( allstars negative control , qiagen ) , which has no known homology to any mammalian genes , was used as a control . to assess the effects of inhibition of gene expression on the uptake of nldl and oxldl by thp-1 macrophages , lipoproteins were labelled with the fluorescent probe dii and uptake was assessed using fluorescence wide - field microscopy . cells were seeded in 24-well plates ( 1.6 10 cells / well ) and incubated with sirna for 4872 h. dii - labelled ldl or oxldl ( 50 g protein / ml ) was then added , and the incubations , continued for periods up to 24 h. cells were fixed and imaged using a leica inverted dmrib wide - field microscope . protein concentrations of cell lysates were determined by the method of lowry and those of nldl and oxldl were obtained using peterson 's modification of lowry 's method . the total cholesterol content of lipoproteins was determined by enzymatic analyses using commercially available kits ( thermo fisher scientific , cramlington , uk ) . statistical analysis was performed using student 's t test or one- or two - way anova ( followed by bonferroni 's multiple comparison test ) , as indicated in the text . to determine whether myo6 and its binding partners are expressed in macrophages , total rna was isolated from untreated thp-1 macrophages and hmdm and then used to generate cdna for amplification of gene products by conventional pcr . bands at the expected product size ( table 1 ) were detected for myo6 ( figure 1(a ) ) and also for dab2 ( figure 1(b ) ) , the large subunit of ap-2 , ap-22 , which was used to detect ap-2 expression ( figure 1(c ) ) and gipc1 ( figure 1(d ) ) . in all cases , no bands were found for any of the genes when pcr was carried out in the absence of either primers ( data not shown ) or cdna . these results demonstrate that both thp-1 macrophages and hmdm express mrna encoding myo6 , dab2 , ap-22 , and gipc1 . although previous studies in mammalian cells have established that myo6 can be expressed as four variants due to alternative splicing of the li and si regions of the tail domain [ 6 , 11 ] , it is not known which splice variants are expressed in macrophages . dance et al . demonstrated the presence of myo6 both with and without the li and si in cos-7 cells , and we have used the same primer sequences to investigate their expression in thp-1 macrophages and hmdm . we found bands identical to those reported by dance et al . for myo6 variants in cos-7 cells ( figure 2 ) , and these were used as markers of band identity . figure 2 shows that bands corresponding to myo6(li ) ( figure 2(a ) ) and myo6(si ) ( figure 2(b ) ) in cos-7 cells were also found in thp-1 macrophages ( figures 2(a ) and 2(b ) , white arrows ) . a band of product size equivalent to myo6(+si ) mrna ( figure 2(b ) , green arrows ) was also detected in thp-1 cells , although at a lower level relative to myo6(si ) in comparison to cos-7 cells . expression of myo6(+li ) was low in cos-7 cells , but a band corresponding to this variant was clearly present in thp-1 macrophages , and these cells also expressed number of other transcripts of larger size ( figure 2(a ) , blue arrows ) . hmdm showed similar patterns of expression of myo6 splice variants to those observed in thp-1 cells , with myo6(li ) and multiple bands of myo6(+li ) ( figure 2(c ) ) and myo6(si ) ( figure 2(d ) ) present ; however , myo6(+si ) was not detected in these cells ( figure 2(d ) ) . immunoblotting indicated the presence of a protein of the molecular weight of dab2 ( 96/67 kda ) in 3 lysates from thp-1 macrophages and in cells from 3 individual donors for hmdm . in addition , a band corresponding to the molecular weight of myo6 was clearly present in 3 lysates of thp-1 cells ( one example is shown in figure 3(b ) ) . for hmdm , a weak band corresponding to myo6 was visible in cells from 2 donors when 45 g protein was loaded ( one example is shown in figure 3(b ) ) but not when a lower amount of protein ( 35 g ) was used ( data not shown ) . the expression of myo6 in hmdm , however , was clear in the immunofluorescence experiments described later . in addition , ap-22 protein could not be detected by immunoblotting , although its presence in thp-1 macrophages was demonstrated by immunofluorescence ( see later ) . immunofluorescence was used to explore the distribution and interactions of myo6 and associated proteins in thp-1 macrophages and hmdm . the proteins were detected using alexa488- ( myo6 , dab2 ) or fitc-(ap-22 ) conjugated secondary antibodies ( green ) and where appropriate , dapi ( blue ) and rhodamine - conjugated phalloidin ( red ) were employed as counterstains for the nucleus and f - actin , respectively . no green fluorescence was observed in negative controls when cells were incubated with fitc- or alexa488-conjugated secondary ab in the absence of the primary antibodies . ap-22 and dab2 were located mainly around the cell periphery in a punctate staining pattern in both thp-1 macrophages ( figures 4(a ) and 4(c ) ) and hmdm ( figures 4(b ) and 4(d ) ) . by contrast , myo6 was found to exhibit a more diffuse staining in both cell types ( figure 5 ) and was found within membrane protrusions and membrane ruffles along the leading edge of the cells ( thp-1 cells figures 5(a ) , and 5(b ) , hmdm , figures 5(c ) and 5(d ) ) . association between myo6 and f - actin was studied using fluorescence labelling techniques in both thp-1 macrophages and hmdm ( figure 6 ) . confocal images showed that , as expected , myo6 appeared to be associated with f - actin , with similar distributions mainly found around the cell periphery in both types of macrophages ( figures 6(a)(ii ) and 6(b ) ) , including possible podosome attachment sites in thp-1 cells ( figure 6(a)(i ) ) . to assess the extent of interaction between myo6 and ap-22 , double immunofluorescence experiments were carried out in thp-1 macrophages in which myo6 was labelled with alexa555-linked secondary antibody to give red fluorescence ( figure 7(a ) ) . ap-22 immunofluorescence was observed mainly at the cell periphery , while that for myo6 was also found intracellularly ( figure 6(a ) ) . regions of overlap were rare and imperfect ( figure 7(a)(ii ) ) , suggesting that the two proteins are not substantially associated within the cells . experiments using an alexa555-linked secondary antibody for dab2 , however , indicated that there was substantial colocalisation between ap-22 and dab2 ( figure 7(b ) ) . the compositions ( protein , total cholesterol , and tbars content ) of the nldl and oxldl preparations used are shown in table 3 . no significant differences were found in the protein or total cholesterol content of nldl as compared to oxldl . the concentration of tbars , however , was approximately 19-fold higher in oxldl , indicating a significantly increased oxidative state . thp-1 macrophages were incubated with or without nldl or oxldl ( 50 g protein / ml ) for 8 h or 24 h , and lysates were then used to determine the abundance of transcripts for myo6 , dab2 , ap-22 , and gipc1 by a combination of rt - qpcr and immunoblotting ( the latter for myo6 and dab2 ) . incubation with nldl caused no significant change in myo6 or dab2 mrna levels at either time point ( figures 8(a ) and 8(b ) ) . mrna abundance for ap-22 , however , was decreased by about 35% after 24 h ( p < 0.001 ) and that for gipc1 by 31% after 8 h ( p < 0.05 ) or 39% after 24 h ( p < 0.001 ) ( figures 8(c ) and 8(d ) ) . oxldl , however , caused a marked decrease in mrna levels for myo6 at both time points ( 8 h , 47% , p < 0.01 ; 24 h , 61% , p < 0.05 ) and also significantly reduced mrna levels for ap-22 ( 39% , p < 0.001 ) and gipc1 ( 22% , p < 0.05 ) compared to control values . myo6 and dab2 protein concentrations showed little change when macrophages were incubated with either nldl or oxldl , with a small decrease in dab2 ( 19% ) in the presence of nldl being the only difference from control values ( figures 8(e)8(h ) ) . myo6 proteins levels , however , were significantly lower after treatment with oxldl as compared to nldl after 8 h ( + 47% , p < 0.01 , figure 8(f ) ) , while dab2 levels were significantly higher ( + 24% , p < 0.01 , figure 8(h ) ) . the effects of inhibition of the expression of myo6 , dab2 , ap-22 , and gipc1 on the uptake of nldl and oxldl by thp-1 macrophages were studied using sirna sequences designed to target their cognate genes . mrna abundance for all test proteins was maximally decreased in thp-1 macrophages 2448 h after transfection with antisense oligonucleotides as compared to scrambled controls ( reduction in abundance was 7585% , myo6 , dab2 , and ap-22 , p < 0.0001 ; 50% gipc , p < 0.001 ) ( figures 9(a)9(d ) ) . after 72 h ( ap-22 ) or 96 h ( myo6 and dab2 ) , however , the inhibitory effect was reduced for all genes except gipc1 . in addition , protein concentrations of myo6 were reduced by 7583% between 48 and 96 h and those for dab2 by > 75% between 24 and 96 h ( p < 0.01 ) ( figures 9(e ) and 9(f ) ) . uptake of nldl and oxldl by thp-1 macrophages was measured using dii - labelled lipoproteins ( 50 g / ml ) and fluorescence microscopy . in non - sirna - treated cells , the area of fluorescence signal increased between 2 h and 24 h , and , as expected , the rate of increase was faster with oxldl as compared to nldl ( figures 10(a ) and 10(b ) ) . comparison of the areas of fluorescence signal in macrophages transfected with scrambled sirna , or sirna targeting myo6 showed no significant differences ( figures 10(c ) and 10(d ) ) ; similar results were obtained when sirna targeting dab2 , ap-22 or gipc1 were used ( data not shown ) . myo6 is an intracellular motor protein found to be associated with f - actin in the cytoskeleton [ 7 , 31 ] ; in cells where it functions in endocytosis , it is also found in association with other proteins that have assigned roles in endocytosis , namely , ap-2 , dab2 , and/or gipc1 . myo6 and the interactive adaptor proteins dab2 , ap-2 , and gipc1 are widely expressed and play diverse , often essential , roles in cellular functioning and signalling . furthermore , these proteins have been shown to function and interact during cme , and myo6 is thought to provide a driving force for vesicle formation and trafficking . however , although the endocytosis of lipoproteins by macrophages to form foam cells is crucial to atherosclerotic development , the potential roles for myo6 and binding partners in this process have not been explored till now . indeed , little information is available about the expression of these proteins or their roles in human macrophages , and nothing is known about their subcellular location or mutual interactions in these cells . we have demonstrated the mrna expression for myo6 , dab2 , ap-2 , and gipc1 in primary human macrophages ( hmdm ) as well as in macrophages derived from the human monocyte cell line , thp-1 ( figure 1 ) . furthermore , the presence of myo6 , dab2 , and ap-2 protein was demonstrated in these two cell types using both immunoblotting and immunofluorescence ( figures 35 ) . previous studies have detected dab2 mrna in mouse bone marrow macrophages and myo6 , dab2 , and ap-2 protein in various murine macrophage cell lines [ 1921 , 32 ] . in addition , myo6 mrna and low levels of gipc1 mrna or protein have been found in human peripheral blood leukocytes . nonetheless , this is the first report of the expression of these proteins in human macrophages , except for one study showing myo6 mrna expression in thp-1 cells . as positive controls , we demonstrated splice variant expression of myo6 in cos-7 cells , previously shown to express myo6 with and without the li and si insert sequences . thp-1 macrophages and hmdm were found to express myo6(li ) , but while only one weak band corresponding to myo6(+li ) was observed in cos-7 cells , numerous bands in this region were seen within the two macrophage cell lines ( figures 2(a ) and 2(c ) ) . dance et al . found a similar pattern of multiple bands for myo6(+li ) in the epithelial cell lines arpe-19 and llc - pk1 and determined that these resulted from alternative splicing within the li region . expression of myo6(si ) was demonstrated in thp-1 macrophages and hmdm in our experiments ( figures 2(b ) and 2(d ) ) , but although myo6(+si ) was clearly present in thp-1 cells , its expression was not detected in hmdm . alternative splice variants of myo6 are differentially expressed and have distinct subcellular locations and functions in various cell types . in polarised epithelial cells , for example , myo6(li ) has been shown to associate with uncoated endocytotic vesicles via gipc1 , while myo6(+li ) is recruited to clathrin coated pits / vesicles via dab2 . thus , the expression of various myo6 splice forms in human macrophages further indicates that myo6 is likely to have multiple functions , including cme , within these cells . in the current study , myo6 was found to be distributed throughout the cytoplasmic pool in both thp-1 macrophages and hmdm , with particular localisation to actin - generated protrusions of the plasma membrane , and possibly membrane ruffles ( figure 5 ) , often expressing a punctuate pattern in the cell periphery indicative of association with vesicular structures , as noted previously [ 6 , 14 , 15 ] . as expected , myo6 appeared to be associated with f - actin , particularly at the apical membrane and at putative podosome attachment sites ( figure 6 ) . similar associations of myo6 to plasma membrane protrusions and membrane ruffles have been reported in a number of migratory cell types other than macrophages , including epithelial , fibroblast , and ovarian border cells [ 13 , 35 ] , suggesting that it plays an important part in the coordination of the various processes required for cell migration , including cell adhesion and directed cell progression . chibalina and colleagues have demonstrated that myo6 functions in endocytosis and vesicle recycling in such cells , and in conjunction with gipc , it has also been shown to participate in the endocytosis of activated 51 integrin , which links it directly to focal adhesion turnover . have also suggested that the association of myo6 with membrane ruffles in fibroblasts may be indicative of a role in macropinocytosis . during cme , ap-2 is known to act in the formation of clathrin - coated pits / vesicles , while dab2 can either associate with ap-2 as an accessory protein or act as an adaptor protein in its own right . dab2 also has numerous functions outside of cme , and so the extent of its association with clathrin - coated vesicles varies between cell types [ 40 , 41 ] . in this study cls fluorescence microscopy was used to show that ap-22 and dab2 ( figure 4 ) are located adjacent to the periphery of both thp-1 macrophages and hmdm in a punctate staining pattern , indicative of vesicle association . to assess the extent of interaction between myo6 , dab2 , and ap-22 in thp-1 macrophages , double immunofluorescence labeling experiments were used . dab2 and ap-22 were shown to be partially colocalised ( figure 7(b ) ) in a punctate pattern , indicative that dab2 , the direct binding partner of myo6 , is associated with clathrin - coated pits / vesicles in thp-1 macrophages . this finding is consistent with previous work showing that dab2 is associated with ap-22 in cos-7 cells . however , although association between myo6 and ap-1 has been reported in other cell types , the present experiments suggest that the two proteins do not have substantially overlapping distributions in macrophages ( figure 7(a ) ) . having shown that myo6 , dab2 , ap-2 , and gipc1 are expressed in human macrophages and that there are some interactions between them , we explored the possibility that they might play a part in macrophage foam cell formation . thp-1 macrophages were used for these studies since there are serious technical difficulties in obtaining sufficient hmdm for the types of experiment needed . these cells have some advantages over hmdm in that , because they are genetically homogeneous , there is less variability in their phenotype , and they can be stored indefinitely at 80c . another factor which was important for our experiments is that transfection efficiency of thp-1 macrophages with sirna is much greater than that with hmdm . the uptake of nldl and oxldl by macrophages is known to be mediated by different receptors : nldl undergoes endocytosis via the ldl receptor ( ldlr ) which is down - regulated when intracellular cholesterol levels increase , while oxldl is recognised by unregulated scavenger receptors , thus its uptake is the main driver of foam cell formation . since numerous studies have demonstrated that nldl may also directly contribute to foam cell formation [ 4345 ] , both nldl and oxldl were used in our studies . the expression of the scavenger receptors cd36 , cd68 and scavenger receptor - a ( sr - a ) has been shown to be upregulated in the presence of oxldl and to a lesser extent nldl [ 4649 ] , while ldlr expression is downregulated in response to cell exposure by nldl [ 50 , 51 ] . in the present work , the effects of nldl and oxldl on the expression of mrna and protein for myo6 , dab2 , ap-2 , and gipc1 in thp-1 macrophages were investigated . compared to control levels , nldl significantly decreased mrna transcript abundance for ap-22 and gipc1 ( figures 8(c ) and 8(d ) ) , while incubation with oxldl resulted in reduced levels of myo6 and ap-22 mrna ( figures 8(a ) and 8(c ) ) . little change was seen at the protein level , however , except for a small decrease in dab2 with nldl ( figures 8(e)8(h ) ) . both myo6 protein and mrna levels were significantly lower after incubation with oxldl compared to nldl ( figures 8(a ) and 8(f ) ) , suggesting that nldl and oxldl have different effects on the endocytic machinery , possibly reflecting their uptake via different receptor - mediated endocytic routes [ 52 , 53 ] . we also examined the effects of knockingdown the expression of myo6 and associated proteins on the uptake of nldl and oxldl . although substantial knockdown of gene expression was achieved ( figure 9 ) , we did not detect any significant effect on the uptake of lipoprotein type as assessed by fluorescence microscopy ( figure 10 ) . these results , together with our findings of relatively small effects of the lipoproteins on mrna and protein expression ( figure 8) , suggest that myo6 and its binding partners do not play a major role in the uptake of nldl or oxldl by macrophages during induction of foam cell formation . the results presented here show that myo6 and its associated proteins dab2 , ap-2 , and gipc are expressed in human macrophages , displaying similar patterns of expression and subcellular location in both the thp-1 cell line and in primary cells ( hmdm ) . myo6 was found to be particularly prominent in protrusions of the plasma membrane and membrane ruffles , where it was associated with f - actin . ap-2 and dab2 appeared to be associated with cell vesicles , and ap-2 , but not myo6 , was colocalised with dab2 . although nldl and oxldl had some effects on the expression of the genes for the proteins investigated in macrophages , attenuation of their expression through the action of cognate sirna molecules did not affect their uptake by the cells . because the nature of these experiments requires large number of cells , however , hmdm could not be used , thp-1 cells being used instead ; for this reason we remain cautious in extrapolating our findings to primary cells , as would be the case with any study involving immortalized cell lines . nevertheless , the findings presented here on expression and subcellular localisation suggest that , in macrophages , myo6 functions in cell adhesion and progression as well as in macropinocytosis . data from the experiments on the uptake of ldl do not support the idea that it plays a major role in foam cell formation .

myosin vi ( myo6 ) functions in endocytosis in conjunction with binding partners including adaptor protein ( ap)-2 , disabled 2 ( dab2 ) , and gaip interacting protein c terminus 1 ( gipc1 ) . this study aimed to investigate the expression and function of myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation . expression of myo6 , ap-2 ( 2 subunit ) , and dab2 in thp-1 macrophages and primary human monocyte - derived macrophages was demonstrated at the mrna and protein level , but gipc1 was only detected at the mrna level . immunofluorescence showed that myo6 was distributed similarly to f - actin in both macrophage types . ap-22 was found to have a similar subcellular distribution to myo6 and dab2 in thp-1 cells . myo6 was located within membrane ruffles and protrusions of the plasma membrane . these results suggest that in macrophages myo6 is required for several functions including cell adhesion , cell progression , and macropinocytosis . low - density lipoprotein ( ldl ) and oxidised ldl ( oxldl ) decreased myo6 and gipc1 mrna expression in thp-1 cells , but uptake of the fluorescence - labelled lipoproteins was unaffected by knockdown of the expression of myo6 or associated proteins with sirna . our findings , therefore , do not support the idea that myo6 plays a major role in foam cell formation .

congenital bochdalek hernia ( cbh ) develops as a result of the inappropriate fusion of the pleuroperitoneal fold and septum transversum at the site of the posterolateral lumbocostal trigone at the sixth week of gestation . this entity was first described by mccauley in 1754 and then by vincent alexander bochdalek in 1848 . this type of diaphragmatic hernia is considered a perinatal disease that presents a few hours after birth , or in early infancy , and it is associated with significant morbidity and mortality . cbh in adults is very rare ; it remains asymptomatic until adulthood and generally presents with thoracic and/or abdominal symptoms aggravated in supine position . we present a case of left - sided congenital bochdalek hernia that remained asymptomatic and was discovered incidentally during secondary cytoreductive surgery for epithelial ovarian cancer ; in this patient , a iatrogenic diaphragmatic defect was found , hyperthermic intraperitoneal chemotherapy ( hipec ) was performed and early postoperative bilateral anterior thalamic infarction developed . a 46-year - old female patient with a bmi of 25 was referred to our tertiary center ( cerrahpasa medical faculty , gynecological oncology unit ) in august 2016 with the diagnosis of platinum - resistant ovarian cancer . positron emission tomography and computed tomography ( ct ) revealed implants on the right diaphragmatic surface after first - line adjuvant chemotherapy . she has never had any thoracic or abdominal symptoms and no history of major trauma . initial preoperative evaluation of the patient revealed no significant sign of diaphragmatic hernia . her physical examination was normal , including chest auscultation sounds and chest x - ray . thoracic ct did not reveal any abnormal image that could be evaluated as a diaphragmatic hernia . she was placed in flank position , and , under general anesthesia , laparotomy was performed . an oval defect with the dimensions of 3 4 cm was seen in the left posterolateral site of the diaphragm ( fig . the most distal portion of the inferior lobe of the left lung was visible through the defect during inspiration and expiration movements . drainage chest tubes were inserted bilaterally via the 6th intercostal spaces to avoid leakage of the chemotherapeutic agents across repaired diaphragm and postoperative massive pleural effusion . the tissue around the congenital left - sided defect was excised circularly and the defect itself was repaired with interrupted nonabsorbable monofilament sutures . after complete cytoreduction had been achieved , hyperthermic intraperitoneal chemotherapy with doxorubicin 35 mg / m and paclitaxel 175 mg / m for 60 min at 42.5c was delivered . no leakage via drainage tubes from the thoracic cavity was noted during the procedure . during the surgery , the patient was managed by standard monitoring , which involved continuous monitoring of electrocardiography , invasive blood pressure , central venous pressure , pulse oximetry , body temperature and hourly urine output . arterial samples were drawn at regular periods for the determination of electrolytes , acid base status and hematocrit . finally , the operation lasted 4 h. at the end of the surgery , the patient remained intubated and was transferred to the intensive care unit for monitoring ongoing resuscitation along with fluid and electrolyte management . the patient was extubated 10 h after completion of surgery , and was transferred to the service on the 2nd postoperative day . prophylactic enoxaparin 4,000 iu was started 6 h after surgery . on the 3rd and 4th postoperative day , excessive sleepiness throughout the day and dyspraxia was noted . bilateral anterior thalamic infarction caused by emboli that block the thalamosubthalamic artery the dose of enoxaparin was changed from 4,000 iu once a day to 800 iu twice a day . follow - up at 4 months after surgery showed no evidence of recurrence and no symptom regarding thalamic infarct . cbh generally presents in newborns with severe respiratory failure or in infancy due to herniation and/or strangulation of the abdominal contents . one of the best studies concerning the incidence of the defect in adults was carried out by mullins et al . , who reviewed 13,138 ct scans . their study revealed an incidence of 0.17% , with 68% being right - sided defects and 77% of the patients being female . since the second study summarized symptomatic cases , demographics identified were different than those in asymptomatic ones . this difference showed that more cases of right - sided cbh in females may remain silent than left - sided cases . these data were supported by several case reports . nevertheless , in our case , the defect was noted on the left side . also , no herniation of intestinal loops was noted , and the patient has never had any thoracic or abdominal symptom that might be related to diaphragmatic hernia . diagnosis of the disease might be carried out by chest ct when there are clinically unexplained thoracic and abdominal symptoms . the chest ct of our patient did not reveal any of the above - mentioned patterns . there are several cases concerning surgical repair of the defect both laparoscopic , via laparotomy , and through the thoracic cavity . we performed upper midline incision for laparotomy , and there was no need for another access to carry out the repair of the diaphragmatic defect . repair of diaphragmatic defect can be performed by various techniques , using absorbable or nonabsorbable sutures in an interrupted or continuous fashion . from our point of view , surgeons may choose any of them provided that they are comfortable with the procedure . the main reason for using bilateral chest tubes was to prevent massive pleural effusion due to hipec . in addition , intra - abdominal pressure increases during hipec , which may subsequently cause a cephalad shift of the diaphragm to increase peak airway pressures and reduce residual capacity . chest drains which were inserted prior to hipec reduce the increase in peak airway pressure , and by this way , the length of the intubation period and hospital stay can be decreased . malignancy itself , hipec , central venous cannulation , the position of the patient 's neck , sudden blood loss and a long duration of the operation were noted as possible risk factors in our case . in conclusion , although congenital bochdalek hernia commonly occurs in the newborn and infancy period , it must not be excluded in the case of unexplained abdominal and/or thoracic symptoms in adults . despite the fact that this kind of rare condition presents with pain , obstruction symptoms or pulmonary symptoms , it can be silent until discovered incidentally . secondly , cerebral embolism should be suspected in the presence of any neurological symptom in postoperative patients .

congenital bochdalek hernia is a defect of the diaphragm and very rare in adults . only around 100 cases have been reported in the literature . herein , we present a case with a recurrent ovarian cancer who underwent secondary cytoreductive surgery and hyperthermic intraperitoneal chemotherapy . an oval defect with dimensions of 3 4 cm was seen in the left posterolateral site of the diaphragm during surgical exploration . in addition , a 6 3 cm iatrogenic right - sided diaphragmatic defect was found and repaired . in the early postoperative period , a bilateral thalamic infarction occurred .

a 4-year - old male neutered domestic shorthair cat was presented to the oregon state university cardiology service for suspected pericardial effusion . cardiac tamponade was documented and pericardiocentesis yielded purulent fluid with cytologic results supportive of bacterial pericarditis . the microbial population consisted of pasteurella multocida , actinomyces canis , fusobacterium and bacteroides species . conservative management was elected consisting of intravenous antibiotic therapy with ampicillin sodium / sulbactam sodium and metronidazole for 48 h followed by 4 weeks of oral antibiotics . re - examination 3 months after the initial incident indicated no recurrence of effusion and the cat remained free of clinical signs 2 years after presentation . bacterial pericarditis is a rare cause of pericardial effusion in cats . growth of p multocida , a canis , conservative management with broad - spectrum antibiotics may be considered when further diagnostic imaging or exploratory surgery to search for a primary nidus of infection is not feasible or elected . pericardial disease in cats is relatively uncommon , with a reported prevalence ranging from 1.0 - 2.3% in post - mortem studies . the most common causes of pericardial effusion in cats results from congestive heart failure secondary to cardiomyopathic disease and neoplasia . other less frequently cited etiologies include trauma , disseminated intravascular coagulation , uremic pericarditis , peritoneopericardial diaphragmatic hernia , feline infectious peritonitis , coagulopathy , hypoalbuminemia and infective pericarditis . infective pericarditis includes viral , bacterial , fungal or parasitic colonization of the pericardium , and is rarely reported in cats . this case report describes a cat with bacterial pericarditis with previously undocumented microorganisms and a favorable clinical response to conservative medical management . a 4-year - old male neutered domestic shorthair cat presented to the primary veterinarian after being found recumbent and dyspneic at home . mild pyrexia ( 39.5c ) , muffled heart sounds and weak femoral pulses were identified on physical examination . thoracic radiographs revealed a globoid cardiac silhouette , and the cat was referred immediately to the oregon state university ( osu ) cardiology service for suspected pericardial effusion ( figures 1 and 2 ) . review of the incoming thoracic radiographs suggested a large - volume pericardial effusion with a small amount of concurrent pleural effusion . the cat s past pertinent history was unremarkable ; it was an indoor cat with no historical altercations with the other household cat . there is a generalized increase in cardiac silhouette size with dorsal deviation of the thoracic trachea . the increased opacity of the cranial mediastinum is most consistent with fat infiltration ventrodorsal radiograph . the cat is positioned obliquely upon presentation to osu , the cat was depressed but responsive . it was hypothermic ( 36.7c ) and tachypneic ( 60 breaths per minute ) with a high - to - normal heart rate ( 220 beats per minute ) . its mucous membranes were blanched and tacky ; no capillary refill time could be obtained . the cat s heart sounds were muffled and femoral pulse quality was poor . echocardiography was performed emergently , confirming a large volume of anechoic pericardial effusion with cardiac tamponade ( figure 3 ; see also supplementary material ) . a small volume of pleural effusion was also present , likely due to impaired filling of the right heart with cardiac tamponade . pericardiocentesis was prioritized and buprenorphine ( 0.15 mg / kg iv , buprenex ; rickitt benckiser healthcare ) was administered for sedation . following aseptic skin preparation of the right fourth sixth intercostal space at the costochondral junction , pericardiocentesis performed with an 18 g intravenous catheter resulted in the removal of 80 ml of malodorous opaque red - tinged fluid flecked with white particles . cytologic evaluation of the pericardial fluid indicated a markedly increased nucleated cell count ( 138,100/l ; international system of units si : 138.1 10/l ) consisting of 20% macrophages and 80% degenerative neutrophils , many with intracellular bacteria of various types . these findings were consistent with a septic suppurative exudate ; consequently , the pericardial fluid was submitted for aerobic and anaerobic bacterial culture and antimicrobial susceptibility . testing for non - bacterial microorganisms ( fungal , viral etiologies ) was not performed . diastolic collapse of the right atrium and ventricle was noted , consistent with cardiac tamponade . the lumens of the right ventricle ( rv ) and left ventricle ( lv ) are labeled . the pericardial effusion is diffusely present around the ventricles ( * ) the signs of cardiogenic shock resolved following pericardiocentesis . the echocardiographic examination was completed with no evidence of structural heart disease or neoplastic masses . cardiac tamponade was abolished and a small amount of residual pericardial and pleural effusions remained after pericardiocentesis . other diagnostics performed included complete blood count ( cbc ) , chemistry panel , urinalysis , feline immunodeficiency virus ( fiv ) and feline leukemia virus ( felv ) antigen testing ( idexx laboratories ) , blood pressure measurement and ecg . the cat s blood pressure was 130 mmhg ( doppler method ) and its ecg indicated sinus rhythm with a left anterior fascicular block - like pattern . the relevant laboratory test abnormalities are listed in table 1 ; abnormalities were consistent with sepsis ( eg , transitional leukogram , mild hyperbilirubinemia ) . the urine sample was obtained several hours after fluid therapy , indicating a urine specific gravity of 1.018 without bilirubin , glucose , protein , cells or bacteria present . wbc = white blood cell diagnostic and therapeutic options were discussed with the cat s owners , including the recommendation to pursue computed tomography ( ct ) to search for a local thoracic or distant source of infection . owing to financial limitations , the owners elected to pursue conservative management with hospitalization and empiric parenteral antibiotic therapy . ampicillin sodium / sulbactam sodium ( 30/kg mg iv q8h ; aurobindo pharma ) , metronidazole ( 15 mg / kg iv q12h ; claris lifesciences ) and intravenous fluid therapy ( lactated ringer s solution ) were initiated . the cat s 5% dehydration deficit was replaced over 6 h , followed by a maintenance fluid rate of 20 ml / h . over the next 48 h , a cbc and chemistry panel were repeated 2 days after presentation ; the relevant results are presented in table 1 . after 2 days of hospitalization , the cat was transitioned to oral medications and discharged with instructions to receive amoxicillin / clavulanic acid ( 20 mg / kg po q8h ; pfizer ) and metronidazole ( 10 mg / kg po q12h ; watson pharma private ) for 4 weeks . at the time of discharge , the pleural effusion was likely either residual pleural fluid from impaired diastolic filling of the right heart during cardiac tamponade , or the result of pericardial effusion leakage following percardiocentesis . the final aerobic culture results yielded heavy growth of both pasteurella multocida and actinomyces canis . aerobic antimicrobial susceptibility testing suggested p multocida was sensitive to ampicillin though resistant to clindamycin and tobramycin . the cat presented to osu for re - evaluation 1 week after discharge and was reported to have normal appetite , energy and demeanor at home . a recheck cbc indicated normalization of the white blood cell count ( 8970/l ; si 8.97 10/l ) with an unremarkable leukogram . furthermore , the chemistry panel indicated complete resolution of the previous electrolyte disturbances , as well as normalization of creatine kinase and alanine aminotransferase . continuation of the antibiotic regimen was recommended and recheck examination was advised following completion of the course . the cat was re - evaluated by echocardiography 3 months after its initial presentation , and no recurrent effusion was noted . feline bacterial pericarditis has been sparsely reported in the veterinary literature . in a large - scale retrospective study of pericardial effusion in 146 cats , several mechanisms can result in septic pericarditis , including pericarditis secondary to localized spread of infection ( eg , pneumonia , suppurative mediastinal lymphadenitis ) , hematogenous infection , extension of endocarditis / myocarditis , or direct inoculation resulting from penetrating wounds , migrating foreign material or surgery . the associated clinical signs are often related to hemodynamic compromise from pericardial effusion or related to systemic infection ( eg , pyrexia , weight loss ) . of the published case reports , some have shown a documented predisposing infection , whereas the inciting etiology was not conclusively identified in others . a cat in one report developed bacterial pericarditis in close temporal proximity to a dental prophylaxis with a common oropharyngeal microorganism , peptostreptococcus . clavulanic acid for approximately 3 weeks and a full recovery was reported 6 weeks after completion of the antibiotic course . another recent case report described bacterial pericarditis in an intact female cat with pyometra and hematogenous spread of escherichia coli . that cat responded well to a 4 week course of enrofloxacin and metronidazole following peri - cardiectomy , and remained clinically stable 1 year later . conversely , another published report of feline bacterial pericarditis identified infection with a mixed population of enterobacteriaceae species and coagulase - negative staphylococcus species without an obvious predisposing cause . an anaerobic culture was not performed , and the cat responded favorably to an 8 week course of broad - spectrum antimicrobial therapy . owing to the multiple organisms involved , direct inoculation via a penetrating wound or foreign material the case reported here did not have a readily apparent systemic or focal infection , or any immune - modulating systemic diseases ; however , an exhaustive search was not pursued . bite wounds are generally associated with the oral flora of the biting animal rather than the skin flora of the victim . the most common aerobic isolate in cat bites is p multocida , and anaerobic isolates commonly include fusobacterium and bacteroides species . though the cat reported here was exclusively housed indoors , another cat was present in the household and may have incited the infection through an unwitnessed altercation . less likely , this infection may have resulted from atraumatic hematagenous spread of bacteria and alternate sources of infection ( eg , odontogenic , esophageal , pleuropulmonary ) were not systematically excluded . while the incidence and causes of septic pericarditis vary across species , the diagnostic approach and therapeutic goals remain the same . identification of the specific pathogen(s ) , related antibiotic susceptibility and source of the infection are paramount . cytology and culture of the pericardial fluid is crucial in the diagnosis of septic cases , unlike most other causes of pericardial effusion . in addition , blood cultures are advised in humans as they are positive in 4070% of cases . aerobic and anaerobic susceptibility testing is also recommended , although most laboratories do not routinely perform susceptibility testing on anaerobic isolates . as anaerobic pericarditis is associated with odontogenic infection , esophageal disease , pleuropulmonary infection , abdominal infection and pelvic infection in humans , a thorough search for adjacent and distant infections owing to the low incidence and small number of published case reports , the ideal therapy for septic pericarditis in cats is unclear . results of susceptibility testing facilitate antibiotic selection , although , if unavailable , broad - spectrum coverage is recommended based on the wide range of bacterial isolates reported . the standard of care in affected humans involves aggressive medical management , including an indwelling pericardial catheter for drainage and targeted antibiotic therapy . in dogs affected by bacterial pericarditis , eradication of the source of the infection is imperative and surgical exploration is indicated in cases that have failed medical management or have suspected abscessation identified on diagnostic imaging . consequently , advanced imaging , exploratory surgery and pericardectomy could be advised in affected cats , although , interestingly , complete recovery has been reported with antibiotics alone in previous cases and the cat reported here . the development of constriction is an important potential sequela to septic and non - septic pericarditis , and should be assessed on patient follow - up . constriction is theorized to occur secondarily to the influx of inflammatory cells into the pericardial space , ultimately leading to proliferation of fibrotic connective tissue and neovascularization . these changes can result in a loss of pericardial elasticity and thus limit diastolic filling . in a prospective human study , 9/500 ( 1.8% ) patients developed constriction over a median follow - up time of 72 months . while overall constriction is a rare complication of human viral or idiopathic pericarditis ( < 0.5% ) , the risk of constriction was highest in the bacterial pericarditis group ( 33% ) . the prevalence of constriction in cats is unknown , although echocardiographic evidence of constrictive physiology was identified in one cat with septic pericarditis . constrictive pericarditis is difficult to definitively diagnose , but echocardiographic findings of a thickened pericardium and respiratory variation of > 25% in mitral inflow velocities can be highly suggestive . in the case presented here , no subjective or clinical evidence of constrictive pericarditis was identified during the limited echocardiograms during recheck examinations ; however , concern exists for its development nonetheless . the cat reported here fully recovered following conservative therapy consisting of pericardiocentesis and broad - spectrum antibiotic treatment . larger retrospective or prospective studies could help further characterize treatment strategies and prognosis in affected cats .

case summarya 4-year - old male neutered domestic shorthair cat was presented to the oregon state university cardiology service for suspected pericardial effusion . cardiac tamponade was documented and pericardiocentesis yielded purulent fluid with cytologic results supportive of bacterial pericarditis . the microbial population consisted of pasteurella multocida , actinomyces canis , fusobacterium and bacteroides species . conservative management was elected consisting of intravenous antibiotic therapy with ampicillin sodium / sulbactam sodium and metronidazole for 48 h followed by 4 weeks of oral antibiotics . re - examination 3 months after the initial incident indicated no recurrence of effusion and the cat remained free of clinical signs 2 years after presentation.relevance and novel informationbacterial pericarditis is a rare cause of pericardial effusion in cats . growth of p multocida , a canis , fusobacterium and bacteroides species has not previously been documented in feline septic pericarditis . conservative management with broad - spectrum antibiotics may be considered when further diagnostic imaging or exploratory surgery to search for a primary nidus of infection is not feasible or elected .

maxillary sinus pneumatization can pose a surgical hazard in terms of oro - antral communications following extraction and endodontic surgery of the antral related teeth . it also increases the risk of introducing foreign bodies , root tips , or teeth displacement into the sinus cavity , and it is well known to influence orthodontic teeth movement [ 57 ] . if the communication remains open or if the infection persists , chronic inflammation of the sinus membrane may result with subsequent permanent epithelization of the oro - antral fistula a situation that further increases the risk of sinusitis . lastly and needless to say that implant - supported rehabilitation of posterior maxilla is jeopardized by the natural tendency of the maxillary sinus to pneumatize bone during life and the inherent bone remodeling , which pursue teeth loss causing rapid alveolar bone resorption . implant insertion within inadequate bone quantity carry risk of oro - antral communication and in such circumstance , maxillary sinus floor elevation is predictable and the possibility of using graft material is not even far . both procedures require extra preoperative planning [ 812 ] . the sound knowledge and preoperative vision of this region will assist the surgeon to be more confident and be familiar with the common anatomic variants and to avoid such serious complications . periapical , panoramic , and conventional ct have been recommended for the preoperative planning . in many clinical situations , the use of three - dimensional imaging proved to be beneficial as compared to two - dimensional imaging and overcomes its limitations . ct scan was developed to overcome the lack of cross - sectional information , superimposition , distortion , and magnification noted in the conventional radiography [ 6,1618 ] . exploring the normal radiographic anatomy of the maxillofacial region has reached areas that were hidden in the past . this is true concerning the maxillary sinus pneumatization especially with the increased reliability of 3d imaging . three - dimensional ct technologies have greatly improved the ability to explore the interior of the cranium and to estimate the volumes of different anatomical compartments such as the maxillary sinus and the nasal cavity . it also facilitated the correlation between these anatomical compartments and the different ethnic groups relative to climate variations . however , the large dose of ionizing radiation delivered by medical ct is crucial and debatable [ 1921 ] . outstandingly , the cbct technology has achieved considerable reduction of absorbed radiation doses compared to medical ct imaging and a bit similar to dental panoramic radiography [ 2224 ] . standard dental protocol scans using traditional ct delivers 1.512.3 times greater radiation than comparable medium field of view dental cbct scans . till that moment , the image quality of cbct was adjudged to be equivalent to that of traditional ct for visualizing the maxillofacial structures . moreover , beam - hardening artifacts due to dental - filling materials and implants are far weaker at cbct than ct [ 2528 ] . considering the anatomical variability related to the maxillary sinus , its intimate relation to the maxillary posterior teeth , and because of all the implications that pneumatization may possess , three - dimensional assessment of maxillary sinus pneumatization is of most usefulness . this is especially the case whenever surgical endodontic apicectomy , periodontal flaps , surgical extraction , implant installation , orthognathic surgeries , or surgical intervention for space occupying lesions involving the maxillary sinus and/or the maxilla are intended . therefore , the aim of this study was to analyze the maxillary sinus dimensions both linearly and volumetrically to assess the maxillary sinus pneumatization . the present study was performed as a retrospective analysis of data stored in a private radiology center . out of respect for doctor patient confidentiality , all personal information concerning the patients as well as the diagnostic cause of the inclusion criteria of patients to the study were based solely on the radiologists interpretation about lack of mucosal thickening in either maxillary sinuses as well as any bone deformities . both sinuses in 15 patients scans were measured giving rise to data from 30 sinuses . images were acquired using the i - cat imaging system ( next generation , imaging sciences international , hatfield , usa ) . the patients were exposed in the sitting position and immobilized using a head band to adjust the head against the head rest and chin cup . the mid - sagittal plane was aligned to be perpendicular to the horizontal plane using vertical and horizontal alignment beams as recommended by the manufacturer . the i - cat is equipped with an amorphous silicon flat panel , and a single 360 degrees scan collects the projection data for reconstruction . the x - ray field size applied was 16 cm diameter 13 cm height , and scanning time was 8.9 s ( fast enough to avoid patient movement , image blurring , and haziness ) . operating parameters were 120 kvp and 5 ma with slice thickness of 0.3 mm ( the standard resolution for scanning at i - cat machine ) . the i - cat s vision software ( imaging sciences international ) was used which allows the recording of linear measurements of images . the measurements were performed by observer ( n a .- w . ) , who has a 15 years experience in oral and maxillofacial radiology . this study was approved by the research ethics committee , faculty of oral and dental medicine , cairo university . the linear measurements were performed according to a protocol that was tested elsewhere for inter- and intraobserver agreement and showed statistically non - significant differences between the observers . since there were no radiopaque markers used in this study , the selection of the cuts for measuring sinus dimensions was based on the presence of certain anatomical landmarks . according to the anatomical fact that the maxillary sinus is pyramidal in shape with an almost square base oriented medially , the measurements of the sinus dimensions were conducted as follows:1.linear measurements of the maxillary sinus length ( craniocaudal extension ; cc ) : on the i - cat vision software , mpr was chosen for interfacing ; adjusting the orientation axis for the axial cut parallel to the occlusal plane at the alveolar crest level ; adjusting the orientation axis of the sagittal cut to be midway between buccal and palatal cortices ; adjusting the coronal cut at area of intended measurement by rotation of the axial image till the orientation axis for the coronal cut becomes perpendicular on buccal cortex . this was repeated at interdental areas between upper first and second premolars , upper second premolar and upper first molar , upper first and second molars , upper second and third molars ; giving rise to 4 craniocaudal measurements : cc 1st and 2nd premolars , cc 2nd premolar and 1st molar , cc 1st and 2nd molars and cc 2nd and 3rd molars , respectively , for each side ( fig . the coronal cut oriented exactly interdental was used ( its axis of orientation in the axial cut was positioned interdentally ) . the measurements were taken from the lowest point of the cortical boundary of orbital floor to the lowest border of the cortical boundary of the sinus floor . to standardize the measurements , they were conducted along the orientation axis apparent on the cut to take the advantage of being automatically adjusted by the software used.2.linear measurements of the maxillary sinus width ( antroposterior dimension ; ap ) and height ( mediolateral dimension ; ml ) were performed on two levels ; along nasal floor and along root of zygoma giving rise to 4 measurements : ap ns , ap zg , ml ns , and ml zg , respectively , on each side . to standardize the axial cut used for measurements , its orientation axis in the coronal cut was adjusted to be exactly passing bilaterally along the inferior cortical boundary of the nasal cavity and root of zygoma , respectively . the measurements were repeated till the maximum antroposterior and mediolateral dimensions were obtained ( figs . 2 and 3 ) . linear measurements of the maxillary sinus length ( craniocaudal extension ; cc ) : on the i - cat vision software , mpr was chosen for interfacing ; adjusting the orientation axis for the axial cut parallel to the occlusal plane at the alveolar crest level ; adjusting the orientation axis of the sagittal cut to be midway between buccal and palatal cortices ; adjusting the coronal cut at area of intended measurement by rotation of the axial image till the orientation axis for the coronal cut becomes perpendicular on buccal cortex . this was repeated at interdental areas between upper first and second premolars , upper second premolar and upper first molar , upper first and second molars , upper second and third molars ; giving rise to 4 craniocaudal measurements : cc 1st and 2nd premolars , cc 2nd premolar and 1st molar , cc 1st and 2nd molars and cc 2nd and 3rd molars , respectively , for each side ( fig . the coronal cut oriented exactly interdental was used ( its axis of orientation in the axial cut was positioned interdentally ) . the measurements were taken from the lowest point of the cortical boundary of orbital floor to the lowest border of the cortical boundary of the sinus floor . to standardize the measurements , they were conducted along the orientation axis apparent on the cut to take the advantage of being automatically adjusted by the software used . linear measurements of the maxillary sinus width ( antroposterior dimension ; ap ) and height ( mediolateral dimension ; ml ) were performed on two levels ; along nasal floor and along root of zygoma giving rise to 4 measurements : ap ns , ap zg , ml ns , and ml zg , respectively , on each side . to standardize the axial cut used for measurements , its orientation axis in the coronal cut was adjusted to be exactly passing bilaterally along the inferior cortical boundary of the nasal cavity and root of zygoma , respectively . the measurements were repeated till the maximum antroposterior and mediolateral dimensions were obtained ( figs . 2 and 3 ) . the selected sites for linear measurements were 16 in number giving rise to a total of 240 readings . ten sinuses for five patients ( out of the original fifteen ) were then selected for further volumetric analysis of the maxillary sinus . right and left volumetric measurements were taken for the five patients.1-volume determination via segmentation technique using simplant software ( simplant , materialise dental nv , leuven , belgium ) .2-volume determination via geometric calculation method according to the geometrical equation : volume of pyramid = base surface area 1/3 height . volume determination via segmentation technique using simplant software ( simplant , materialise dental nv , leuven , belgium ) . volume determination via geometric calculation method according to the geometrical equation : volume of pyramid = base surface area 1/3 height . volume of maxillary sinus ( pyramid ) = antroposterior ( width ) craniocaudal(length ) mediolateral(height)/3 . in order to obtain the width , length , and height of the sinus , the coronal and axial cuts were sequentially reviewed to get maximum height of the sinus [ mediolateral dimension ] and the sagittal cuts were sequentially reviewed to get the maximum sinus base width [ antroposterior dimension ] and length [ craniocaudal dimension ] ( fig . the amount of the maxillary sinus pneumatization was calculated relative to the highest level of the sinus floor expected at the distal side of the 1st premolar ( where the sinus floor is supposed to start ) by subtracting this value from the other measured craniocaudal values ; e.g. , sinus pneumatization between the 2nd and 3rd molars = craniocaudal dimension between the 2nd and 3rd molars craniocaudal dimension between the 1st and 2nd premolars . in cases where the sinus was absent at the area between 1st and 2nd premolars , the craniocaudal dimension between the 2nd premolar and 1st molar was used in the subtraction equation . descriptive statistics were used for all the measurements . the average , standard deviation , coefficient of variation , and 95% confidence interval values for the craniocaudal , mediolateral , and antroposterior dimensions were calculated for the right and left sides of each patient separately . the pearson s correlation coefficient was used to evaluate the correlation between right and left 2d linear measurements for sinus symmetry as well as the correlation between 3d simplant and geometrically derived volumetric measurements . student s paired t - test was also performed for the linear measurements ( sample size = 15 ) . the linear measurements were performed according to a protocol that was tested elsewhere for inter- and intraobserver agreement and showed statistically non - significant differences between the observers . since there were no radiopaque markers used in this study , the selection of the cuts for measuring sinus dimensions was based on the presence of certain anatomical landmarks . according to the anatomical fact that the maxillary sinus is pyramidal in shape with an almost square base oriented medially , the measurements of the sinus dimensions were conducted as follows:1.linear measurements of the maxillary sinus length ( craniocaudal extension ; cc ) : on the i - cat vision software , mpr was chosen for interfacing ; adjusting the orientation axis for the axial cut parallel to the occlusal plane at the alveolar crest level ; adjusting the orientation axis of the sagittal cut to be midway between buccal and palatal cortices ; adjusting the coronal cut at area of intended measurement by rotation of the axial image till the orientation axis for the coronal cut becomes perpendicular on buccal cortex . this was repeated at interdental areas between upper first and second premolars , upper second premolar and upper first molar , upper first and second molars , upper second and third molars ; giving rise to 4 craniocaudal measurements : cc 1st and 2nd premolars , cc 2nd premolar and 1st molar , cc 1st and 2nd molars and cc 2nd and 3rd molars , respectively , for each side ( fig . the coronal cut oriented exactly interdental was used ( its axis of orientation in the axial cut was positioned interdentally ) . the measurements were taken from the lowest point of the cortical boundary of orbital floor to the lowest border of the cortical boundary of the sinus floor . to standardize the measurements , they were conducted along the orientation axis apparent on the cut to take the advantage of being automatically adjusted by the software used.2.linear measurements of the maxillary sinus width ( antroposterior dimension ; ap ) and height ( mediolateral dimension ; ml ) were performed on two levels ; along nasal floor and along root of zygoma giving rise to 4 measurements : ap ns , ap zg , ml ns , and ml zg , respectively , on each side . to standardize the axial cut used for measurements , its orientation axis in the coronal cut was adjusted to be exactly passing bilaterally along the inferior cortical boundary of the nasal cavity and root of zygoma , respectively . the measurements were repeated till the maximum antroposterior and mediolateral dimensions were obtained ( figs . 2 and 3 ) . linear measurements of the maxillary sinus length ( craniocaudal extension ; cc ) : on the i - cat vision software , mpr was chosen for interfacing ; adjusting the orientation axis for the axial cut parallel to the occlusal plane at the alveolar crest level ; adjusting the orientation axis of the sagittal cut to be midway between buccal and palatal cortices ; adjusting the coronal cut at area of intended measurement by rotation of the axial image till the orientation axis for the coronal cut becomes perpendicular on buccal cortex . this was repeated at interdental areas between upper first and second premolars , upper second premolar and upper first molar , upper first and second molars , upper second and third molars ; giving rise to 4 craniocaudal measurements : cc 1st and 2nd premolars , cc 2nd premolar and 1st molar , cc 1st and 2nd molars and cc 2nd and 3rd molars , respectively , for each side ( fig . the coronal cut oriented exactly interdental was used ( its axis of orientation in the axial cut was positioned interdentally ) . the measurements were taken from the lowest point of the cortical boundary of orbital floor to the lowest border of the cortical boundary of the sinus floor . to standardize the measurements , they were conducted along the orientation axis apparent on the cut to take the advantage of being automatically adjusted by the software used . linear measurements of the maxillary sinus width ( antroposterior dimension ; ap ) and height ( mediolateral dimension ; ml ) were performed on two levels ; along nasal floor and along root of zygoma giving rise to 4 measurements : ap ns , ap zg , ml ns , and ml zg , respectively , on each side . to standardize the axial cut used for measurements , its orientation axis in the coronal cut was adjusted to be exactly passing bilaterally along the inferior cortical boundary of the nasal cavity and root of zygoma , respectively . the measurements were repeated till the maximum antroposterior and mediolateral dimensions were obtained ( figs . 2 and 3 ) . the selected sites for linear measurements were 16 in number giving rise to a total of 240 readings . ten sinuses for five patients ( out of the original fifteen ) were then selected for further volumetric analysis of the maxillary sinus . right and left volumetric measurements were taken for the five patients.1-volume determination via segmentation technique using simplant software ( simplant , materialise dental nv , leuven , belgium ) .2-volume determination via geometric calculation method according to the geometrical equation : volume of pyramid = base surface area 1/3 height . volume determination via segmentation technique using simplant software ( simplant , materialise dental nv , leuven , belgium ) . volume determination via geometric calculation method according to the geometrical equation : volume of pyramid = base surface area 1/3 height . volume of maxillary sinus ( pyramid ) = antroposterior ( width ) craniocaudal(length ) mediolateral(height)/3 . in order to obtain the width , length , and height of the sinus , the coronal and axial cuts were sequentially reviewed to get maximum height of the sinus [ mediolateral dimension ] and the sagittal cuts were sequentially reviewed to get the maximum sinus base width [ antroposterior dimension ] and length [ craniocaudal dimension ] ( fig . the amount of the maxillary sinus pneumatization was calculated relative to the highest level of the sinus floor expected at the distal side of the 1st premolar ( where the sinus floor is supposed to start ) by subtracting this value from the other measured craniocaudal values ; e.g. , sinus pneumatization between the 2nd and 3rd molars = craniocaudal dimension between the 2nd and 3rd molars craniocaudal dimension between the 1st and 2nd premolars . in cases where the sinus was absent at the area between 1st and 2nd premolars , the craniocaudal dimension between the 2nd premolar and 1st molar was used in the subtraction equation . descriptive statistics were used for all the measurements . the average , standard deviation , coefficient of variation , and 95% confidence interval values for the craniocaudal , mediolateral , and antroposterior dimensions were calculated for the right and left sides of each patient separately . the pearson s correlation coefficient was used to evaluate the correlation between right and left 2d linear measurements for sinus symmetry as well as the correlation between 3d simplant and geometrically derived volumetric measurements . student s paired t - test was also performed for the linear measurements ( sample size = 15 ) . the maximum craniocaudal extension of the maxillary sinus was located around the 2nd molar in 28 sinuses out of 30 ( 93% ) . maximum craniocaudal extension of the maxillary sinus was located distal to the 2nd molar in 15 sinuses out of 30 ( 50% ) followed by the mesial side of the 2nd molar ( 11 sinuses out of 30 = 36% ) . in only two sinuses , the craniocaudal extension of the maxillary sinus was equal on both sides around the 2nd molar . the maximum craniocaudal extension of the maxillary sinus was seen around the 1st molar in one sinus only and it was equal on both the distal and the mesial sides . in another sinus , the maximum craniocaudal extension of the maxillary sinus was seen between the 2nd premolar and 1st molar . almost in all cases , 4 sinuses ( bilateral in 2 patients ) began more distally at the area of the 2nd premolar and one sinus did not reach beyond the level of 2nd molar . the largest average for craniocaudal dimensions was mesial to the 2nd molar ( 35.54 3.96 mm ) ( table 1 ) . the maximum mediolateral extension of the maxillary sinus was located at the level of root of zygomatic complex in 90% of sinuses ( 27 sinuses out of 30 ) . the maxillary sinus was located at a higher level than the nasal floor in 3 sinuses ( 10% of sinuses ) , 2 of these sinuses were in the same patient . the maximum antroposterior extension of the maxillary sinus was seen at the level of the root of zygomatic complex in 90% of sinus ( 27 sinuses out of 30 ) . in 3 sinuses , the maximum antroposterior extension was seen at the level of the nasal floor , 2 of them were in the same patient . the sinus was bilaterally absent at the level of nasal floor in the same case that did not pneumatize distal to the 2nd molar and was absent in the right side of another case . the largest average for mediolateral and antroposterior dimensions was at the zygomatic complex level with amounts of 20.43 2.62 mm and 31.54 3.2 mm , respectively ( table 2 ) . the amount of sinus pneumatization was calculated relative to the craniocaudal extension of the sinus between the 1st and 2nd premolars . in the two cases where the sinus anatomically started at the 2nd premolar level , the amount of pneumatization was calculated relative to the craniocaudal extension at the 2nd premolar . thus , in these two cases , no value was recorded for sinus pneumatization between 2nd premolar and 1st molar . in a single case , the extension of the sinus did not reach the 3rd molar so no value was recorded for this site . negative pneumatization values denote higher sinus floor level at these sites ( lesser craniocaudal dimension ) compared to the sinus floor level at the site of the 1st premolar . the largest average sinus pneumatization was mesial to the 2nd molar ( 14.04 3.5 mm ) , while the average pneumatization around the 1st molar was 9.21 3.3 mm and 13.76 3.84 mm for the left side and 9.1 2.77 mm and 14.04 3.5 mm for the right side relative to the 1st premolar ( table 3 ) . there was a high correlation between the linear measurements of the right and left sides , where the antroposterior extension of the sinus at level of the nasal floor had the largest correlation ( r = 0.89 ) . the calculated p - values were all statistically non - significant denoting lack of difference between the two sides . there was also a high correlation between the simplant and geometric derived maxillary sinus volumes for both right and left sides ( r = 0.98 and 0.96 , respectively ) ( table 4 ) . the anatomical pneumatization and relations of the maxillary sinus through the alveolar bone are complex , due to the variable extensions of the sinus . the relations between the teeth and the sinus floor are critical elements for diagnosis , dental treatments , and any surgical intervention of dento - antral complex . several studies have investigated the maxillary sinus volume , dimensions , and the relative positions of the maxillary sinus to teeth . there is a wide range of maxillary sinus dimensions in different studies that may reflect the influential effects like human and race variability and triggering of pneumatization . the selected sites for linear measurements were 16 in number giving rise to a total of 240 readings . increasing the number of readings allowed having average values that were comparable to other studies results . the maxillary sinus is anatomically pyramidal in shape with its apex located at the zygoma . accordingly , the linear measurements performed in this study were conducted so that the mediolateral extension represented the height of the maxillary sinus geometrically from its base till its apex , while the antroposterior and the craniocaudal extensions represented the length and the width of the sinus base . in this study , it was found that the largest average craniocaudal , mediolateral , and antroposterior extensions of the maxillary sinus using cbct were 35.54 3.96 , 20.43 2.62 , and 31.54 3.2 mm , respectively . a comparable average dimension of the sinus was that of tiwana et al . . they stated that 33 mm high , 23 mm wide , and 34 mm in an anterior posterior length are the average dimension of the maxillary sinus . moreover , the analysis of maxillary sinus by the application of high - resolution ct in shahbazian et al . study revealed that the antroposterior and mediolateral dimensions of maxillary sinus were in the range of 38 mm ( sd 5.2 ) and 23.5 mm ( sd 5.1 ) , respectively . yet , their study did not mention information about the craniocaudal extension of the sinus . the maxillary sinus in the adult consists of a pyramid shaped cavity with its base at the lateral nasal wall and its apex extending into the zygomatic process of the maxilla . the results obtained in this study revealed that the height of this pyramid ( mediolateral ) is the smallest dimension , while the antroposterior and craniocaudal dimensions of its base are nearly equal . the results obtained in the current study furthermore assure that the maximum craniocaudal extension of the maxillary sinus was located around the 2nd molar ( 28 cases out of 30 = 93% ) . this result strongly matches that of nimigean et al . , who found that the lowest point of the sinus floor was related to the 2nd molar in 93.9% of cases . on the contrary , koppe et al . found in 50% of the examined skulls that the apices of the upper first and second molars gave rise to prominences on maxillary sinus floor and ariji et al . showed that the roots of the maxillary first molar were close to the sinus floor in 60% of the studied specimens . the frequency of greatest craniocaudal extension of the maxillary sinus relative to posterior maxillary teeth surfaces was 50% for the distal surface of the 2nd molar , 36% for the mesial side of the 2nd molar , and 3.3% for the mesial side of the 1st molar . a similar relation between the sinus and the teeth with different frequency was that of killey and kay . their frequency of close proximity between the roots of the posterior maxillary teeth and the sinus floor was 45.5% for the 2nd molars , 30.4% for the 1st molars , and 19.7% for the 2nd premolars . . also found that the distance between sinus floor and root tip was longest for the 1st premolar root tip and shortest for the 2nd molar buccodistal root tip for both right and left sides . moreover , nimigean et al . concluded that the danger of antral penetration is greater at the level of the buccal roots of the 1st and 2nd molars followed by the 2nd premolar . they also considered these sinusal roots and inferred that variations of the sinus floor s depth can depend on sinuses dimensions , their size , and pneumatization . variations of the pneumatization of the maxillary alveolar process could take place due to the craniofacial morphological modifications through evolution that is influenced by dentition , chewing force , breathing movements and craniofacial growth factors that also control the pneumatization of the maxillary alveolar processes . this study showed that the largest average sinus pneumatization was mesial to the 2nd molar ( 14.04 3.5 mm ) , while the average pneumatization around the 1st molar was 9.21 3.3 and 13.76 3.84 mm for the left side and 9.1 2.77 and 14.04 3.5 mm for the right side relative to the sinus pneumatization between 1st and 2nd premolars . unlike most of the studies that estimate sinus pneumatization of alveolar bone relative to the horizontal level of the nasal floor , the present study calculated the sinus pneumatization relative to the highest level of the sinus floor proposed to be located between the 1st and 2nd premolar , where the sinus floor starts to assume a more horizontal level . although the current study did not calculate the amount of bone remaining between sinus floor and root apices , yet it is obvious that the maximum amount of sinus pneumatization around the 2nd molar obtained in our study goes in agreement with the least amount of bone remaining above the 2nd molar in different studies . the inverse relation between sinus pneumatization and remaining alveolar bone is well known and is further strengthened by nimigean et al . . their study inferred that the antral floor depends upon the dental scaffold that constitutes the main factor during development and will transform in relation with the normal / pathological status of the dento - periodontal apparatus , for which they concluded that the available bone is lost from the inferior expansion of the sinus after teeth loss . in a striking observation , the average linear craniocaudal , antroposterior , and mediolateral measurements were almost bilaterally matching in all cases . the student s paired t - test also revealed that there was a non - significant statistical difference between the right and left sides . study revealed symmetric morphology of maxillary sinus in 83% of patients , while the remaining patients ( 17% ) showed a predominant asymmetric morphology . radiologically compared the depth of the sinus floor and did not observe statistical differences between the right and the left sides . in this study , the 3d volumetric measurements of the maxillary sinus obtained for only five patients using the simplant software highly correlated with the mathematically obtained volumes by the geometric calculation ( 0.98 for the right and 0.96 for left side ) . thus , for 3d volumetric measurements of maxillary sinus , the need for simplant software should be questioned regarding the cost effectiveness . although different software s for volumetric analysis seem attractive , illustrative and more diagnostic , yet the geometric method offered a much cheaper , easier , and less sophisticated substitute . the relations of the sinus floor can be accurately assessed on the different orthogonal images obtained through 3d cbct scan . the geometric method for volumetric analysis offered a much cheaper , easier , and less sophisticated substitute ; therefore , with the availability of software , 3d volumetric measurements are more facilitated . cbct showed a great potential for proper preoperative planning and is an indispensable alternative for ct when 3d imaging is mandatory for all dental practitioners . the decision about the imaging technique that is most appropriate for each clinical situation should be based upon the radiation dose , the cost , and the reliability of each technique .

considering the anatomical variability related to the maxillary sinus , its intimate relation to the maxillary posterior teeth and because of all the implications that pneumatization may possess , three - dimensional assessment of maxillary sinus pneumatization is of most usefulness . the aim of this study is to analyze the maxillary sinus dimensions both linearly and volumetrically using cone beam computed tomography ( cbct ) to assess the maxillary sinus pneumatization . retrospective analysis of 30 maxillary sinuses belonging to 15 patients cbct scans was performed . linear and volumetric measurements were conducted and statistically analyzed . the maximum craniocaudal extension of the maxillary sinus was located around the 2nd molar in 93% of the sinuses , while the maximum mediolateral and antroposterior extensions of the maxillary sinus were located at the level of root of zygomatic complex in 90% of sinuses . there was a high correlation between the linear measurements of the right and left sides , where the antroposterior extension of the sinus at level of the nasal floor had the largest correlation ( 0.89 ) . there was also a high correlation between the simplant and geometric derived maxillary sinus volumes for both right and left sides ( 0.98 and 0.96 , respectively ) . the relations of the sinus floor can be accurately assessed on the different orthogonal images obtained through 3d cbct scan . the geometric method offered a much cheaper , easier , and less sophisticated substitute ; therefore , with the availability of software , 3d volumetric measurements are more facilitated .

many rnas function as folded , structured molecules rather than as protein - encoding messengers . in fact , highly conserved , structured non - coding rnas ( ncrnas ) essential to basic cellular processes represent the majority of a cell 's rna . such ncrnas are responsible for translation , pre - mrna splicing , histone mrna maturation , guiding rna modifications , and other essential cellular processes . recent genome - wide transcriptome analyses in multiple organisms indicate that many regions of the genome are transcribed into ncrnas , leading to discoveries of low - abundance , functional rnas that were previously missed . several new classes have emerged in the last decade , such as micrornas , large intergenic non - coding rnas ( lincrnas ) , and promoter- or termini - associated short rnas . the functions of most of these ncrnas remain undiscovered . because many abundant ncrnas function as folded structures , it is likely that some of these less abundant ncrnas also fold to perform their cognate functions . determination of rna structure is largely performed by biochemical experiments that probe one rna sequence in solution . chemical agents or nucleases that react with rna bases depending on their structural context can help distinguish between bases that participate in base pairing and other stabilizing interactions versus bases that do not . recent advances in probing by selective 2-hydroxyl acylation analyzed by primer extension ( shape ) enable faster , higher - quality probing , but still focus on just one rna sequence per experiment . in contrast , computational structure prediction methods allow rapid , large - scale analyses of many rna sequences . in addition to methods rooted in comparative sequence analysis , which require several rna sequences with a conserved structure , there exist methods that predict structure from a single sequence and are useful for rnas for which structural homologs are not known or that undergo lineage - specific structure changes and thus lack structure conservation . such methods provide theoretical folds for a rna sequence , usually using thermodynamic models . while generally powerful , they often suffer from ambiguity since they can predict several different structures for a sequence , necessitating biochemical data to choose amongst candidate folds . to draw on both the speed of computational methods and the quality of rna probing experiments , we developed fragseq ( fragmentation sequencing ) , a method that uses a nuclease specific for single stranded rna on a complex rna mixture followed by high - throughput sequencing and bioinformatic analysis to deduce cut sites ( phosphate backbone scissions ) . this analysis provides an rna accessibility profile , akin to dnase hypersensitivity assays on chromatin . we apply fragseq to naked rnas from the mouse nuclear transcriptome and deduce structure data for known and novel ncrnas . we chose nuclear rna from undifferentiated mouse embryonic stem cells ( undiff ) or cells differentiated into neural precursors ( d5np ) to assess whether our method gave reproducible results for rnas present in both samples . the nucleus contains many rnas in the 70 - 300 nucleotide range ; nuclease treatment yielded fragments in the 20 - 100 nucleotide range required for the high - throughput sequencing protocol used ( fig . to specifically clone rna fragments derived from nuclease cuts and not those derived from random hydrolysis , we used endonuclease p1 ( ec 3.1.30.1 ) , which has preference for single - stranded dna and rna and yields 5 monophosphate and 3 oh products . in our buffer conditions , p1 specifically cut single - stranded regions of well - characterized rnas ( u1a snrna and 5s rrna ) . importantly , we tested whether addition of mouse total nuclear rna to u1a or 5s rrna in vitro transcripts would influence the pattern of digestion , implying trans interactions . when performing the reactions at dilute rna concentrations , both rnas had an identical pattern of digestion whether probed in homogenous or complex mixture ( supplementary fig . 1 ) . we either gel - isolated intact nuclear rnas of 20 - 100 nucleotides directly or first performed a limited p1 nuclease digestion before gel isolation . the control treatment without nuclease digestion allowed us to estimate the occurrence of fragments with an endogenous 5 phosphate , as opposed to fragments with a 5 phosphate produced by nuclease cleavage . additionally , we treated an equal mass of input 20 - 100 nucleotide rnas with polynucleotide kinase ( pnk treatment ) and atp , catalyzing 5 phosphorylation and 3 cyclic phosphate removal , which allowed us to examine endogenous breaks that do not leave a 5 phosphate and 3 oh . after gel isolation of these three parallel treatments , adapters were ligated directly to rna fragments in a manner requiring both a 5 phosphate and 3 oh on each rna , thus preserving orientation information for each fragment . after subsequent reverse transcription , the libraries were individually barcoded during pcr , pooled and sequenced using the abi solid3 platform , then mapped to the mouse genome using the abi small rna analysis pipeline ( http://solidsoftwaretools.com ) . sequencing summary statistics of the barcoded samples ( supplementary table 1 ) 2 ) and the coverage of individual rnas in nuclease versus control treatment ( supplementary fig . 3 ) are consistent with our experimental design and show that we obtained good coverage of ncrnas . most known ncrnas longer than 100 bases have higher coverage in the nuclease sample than in the control because their native form is too long to sequence and does not contain an endogenous 5 phosphate ; whereas a single nuclease cleavage creates the 5 phosphate required for cloning and brings the rna into sequencing size range ( supplementary fig . the exceptions are short c / d box snornas , which tend to have native 5 phosphates and fall within our sequencing size range ; indeed , they occupy a greater fraction of read mappings in the control sample than in the nuclease or pnk samples , indicating we are correctly enriching for 5 phosphate products . 1b ) takes genome - mapped reads from the nuclease and control treatments , as well as a set of transcript coordinates , and outputs cutting scores for each site within each transcript . a site is the phosphate backbone between two adjacent bases where scissions can occur ; a cutting score is a value ( greater than zero ) that reflects the preference of the nuclease for catalyzing scissions at that site relative to other sites in the same rna . briefly , the cutting score is the log ratio of probabilities of observing a break in the nuclease treatment versus the control treatment , after correcting for abundance differences and missing / low - valued data ( see supplementary note 1 for the exact algorithm definition and supplementary note 2 for design rationale ) . because p1 cuts 3 of an unpaired base , a high cutting score at a site indicates that the upstream base is unlikely to be involved in base pairing or tertiary interactions . these cutting scores form the basis of our subsequent analysis ( see also supplementary discussion ) . we show the flow of data through the algorithm , from genome - mapped reads to cutting scores , for the example rna u1a ( fig . for each site along the transcript , we counted how many reads begin there , and how many trim reads ( defined in supplementary notes 1 and 2 ) end there , summing them to get counts of observed breaks in each sample ( fig . 4 ) . we corrected these counts for missing data and normalized to get probabilities of observing breaks at each site in each rna in each sample ( fig . 2d ) , which are used to compute cutting scores for each site ( fig . high cutting scores tend to occur only in regions of single - stranded rna ( figs . 2e ) with pearson correlation coefficients of 0.889 , 0.813 , and 0.817 for u1a , c / d box snorna u3b , and spliceosomal snrna u5 , respectively ( supplementary fig . 5 ) . this indicates that our method obtains similar structure data in biological samples with different transcriptional profiles . fragseq cutting scores are in good agreement with known secondary structures of u1a ( fig . 3 ) , as well as several other ncrnas whose secondary structures have been examined ( fig . our method is particularly good at locating stem - loops and hinge regions , producing consecutive high cutting scores in those areas . this is expected , as p1 has been shown to prefer a minimum of 3 consecutive ssdna bases to catalyze scission , but operates most optimally on runs of 4 - 6 bases of ssdna , and likely has the same preference for ssrna . we occasionally observe weak cutting scores in regions believed to be dsrna , but this signal is generally not above the spurious level of other probing agents observed in conventional probing experiments . we examined whether the extent of p1 cutting as inferred by our assay correlates with susceptibility to ssrna probing chemicals and enzymes in previous studies , to show that fragseq can capture information about the susceptibility of a site uncovered by conventional methods , but in a high - throughput manner . we compared our cutting scores to probing performed on human u3 and human u5 which are sufficiently similar to the mouse homologues ( u3b : 87% identity , u5 : 95% identity ) . like our study , these studies probed naked rna in solution after purification from cell lysate , so they contained endogenous base editing and modifications . for u3 , we focused on the mouse u3b homolog , which has 3.3 to 5.4 times more reads than homolog u3a across our samples and treatments . we find that for both rnas , previously determined regions of high reactivity towards probes specific for unpaired bases ( fig . 3a for u3b , 3c for u5 ) correlate with high fragseq cutting scores ( fig . stem - loops sl1 and sl2 and the hinge region in u3b and stem - loop sl1 in u5 have strong reactivity in all studies including ours . for large interior loops , moderate to strong reactivity in prior studies is also seen in our studies , except for il5 in u3b ; however , it contains b and c boxes that may form base - pairs and non - canonical k - turn interactions that could prevent cleavage by p1 . it should also be mentioned that p1 is a far larger enzyme ( 45 - 50 kda ) than other single stranded ribonucleases like rnase a and t1 ( 14 and 11 kda , respectively ) . this difference could account for reactivity at certain internal sites where steric clashes may play a role . we wanted to validate fragseq results on previously unprobed rnas using conventional techniques to ensure that our algorithm was not over - fit towards rnas with previously known structures . unlike canonical c / d box snornas that guide 2-o - methylation in a rnp complex and are therefore thought to lack structure in the absence of protein partners , the long c / d box snornas u3 ( fig . the boxes , guides , and other features of a canonical c / d box snorna generally do not comprise more than 80 bases , so it is unclear what structural role the remaining sequence performs in uncharacterized long snornas . we examined cutting scores for all c / d box snornas over 120 bases ( fig . 4b ) , and selected u15b which has a predicted 2-o - methylation target , u22 , required for processing of 18s rrna by an unknown mechanism , and u97 , which has no predicted target , for follow - up probing with conventional methods . these examples also span a wide range of read coverage in our data , which allowed us to examine how well fragseq performs at different coverage levels . we carried out enzymatic probing of these rnas , transcribed in vitro , with rnases v1 , which prefers stacked bases , and t1 , a , and p1 , which prefer ssrna ( supplementary fig . 6 ) . 8a ) that regions that behave as ssrna on the fragseq assay also tend to behave as ssrna in our follow - up probing , indicating that moderate to high cutting scores are accurate evidence of ssrna ( supplementary note 3 ) . when compared to follow - up probing , u15b and u22 have more reliable cutting scores than u97 , probably because the coverage for u97 is the lowest ( see supplementary discussion ) . for example , we did not detect breaks at u15b bases 116 to 126 in any samples ( data not shown ) , although they are highly reactive in follow - up probing . this is probably because cuts in that region would produce fragments that are outside of the 20 - 100 base size selection range . we constructed structure models for these three snornas using computational methods , phylogeny information , and data from our follow - up probing ( fig . 5b , supplementary fig . 8b , supplementary note 3 ) . superimposing the cutting scores on these secondary structure models ( fig 8c ) shows that fragseq data agrees with models derived using conventional techniques because high cutting scores tend to occur in ssrna regions . we chose nuclear rna from undifferentiated mouse embryonic stem cells ( undiff ) or cells differentiated into neural precursors ( d5np ) to assess whether our method gave reproducible results for rnas present in both samples . the nucleus contains many rnas in the 70 - 300 nucleotide range ; nuclease treatment yielded fragments in the 20 - 100 nucleotide range required for the high - throughput sequencing protocol used ( fig . to specifically clone rna fragments derived from nuclease cuts and not those derived from random hydrolysis , we used endonuclease p1 ( ec 3.1.30.1 ) , which has preference for single - stranded dna and rna and yields 5 monophosphate and 3 oh products . in our buffer conditions , p1 specifically cut single - stranded regions of well - characterized rnas ( u1a snrna and 5s rrna ) . importantly , we tested whether addition of mouse total nuclear rna to u1a or 5s rrna in vitro transcripts would influence the pattern of digestion , implying trans interactions . when performing the reactions at dilute rna concentrations , both rnas had an identical pattern of digestion whether probed in homogenous or complex mixture ( supplementary fig . 1 ) . we either gel - isolated intact nuclear rnas of 20 - 100 nucleotides directly or first performed a limited p1 nuclease digestion before gel isolation . the control treatment without nuclease digestion allowed us to estimate the occurrence of fragments with an endogenous 5 phosphate , as opposed to fragments with a 5 phosphate produced by nuclease cleavage . additionally , we treated an equal mass of input 20 - 100 nucleotide rnas with polynucleotide kinase ( pnk treatment ) and atp , catalyzing 5 phosphorylation and 3 cyclic phosphate removal , which allowed us to examine endogenous breaks that do not leave a 5 phosphate and 3 oh . after gel isolation of these three parallel treatments , adapters were ligated directly to rna fragments in a manner requiring both a 5 phosphate and 3 oh on each rna , thus preserving orientation information for each fragment . after subsequent reverse transcription , the libraries were individually barcoded during pcr , pooled and sequenced using the abi solid3 platform , then mapped to the mouse genome using the abi small rna analysis pipeline ( http://solidsoftwaretools.com ) . sequencing summary statistics of the barcoded samples ( supplementary table 1 ) 2 ) and the coverage of individual rnas in nuclease versus control treatment ( supplementary fig . 3 ) are consistent with our experimental design and show that we obtained good coverage of ncrnas . most known ncrnas longer than 100 bases have higher coverage in the nuclease sample than in the control because their native form is too long to sequence and does not contain an endogenous 5 phosphate ; whereas a single nuclease cleavage creates the 5 phosphate required for cloning and brings the rna into sequencing size range ( supplementary fig . the exceptions are short c / d box snornas , which tend to have native 5 phosphates and fall within our sequencing size range ; indeed , they occupy a greater fraction of read mappings in the control sample than in the nuclease or pnk samples , indicating we are correctly enriching for 5 phosphate products . 1b ) takes genome - mapped reads from the nuclease and control treatments , as well as a set of transcript coordinates , and outputs cutting scores for each site within each transcript . a site is the phosphate backbone between two adjacent bases where scissions can occur ; a cutting score is a value ( greater than zero ) that reflects the preference of the nuclease for catalyzing scissions at that site relative to other sites in the same rna . briefly , the cutting score is the log ratio of probabilities of observing a break in the nuclease treatment versus the control treatment , after correcting for abundance differences and missing / low - valued data ( see supplementary note 1 for the exact algorithm definition and supplementary note 2 for design rationale ) . because p1 cuts 3 of an unpaired base , a high cutting score at a site indicates that the upstream base is unlikely to be involved in base pairing or tertiary interactions . these cutting scores form the basis of our subsequent analysis ( see also supplementary discussion ) . we show the flow of data through the algorithm , from genome - mapped reads to cutting scores , for the example rna u1a ( fig . for each site along the transcript , we counted how many reads begin there , and how many trim reads ( defined in supplementary notes 1 and 2 ) end there , summing them to get counts of observed breaks in each sample ( fig . 2c , supplementary fig . we corrected these counts for missing data and normalized to get probabilities of observing breaks at each site in each rna in each sample ( fig . 2d ) , which are used to compute cutting scores for each site ( fig . high cutting scores tend to occur only in regions of single - stranded rna ( figs . 2e ) with pearson correlation coefficients of 0.889 , 0.813 , and 0.817 for u1a , c / d box snorna u3b , and spliceosomal snrna u5 , respectively ( supplementary fig . 5 ) . this indicates that our method obtains similar structure data in biological samples with different transcriptional profiles . fragseq cutting scores are in good agreement with known secondary structures of u1a ( fig . 3 ) , as well as several other ncrnas whose secondary structures have been examined ( fig . our method is particularly good at locating stem - loops and hinge regions , producing consecutive high cutting scores in those areas . this is expected , as p1 has been shown to prefer a minimum of 3 consecutive ssdna bases to catalyze scission , but operates most optimally on runs of 4 - 6 bases of ssdna , and likely has the same preference for ssrna . we occasionally observe weak cutting scores in regions believed to be dsrna , but this signal is generally not above the spurious level of other probing agents observed in conventional probing experiments . we examined whether the extent of p1 cutting as inferred by our assay correlates with susceptibility to ssrna probing chemicals and enzymes in previous studies , to show that fragseq can capture information about the susceptibility of a site uncovered by conventional methods , but in a high - throughput manner . we compared our cutting scores to probing performed on human u3 and human u5 which are sufficiently similar to the mouse homologues ( u3b : 87% identity , u5 : 95% identity ) . like our study , these studies probed naked rna in solution after purification from cell lysate , so they contained endogenous base editing and modifications . for u3 , we focused on the mouse u3b homolog , which has 3.3 to 5.4 times more reads than homolog u3a across our samples and treatments . we find that for both rnas , previously determined regions of high reactivity towards probes specific for unpaired bases ( fig . 3a for u3b , 3c for u5 ) correlate with high fragseq cutting scores ( fig . stem - loops sl1 and sl2 and the hinge region in u3b and stem - loop sl1 in u5 have strong reactivity in all studies including ours . for large interior loops , moderate to strong reactivity in prior studies is also seen in our studies , except for il5 in u3b ; however , it contains b and c boxes that may form base - pairs and non - canonical k - turn interactions that could prevent cleavage by p1 . it should also be mentioned that p1 is a far larger enzyme ( 45 - 50 kda ) than other single stranded ribonucleases like rnase a and t1 ( 14 and 11 kda , respectively ) . this difference could account for reactivity at certain internal sites where steric clashes may play a role . we wanted to validate fragseq results on previously unprobed rnas using conventional techniques to ensure that our algorithm was not over - fit towards rnas with previously known structures . unlike canonical c / d box snornas that guide 2-o - methylation in a rnp complex and are therefore thought to lack structure in the absence of protein partners , the long c / d box snornas u3 ( fig . the boxes , guides , and other features of a canonical c / d box snorna generally do not comprise more than 80 bases , so it is unclear what structural role the remaining sequence performs in uncharacterized long snornas . we examined cutting scores for all c / d box snornas over 120 bases ( fig . 4b ) , and selected u15b which has a predicted 2-o - methylation target , u22 , required for processing of 18s rrna by an unknown mechanism , and u97 , which has no predicted target , for follow - up probing with conventional methods . these examples also span a wide range of read coverage in our data , which allowed us to examine how well fragseq performs at different coverage levels . we carried out enzymatic probing of these rnas , transcribed in vitro , with rnases v1 , which prefers stacked bases , and t1 , a , and p1 , which prefer ssrna ( supplementary fig . 6 ) . 8a ) that regions that behave as ssrna on the fragseq assay also tend to behave as ssrna in our follow - up probing , indicating that moderate to high cutting scores are accurate evidence of ssrna ( supplementary note 3 ) . when compared to follow - up probing , u15b and u22 have more reliable cutting scores than u97 , probably because the coverage for u97 is the lowest ( see supplementary discussion ) . for example , we did not detect breaks at u15b bases 116 to 126 in any samples ( data not shown ) , although they are highly reactive in follow - up probing . this is probably because cuts in that region would produce fragments that are outside of the 20 - 100 base size selection range . we constructed structure models for these three snornas using computational methods , phylogeny information , and data from our follow - up probing ( fig . 5b , supplementary fig . 8b , supplementary note 3 ) . superimposing the cutting scores on these secondary structure models ( fig 8c ) shows that fragseq data agrees with models derived using conventional techniques because high cutting scores tend to occur in ssrna regions . due to read length limitations , most rna - seq studies turn to random hydrolysis of the sample before sequencing . instead , we fragmented rna in a structure - specific manner , reporting on nuclease susceptibility along each transcript . fragseq will not generate the uniform coverage across a transcript needed for accurate abundance estimates or alternative splicing characterization . instead , quantitative comparisons along each transcript , expressed as cutting scores , are made between enzyme - treated samples versus control samples , yielding information about rna structure . for analysis of a novel transcriptome , the fragseq preparation can be done in parallel with other preparations that quantify abundance , barcoding the samples for analysis in a single sequencing run . by using nuclease p1 , we were able to specifically enrich for its products and avoid products of spontaneous or canonical rnase degradation . using the parallel pnk treatment where these latter products were converted to clonable rnas showed how sequencing multiple treatments yields insights into naturally labile sites . in parallel with this manuscript , a similar technique for high - throughput rna structure probing was introduced . that study utilized nuclease s1 , which has similar properties to p1 , and rnase v1 , which cleaves stacked bases . their readout of structure is reported as a ratio of susceptibilities of each rna site to the two nucleases , whereas fragseq monitors one nuclease with respect to a control run without nuclease . we favor cutting scores that are log ratios of data from nuclease versus control treatments because they describe , for each site , its nuclease susceptibility relative to its natural degradation susceptibility in the cell or during the preparation . cut counts per site in the nuclease - treated sample alone do not provide data as informative as cutting scores ( compare fig . we provide configurable software to compute cutting scores from mapped sequencing reads , outputting them and intermediate analysis data in formats compatible with the ucsc genome browser ( http://genome.ucsc.edu ) , allowing visualization of structure data in a genomic context . we also modified the well - established rnastructure software to allow input of fragseq data to guide computational structure prediction ( supplementary discussion ) . we do not observe single - hit kinetics for which probing studies generally aim , as many ncrna reads do not contain the native 3 ends of the rna from which they originate ( supplementary fig . 9 ) . we also do not observe native 5 ends for those rnas , but that is due to the trimethylguanosine cap blocking adapter ligation . we have not determined whether multiple cuts by p1 in solution are indeed the general case , or whether our size selection step enriches for products of multiple hits . perhaps calibrating p1 for single - hit kinetics on in vitro transcribed test rnas did not translate to single - hit kinetics in the nuclear transcriptome where many ncrnas are highly modified . in addition , the test rnas in our probing experiment were all intact at the beginning of digestion , whereas a portion of the ncrnas in the nuclear sample may be partially degraded . in any case , it is clear that reads produced by multiple cuts are providing reliable structure data . this is likely because p1 prefers to cut in stem - loops or hinge regions and these cuts are unlikely to cause the closing helix to denature under our salt conditions , so the original structure may not change before subsequent cuts . as hinge regions often connect domains that fold separately , cuts there would not lead to refolding of those independent domains . this may not be true for larger structured rnas with long - range tertiary interactions , but these rnas fall outside of the scope of our current method . rather than comparing to conventional single - hit probing , it is more fitting to liken fragseq nuclease data to dnase hypersensitivity assays on chromatin in that it gives a global perspective of rna structure ( e.g. stem - loop positions ) rather than fine details ( e.g. bulges in a helix ) . we envision several areas of rna biology where refinement of a fragseq protocol might prove fruitful . one topic of particular interest is riboswitches , rna molecules that change structure upon the binding of a metabolite ligand . using parallel sequencing runs with and without the ligand of interest could yield a differential pattern of cutting scores along such rnas that would serve as a signature of a conformational change . additionally , nuclease protection assays could be scaled up to whole transcriptomes by performing parallel nuclease digestions with and without an rna - binding protein pre - incubated with the whole - cell rna . identifying differentially protected regions would hone in on the rna binding protein 's specificity for sequence or structural context . likewise , such digestions could be carried out on whole cell or nuclear extracts with proteins still bound . nuclease p1 would be a good candidate for these digestions since the buffer conditions for extracts are usually similar to the relatively physiological ph and salt concentrations used in this study . nuclease p1 is also stable at high temperatures so we envision that fragseq could be another way to monitor thermal denaturation of rna domains . by parallel sequencing from nuclease reactions performed at different temperatures , the single - stranded character of a given transcript could be monitored and act as a proxy for unfolding . though we focused on one enzyme here , our experimental pipeline and software could be easily adapted to other enzymatic or chemical probes , so long as a proper control is carried out in parallel . fragseq , combined with methods developed in previous rna - seq studies , enables researchers to take high - throughput transcriptome analysis beyond one - dimensional sequence to reveal structural features of rnas and provide clues to their underlying biology .

previous efforts to determine structures of non - coding rna ( ncrna ) probed only one rna at a time with enzymes and chemicals , using gel electrophoresis to identify reactive positions . to accelerate rna structure inference , we have developed fragseq , a high - throughput rna structure probing method that uses high - throughput rna sequencing on fragments generated by nuclease p1 , which specifically cleaves single stranded nucleic acids . in experiments probing the entire mouse nuclear transcriptome , we show that we can accurately and simultaneously map single - stranded regions ( ssrna ) in multiple ncrnas with known structure . we carried out probing in two cell types to demonstrate reproducibility . we also identified and experimentally validated structured regions in ncrnas never previously probed .

a 45-year - old iranian woman was referred to the private clinic in the city of hamadan for implant consultation . her past medical history was not notable , and there was no evidence of systemic disease . she had no history of trauma to the mandible . in the extra - oral examination , intra - oral examination revealed normal oral mucosa , the absence of soft tissue expansion , and teeth of a normal color . all of the teeth were asymptomatic , with no pain or tenderness on percussion or palpation . the involved teeth were vital in an electric stimulation test . for assessment before implant insertion , cbct had been ordered . during evaluation of the implant insertion areas on promax3d cbct ( planmeca oy , helsinki , finland ) , a radiolucent - radiopaque mixed lesion located on the apices of the lower incisors was observed . on the axial , sagittal , and coronal cbct images , the extension of the lesion was observed from the mesial side of the right mandibular lateral incisor to the distal side of the left mandibular lateral incisor . it was a multifocal lesion in which solitary lesions were reached together and made a larger lesion . the total dimension of the lesion was about 16.6 mm in the mesiodistal direction and 6.9 mm in the longest superior - inferior direction . on the panoramic reconstructed cbct image , the lesion associated with the left mandibular lateral incisor was radiolucent , whereas the lesion on the apex of the left central incisor was mixed radiolucent - radiopaque and the lesion associated with the right central incisor was radiopaque with a radiolucent rim around the lesion of this tooth ( fig . the state of the lesion relative to the buccal and lingual cortical plates could be assessed , which might not be possible on the conventional radiographs . on the axial image , one of them was located at the mesial side of the right canine and the other between the left central and lateral incisors ( fig . cross - sectional images , the discontinuity of the lingual cortex was found at the area between the two central incisors on several consecutive sectional images ( fig . this cortical discontinuity was more obvious on the three - dimensional ( 3d ) cbct images ( fig . however , it should be considered that the cortical bone could be seen to have destruction on 3d images , even though it would have been thin without discontinuity on the cross - sectional images . on the digital periapical radiograph that was taken for periodontal ligament space widening was found , especially around the root of the left lateral incisor . based on the patient clinical and radiographic findings , a diagnosis of multifocal periapical cemento - osseous dysplasia was made . pcod is a specific lesion within this group of conditions that usually occurs in middle - aged black women.4 a systematic review of the literature on cod indicated an ethnic distribution of 59% , 37% , and 3% for blacks , asians ( japanese , chinese , and korean ) , and caucasians including indian cases , respectively , in case reports.8 zegarelli et al reported the incidence of pcod in the general population to be 2 - 3/1000.9 periapical cemento - osseous dysplasia generally does not cause cortical bone expansion or perforation.9 - 12 alsufyani and lam reviewed the clinical and radiographic characteristics of 118 patients with cod.13 they showed that 71.6% of 118 patients had no cortical expansion , 76% had intact lamina dura , and 93% had a normal periodontal ligament space . modern advances in imaging help the dentist to use the cbct with the appropriate field of view and spatial resolution ; therefore , the internal mineralized structure of the pathologic lesions can be investigated with a low radiation dose.14 axial cbct images clearly demonstrate the location and extension of the lesion . the expansion and perforation of the cortical plates can be evaluated on cbct even if they are slight . this report showed the discontinuity of the lingual cortex on some axial and cross - sectional cbct images . this characteristic of the lesion was an unusual feature among the cases of pcod reported up to now that may be due to its large size and needed to be checked in further regular follow - ups . this feature also could be found in an exaggerated fashion on the 3d cbct images . although 3d imaging could help the radiologist in diagnosing the lesion for the first look , it should be noted that it is not a reliable viewing modality for assigning the real status of the lesion in relation to its surrounding bone structures . in many cod cases that have been misdiagnosed and/or mismanaged , the lesions were identified in their early stages as a periapical rarefying osteitis such as periapical abscess , granuloma , or cyst , and unnecessary endodontic treatment was performed . therefore , vitality tests are especially important for differential diagnosis . in the radiolucent - radiopaque mixed stage and the radiopaque stage , the differential diagnosis might include chronic sclerosing osteomyelitis , cementoossifying fibroma , odontoma , cementoblastoma , and osteoblastoma.13 periapical cemento - osseous dysplasia is usually detected on routine radiological examinations . however , the differential diagnosis of our case could be calcifying cystic odontogenic tumor and cemento - ossifying fibroma . by using cbct discrimination of pcod from these lesions that exhibit similar internal calcification on conventional radiography high density mass in pcod which is centered in low density area is different from findings of calcifying cystic odontogenic tumor in which calcification is observed at or near the cyst wall.15 also , cemento - ossifying fibroma has more obvious concentric buccolingual expansion on multiplanar cbct image . unless pcod is symptomatic , treatment is usually not needed because development and maturation of the lesion is self - limiting . intervention may cause secondary infection of the cementum - like radiopacities , which may in turn induce osteomyelitis in these lesions.13 however , if pcod demonstrates unusual changes or becomes symptomatic , surgical intervention would then be needed.7 this case was considered as no treatment with only periodic follow - up check .

periapical cemento - osseous dysplasia ( pcod ) is a subtype of cemento - osseous dysplasia that usually occurs in middle - aged black women . this report described a case of a 45-year - old iranian woman who was diagnosed with pcod on the basis of cone beam computed tomographic ( cbct ) findings . cbct enabled detailed visualization of the bone changes . this report described the special radiographic characteristics of pcod , including discontinuity of the lingual cortex on the cbct sectional and three - dimensional images .

worldwide , the population with diabetes is currently estimated at 366 million and is expected to exceed half a billion by 2030 . foot ulcers are the principal cause of severe complications and hospitalization among patients with diabetes , substantially increasing the costs with this disease . in the united states , the annual cost of foot ulcers is estimated at us$11 billion . in brazil , the population aged 30 years and over with type 2 diabetes is estimated at 6.5 million . among these , roughly 323 000 cases of foot ulcers are reported annually , 97 000 of which require hospitalization . adding to the costs of managing infection , patients with diabetes are confronted with the risk of limb amputation , with rates 30 to 40 times higher than in individuals without the disease . studies have shown the incidence of diabetic foot to be on the order of 3% to 4% , accounting for roughly 11 million patients with this condition in 2014 [ 5 , 6 ] . peripheral neuropathy , ulceration , infection , and peripheral vascular disease are the principal factors for ulcer complications and loss of a lower limb in diabetic patients [ 7 , 8 ] . nonetheless , ambiguity remains as to which factors are most conducive to amputation outcomes and how strongly they affect these events . structured healthcare is one of the most effective approaches to reducing the indicators for diabetic foot amputation , and studies have shown that these can be reduced by as much as 75% . factors such as low socioeconomic status , smoking [ 10 , 11 ] , gender , renal impairment , ischemia , diabetic neuropathy , and high levels of glucose and triglycerides have been reported as importantly associated with the risk of foot amputation . this study evaluated the effect that clinical , biochemical , epidemiological , and patient - behavior - related predictors have on amputation outcomes in patients with diabetic foot . knowledge of these factors and their influence on this outcome is critical to enable multidisciplinary teams to develop management and treatment plans for diabetic patients so as to prevent the need for foot amputation . this cross - sectional study comprised 100 patients with diabetic foot hospitalized at the vascular surgery clinic of the conjunto hospitalar de sorocaba , in sorocaba county , so paulo state , southeastern brazil . inclusion criteria were minimum age of 18 years , diagnosis of diabetes , presence of infected ulcers on a lower limb , and agreement to participate ( expressed by signing a consent form ) . the project was approved by the research ethics committee of the universidade de sorocaba ( opinion 0028/10 ) and complied with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . the patients responded to a structured questionnaire about their sociodemographic status , knowledge of the disease , previous antibiotic use , and compliance with diabetes treatment . data on the clinical characteristics and health status of patients were collected from medical records . the clinical and laboratory evaluations were performed at the laboratory for diabetes conjunto hospitalar de sorocaba . comorbidities had been evaluated by a group of specialists , based on consensus and guidelines [ 1519 ] . compliance with outpatient treatment for diabetes was evaluated using the morisky test , which consists of four simple questions . sometimes , if you feel worse when you take you medication , do you stop taking it ? the higher the score , the more adherent the patient . given the high prevalence of limb amputation , we estimated prevalence ratios and their respective confidence intervals ( 95% ci ) for the univariate analysis of the relationships between variables and outcomes , using shapiro - wilk test , student 's t - test , or mann - whitney test . the variables with p values of less than 0.25 were selected for multivariate analysis using the cox regression model with robust variance . table 1 shows that age of the patients ( n = 100 ; 32 women , 68 men ) ranged from 31.9 to 89.7 years ( median : 62 years ) , with 55% of patients older than 60 . most patients were male ( 68% ) , caucasian ( 78% ) , poorly educated ( 69% ) , nonsmokers ( 81% ) , and alcoholics ( 84% ) and had type 2 diabetes ( 99% ) . of the total , 22% had been diabetic for less than five years , 24% from five to 10 years , 17% from 10 to 15 years , 16% from 15 and 20 years , and 21% for more than 20 years . in most patients , most had attended annual medical appointments ( 73% ) and , over the past year , had attended more than three appointments ( 67% ) and tested for blood glucose levels ( 86% ) . glucose levels at admission ranged from 4.10 to 28.7 mmol / l ( mean : 12.43 5.03 mmol / l ) . the most frequent chronic complications were neuropathy ( 91% ) , hypertension ( 72% ) , vascular peripheral disease ( 63% ) , retinopathy ( 42% ) , dyslipidemia ( 41% ) , nephropathy ( 26% ) , coronary insufficiency ( 23% ) , and cerebrovascular insufficiency ( 16% ) . on admission , 75% of patients had grade 4 ulcers , while 20% had grade 3 and 5% had grade 2 ulcers . less than half of the patients had undergone a prior conventional , nonsurgical procedure ( debridement ) ( 45% ) or amputation ( 32% ) . for 74% , most , however , had an ulcer of less than 2 cm ( 84% ) , gangrene ( 76% ) , and a neuroischemic diabetic foot ( 86% ) . most patients showed signs of inflammation ( 89% ) and had osteomyelitis ( 52% ) , which was also present with the high incidence of grade 4 ulcers ( 75% ) . compliance with treatment was poor in 72% of patients ( score 2 for 35 individuals , score 3 for 15 , and score 4 for 15 ) , while 27 were considered compliant ( score 0 for 23 patients and score 1 for five ) . no statistically significant differences were observed in the prevalence of diabetic foot amputation with regard to gender , ethnicity , schooling , monthly income , alcohol consumption , or smoking . no statistically significant differences in the prevalence of diabetic foot amputation were detected based on the occurrence of comorbidities . however , 75% of patients with two or three previous hospital admissions for chronic complications required foot amputation , whereas only 52.6% of those with one single admission experienced this outcome ( p = 0.043 ; table 2 ) . table 3 shows that 78.6% of poor compliers ( morisky scores 0 or 1 ) had a foot amputated , whereas only 54.2% of compliant patients ( scores 24 ) did so ( p = 0.012 ) . patients with a history of conservative procedures had a lower prevalence of amputation than those not subjected to this procedure ( p < 0.001 ) . however , previous amputation was unrelated to an amputation outcome ( p = 0.255 ) . also , amputations were more frequent in patients with osteomyelitis than those lacking this condition ( p < 0.001 ; table 4 ) . to identify variables independently associated with progression to amputation , cox multiple regressions ( with robust variance ) the association between ulcer grade ( wagner criteria ) and treatment compliance score ( morisky test ) was statistically significant ( p = 0.014 , chi - squared test ) . the prevalence of gangrene in patients with higher treatment compliance was 68.1% , rising to 92.8% in less compliant individuals ( morisky scores 0 or 1 ; wagner grade 4 ) . therefore , two models were found on multivariate analysis : one using the morisky test ( table 5 ) and the other employing wagner criteria ( table 6 ) . amputation outcomes proved independently associated with previous conservative procedures , previous use of antibiotics , and morisky test scores or wagner criteria ( tables 5 and 6 ) . the risk of foot amputation for patients who had received conservative treatment was 63% lower than for those with a previous amputation ( p < 0.001 ; table 5 ) , while for individuals previously treated with antibiotics the risk of foot amputation was 42% higher than for patients not subjected to this drug therapy ( p = 0.026 ) . considering wagner grades , the risk of foot amputation was 61% lower in individuals who had previously undergone conservative procedures than in those who had not ( p < 0.001 ) , table 6 . among those previously treated with antibiotics , this risk was 36% higher than for those without antibiotic therapy ( p = 0.042 ) . furthermore , for each unit increment in wagner grade , there was a 65% increase in the risk of foot amputation in patients admitted with infectious complications in a lower limb ( p = 0.018 ) . in most subjects ( 81% ) , blood glucose levels ranged from 5.55 to 16.65 mmol / l . glucose levels below 11.09 mmol / l at admission are associated with lower morbidity and mortality , and proper glycemic control is a critical factor for the infection eradication and ulcer healing . chronic hyperglycemia is the most frequent etiological factor for complications of diabetes mellitus [ 2225 ] . neuropathy was reported in 91% of patients , coinciding with published data indicating a high prevalence of neuropathy in diabetic patients hospitalized for foot injuries . retinal impairment and nephropathy are the two most common microvascular complications , both of which were present in the study population ( at 42% and 26% , resp . ) . in patients with diabetes , nephropathy is a marker for generalized vascular disease , and these patients are probably more susceptible to developing peripheral vascular disease . recent studies also suggest that the incidence of diabetic foot ulcers is more frequent in individuals with micro- and macroalbuminuria [ 2830 ] . patients who reported prior use of antibiotics had a 42% higher risk of major amputation than those not receiving antibiotic therapy . previous prolonged use of antibiotics selects for resistant microorganisms , making treatment more difficult and increasing the risk of amputation . the present data suggest an increased risk of amputation in patients less compliant with drug therapy . adherence to the prescribed therapy has led to significant improvements in the health and quality of life of patients with diabetes [ 7 , 3337 ] . compliance with medication is essential in chronic diabetes , improving control of disease progression and attenuating the severity of chronic complications . reinforcement of guidelines on diabetes care and the importance of medication , both of which can increase treatment compliance , are facilitated when patients have more than three medical appointments per year . in the present investigation , patients with a history of antibiotic use had an increased risk of progressing to amputation . each unit increment in ulcer severity ( measured in wagner grades ) increased the risk of amputation . similar results were found in a brazilian study that demonstrated a directly proportional relationship between wagner grade and risk of limb amputation . it is worth noting , however , that the morisky test was originally developed for hypertension but has been used to evaluate drug treatment in patients with diabetes , a feature that may constitute a limitation of the present study . another limitation is that information on previous use of antibiotics was self - reported rather than collected from medical records . studies evaluating the extent of problems related to diabetic foot can provide elements for intervention policies and prevention programs particularly in government - funded healthcare services involving multidisciplinary teams specialized in diabetic foot care , ultimately ensuring improved treatment with more efficient use of resources . the present findings highlight that antimicrobial therapy protocols for outpatients with diabetic foot need reviewing . control of the disease before hospitalization can significantly reduce amputations in patients with diabetic foot . knowledge of these factors and their influence on amputation outcomes is critical to allow multidisciplinary teams to develop management and treatment protocols for patients with diabetes . the present findings show that limb amputation outcomes were strongly lowered by conservative treatment and compliance with diabetes drug therapy . implemented in a preventive manner , these two measures can significantly reduce lower limb amputation in patients with diabetes .

the aim of this study was to identify and quantify risk factors for amputation in diabetic patients hospitalized for foot infections . this cross - sectional study comprised 100 patients with diabetic infectious complications in the lower limbs . the variables investigated were related to diabetes , infection , and treatment compliance . multiple cox regression analysis was performed to identify the variables independently associated with the outcome of amputation . the most prevalent chronic complications were neuropathy and hypertension . most patients presented with a neuroischemic foot ( 86% ) . the morisky test showed that 72% were not compliant with diabetes treatment . regarding patient outcome , 61% progressed to amputation , 14% to debridement , and 9% to revascularization . the results showed a 42% higher risk for progression to amputation in patients with previous use of antimicrobials . also , the amputation risk was 26% higher for those less compliant with diabetes treatment . an increase of one point in the wagner ulcer classification criteria corresponded to a 65% increase in the risk of amputation . undergoing conservative , nonsurgical procedures prior to admission provided a 63% reduction in the risk of amputation . knowledge of these factors is critical to enable multidisciplinary teams to develop treatment plans for these patients so as to prevent the need for amputation .

microcystic adnexal carcinoma ( mac ) is a rare malignant cutaneous neoplasm with pilar and eccrine gland differentiation . mac was first described as a separate clinical entity by goldstein et al in 1982 . it is locally aggressive but rarely metastasizes , usually presenting as a slow growing asymptomatic lesion on the head and neck . fewer than 300 cases have been reported worldwide , according to an analysis by yu et al . a 67-year - old man presented to a primary care skin cancer clinic in melbourne , australia for a routine six - month skin cancer examination . there was a long history of recreational sun exposure . seven separate basal cell carcinomas had required excision from his forehead , nose , pinna , posterior neck , mid back calf in the last decade . most recently a moderately differentiated squamous cell carcinoma had been excised from his right upper forehead some six months previous . a whole body skin examination was undertaken with the aid of a heine delta 20 non - polarizing dermatoscope ( heine optotechnik , herrshing , germany ) . digital clinical and dermatoscopic images were taken with a medicam 800 fotofinder non - polarizing camera ( fotofinder systems gmbh , aichner , birnbach , germany ) , the dermatoscopy images being at 20 magnification . examination confirmed fitzpatrick skin type 2 with severe actinic damage to the skin of his face , upper trunk and distal limbs with multiple solar lentigines and actinic keratoses . significant actinic damage to the lower lip ( actinic cheilitis ) was apparent . during the examination the patient pointed out a white , scar - like lesion on his mid left cheek measuring 8 4 mm in diameter . it was non - pigmented and was composed of a clearly demarcated flat white plaque ( figure 1 ) . dermatoscopically the lesion exhibited a dense white structureless area with fine linear branched blood vessels centrally . a notable feature was the white clods of variable diameter superiorly ( figure 2 ) . eccrine syringoid carcinoma , a rare malignant cutaneous adnexal tumor , should also be included in the differential diagnosis . an excisional biopsy was performed using an elliptical excision , and the specimen was submitted for assessment by a specialist dermatopathologist . examination of the histological sections revealed a deeply invasive dermal neoplasm composed superficially of keratin filled cysts with calcification , and in the underlying reticular dermis , of infiltrative aggregates of basaloid cells in slender strands and syringomatoid aggregates . the differential diagnosis was between a microcystic adnexal carcinoma and a fibrosing basal cell carcinoma with follicular differentiation . this staining pattern , although not entirely specific , was more in favour of microcystic adnexal carcinoma than fibrosing basal cell carcinoma . ber - ep4 expression has been noted in 38% of microcystic adnexal carcinomas and 100% of basal cell carcinomas . however ck15 is expressed in 92% of microcystic adnexal carcinomas , whereas basal cell carcinomas are negative . hence , in conjunction with the positive cea , the findings favoured a microcystic adnexal carcinoma ( figures 3 to 11 ) . a search of the literature has not discovered any previously published dermatoscopy images of a microcystic adenexal carcinoma . the two most notable dermatoscopic features of the lesion we present were the dense white structureless area centrally and the white clods of variable diameter peripherally . white clods of this pattern have also been observed in the more common adnexal skin tumor trichoepithelioma and may represent keratin retention cysts . microcystic adenexal carcinoma is currently considered a rare tumor . however , such rarities will present more often as the world population increases in age and has increased access to modern medicine . the authors feel it is important to publish such dermatoscopic images as ours to as wide an audience as possible to aid clinical diagnosis in future .

we present a case report of a microcystic adnexal carcinoma on the cheek of a 67-year - old man . clinical , dermatoscopic and dermatopathologic images are presented . a search of the literature has not discovered any previously published dermatoscopy images of microcystic adenexal carcinoma .

neurofibromatosis type-1 ( nf-1 ) is a multisystem , autosomal dominant disorder of peripheral nerves affecting nearly 1/3000 individuals worldwide.1 it was first described by a german pathologist , friedrich daniel von recklinghausen . inherited or spontaneous mutation of the neurofibromin gene located on chromosome 17 is responsible for this diverse disorder . common skeletal manifestations of nf-1 include spinal deformities , congenital tibial dysplasia ( congenital bowing and pseudarthrosis ) , sphenoidal dysplasia and cystic lesions in bones . pathological fracture of the acetabulum with anterior dislocation of hip secondary to osseous involvement of the acetabulum , femoral head , and pubic rami has never been documented in a case of nf-1 . a 16-year - old boy presented with the complaints of pain in the left hip associated with the inability to bear weight following a trivial fall . on examination , the affected limb was 1.5 cm short , abducted and externally rotated . on general examination , patient had 8 caf au lait spots over the body , bilateral axillary freckles and multiple palpable neurofibromas in the subcutaneous tissues of forearm , thighs and back [ figure 1 ] . patient met three out of seven criteria described for the diagnosis of nf-1 [ table 1].2 plain radiograph and computed tomography scan of pelvis revealed an ill - defined lytic lesion causing pathological fracture - dislocation of the left hip [ figure 2 ] . magnetic resonance imaging ( mri ) showed additional soft tissue involvement and joint effusion [ figure 3 ] . ultrasound guided fine - needle aspiration cytology showed scanty cellularity with round to oval cells having minimal pleomorphism ; hyperchromatic nucleus and moderate cytoplasm with spindle cells and osteoblasts . clinical photograph showing skin lesions - caf au lait spots ( black arrows ) and axillary freckling ( white arrow ) criteria for diagnosis of nf-1 ( at least 2 or more features ) preoperative radiograph anteroposterior view ( a ) and computed tomography scan ( b ) of pelvis showing an ill - defined lytic lesion destroying anterior column of acetabulum , pubic rami and part of the femoral head coronal ( a ) and axial ( b ) sections of magnetic resonance imaging of pelvis showing expansile lytic lesion of acetabulum and pubic rami with soft tissue involvement and joint effusion a wide local excision followed by arthrodesis of the joint was planned . considering the extent of bony and soft tissue involvement , we used a modification of the ilioinguinal and iliofemoral approach to have a wide exposure . we used the conventional ilioinguinal incision and combined it with femoral part of the iliofemoral incision [ figure 4a ] . on exposing the pelvis , anatomy was distorted . the deformed femur head and acetabulum with deficient pubic rami there was extensive soft tissue involvement adjacent to the acetabulum and lower part of the ilium . the entire acetabulum with 2 - 3 cm clear margin of the ilium was resected along with the abnormal soft tissue . iliofemoral arthrodesis was done using a 14 hole stainless steel dynamic compression plate [ figures 4c d , and 5 ] . intraoperative photographs showing ( a ) skin incision ( b ) deformed femoral head ( c ) 14 hole dcp ( d ) iliofemoral arthrodesis postoperative radiograph after tumor resection and iliofemoral arthrodesis histopathology revealed dense collagenous tissue cores with spindle cells having blunt nuclei with minimal atypia and no mitosis or necrosis [ figure 6a ] . on immunohistochemistry , cells were s-100 positive and of neural origin [ figure 6b ] . partial weight bearing was allowed at 6 weeks and full weight bearing at 10 weeks . at 1-year followup , the patient was comfortable , pain free , able to ambulate unassisted , stand on one limb , sit and climb stairs without any difficulty [ figure 7 ] . ( a ) histopathological photomicrograph showing dense collagenous tissue cores and spindle cells with blunt nuclei . ( b ) immunohistochemistry showing neural marker s-100 positivity clinical photographs showing functional outcome at 1-year followup type 1 neurofibromatosis or von recklinghausen disease , is a multisystem disorder that primarily affects the cell growth of neural tissue and characterized by involvement of skin , peripheral nerves , subcutaneous tissue , eyes , and skeletal system . although involvement of the musculoskeletal system is common , there have been only a few cases of subluxation / dislocation of hip in patients with nf [ table 3].34567891011121314 on reviewing the literature , the etiology of hip instability leading to pathological subluxation / dislocation in patients with nf-1 can be classified as local and remote . most of the cases are secondary to local ( intra and peri - articular ) neurofibromas , which can result in mass effect , bony erosions ( ilium , acetabulum , and femoral neck ) , acetabular dysplasia , narrowing of the femoral neck , coxa valga , increased femoral neck offset , capsuloligamentous laxity , and synovial membrane proliferation.3458101113 remote causes of hip instability include intra spinal neurofibromas / schwannomas leading to motor deficit ( hip abductor weakness ) or sensory deficit ( charcot 's neuropathic arthropathy ) , limb length discrepancies secondary to hemi - hypertrophy of lower limb and abnormal biomechanical alteration in the spinopelvic alignment due to scoliosis.6791011 endo et al . described anterior subluxation of hip secondary to decreased femoral head coverage resulting from decreased lumbosacral lordosis and posterior pelvic inclination following scoliosis correction.11 until date , there has been no case of nf-1 reported in the literature with pathological fracture of the acetabulum with anterior dislocation of hip attributable to a neurofibroma involving the acetabulum , pubic rami and femoral head . orthopedic manifestations of nf-1 comprehensive literature review on published cases of hip dislocation / subluxation in nf-1 the various treatment options described for pathological hip dislocations in nf-1 include closed reduction , open reduction , shelf operation with fascia lata tenorraphy , rotational acetabular osteotomy with femoral varus osteotomy , girdle stone resection , total hip replacement with the trochanteric distalisation.791114 the rate of re dislocation is very high in most of the cases , subsequently requiring a secondary surgical procedure for stabilization . since only a handful of cases have been described in the literature , it is difficult to comment upon the best line of management . in our case , arthrodesis was the best possible option since the bone stock after tumor resection was so inadequate that none of the above mentioned procedures could be tried . moreover , the fear of redislocation , which might necessitate repeated surgeries , was negated . a combination of ilioinguinal and iliofemoral approach was employed to have a better exposure of the hip joint . the neurofibromatous tumors associated with nf-1 are usually benign ; however , there is a 2 - 5% chance of malignant transformation , especially with plexiform neurofibromas.15 plexiform neurofibromas are diffuse , poorly defined nerve sheath tumors arising from multiple nerve fascicles and surrounding tissues . they are more prone for hemorrhage , dysfunction , pain , disfigurement , and malignant transformation.16 there was no clinical or radiological evidence of recurrence or malignant transformation in our patient at 1-year followup . subtle clinical signs such as skin patches , axillary freckling , and subcutaneous neurofibromas can be easily missed . orthopedic surgeons must be aware about the various management options available and tailor them as per the needs of their patient . iliofemoral arthrodesis offered a good functional outcome with improved quality of life in our case .

skeletal neurofibromatosis ( nf ) commonly manifests as scoliosis and tibial dysplasias . nf affecting the pelvic girdle is extremely rare . pathological fracture of the acetabulum leading to anterior hip dislocation in a patient with nf-1 has never been reported in the literature . the paper presents the clinical symptomatology , the course of management and the successful outcome of such a rare case of nf-1 . histopathological and immunohistochemistry studies showing abundant spindle cells , which are s-100 positive and of neural origin are the classical hallmarks of neurofibromatous lesions . tumor resection and iliofemoral arthrodesis can be considered as a valid option in young patients with pathological fracture dislocation of the acetabulum .

recent papers by and show the application of fractional calculus to pharmacokinetics ( pk ) . leffler function : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{\alpha } \left ( z \right ) = \sum\limits_{i = 0}^{\infty } { { \frac{{z^{i } } } { { \upgamma \left ( { \alpha i + 1 } \right ) } } } } $ $ \end{document}in place of the mono - exponential:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e^{z } = \sum\limits_{i = 0}^{\infty } { { \frac{{z^{i } } } { i ! } } } = \sum\limits_{i = 0}^{\infty } { { \frac{{z^{i } } } { { \upgamma \left ( { i + 1 } \right ) } } } } = e_{1 } \left ( z \right ) $ $ \end{document}if drug is given as a bolus dose in a venous site , drug concentration in plasma at a time t after the dose administration , c(t ) , can be represented using a response function of the form:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = dose \ , \left [ { \theta e_{\alpha } \left ( { - \lambda t^{\alpha } } \right ) } \right ] $ $ \end{document}where dose is the amount of drug given , and 1 have the interpretation of the elimination rate constant and the reciprocal of volume of distribution of the plasma compartment , respectively , if model ( 3 ) is seen as solution corresponding to a single compartment model described by a ( caputo ) fractional differential equation of order , see , e.g. , [ 5 , 6 ] . the paper of shows solutions for some specific two- and three - compartmental structures described by fractional order kinetics . however , as pointed out by , while the connection between response function and compartmental structure is immediate for the single compartment case , this is less so for the case of multi - compartmental ones . the purpose of this communication is to discuss this connection , and to do so we will ( 1 ) clarify the distinction between different types of systems of fractional differential equations , in particular discussing the difference between commensurable and non - commensurable ones , ( 2 ) show solutions for the corresponding response functions , and ( 3 ) discuss their application to the modeling of pk data . commensurate fractional order linear systems are described by a system of linear fractional differential equations ( fde ) of the form : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \frac{{d_{\blacktriangle } ^{{^{\alpha } } } } } { { { \text{d}}t}}}{\mathbf{x}}(t ) = \left ( { \begin{array}{*{20}c } { { \frac{{d_{\blacktriangle } ^{{^{\alpha } } } } } { { { \text{d}}t}}}x_{1 } ( t ) } \\ \ldots \\ { { \frac{{d_{\blacktriangle } ^{{^{\alpha } } } } } { { { \text{d}}t}}}x_{m } ( t ) } \\ \end{array } } \right ) = \left ( { \begin{array}{*{20}c } { a_{11 } } & \ldots & { a_{1 m } } \\ \ldots & \ldots & \ldots \\ { a_{m1 } } & \ldots & { a_{mm } } \\ \end{array } } \right){\mathbf{x}}(t ) + { \mathbf{f}}(t ) = a{\mathbf{x}}(t ) + { \mathbf{f}}(t ) $ $ \end{document}with initial conditions\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{x}}(0 ) = { \mathbf{x}}_{0 } $ $ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d_{\blacktriangle } ^{{^{\alpha } } } $ $ \end{document } is the caputo fractional differential operator of order > 0 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{f}}(t ) $ $ \end{document}is the ( vector valued ) input function to the system . these systems are called commensurate because all the differential equations are of the same fractional order , . as a consequence t can be shown that the solution to the system of fde represents the entire state of the system at any given time . in particular , a compartmental system can be obtained , for 0 < 1 , exactly as for a system of ode , by introducing mass - balance constrains:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{ji } \ge 0,\quad i \ne j $ $ \end{document}and the following:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \text{a}}_{ii } \le 0 \\ \left| { { \text{a}}_{ii } } \right|\ge \sum\limits_{\begin{array}{l } j = 1 \\ j \ne i\end{array } } ^{m } { a_{ji } } \hfill \\ \end{array } $ $ \end{document}which guarantee that all states are non - negative . it can be shown ( see e.g. ) that the solutions to the system of linear fde ( 4 ) depend on the eigenvalues of its characteristic equation , that is , in the laplace domain , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \det \left ( { b(s ) = a(s ) - si } \right ) $ $ \end{document}. in particular if the eigenvalues are real and distinct the solution to eq . 4 takes the form1:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{x}}(t ) = b_{1 } { \mathbf{u}}_{1}^{(1 ) } e_{\alpha } ( \lambda_{1 } t^{\alpha } ) + b_{2 } { \mathbf{u}}_{2}^{(2 ) } e_{\alpha } ( \lambda_{1 } t^{\alpha } ) + \cdots + b_{m } { \mathbf{u}}_{m}^{(m ) } e_{\alpha } ( \lambda_{1 } t^{\alpha } ) $ $ \end{document}where b1 , b2 , , bm are constants , 1 , 2 , , m and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{u}}_{1}^{(1 ) } , { \mathbf{u}}_{2}^{(2 ) } , \ldots , { \mathbf{u}}_{m}^{(m ) } $ $ \end{document } are the eigenvalues and eigenvectors of the characteristic equation for ( 4 ) . it is immediate from ( 7 ) that for a bolus input in the j - th compartment the solution for drug concentration in the same compartment takes the form:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{j } ( t ) = dose\left [ { \theta_{1 } e_{\alpha } ( \lambda_{1 } t^{\alpha } ) + \theta_{2 } e_{\alpha } ( \lambda_{2 } t^{\alpha } ) + \cdots + \theta_{m } e_{\alpha } ( \lambda_{m } t^{\alpha } ) } \right ] $ $ \end{document}which establishes a direct connection with the familiar multi - exponential response function corresponding to ordinary multi - compartment linear systems with distinct eigenvalues . a non - commensurate fractional order linear system is described by : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { \begin{array}{*{20}c } { { \frac{{d_{\blacktriangle } ^{{^{{\alpha_{1 } } } } } } } { { { \text{d}}t}}}x_{1 } ( t ) } \\ \ldots \\ { { \frac{{d_{\blacktriangle } ^{{^{{\alpha_{m } } } } } } } { { { \text{d}}t}}}x_{m } ( t ) } \\ \end{array } } \right ) = a{\mathbf{x}}(t ) + { \mathbf{f}}(t ) $ $ \end{document}where now 1 , 2 , , m are distinct ( real positive ) numbers indicating the fractional order for each equation . as remarked by , in reference to the systems of compartments shown in ( 2 ) , the equations in do not satisfy mass - balance even if conditions ( 5 ) are satisfied , and in general the solution to the system of fde ( 9 ) does not represent the states of the system . non - negativity is also no longer guaranteed by the relationships ( 6 ) ( and there are non - trivial issues associated with demonstrating the stability , observability and reach - ability of such systems , see . ) numerical methods must be employed to find the solution to ( 9 ) , since a close form solution equivalent to ( 7 ) does not exist [ 10 , 11 ] . however , solutions can be obtained if is assumed that the fractional orders are rational numbers , that is i = pi / qi where pi , qi are integers , i = 1 , , m ( 12).2 the mathematics necessary to obtain the general solution are quite involved , and for the purpose of this paper we only show a subset of the possible solutions , in particular for c(t ) ( see [ 1113 ] for more general results ) . for a non - commensurate system , it can be shown that a solution for drug concentration in the j - th compartment takes the form:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{j } ( t ) = dose\left\ { { t^{{\gamma - \alpha_{j } } } \left [ { \theta_{1 } e_{{\gamma , \gamma - \alpha_{j } + 1 } } ( \lambda_{1 } t^{\gamma } ) + \theta_{2 } e_{{\gamma , \gamma - \alpha_{j } + 1 } } ( \lambda_{2 } t^{\gamma } ) + \cdots + \theta_{m } e_{{\gamma , \gamma - \alpha_{j } + 1 } } ( \lambda_{m } t^{\gamma } ) } \right ] } \right\ } $ $ \end{document}where = 1/q , q = m.c.d(q1 , , qm ) , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{\alpha , \beta } \left ( z \right ) = \sum\limits_{i = 0}^{\infty } { { \frac{{z^{i } } } { { \upgamma \left ( { \alpha i + \beta } \right ) } } } } $ $ \end{document } is the two - parameters mittag leffler function ( 5 ) . one notices two important facts : first , this solution only depends on the fractional order for compartment j , j , and , second in direct analogy to the solution ( 8) above , the mittag leffler function exponents are determined by the eigenvalues of the characteristic equation for the system . we now have the ingredients to show a simple example of applications of multi - terms mittag leffler response functions to fit pk data . we consider the case m = 2 , which for a standard ode system generates the response function:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = dose\left ( { \theta_{1 } e^{{\lambda_{1 } t } } + \theta_{2 } e^{{\lambda_{2 } t } } } \right ) $ $ \end{document}for a commensurate fde system obtains:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = dose\left ( { \theta_{1 } e_{\alpha } ( \lambda_{1 } t^{\alpha } ) + \theta_{2 } e_{\alpha } ( \lambda_{2 } t^{\alpha } ) } \right ) $ $ \end{document}and for a non - commensurate fde system\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = dose \ , t^{\gamma - \alpha } \left ( { \theta_{1 } e_{\gamma , \gamma - \alpha + 1 } ( \lambda_{1 } t^{\gamma } ) + \theta_{2 } e_{\gamma , \gamma - \alpha + 1 } ( \lambda_{2 } t^{\gamma } ) } \right ) $ $ \end{document}the parameters 1 , 2 , 1 , 2 , , are estimated from the data , with the constraints 1 , 2 > 0 , 1 , 2 < 0 , and 0 < , 1 , which guarantee that eq . 13 is non - negative and non - increasing ( strictly monotone ) for t 0.3 to evaluate the single and two - parameters mittag leffler function we implemented a fortran 90 version the algorithm reported in . we used the computer program nonmem to obtain the parameters estimates . as a check , fig . 11 of , = 1 and 0 < 2 . contrary to , which has a strong influence on the overall shape of the curve , the parameter has its most pronounced influence on the value of the function at t = 0.fig . 1the mittag leffler function e,(t ) for = 1 and ( 0 , 2 ) the mittag leffler function e,(t ) for = 1 and ( 0 , 2 ) figure 2 shows the fit of models ( 11 ) ( solid line ) , ( 12 ) ( widely dashed line ) , and ( 13 ) ( dashed line ) , to error corrupted data simulated using an eight compartments mammillary model . note the added flexibility introduced by use of a sum of single- and two - parameters mittag leffler functions in respect to exponentials : the values of minus twice log - likelihood for the fit of the simulated data were 316.895 , 381.974 , and 406.354 , for models ( 11)(13 ) , respectively . of course , this is just an example to show the feasibility of the approach : for this simulation , a sum of exponentials would fit the simulated data perfectly well.fig . 2simulated data ( circles ) with superimposed the fit of the response function for a second order ordinary system , eq . 11 in the text ( solid line ) , commensurate fde , eq . 12 ( widely dashed line ) , and non - commensurate fde , eq . 13 ( dashed line ) simulated data ( circles ) with superimposed the fit of the response function for a second order ordinary system , eq . 11 in the text ( solid line ) , commensurate fde , eq . 12 ( widely dashed line ) , and non - commensurate fde , eq . 13 ( dashed line ) following up on the papers of and , and the commentary by , the first purpose of this commentary is to further clarify the nature of systems of fde , and in particular to point out the distinction between commensurate and non - commensurate ones . commensurate systems of fde have a direct relationship with system of ode , and in particular when formulated in terms of compartmental models ( that is , satisfying mass balance and non - negativity constraints , see eqs . 5 , non - commensurate systems of fde do not , in general , represent the state of a system . leaving to the side the issue of what exactly system ( 9 ) represents , one is still justified in using it as a black - box type model for single - input / single - output experiments , as long as physical constraints ( non - negativity in particular ) are satisfied . to do so response functions are a convenient tool , and we show that , for commensurate and non - commensurate fde , relatively simple ones can be derived which satisfy such requirements . the solutions for a system of commensurate fde takes the form of the sum of mittag leffler functions , with a single parameter , eq . 8 , while solutions for a system of non - commensurate fde can be expressed by a sum of two - parameters mittag leffler functions , such as eq . , one can establish a direct analogy between the familiar sum of exponentials used in pk , and importantly all the relationships between compartmental transfer rate constants and the intercepts and exponents of the corresponding response functions found in classic textbooks on pk [ 17 , 18 ] , carry forward to fractional differential equations . in conclusion , while insight into the physiological interpretability of fde system might be gained in the future , and the formulation of non - commensurate systems of fde to represent the states of a system ( required by , e.g. , a physiological flow model ) requires further investigation , the response functions ( 8) and ( 10 ) can be used to investigate the existence of pk data sets which might actually show complex fractional kinetics.4

systems of fractional differential equations ( sfde ) have been increasingly used to represent physical and control system , and have been recently proposed for use in pharmacokinetics ( pk ) by ( j pharmacokinet pharmacodyn 36:165178 , 2009 ) and ( j phamacokinet pharmacodyn , 2010 ) . we contribute to the development of a theory for the use of sfde in pk by , first , further clarifying the nature of systems of fde , and in particular point out the distinction and properties of commensurate versus non - commensurate ones . the second purpose is to show that for both types of systems , relatively simple response functions can be derived which satisfy the requirements to represent single - input / single - output pk experiments . the response functions are composed of sums of single- ( for commensurate ) or two - parameters ( for non - commensurate ) mittag leffler functions , and establish a direct correspondence with the familiar sums of exponentials used in pk .

it is caused by a complex interplay of motor , sensory , and cognitive impairments2 . these neurological deficits are the prime cause of reduced quality of life and social participation3 . thus , gait and balance recovery is regarded as a chief goal in stroke rehabilitation4 . until now , various exercise programs such as progressive exercise5 , 6 , muscle strength exercise7 , rhythmic pattern exercise8 , 9 , and virtual training10 have been used to regain the balance , mobility , and endurance of stroke patients in clinical and research settings . among these interventions , repetitive motor training can alter brain representation maps and is mainly and basically used for managing the motor function recovery in stroke patients11 . skilled activity is necessary to drive brain changes that might lead to improvements in functional activities such as gait12 . stationary cycling , which requires less balance capability , has been used for training patients with or without nervous system disorders who have difficulty in maintaining balance and independent gait13 . cycling and walking share similar locomotor patterns of reciprocal flexion and extension movements and alternating muscle activation of antagonists14 . cycling can improve functional mobility and acts as a pseudo walking task - oriented exercise2 . besides improving muscle strength , cycling exercise also facilitates muscle control of the lower limbs , which may allow putting more weight on the affected leg while standing . for this reason , stationary cycling exercise has been used with various other interventions in the clinical environments15 . however , the pure effect of cycling exercise is uncertain in chronic stroke patients . therefore , this study investigated the effects of stationary cycling exercise on the balance and gait abilities of chronic stroke patients . this study was designed as a randomized , double - blind , pretest - posttest controlled trial . in this study , all experimental procedures and contents were explained to each participant , who provided written informed consent thereafter . all of the experimental procedures were approved by the institutional review board of sahmyook university . the inclusion criteria were as follows : presence of hemiparesis secondary to stroke that had occurred in the past 6 months ; ability to walk 10 m independently with or without an assistive device ; ability to communicate and understand , with a mini - mental status examination score of more than 21 points ; no visual disorders or visual field deficit ; and no known musculoskeletal conditions that would affect the ability to walk safely . the 6 subjects who refused to participate in the present program or did not meet the inclusion criteria were excluded from the study . the subjects were randomly assigned to the following 2 groups by using a table of random sampling numbers : the experimental and the control group . both the subjects and the therapist were blinded to group assignments of the patients . all of the participants were evaluated before training and at the end of the 4-week training period . the patients in the experimental group performed the cycling exercise 30 minutes a day , 5 times a week for 4 weeks . both groups received traditional therapy for 30 minutes per session , 5 times a week for 4 weeks . stationary bicycle training has been used in order to improve the balance and walking abilities of stroke patients . in this study , only the lower extremity part of a dual - extremity ergometer ( super dynamic 3000 , shingwang medical ) was used . to perform the exercise , the patient mounted a stationary bicycle safely under supervision , and the therapist adjusted the position of the seat and tied the ankles and calf to the pedals . the therapist was fully aware of the order and method of the stationary bicycle training , and the patients performed the exercise after receiving instructions and familiarizing themselves with the ergometers , including test runs . the resistance was set at 1 of 4 , which corresponded to 2530 w on the stationary bicycle . the patient was riding the bicycle at 5060 rpm without stopping , for 30 min , 5 times a week for 6 weeks . the subjects balancing skills were rated using the berg balance scale ( bbs ; range , 056 ) , and the timed up - and - go ( tug ) test was used to evaluate dynamic balance abilities . the patients were asked to stand up , walk at a comfortable speed to a point marked 3 m away from their chair , turn around , walk back , and sit down in the chair . the cut - off tug test score that indicated normal versus below normal performance was 12 seconds . the intra - rater ( r = 0.99 ) and inter - rater ( r = 0.98 ) reliability values were high16 . the bbs and tug test scores were obtained by an experienced physical therapist blinded to the group assignment . gait ability was measured by using the timed 10-m walking test ( 10mwt ) . for the 10mwt , 10 m was measured on the floor by using a tape measure , and the start and end points were marked with tape . in order to provide sufficient distance for acceleration and deceleration , intervals of 2 m were added before the start and after the end marks . the 10-m walking time was measured with a stopwatch for the period from the moment the subject s feet passed the starting line to the moment they crossed the finish line . the subjects practiced once , and all measurements were made 3 times , using the average value of the 3 measurements in the analysis . . for the 10mwt , the test - retest and inter - rater reliability have been reported to be 0.95 and 0.90 , respectively ; both of these values are very high17 . the statistical package for the social sciences ( spss ) ver . 18.0 was used for data analysis . descriptive statistics was used to analyze the general characteristics of the subjects . in order to examine the effects of the intervention in each group , a paired t test was conducted . in order to investigate differences between the groups , the demographic characteristics of the participants are shown in the table 1table 1.general characteristics of the subjectsexperimental group(n = 16)control group(n = 16)gender ( male / female)12/413/3age ( years)65.2 6.461.7 6.1height ( cm)165.0 7.9169.0 6.1weight ( kg)69.5 10.466.8 10.0lesion side ( right / left)7/910/6mmse ( score)26.10 1.7425.80 2.12data are expressed as mean sd . mmse : mini - mental state examination as shown in table 2table 2.changes in balance and gait abilitiesexperimental groupcontrol groupbbs scorepretest36.15 5.9837.06 5.61posttest37.90 5.65 * 37.44 5.62postpre1.75 1.520.40 0.88tug ( sec)pretest25.11 5.4024.19 3.47posttest16.74 3.07 * 19.48 3.90*postpre8.4 4.354.71 4.86 * 10mwt ( sec)pretest44.75 18.4045.93 13.22posttest37.74 15.70 * 43.96 12.04*postpre7.02 7.021.96 3.13*bbs : berg balance scale ; tug : timed up - and - go ; 10mwt : 10-m walking test . significant difference , paired t test : * p < 0.05 ; significant difference , independent t test : p < 0.05 , the experimental group showed significant improvements in bbs , tug test , and 10mwt scores after the intervention ( p < 0.05 ) , whereas the control group showed significant improvements in their tug test and 10mwt scores , but not in their bbs score ( p < 0.05 ) . moreover , the experimental group showed greater improvement than the control group in 3 outcome measurements ( p < 0.05 ) . bbs : berg balance scale ; tug : timed up - and - go ; 10mwt : 10-m walking test . significant difference , paired t test : * p < 0.05 ; significant difference , independent t test : p < 0.05 the aim of this study was to test the effects of cycling exercise on the balance and gait abilities of chronic stroke patients . the results demonstrated that the stationary cycling exercise supplemented with conventional therapy led to better balance and gait abilities than the conventional therapy alone . first , the stationary cycling training was found to have a positive effect on dynamic balance as measured by using the tug test . the results are similar to those obtained in a previous study by kim et al . , who compared ergometer bicycle training with treadmill walking training in stroke patients and reported significant improvement in tug test scores in both groups , although the differences between the groups were not significant18 . this suggests that the effectiveness of cycling training in improving locomotor function is similar to the effectiveness of treadmill exercise in stroke patients . preliminary evidence suggests that cycling training programs reduce musculoskeletal impairment after stroke . in terms of muscle strength , a study by kuo and zajak suggested that the muscles that may be particularly important for this purpose are the hamstrings , rectus femoris , gastrocnemius , and tibialis anterior . these were all activated during the cycling task , which requires reciprocal flexion and extension movements of the hip , knee , and ankle19 . it is interesting that lustosa et al . reported a significant correlation between muscle strength and improvement in tug test score . therefore , we assumed that in this study the mechanism of tug test score improvement in stroke patients was muscle strengthening20 . repetitive bilateral training and treadmill walking with or without suspension have a positive effect on walking ability21 . repetitive practice is known to be important for motor learning , as the repetitions enable the system to coordinate muscle synergies5 . cycling and walking share similar locomotor patterns of repetitive reciprocal flexion and extension movements . hence , stationary cycling exercise , which employs reciprocal movement of the lower limbs and requires coordination of corresponding muscles , effectively increased the gait ability . cycling training stimulates motor regions in the central nervous system and activates the cerebral cortex which eventually improves motor learning and balance . based on the results , stationary cycling training can be effective in rehabilitation of stroke patients with gait and dynamic balance deficits . this study has the following limitations that we plan to address in future studies , including the small sample size and relatively short intervention duration . further large - scale , long - term controlled clinical studies are required to verify the clinical benefits of stationary cycling exercise training .

[ purpose ] the objective of this study was to investigate the effects of stationary cycling exercise on the balance and gait abilities of chronic stroke patients . [ subjects ] thirty - two chronic stroke patients were randomly assigned to an experimental group ( n=16 ) or a control group ( n=16 ) . [ methods ] all of the subjects received the standard rehabilitation program for 30 minutes , while the experimental group additionally participated in a daily session of stationary cycling exercise for 30 minutes , 5 times per week for 6 weeks . to assess balance function , the berg balance scale and timed up - and - go test were used . the 10-m walking test was conducted to assess gait function . [ results ] both groups showed significant improvements in balance and gait abilities . the improvements in the berg balance scale and timed up - and - go test scores ( balance ) , and 10-m walking test score ( gait ) in the stationary cycling exercise group were significantly greater than those in the control group . [ conclusion ] this study demonstrated that stationary cycling exercise training is an effective intervention for increasing the balance and gait abilities of chronic stroke patients . therefore , we suggest that stationary cycling training is suitable for stroke rehabilitation and may be used in clinical practice .

since the particle size is a key consideration in nuclear transport , three nps of different sizes ( 15.0 1.2 , 8.5 0.8 , and 5.5 0.8 nm , respectively ) were synthesized to study the size effect of the nanoparticle on nuclear internalization ( see tem images in figure s1 ) . after modification with a dense shell of the das , the hydrodynamic diameters of these nps ( termed 15-np , 8.5-np , and 5.5-np ) , as measured by dynamic light scattering ( dls ) , were 37.8 1.3 , 18.2 1.2 , and 11.7 1.5 nm , respectively . to prevent mutual interference between tumor targeting and nanodrug capture , a directional modification was performed on the nrs ( 65 nm 20 nm ) , which means the aptamer and the cs were separately modified on the end faces and the side face of the nr , rather than being randomly spread . the principle of this selective modification process was based on the preference of ctab to accumulate on the side face of nr , which makes its end faces more reactive to thiolated dnas . specifically , the aptamer at a relatively low concentration was first used to occupy the nr ends . subsequently , the cs at a high concentration was added to replace ctab on the nr side face . to make the resulting nanomaterial biocompatible , thiolated peg was used , followed by several washing steps . via dna hybridization , as characterized by tem ( figure 1 ) , nps were exactly immobilized onto the nr sides , leaving the nr ends bald , indicating successful fabrication of the side - assembly nanostructures ( termed 15-np / nr , 8.5-np / nr , and 5.5-np / nr ) . the self - assembly process was further investigated with dls and uv vis absorption spectroscopy . the results from different angles all demonstrated the successful assembly of nps and nrs resulting from dna hybridization ( see details in figures s2a and s2b ) . tem images of 15-np / nrs , 8.5-np / nrs , and 5.5-np / nrs . 15-nps , 8.5-nps , and 5.5-nps mostly attach onto the nr sides , but not the nr ends , indicating the successful fabrication of the side - assembly nanostructures . the scale bar represents 50 nm . after synthesizing and characterizing these nanoassemblies , we next investigated their nir - response and drug - carrying capability . the efficient photothermal effect of nrs was verified by the rapid temperature rise of the nr medium when irradiated by the nir laser ( see details in figure s3a ) . then the nir - activated release of nps from nrs was demonstrated by the reduced average size ( dls ) and the disassembled structure ( tem ) of the 15-np / nrs after exposure to the laser ( figure s3b ) . to fabricate the nuclear - uptake nanodrug delivery system , doxorubicin ( dox ) , a widely used anticancer drug , was chosen as the model and loaded on the nps by intercalating into the ( cgt)6/(acg)6 duplexes of the das ( table s1 ) . the drug payload of nps was measured by monitoring the dox fluorescence from the supernatant after nps were centrifuged down . as shown in figure 2a , a gradual decrease of dox fluorescence was observed when increasing the molar ratio of nps . by interpolating from a standard calibration curve , the dox payload of each 15- , 8.5- , and 5.5-np was 450 19 , 277 28 , and 40 13 molecules , respectively . the stability of np / dox complexes was evaluated via a drug leakage experiment using mini dialysis units . as shown in figure 2b , the release of dox from nps was rather slow , with less than 30% of the entire payload detected in the solution after 60 h , in comparison to the rapid diffusion of free dox , indicating high stability of the np / dox complexes . furthermore , the nanoasemblies stored at 4 c for 8 weeks remained mostly intact . ( a ) dox fluorescence spectra of the supernatant after centrifuging to precipitate the das - modified nps , the dox concentration was fixed at 2 m when increasing the 15-np - das / dox mole ratio . ( b ) dox leakage dynamics from np - dox complexes ( dox : 10 m ) . ( c ) clsm images of cem cells after incubation with 15-nps , 8.5-nps , and 5.5-nps ( which represent the das - modified nps of 15 , 8.5 , and 5.5 nm , respectively ) at 37 c for 10 or 22 h. the green and blue fluorescence arises from the tamra fluorophore labeled on the 3 end of das and hoechst 33342 , respectively . the scale bar represents 5 m . having confirmed the potential of using the np / nr nanoassemblies as nir - responsive drug nanocarriers in buffer solution , we proceeded to test their performance in cells . a nondrug - resistant leukemia cell line , cem , was first used as the cell model . to monitor the cellular uptake and intracellular distribution of nps of different sizes , the das was labeled with a 5-end tamra fluorophore ( das - tmr ) . after modification with das - tmr , the fluorescent particles were incubated with the cem cells at 37 c for different lengths of time . then confocal laser scanning microscopy ( clsm ) measurements were performed , and the results are shown in figure 2c . nps of 15 nm were mainly localized in the cytoplasm , even after 22-h incubation , as indicated by the tamra fluorescence signal outside the nucleus . in contrast , 5.5-nps accumulated in the nucleus after incubation for 10 h , demonstrating rapid nuclear uptake . for 8.5-nps , no obvious signal was observed in the nucleus for 10-h incubation , but the nucleus emitted tamra fluorescence after incubation for 22 h , indicating that 8.5-np - dass can enter the nucleus starting from 10 h. to strike a balance between drug loading capability and nuclear translocation efficiency , the 8.5-np was used as a model nuclear - uptake nanoscaffold of dox in the following study . to achieve active tumor targeting , sgc8 , an aptamer that can specifically bind to protein tyrosine kinase 7 ( ptk7 , kd = 0.8 nm ) which is overexpressed on the membrane of cem cells but not ramos cells , the specific binding of sgc8 , nr - sgc8 , and np / nr - sgc8 to the target cem cells , rather than nontarget ramos cells , was demonstrated by flow cytometry ( figure 3a ) . also , the specific cellular uptake and cytoplasmic location of nr - sgc8s were visualized with clsm ( figure s4 ) . moreover , the amount of the internalized nps delivered by the nr - sgc8s was higher than that of equivalent free nps ( figure s5 ) , which may have resulted from the high payload and the favorable cell - uptake size of the nanoassembly , as well as the promotion of the cell - internalizing aptamer . specific cell binding and photocontrolled intracellular distribution of np / nr - sgc8s.(a ) flow cytometry assay proving the specific binding of sgc8 , nr - sgc8 , and np / nr - sgc8 to target cem cells not to nontarget ramos cells ( lib represents a random library sequence ) . ( b ) clsm images of cem cells after treatment with 15-np / nr - sgc8s without ( i ) and with ( ii ) nir irradiation or after treatment with 8.5-np / nr - sgc8s without ( iii ) and with ( iv ) nir irradiation . from left to right : fluorescence image for np - tmr , nr - cy5 , and overlay of the np - tmr , nr - cy5 , and hoechst 33342 fluorescence channels plus the bright field channel . the scale bar represents 5 m . the nir - responsive behavior of 8.5-np / nr - sgc8s and 15-np / nr - sgc8s in cem cells was investigated with clsm . without laser treatment , the 8.5-np signal was observed in the cytoplasm and overlapped with the nr - sgc8 signal ( figure 3b ) . however , upon nir irradiation , 8.5-nps were found in the nuclei , while nr - sgc8s remained in the cytoplasm , indicating that 8.5-nps were released from nrs after exposure to the laser and then diffused into the nuclei . the 15-nps remained in the cytoplasm irrespective of nir irradiation , corresponding well with their inability to undergo nuclear internalization . the photocontrolled nuclear internalization of 8.5-nps was further confirmed by using inductively coupled plasma atomic emission spectrometry ( icp - aes , figure s6 ) . on the basis of the fluorescence quenching of dox by intercalating into the gc duplex , the intracellular distribution of dox was investigated by treating cem cells with nonfluorophore - labeled 8.5-np - dox / nr - sgc8s . after nir irradiation and then incubation for another 22 h , the recovered fluorescence of dox was highly accumulated in the nuclei , with a relatively small amount in the cytoplasm , indicating that most dox were released in the nuclei ( figure s7 ) . however , without nir treatment , the intranuclear dox fluorescence was rather weak , showing a slow and sustained release of dox in the cytoplasm . as a control , cells were treated with free dox , and the dox signal was found throughout the cells , resulting from concentration - gradient diffusion . furthermore , the negligible influence of the laser irradiation on the stability of the np - dox complex was confirmed by the small dox signal change from the 8.5-np - dox / nr - sgc8s - treated cem cells before and right after laser exposure ( figure s8 ) . these results show great potential of the 8.5-np - dox / nr - sgc8 system for nir - controlled intranuclear drug delivery . the therapeutic effect of this 8.5-np - dox / nr - sgc8 system on cem and ramos cells was tested by mts assay . as shown in figure 4 and figure s9 , the nanomaterials themselves and the pure nir irradiation had little negative impact on either cem or ramos cells ( the cell viability of both cell lines remained above 95% ) , indicating excellent biocompatibility of these nanomaterials and the laser . for free dox , a dose - dependent cytotoxicity was observed on both cem and ramos cells . however , when treated with 8.5-np - dox / nr - sgc8s , only cem cells showed dose - dependent cell inactivity , indicating the selective cytotoxicity of dox delivered by this nanoassembly platform . after nir irradiation , a dramatic decrease of cell viability on cem cells was caused by 8.5-np - dox / nr - sgc8s with a nearly 3-fold lower ic50 of 0.36 m compared to that without nir irradiation ( 1.22 m ) . to verify whether the enhanced therapeutic efficacy originated from the synergy of dox , photothermal effect , and np / nr nanocomplexes , cem cells were incubated with free dox and non - dox - loaded 8.5-np / nr - scg8c ( the dox loading site of das was replaced with a common dna duplex ) together and then irradiated with the nir laser . the therapeutic effect of this case was lower than that of the nir - activated 8.5-np - dox / nr - scg8c , indicating that the synergistic effect was not an important consideration in this system ( figure s10 ) . thus , it is reasonable to attribute the enhanced killing efficiency to the released 8.5-np - dox from the nanotruck . to ensure that the nuclear accumulation of nanodrug induced higher cell apoptosis , the nir - activated 15-np - dox / nr - sgc8 system was used as a non - nuclear - uptake control . as shown in figure s11 , with nir irradiation , the cytotoxicity of 8.5-np - dox / nr - sgc8s was 24% higher than that of 15-np - dox / nr - scg8c , while no obvious difference was observed in either case without nir irradiation . these results demonstrate that this nuclear - uptake nanodrug delivery system can greatly enhance the therapeutic efficacy on the target cancer cells . viability of cem cells ( a ) and ramos cells ( b ) with different treatments . the error bars represent the standard deviation of three independent experiments . to investigate the ability of this nuclear - uptake nanodrug delivery system to address the mdr problem , k562/d , a drug - resistant cancer cell line with overexpression of p - gp ( figure s12 ) , was used , while its specific internalizing aptamer , kk1b10 , was used as the targeting ligand to functionalize the nanotruck . the specific binding of kk1b10 , nr - kk1b10 , and np / nr - kk1b10 with k562/d cells was proven by flow cytometry ( figure s13 ) . as shown in figure 5 and figure s14 , enhanced killing efficiency was achieved by incubating k562/d cells with 8.5-np - dox / nr - kk1b10s with nir irradiation , while a much lower therapeutic effect of free dox was detected for this cancer cell line . to confirm that the enhanced therapeutic effect originates from the intracellular accumulation of dox by this nir - responsive nanodrug delivery system , flow assay was performed to measure the dox signal in k562/d cells under different treatments . upon nir irradiation , the dox fluorescence from the sample incubated with 8.5-np - dox / nr - kk1b10s was 2.5-fold higher than that from the sample incubated with free dox ( figure s15 ) . in contrast , without laser treatment , the cells incubated with 8.5-np - dox / nr - kk1b10s produced a modest dox signal . these results demonstrate that this nuclear - uptake nanodrug delivery system can recover the chemotherapeutic sensitivity of k562/d to dox by bypassing cell membrane - expressed p - gp . viability of k562/d cells treated with free dox or nir - activated 8.5-np - dox / nr - kk1b10s . in summary , we have developed a dna - based nanoassembly platform for cancer therapy . unlike traditional intranuclear transport strategies of nanoparticles , no nuclear localization signal ( nls ) peptides are required in our design , and the nuclear uptake of nanodrugs is mainly attributed to small particle size , thus avoiding complicated nls modification processes and maintaining the valid occupancy of drug - loading probes on the np surface . by using this photocontrolled , size - transformable nanosystem , nanodrugs can be efficiently transported across the cell membrane and enter the nucleus in a coordinated and harmonious manner . furthermore , this dna - based nanoassembly platform can accumulate chemotherapeutic drugs in the nuclei , thus greatly enhancing their therapeutic efficacy against drug - resistant cancer cells by effectively bypassing p - gp . this proof - of - concept structure also opens a new door in the use of nanoassemblies for the design of drug delivery systems for biological and clinical research . to comprehensively evaluate the superiority of this nuclear - uptake nanodrug delivery system , further efforts are being made on the testing in tumor - bearing mice models . on the other hand , since the tissue penetration of the nir laser is limited to around ten millimeters , an alternative strategy for activatable dissociation of the nanoassemblies are needed to apply this system to treatment of drug - resistant metastatic tumors . all dna strands were synthesized on an abi 3400 dna synthesizer ( applied biosystems , foster city , ca , usa ) , and the specific sequences are listed in table s1 . both the synthesis and the deprotection processes were conducted as described by the reagent manufacturers . then , the dnas were precipitated by high - salt ethanol in a freezer at 20 c for 30 min and collected by centrifugation at 4000 rpm for 30 min . subsequently , the dna precipitates were dissolved with 400 l of 0.2 m triethylamine / acetate ( glen research corp ) . the purification step was performed by hplc ( prostar , varian , walnut creek , ca , usa ) with a c18 column ( 5 m , 250 mm 4.6 mm , alltech ) using acetonitrile and 0.1 m triethylammonium acetate ( teaa ) aqueous solution as the mobile phase . after being dried by a rotary evaporator , the purified dnas were detritylated with 80% acetic acid , precipitated with cold salted ethanol , collected by centrifugation , and dried by vacuum . finally , the dna products were obtained , and their concentrations were measured with a uv vis spectrometer ( cary bio-300 , varian ) . nps of 5.5 and 8.5 nm were synthesized by a seed - mediated growth method and then washed by centrifugation to remove hexadecyltrimethylammonium bromide ( ctab ) . the modification of nps with the drug - loading / attaching dna strand ( das ) was conducted following a reported protocol . the thiolated das ( 0.2 mm , 20 l ) was deprotected by 10 mm tris(2-carboxyethyl ) phosphine ( tcep , neutral ph , thermo scientific ) at room temperature for 60 min and then mixed with 1 ml of nps ( 20 nm ) . after incubation for 16 h , the mixture was salt - aged by slowly adding 200 l of nacl ( 1 m ) and allowed to incubate for 16 h. then , the excess dnas were twice removed by centrifugation , and the precipitate was resuspended in 250 l of 1 pbs . silver nanorods were synthesized and washed according to the procedure in our previous report , and the concentration was determined through the longitudinal absorption band of the uv vis spectrum . the selective modification of nr was performed according to a reported protocol with some adjustment . briefly , nrs were incubated with targeting aptamers ( nr / aptamer ratio was 1:100 ) in 2 mm ctab solution for 12 h. then , the capture strands ( cs ) were added ( nr / cs ratio was 1:500 ) and allowed to incubate for another 12 h. the adsorbed ctab was further displaced with thiolated peg . after that , the modified nr was salt aged by slowly adding 1 m nacl to give a final 0.3 m concentration of na , and the mixture was held at room temperature for at least 12 h. after washing 5 times by centrifugation , the resultant nrs were resuspended in 1 pbs for further use . to fabricate the self - assembly nanocomplexes , the das - modified nps of 5.5 , 8.5 , and 15 nm were mixed with the modified nrs at a np / nr ratio of 20:1 , 15:1 , and 10:1 , respectively , and incubated at room temperature for at least 24 h. the mixture was then gently centrifuged to remove unbound nps . for dox loading , nps were modified with das whose 3-end portion can form a ( cgt)6/(acg)6 duplex with a corresponding cdna . the dox loading was conducted by mixing dox with np - dass , incubating at room temperature for 30 min , and then centrifuging to collect the np / dox complexes . the number of dox per np was determined by measuring the fluorescence intensity of unbound dox in the supernatant and then interpolating from a standard linear calibration curve . for the dox leakage assay , 150 l of np / dox complexes was added to a mini dialysis unit [ 3.5 molecular weight cutoff ( mwko ) , thermo scientific ] , and the equivalent of free dox was used as a control . each unit was immersed in 3 ml of 1 pbs in a 5 ml beaker with gentle stirring at 450 rpm . at each given time point , a 100-l aliquot of the dialysis solution was collected for dox fluorescence measurement . after that , the collected solution was returned to the corresponding beaker . cem ( human acute lymphoblastic leukemia ) and ramos ( human burkitt s lymphoma ) were purchased from american type culture collection . k562/d ( doxorubicin - resistant chronic myelogenous leukemia ) was generously provided by dr . troy a. a. harkness of the department of anatomy and cell biology , college of medicine , university of saskatchewan . cem , ramos , and k562/d cells were cultured in rpmi 1640 , rpmi 1640 , and imdm medium , respectively , supplemented with 10% fbs ( heat - inactivated ; gibco ) and 100 iu / ml penicillin cells ( 5 10 in 100 l of medium ) were incubated with free dox , fluorescent nanoparticles , or nanocomplexes at 37 c with 5% co2 for different time lengths . after several centrifugation / washing steps , the cells were suspended in 1 pbs . for photoactivation , cells were irradiated with nir laser ( 600 mw / cm ) for 10 min . bisbenzimide hoechst 33342 ( sigma - aldrich ) was used for nuclear staining by incubating with cells at 37 c for 20 min . fluorescence imaging was performed on a leica tcs sp5 confocal microscope ( leica microsystems ) with a 63 oil - immersion objective . in most cases , the red color represents the fluorescence of cy5 ( em = 670 nm ) , the pink color represents the fluorescence of dox ( em = 600 nm ) , and the green color represents the fluorescence of tamra ( em = 570 nm ) . cem cells ( 1 10 ) were incubated with 8.5-np / nr - sgc8s or 15-np / nr - sgc8s at 37 c with 5% co2 for 6 h. then the cells were washed with pbs three times , irradiated with nir laser for 0 or 10 min , and incubated at 37 c with 5% co2 for another 22 h. to extract nuclei , the cells were collected by centrifugation , resuspended in 10 mm tris - hcl buffer ( ph 7.4 ) containing 100 mm nacl , 1 mm edta , and 1% triton x-100 at 4 c for 10 min and finally centrifuged at 1000 g for 3 min . after several centrifugation / washing rounds to remove the adsorbed nanoparticles on the nuclear membrane , the collected nuclei were lysed by a lysis solution containing 0.5% triton x-100 and 1 m naoh with sonication . subsequently , the nanoparticles from the nuclei were digested by incubating with aqua regia at 65 c overnight and diluted in 2% hno3 solution . nps accumulated in nuclei were measured by quantifying au element by inductively coupled plasma atomic emission spectrometry ( icp - aes ) . to evaluate the cell binding affinity of different nanocarriers , the aptamer was labeled with a fluorescein ( fitc ) . cells were incubated with free aptamers , nr - aptamers , or 15-np / nr - aptamers at 4 c for 30 min . after removal of unbound materials by several centrifugation / washing steps , the cells were analyzed on a facscan cytometer ( accuri c6 ) by counting 20 000 events . the cell viability under different treatments was determined by celltiter 96 cell proliferation assay ( promega ) . cells were incubated with free dox , np / nrs , or np - dox / nrs at 37 c with 5% co2 for 2 h and then centrifuged to precipitate . the supernatant ( 80% ) was removed , followed by adding equivalent fresh culture medium ( 10% fbs ) . for nir - responsive regulation , cells were irradiated with a nir laser ( 808 nm , 600 mw / cm ) for 10 min , followed by additional incubation to allow further growth for 48 h. then , 80% of the medium was removed and replaced with 20 l of mts reagent diluted in 100 l of rpmi 1640 . the resulting cell samples were incubated at 37 c for 12 h. finally , the absorbance at 490 nm was collected using a tecan safire microplate reader , and cell viability was calculated using the equation provided by the manufacturer .

the development of multidrug resistance ( mdr ) has become an increasingly serious problem in cancer therapy . the cell - membrane overexpression of p - glycoprotein ( p - gp ) , which can actively efflux various anticancer drugs from the cell , is a major mechanism of mdr . nuclear - uptake nanodrug delivery systems , which enable intranuclear release of anticancer drugs , are expected to address this challenge by bypassing p - gp . however , before entering the nucleus , the nanocarrier must pass through the cell membrane , necessitating coordination between intracellular and intranuclear delivery . to accommodate this requirement , we have used dna self - assembly to develop a nuclear - uptake nanodrug system carried by a cell - targeted near - infrared ( nir)-responsive nanotruck for drug - resistant cancer therapy . via dna hybridization , small drug - loaded gold nanoparticles ( termed nanodrugs ) can self - assemble onto the side face of a silver gold nanorod ( nr , termed nanotruck ) whose end faces were modified with a cell type - specific internalizing aptamer . by using this size - photocontrollable nanodrug delivery system , anticancer drugs can be efficiently accumulated in the nuclei to effectively kill the cancer cells .

type 1 diabetes ( t1d ) , is a chronic autoimmune disease and it is common in children the body 's own immune system attacks the beta - cells in the islets of langerhans of the pancreas , destroying or damaging them . it can lead to long - term complications including cardiovascular disease , blindness and kidney failure . the damage of the beta - cells in the islets of langerhans which caused by cytotoxic lymphocytes results in insulin deficiency and hyperglycemia . autoreactive t - cells that recognize islet autoantigens have been identified and are thought to play a direct role in t1d immunopathogenesis . the breakdown of beta cell - specific self - tolerance by t lymphocytes involves a number of dysregulated events intrinsic and extrinsic to t cells . the peripheral tolerance to self antigens t regulatory cells are minor population of cd4 + t cells express high levels of cd25 . it has an important role in the control of immune reactivity against self antigens , and probably plays a role in pathogenesis of t1d . different studies[169 ] had recently reported findings related to the frequency and function of regulatory t cells in t1d , but their results represents a somewhat conflicting body of findings . we aimed to estimate the frequency of regulatory t cells in recently diagnosed diabetes in children attending assiut children university hospital , egypt . twenty patients meeting the diagnostic criteria of t1d were recruited consecutively at the pediatric clinical endocrinology unit , children hospital of assiut university , faculty of medicine . in addition to twenty children with age and sex - matched , none of whom had either a personal or family history of diabetes or other autoimmune pathologies as control were included in the study . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas.on insulin replacement therapy.age range 2 - 16 years.diabetic duration less than 12 weeks . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas . on insulin replacement therapy . children with secondary diabetes mellitus ( dm).children with type 2 dm.evidence of active infection requiring antibiotic therapy or other concurrent diseases.other autoimmune disease.age < 2 years > 16 years . evidence of active infection requiring antibiotic therapy or other concurrent diseases . other autoimmune disease . twenty patients meeting the diagnostic criteria of t1d were recruited consecutively at the pediatric clinical endocrinology unit , children hospital of assiut university , faculty of medicine . in addition to twenty children with age and sex - matched , none of whom had either a personal or family history of diabetes or other autoimmune pathologies as control were included in the study . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas.on insulin replacement therapy.age range 2 - 16 years.diabetic duration less than 12 weeks . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas . on insulin replacement therapy . children with secondary diabetes mellitus ( dm).children with type 2 dm.evidence of active infection requiring antibiotic therapy or other concurrent diseases.other autoimmune disease.age < 2 years > 16 years . evidence of active infection requiring antibiotic therapy or other concurrent diseases . other autoimmune disease . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas.on insulin replacement therapy.age range 2 - 16 years.diabetic duration less than 12 weeks . definite diagnosis of t1d according to the definition of the world health organization criteria that defines this form of diabetes with permanent insulinopenia prone to ketoacidosis , result from a cellular - mediated autoimmune destruction of the beta cells of the pancreas . on insulin replacement therapy . children with secondary diabetes mellitus ( dm).children with type 2 dm.evidence of active infection requiring antibiotic therapy or other concurrent diseases.other autoimmune disease.age < 2 years > 16 years . evidence of active infection requiring antibiotic therapy or other concurrent diseases . other autoimmune disease . all cases were subjected to : full history including demographic factors : age , sex , residence , family history of diabetes.full clinical examination.complete blood count ( celltac e automated hematology analyzer , tokyo , japan).serum insulin c - peptide levels were measured by radioimmunoassay using commercial kits ( diagnostic systems laboratories inc , webster , texas ) . fasting normal insulin c peptide=0.78 - 5.19 ng / ml.flow cytometric detection of regulatory t cells , b - lymphocytes and t - lymphocytes . full history including demographic factors : age , sex , residence , family history of diabetes . full clinical examination . serum insulin c - peptide levels were measured by radioimmunoassay using commercial kits ( diagnostic systems laboratories inc , webster , texas ) . fasting normal insulin c peptide=0.78 - 5.19 ng / ml . flow cytometric detection of regulatory t cells , b - lymphocytes and t - lymphocytes . cd4cd25foxp3 regulatory t cells in whole blood samples were enumerated using fluoroisothiocyanate ( fitc)-conjugated forkhead box protein 3 ( foxp3 ) ( e bioscience , usa ) , phycoerythrin ( pe ) conjugated cd25 ( iq product , the netherland ) and peridinium - chlorophyll - protein ( per - cp)-conjugated cd4 ( becton dickinson , bioscience , usa ) . fifty l of blood sample was incubated with 10 l of cd4 , cd25 for 15 minutes at room temperature in the dark . following incubation , rbc lysis , washing with phosphate buffer saline ( pbs ) , addition of fixed solution to fix the cells and incubation for 10 minutes were done . after incubation , cells were washed with pbs , and then permelized solution and 10 l of foxp3 were added and incubated for 30 minutes at room temperature . for detection of b- and t - lymphocytes , 50 l of blood sample was stained with 10 l of fitc - conjugated cd19 and pe - conjugated cd3 ( becton dickinson biosciences , usa ) . flow cytometric analysis was done by facscalibur flow cytometry with cellquest software ( becton dickinson biosciences , usa ) . forward and side scatter histogram was used to define the lymphocyte population ( r1 ) . total cd4cd25 , cd4cd25 , cd4cd25 ( defined as the population of cd4 positive t cells whose cd25 expression exceeded the level of cd25 positivity seen in the cd4 negative t cells ) and cd4cd25 foxp3 regulatory t cells was evaluated as a percentage of total lymphocytes and of cd4 as shown in figure 1 . the expression of foxp3 in cd4cd25 and in cd4cd25 cells was expressed as geometric mean of fluorescence intensity ( mfi ) . ( a ) forward and side scatter histogram was used to define the lymphocytes population ( r1 ) . ( b , c ) the expression of cd4 and cd25 in total lymphocytes ( r1 ) was detected , compared with the negative isotype control and and different gates were drown to define cd4 cd25cells ( r2 ) , cd4cd25 cells ( r3 ) , and cd4cd25 cells ( r4 ) . ( d ) the percentage of cd4cd25 foxp3cells ( r5 ) in total lymphocytes was determined . ( e - g ) show the analysis of regulatory t cells in cd4 cells ( r 6 ) . cd4 cd25cells ( r7 ) , cd4cd25 cells ( r8 ) , and cd4cd25 cells ( r9 ) . ( h ) show the expression as a geometric mean of fluorescence intensity ( mfi ) of foxp3 in cd4cd25 cells . the positivity was defined as fluorescence ( red histogram ) higher than that of the isotype control ( open histogram ) statistical package for social sciences ( spss ) , version 16 was used for data analysis . all data were expressed as the mean standard error of mean ( sem ) . due to the small sample size and a propensity for outliers in some of the variables , cd4cd25foxp3 regulatory t cells in whole blood samples were enumerated using fluoroisothiocyanate ( fitc)-conjugated forkhead box protein 3 ( foxp3 ) ( e bioscience , usa ) , phycoerythrin ( pe ) conjugated cd25 ( iq product , the netherland ) and peridinium - chlorophyll - protein ( per - cp)-conjugated cd4 ( becton dickinson , bioscience , usa ) . fifty l of blood sample was incubated with 10 l of cd4 , cd25 for 15 minutes at room temperature in the dark . following incubation , rbc lysis , washing with phosphate buffer saline ( pbs ) , addition of fixed solution to fix the cells and incubation for 10 minutes after incubation , cells were washed with pbs , and then permelized solution and 10 l of foxp3 were added and incubated for 30 minutes at room temperature . for detection of b- and t - lymphocytes , 50 l of blood sample was stained with 10 l of fitc - conjugated cd19 and pe - conjugated cd3 ( becton dickinson biosciences , usa ) . flow cytometric analysis was done by facscalibur flow cytometry with cellquest software ( becton dickinson biosciences , usa ) . forward and side scatter histogram was used to define the lymphocyte population ( r1 ) . total cd4cd25 , cd4cd25 , cd4cd25 ( defined as the population of cd4 positive t cells whose cd25 expression exceeded the level of cd25 positivity seen in the cd4 negative t cells ) and cd4cd25 foxp3 regulatory t cells was evaluated as a percentage of total lymphocytes and of cd4 as shown in figure 1 . the expression of foxp3 in cd4cd25 and in cd4cd25 cells was expressed as geometric mean of fluorescence intensity ( mfi ) . ( a ) forward and side scatter histogram was used to define the lymphocytes population ( r1 ) . ( b , c ) the expression of cd4 and cd25 in total lymphocytes ( r1 ) was detected , compared with the negative isotype control and and different gates were drown to define cd4 cd25cells ( r2 ) , cd4cd25 cells ( r3 ) , and cd4cd25 cells ( r4 ) . ( d ) the percentage of cd4cd25 foxp3cells ( r5 ) in total lymphocytes was determined . ( e - g ) show the analysis of regulatory t cells in cd4 cells ( r 6 ) . cd4 cd25cells ( r7 ) , cd4cd25 cells ( r8 ) , and cd4cd25 cells ( r9 ) . ( h ) show the expression as a geometric mean of fluorescence intensity ( mfi ) of foxp3 in cd4cd25 cells . the positivity was defined as fluorescence ( red histogram ) higher than that of the isotype control ( open histogram ) statistical package for social sciences ( spss ) , version 16 was used for data analysis . all data were expressed as the mean standard error of mean ( sem ) . due to the small sample size and a propensity for outliers in some of the variables , some demographic and clinical data of diabetic children and controls were presented in table 1 . some demographic and clinical data of diabetic children and control there were no significant difference in white blood cells count , platelet count and hemoglobin concentration between diabetic patients and controls [ table 2 ] . the level of insulin c peptide was significantly lower in children with / t1d compared with controls with p < 0.000 . some laboratory characteristics of diabetic patients and controls there were no significant difference in the percentages of t lymphocytes ( cd3 ) , b lymphocytes ( cd19 ) and t helper cells ( cd4 ) between patients than controls [ table 3 ] . the percentages of total cd4cd25 and cd4cd25 in total lymphocytes were not significantly different between patients and controls . the percentages of cd4cd25 and cd4cd25 foxp3 in total lymphocytes were significantly decreased in patients than controls . similar results were observed when these cells were analyzed as a percentage of cd4 t cells . regulatory t cells in diabetic patients and controls the mfi of foxp3 expression in cd4cd25 foxp3 cells was significantly decreased in patients than controls , while mfi of foxp3 expression in cd4cd25 cells was not significantly different between patients and controls [ table 3 ] . the frequency of cd4cd25 foxp3 was positively correlated with age of the patients ( r = 0.585 , p < 0.000 ) , and the level of insulin c peptide ( r = 682 , p < 0.000 ) [ figures 2 and 3 ] . correlations between the frequency of cd4cd25foxp3 and age of diabetic patients correlations between the frequency of cd4+cd25foxp3 and level of insulin c peptide type 1 diabetes is a well - known autoimmune disease ; however there are still some processes in its pathogenesis to be elucidated . t regulatory cells are essential for maintaining peripheral tolerance , preventing autoimmune diseases and limiting chronic inflammatory diseases . these cells modulate the intensity and quality of immune reactions through attenuation of the cytolytic activities of reactive immune cells . in this study , cd4cd25 foxp3 cells were considered as regulatory t cells , as the suppressive capacity of regulatory t cells in humans seems to be confined to cd4cd25 cells with the highest expression of cd25 ( cd4cd25 , whereas cd4 t cell with intermediate expression of cd25 might also contain recently activated t cells and effector t - cells without regulatory function.[1517 ] also , the identification of foxp3 as a regulatory lineage specific factor provided a useful phenotypic and optimal marker for regulatory t cells,[1820 ] and the suppressive phenotype and the development of regulatory function depend on the expression of foxp3.[2124 ] indeed , recent results indicate that foxp3 behaves as a master regulator of the regulatory t cells phenotype . we found the frequency of cd4 cd25 and cd4 cd25 foxp3 both in total lymphocytes and in cd4 cells were significantly decreased in diabetic patients than controls while the frequency of total cd4cd25 and cd4 cd25 both in total lymphocytes and in cd4 cells were not significantly different in patients and controls . this decline in the frequency of cd4 cd25 and cd4 cd25 foxp3 t cells in our patients could implies that the deficiency of regulatory t cells may has a role in the pathogenesis of type 1 diabetes . in accordance with our results , luczyski et al . , found a statistically significant decrease of t regulatory cells in children with newly diagnosed / t1d . ryba et al . , also reported lower percentage of regulatory t cells in children with / t1d . luczyski et al . , reported that percentage of cd4cd25 was decreased in diabetic patients than controls , the same as our study , while the percentages of cd4+cd25highcd127dim/ were very low and did not differ between t1d and control children and this difference could be due to the of different markers of regulatory t cells they used , cd127dim/ and not foxp3 as our study . lindley et al . , reported that there is no difference in the level of regulatory t cells between patients with / t1d and healthy controls . however , in these studies , the patients were adult or have long lasting diabetes . in brusko et al . , their patients and controls are older than our patients , and their controls from considerably older than their patients . glisic - milosavljevic et al . , reported that there is a higher level of ongoing apoptosis in cd4+cd25 + t cells in recent - onset t1d subjects and in subjects at high risk for the disease . on the contrary , in long - standing / t1d and / t2d subjects , cd4+cd25 + t cell apoptosis is at the same level as in control subjects . this high level of cd4+cd25 + t - cell apoptosis could explain the decrease of regulatory t cells in our patients . foxp3 , is a critical molecular switch for the genetic programming of natural regulatory t cell development and function . in this study ; the mfi of foxp3 expression in cd4cd25 cells were 44.68 2.34 and 74.81 3.47 in patients and controls respectively , while the mfi of foxp3 expression in cd4cd25 were 29.05 2.49 and 37.09 3.50 in patients and controls respectively . the expression of foxp3 in cd4cd25 foxp3 cells was significantly decreased in diabetic patients than controls , while its expression in cd4cd25 cells was not significantly different between patients and controls . lawson et al . reported that there was no difference in foxp3 expression on cd4cd25 t cells in patients with / t1d , but in contrast to our study the patients had long standing diabetes , and both patients and controls were adult subjects . in the present study , cd4cd25foxp3 was positively correlated with age of diabetic children . brusko et al . , reported in their study that increasing age was associated with an increase in total cd4cd25 frequency . in the present study , insulin c peptide level was significantly lower in children with / t1d compared with control . in addition , the frequency of cd4cd25 foxp3 was positively and significanty correlated with the level of insulin c peptide . insulin c - peptide level is the most reliable factor in evaluation of the endogenous insulin secretion in patients with / t1d . autoimmune destruction of the beta cells of pancreas results in deficiency of both insulin and insulin c - peptide . this study concluded that children with / t1d have lower percentages of t regulatory cells in the peripheral blood which correlated positively with age of patients and the level of insulin c peptide . the percentages of regulatory t cells were assessed in the peripheral blood but not at the site of affection ( pancreas and/or draining lymph nodes).the distinction of regulatory t cells by a flow cytometry including - high expression of cd25 antigen is very subjective and can result in different findings from different laboratories . the percentages of regulatory t cells were assessed in the peripheral blood but not at the site of affection ( pancreas and/or draining lymph nodes ) . the distinction of regulatory t cells by a flow cytometry including - high expression of cd25 antigen is very subjective and can result in different findings from different laboratories . the percentages of regulatory t cells were assessed in the peripheral blood but not at the site of affection ( pancreas and/or draining lymph nodes).the distinction of regulatory t cells by a flow cytometry including - high expression of cd25 antigen is very subjective and can result in different findings from different laboratories . the percentages of regulatory t cells were assessed in the peripheral blood but not at the site of affection ( pancreas and/or draining lymph nodes ) . the distinction of regulatory t cells by a flow cytometry including - high expression of cd25 antigen is very subjective and can result in different findings from different laboratories .

background : regulatory t cells have an important role in the control of immune reactivity against self antigens and probably play a role in pathogenesis of type 1 diabetes ( t1d ) . we aimed to determine the frequency of regulatory t cells in recently diagnosed children with / t1d.materials and methods:20 children with / t1d and 20 healthy children of matched age and sex as controls were enrolled in this study . all cases were subjected to a thorough history taking , full clinical examinations and investigations which include ; insulin c peptide levels and flow cytometric detection of b- , t - lymphocytes and regulatory t cells.results:insulin c peptide level was significantly lower in children with/ t1d compared with controls . the percentages of b and t - lymphocytes were not significantly different between patients and controls . the percentages of cd4+cd25+high and cd4+cd25+high foxp3 + cells both in total lymphocytes and in cd4 + lymphocytes were significantly decreased in patients than controls , while the percentages of total cd4+cd25 + and cd4+cd25+intermediate both in total lymphocytes and in cd4 + lymphocytes were not significantly different between patients and controls . the geometric mean of fluorescence intensity ( mfi ) of foxp3 + expression in cd4+cd25+high cells was significantly decreased in patients than controls . positive correlations were observed between both age and insulin c peptide and frequency of cd4+cd25+high foxp3.conclusion:the percentage of regulatory t cells ; cd4+cd25+high foxp3 was decreased in children with recent t1d and may have a role in its pathogenesis . their role as a prognostic signifi cance and their relation to various complications should be explored .

an increased man - made radiation exposure - risk from the use of high - dose imaging modalities such as computed tomography and angiographic suites is now being observed in many health - care centers with over 3.6 million diagnostic examinations performed annually worldwide . interventional procedures are performed by cardiologists , radiologists , endovascular surgeons , operation theater staff , etc . , due to the well - known benefits in medicine . however , it is crucial for the referring clinician and the interventionalist ( radiologist / cardiologist / clinician in the operation theater ) to assess the potential benefit - risk ratio for various interventional procedures as some of the procedures involve a high radiation exposure due to prolonged fluoroscopic screening . all interventional cardiological procedures are invariably performed using dedicated fluoroscopy and angiography suites equipped with either image intensifier ( ii)-based or flat panel detector ( fpd)-based systems . the ii - based systems have been used for fluoroscopy for more than two decades . on the other hand , the fpd - based systems for medical imaging emerged in the 1990s initially for two - dimensional ( 2d ) projection x - ray image , and subsequently for a real - time interventional angiography suites equipped with ii or fpd have the potential to impart high radiation doses to patients if optimization strategies are not well - implemented . stringent optimization involves orientation of staff , consistent restriction of frame rates during image acquisition , using low dose settings , judicious use of magnification , etc . it is also necessary to understand the potential risks due to radiation from different interventional procedures . for this reason , it is necessary that one should be knowledgeable in the magnitude of radiation dose associated with each intervention . this can be achieved by measuring real - time doses using devices such as a dose area product ( dap ) meter . most of the newer angiography machines are equipped with a dap meter fitted on the collimator assembly of the machine . dap is particularly a useful method for assessing and comparing the radiation dose from screening procedures and acts as a surrogate for radiation risk . entrance surface dose ( esd ) is also used for measuring radiation doses . from the dose descriptors , it is possible to estimate organ doses as well as effective doses for each procedure . radiation doses from interventional procedures have been widely reported in literature , with more emphasis on doses from ii - based systems . however , there are only a few reports on radiation doses from fpd systems as it may be a transition period from ii to fpd for most of the interventional users . some patient and phantom - based studies reported in literature state that doses from fpd are higher than ii systems . few other studies report that radiation doses from fpd are lower than ii systems . in comparing conventional and digital systems , hence , it is not clear whether fpd imparts lower radiation dose than ii and whether there would still be a need to further optimize doses in the newer fpd systems . the purpose of this article is to review and compare radiation doses from ii and fpd - based systems in interventional cardiology in a tertiary referral center that has introduced fpd system recently . it is anticipated that this information will be useful for those performing cardiological interventions and for those who are on a transition from ii to fpd . cardiovascular interventions were performed using two dedicated catheterization labs , each equipped with philips allura fd10 flat panel system ( netherlands ) . the dose area product ( dap ) values for coronary angiography ( cag , n = 222 ) and percutaneous transluminal coronary angioplasty ( ptca , n = 75 ) procedures performed during a one year period 20122013 were prospectively recorded using a built - in calibrated dap meter ( transmission ionization chamber ) . the ptca procedure was invariably performed by a senior interventionalist assisted by two junior cardiologists , while the cag was performed either by the senior interventionalist or by junior cardiologists . for a similar comparison of clinical protocols adopted in the institution , dap values from cag and ptca performed using philips integris h3000 and h5000 ii - based systems ( netherlands ) reported earlier all the x - ray systems were on periodic qa programs and conformed to the manufacturer 's specifications . the ii and fpd systems had low- , normal- , and high - dose settings , respectively , for fluoroscopy . these machines incorporated a total filtration of 2.5 mmal with possibility of selecting spectral filters such as 0.1 mm , 0.2 mm , 0.4 mm cu for dose reduction . during the course of the study , low dose setting with 0.4 mm cu filter was invariably selected during fluoroscopy . in the earlier work using ii - based system , a 23-cm image intensifier format ( iif ) was used during fluoroscopic screening in cag and ptca procedures for tracing the path of the catheter from the region of arterial puncture and to the screening of the cardiac valve region . a 17-cm iif was used for the oblique , caudal , cranial , and lateral projections delineating the coronary anatomy . in the fpd system , 25 cm fluoro format was used during screening and 20 cm was used for other projections to delineate coronary anatomy . a transition from ii to fpd system for a catheterization lab would require adequate justification in terms of radiation dose , image quality , maintenance , and investment . it has been reported that the fpds designed specifically for fluoroscopic purposes provide superior image quality and dose efficiency compared to the ii systems , except at the lowest fluoroscopic dose levels . prieto et al . , reports that even after upgrading to the fpd from ii , significant increase in patient doses were observed though the fluoroscopic time and number of images remaining the same in both cases during the initial transition period . as only a few studies on radiation doses are available for fpd systems ; more work is required on optimization strategies in the fpd systems . table 1 shows the dap values and fluoroscopic time duration for ii and fpd systems from the referral center where the study was conducted . the dap values for ii - based systems represented in table 1 is from the use of optimized protocol as reported in the previous published article from the same referral center where the study was conducted . prior to optimization in ii systems , the doses were above 50% ; however , it was possible to optimize dose by halving the entrance dose ratesby selecting 0.4 mm cu filtration ( generally recruited in pediatric protocols ) during fluoroscopy . selection of 0.4 mm cu filtration did not suffer significant loss of image quality ; however , tube potentials were increased from 80 kvp to 103 kvp during fluoroscopy . in the fpd system , tube potentials ranged from 90 kvp110 kvp when 0.4 mm cu filtration was selected with low dose settings during fluoroscopy . having adhered to the same optimization strategies in both systems , doses were similar owing to the fact that further optimization is warranted in fpd system . reported dap values of 31 and 33 gycm from cag with the use of ii and fpd system , respectively . they have also reported that the total dap from fluoroscopy and cine for ii and fpd are not significantly different and the image quality from fpd is better than ii in cine mode with no difference in the imaging performance in fluoroscopy mode . fluoroscopic time and dose area product values from ii and flat panel detector ( fpd)-based systems from cardiological interventions tables 2 and 3 shows dap values for cag and ptca procedures from various studies in literature . the arithmetic mean dap and fluoroscopic time duration using ii system as reported in literature was 39 gycm for 6.6 min and 61.2 gycm for 17 min for cag and ptca respectively [ table 2 ] . with the use of fpd , mean dap and fluoroscopic time duration were 28.4 gycm for 7 min and 61 gycm for 18.05 min for cag and ptca , respectively [ table 3 ] . from tables 2 and 3 , radiation doses from fpd were significantly low for cag but were similar for ptca when compared to ii systems . it should be noted that time duration for cag and ptca was not available for some reported studies in tables 2 and 3 . wide variation of doses is observed from these studies which may be attributed to the angiographic system used , time duration of the procedure and work environment . doses of the order of 492 gycm were recorded in ptca procedure from fpd system which was higher than the ii systems . chida et al . , conducted studies from various ii and fpd system and the average entrance doses of cine angiography and fluoroscopy in fpd systems were not significantly different . though fpds possess good detective quantum efficiency , they did not inherently reduce the radiation dose . jensen et al . , observed that patient doses from fpd were lower than ii systems ; however , the eye lens doses for radiologists were higher in fpd than in ii due to the use of high filtration and recruitment of high tube potentials . in our study , high tube potentials were recruited when low dose settings involving high filtration were selected , which may have the potential to increase staff doses . radiation doses from cardiological interventions performed using image intensifier - based systems radiation doses from cardiological interventions performed using flat panel detector - based systems it is prudent to adopt stringent optimization measures in fpd as the dose may be higher than the ii systems as reported by prieto et al . and dose reduction is possible in either ii or fpd systems . during the initial stages of our study , the doses from fpd were similar to the ii system though high filtration was used . reports that it is possible to achieve mean dap of 6.2 and 10.4 gycm for cag and ptca procedures performed using ii system . they further report that the reduction of doses was by influencing the quality of fellowship training , consistent restriction to mean values of 171 frames per cag , 165 frames per ptca , low - level fluoroscopy , training in the use of fluoroscopy - free blind positioning to the region of interest , restrictions to achieve lower ii entrance dose for adequate image quality . from tables 1 and 3 , the mean dap for cag from fpd were higher than those reported by kuon et al . tsapaki et al . , reported the doses from fpd were increased by 35% compared to ii when fluoroscopy levels were changed from low to high mode ; they also recorded a minimum dap value of 6.1 and 14.3 gycm for cag and ptca , respectively , with the use of low dose fluoroscopy settings in fpd . dekker et al . , reported that the new generation fpds incorporated with good image processing and noise reduction techniques resulted in reducing patient doses by 43% without compromising image quality and staff doses by 50% during electrophysiological interventions . though fpd has reduced entrance dose rates , it does not automatically reduce radiation doses in clinical practice . further work is necessary to study the possibilities of dose reduction in fpd so as to be implemented in the clinical set up . the patient dose and image quality in any newer modality needs to be permanently monitored and transition from ii to fpd requires careful attention . this is a preliminary study as the institution where the study was conducted recently moved from ii to fpd - based systems . though radiation doses for cardiological interventions from fpd were similar to the ii - based system achieved after optimization , the advantages of fpd in terms of good image uniformity , improved patient imaging accessibility due to smaller size , absence of geometric distortion / veiling glare or vignetting make the fpd superior to the ii systems . to achieve improved patient dose reduction , it is advisable to strictly adhere to low dose protocols with high filtration in fpd systems . in addition , more attention for staff doses is warranted especially for interventionalists when this stringent patient dose reduction is employed . it is recommended to follow stringent dose reduction strategies right from the period of initial installation when there is a transition from ii to fpd systems . further studies are required to develop dose optimization in fpd , though use of high filtration is already in place .

flat panel detector ( fpd ) technology in interventional cardiology is on the increase due to its varied advantages compared to the conventional image intensifier ( ii ) systems . it is not clear whether fpd imparts lower radiation doses compared to ii systems though a few studies support this finding . this study intends to compare radiation doses from ii and fpd systems for coronaryangiography ( cag ) and percutaneous transluminal coronary angioplasty ( ptca ) performed in a tertiary referral center . radiation doses were measured using dose area product ( dap ) meter from patients who underwent cag ( n = 222 ) and ptca ( n = 75 ) performed using fpd angiography system . the dap values from fpd were compared with earlier reported data using ii systems from the same referral center where the study was conducted . the mean dap values from fpd system for cag and ptca were 24.35 and 63.64 gycm2 and those from ii system were 27.71 and 65.44 gycm2 . transition from ii to fpd system requires stringent dose optimization strategies right from the initial period of installation .

a pure ground - glass nodule ( ggn ) in the lung may represent an early lung malignancy , such as adenocarcinoma in situ ( ais ) or minimally invasive adenocarcinoma ( mia ) ( 1 ) . approximately 20 - 30% of patients with adenocarcinoma and a predominantly pure ggn who undergo surgery have additional ggn lesions , either in the same or different lung lobes ( 2 ) . in such cases , multiple limited resection may be an appropriate treatment if a few pure ggn lesions are scattered in multiple lobes or peripherally in one lobe , and the patient has sufficient pulmonary functional reserve ( 3 ) . however , alternative options should be considered if several ggn lesions are scattered in both lungs with multiple lobes involved , and there is a risk for insufficient pulmonary functional reserve after resection . chemotherapy has been suggested as an additional therapeutic option , if surgery is not possible due to a lack of pulmonary functional reserve ( 4 ) . another strategy is to rely on serial computed tomography ( ct ) scans until the nodule develops a definite solid portion . if curative therapy is not performed , there remains a significant chance for progression ( 1 ) . cryoablation has been explored as an option for treating metastatic lesions in patients with non - small cell lung cancer who can not undergo surgery or chemotherapy ( 5 ) . however , no clinical reports of cryoablation techniques applied to multiple ggns in the lung have been published . here , we describe a patient with lung cancer and a pure ggn in the left lower lung lobe that persisted after several limited resections and was successfully treated with cryoablation . a 59-year - old woman visited our hospital due to abnormal chest radiography findings on a health maintenance exam at another hospital . all pre- and post - procedural ct scans were performed using a multi - detector ct scanner ( somatom definition flash , enlargen siemens , forchheim , germany ) . the scanning parameters were : 120 kvp , 200 mas , 0.75-mm collimation , 2-mm reconstruction intervals , smooth algorithm , and pitch of 1 . several ggns were detected by ct scan : a 12-mm - sized , partially solid nodule in the right middle lobe ( fig . 1a ) , a 7-mm - sized pure ggn in the right upper lobe , and two 5-mm - sized pure ggns in each of the lower lobes ( fig . the patient underwent a right middle lobectomy and wedge resection of the right upper lobe using video - assisted thoracoscopic surgery . the partially solid nodule in the right middle lobe was diagnosed as adenocarcinoma , and the pure ggn in the right upper lobe was diagnosed as a mia . the two pure ggns in both lower lobes remained without significant change on a 10-month follow - up ct scan . an additional wedge resection was performed for the pure ggn in the right lower lobe , and this nodule was diagnosed as ais . after these surgeries , the patient did not have sufficient pulmonary functional reserve to tolerate any additional surgery . thus , we decided to perform cryoablation on the pure ggn in the left lower lobe . the patient fasted for 6 hours before the procedure , and her blood coagulation parameters were checked . we used an argon and helium gas - based system ( seednet , galil medical , yokneam , israel ) , a 1.5-mm 17-g cryoprobe , and a thermal sensor . the procedure was performed in a fluoroscopy room by an interventional radiologist . after placing the patient in the prone position on the table , a cross - shaped radio - opaque skin marker was applied to determine the needle insertion site under fluoroscopy . subsequently , a c - arm cone - beam ct ( cbct ) scan ( dynact , enlargen siemens ) was taken ( fig . 1d - f ) to obtain 1.5-mm thick axial , coronal , and sagittal images . based on these images , we measured the distance from the center of the skin marker to the target ggn . the area of the skin marker was draped , and a 1% lidocaine solution was injected into the skin down to the pleural surface . after localizing the needle tip , a cbct scan was performed to confirm the probe insertion site . a 17-g cryoprobe ( seednet , galil medical ) was carefully introduced into the pure ggn under fluoroscopic guidance . we used a fluoroscope and cbct to visualize the size of the ice ball during the procedure . we took a ct scan to confirm that the ice ball had reached sufficient size , as indicated by a low attenuation region with an envelope of ground - glass opacity and a margin of 1 cm beyond the ggn . a small pneumothorax was noted after removing the cryoprobe ; however , the patient was asymptomatic with stable vital signs . we performed a follow - up ct scan 1 day after cryoablation to assess any initial changes in the ablated lesion and procedural - related side effects ( fig . the scan showed a 3-cm sized ablated zone in the left lower lobe with no major procedure - related complications , such as hemothorax . the size of the ablated zone had decreased markedly on a follow - up ct scan 2 months later . the pure ggn in the left lower lobe was judged to have been successfully ablated without recurrence on a 6-month follow - up ct scan ( fig . our results show the potential of cryoablation as a new treatment method for ggn , particularly in patients in whom surgery is contraindicated . some controversy exists as to whether some nodules should be resected or followed by serial ct scans . kim et al . ( 6 ) noted that several pure ggn lesions detected in patients undergoing surgery for bronchioloalveolar carcinoma did not change in size or features during follow - up . they suggested that surgical resection should be considered in selected cases in which nodules exhibit significant changes in size or appearance during follow - up . ( 7 ) found that all ggn lesions > 10 mm were carcinomas and insisted that these lesions should be resected rather than followed . partially solid ggn lesions should be resected regardless of size because they represent more invasive lesions than pure ggns . thus , a non - surgical alternative is needed for patients with insufficient pulmonary functional reserve or in patients in whom surgery is technically not feasible . minimally invasive ablation techniques , such as radiofrequency ablation ( rfa ) and cryoablation , may be useful adjunctive treatment options for lung cancer or metastatic lesions and preserve pulmonary function ( 5 , 9 , 10 ) . minimally invasive ablation techniques for lung cancer have several advantages , including selective damage , minimal morbidity and mortality , minimal loss of lung function , repeatability , low cost , excellent monitoring during treatment , less pain , and shorter hospital stay ( 11 ) . ablating lung tumors is currently used for curative treatment of primary lung cancer and metastatic lung malignancies or cytoreduction ( 12 ) . ablation is also an option in patients with lung metastases from colorectal and renal cell carcinoma , melanoma , hepatocellular carcinoma , or primary sarcoma ( 13 ) . although rfa is a contemporary tumor ablation technique , it may cause serious complications , such as air embolism or uncontrolled pain ( 9 , 14 ) . cryoablation is a thermoablational technique that consists of alternating cycles of decreasing ( freezing ) and increasing ( thawing ) temperatures , leading to direct cellular and vascular injury . cryoablation works by forming an ice ball , which causes increased extracellular osmolarity and , as a consequence , water diffuses from the intracellular space into the extracellular space . thus , cryoablation may be a better option for patients with extensive emphysema ( 10 ) . however , percutaneous or surgical biopsy was difficult due to the small size and multiplicity of the ggn lesions . nevertheless , localized ggns can include invasive disease , such as ais or mia ( 1 ) . in our case , two similar nodules in the right upper and right lower lobes were identified during surgery as mia and ais , respectively . cryoablation may reduce the likelihood that a remnant ggn will progress to advanced cancer and does not require additional surgery . thus , long - term follow - up may be necessary to determine the outcome of cryoablation for treating pure ggns . in conclusion

treatments for pure ground - glass nodules ( ggns ) include limited resection ; however , surgery is not always possible in patients with limited pulmonary functional reserve . in such patients , cryoablation may be a suitable alternative to treat a pure ggn . here , we report our initial experience with cryoablation of a pure ggn that remained after repeated surgical resection in a patient with multiple ggns . a 5-mm - sized pure ggn in the left lower lobe was cryoablated successfully without recurrence at the 6-month follow - up .

despite positive changes in professional definitions , such as removal of homosexuality from dsm in 1973 , deviations from gender specific heterosexual development still constitute a reason for concern for most of the parents . unique stressors associated with homosexuality such as stigma , discrimination and bully victimization may adversely affect mental health of young individuals having same - sex attraction . review of relevant literature reveals that people having same - sex attraction are at higher risk of mental disorder , suicidal ideation , substance misuse , and deliberate self - harm than heterosexual people . gay youths are more likely to engage in school refusal , runaway behavior and subsequent homelessness , prostitution and sexually transmitted diseases . while it is true that a great majority of people with same - sex attraction do not report any problem relating to their sexual behavior , there is also a significant amount of suffering as well . it is important to bear in mind that human history is loaded with accounts of severe persecution of individuals with same - sex attraction . it was not until the second half of the 20th century that most influential european countries decriminalized homosexual activity . even today , in at least 76 countries , consensual same - sex relationships are considered as criminal offences punishable by imprisonment and even death , in at least five countries . thus , the medical profession has a duty to alleviate this suffering in all legitimate ways possible . besides fostering greater tolerance of homosexuality on behalf of society , clinicians are required to prevent negative health outcomes associated with it which may be better addressed by understanding the roots of homosexual behavior . there is a great deal of research that tries to identify biological factors influencing same - sex orientation . it is suggested that an individual s sexual orientation is determined by early biological events generated by genes and prenatal sex hormones , both of which hardwire brain morphology . in regard to heritable factors , despite several family , twin and molecular studies , much remains to be known about the genetics of human homosexuality . although natural selection favors heterosexuality for transmission of genes from one generation to the next , persistence of homosexuality in a small but consistent percentage of the human population is of question . the prenatal androgen model , which has become a clich in the field , argues that homosexuality in men is due to under - exposure to prenatal androgens and in women , due to over - exposure . attempts to support the model included hormone measurements , animal experimentations and studies of rare disorders ( i.e. , congenital adrenal hyperplasia and testicular feminization ) all of which revealed inconsistent results . in the following surge of research in prenatal androgen model , indirect consequences of prenatal hormone impact , such as digit ratio , fingerprint patterns and oto - acoustic emissions have been studied , without reaching definitive conclusions . it is warranted that prenatal androgenization is essential for male gender identity development , but apparently not decisive . thus , researchers and clinicians should think about what else may play a role in this puzzling area . the thyroid gland , located in front of the neck , produces two related hormones , thyroxine ( t4 ) and triiodothyronine ( t3 ) . these hormones have a critical role in cell differentiation during development and help maintain thermogenic and metabolic homeostasis in the adults . thyroid disorders are highly prevalent in the population and mostly affect women in childbearing age . in pregnancy , thyroid function is altered by 4 factors in pregnancy : i ) the transient increase in human chorionic gonadotropin ( hcg ) during the first trimester , which cross - reacts with thyroid - stimulating hormone ( tsh ) receptor ; ii ) the estrogen - induced rise in thyroxine - binding globulin ( tbg ) which is the major transport protein for thyroid hormone ; iii ) increased autoimmunity ; and iv ) increased urinary iodide clearance , which can cause impaired thyroid hormone production in areas of low iodine supply . consequently , the levels of both t3 and t4 , the major hormones released by the thyroid , increase by ~50% and serum tsh levels decrease in the first trimester and increase in the second and third trimesters . hypothyroidism may affect up to 3% of all pregnant women , while hyperthyroidism is less than 1% . subclinical forms of thyroid disorders is also a growing concern as undiagnosed cases may result in serious consequences . it is a well - established fact that thyroid hormones are essential for both fetal and post - natal neurodevelopment and for the regulation of neuropsychological function in human beings . either due to iodine deficiency or autoimmune thyroid dysfunction , reduction of circulating maternal thyroid hormones has been found to result in lower iq in infants and young children in several retrospective and prospective studies . during gestation , this is expected to include behavior relating to sexual attraction , however , there is no study focusing on this issue . in one related study by ellis and hellberg , fetal exposure to thyroid medications and diet pills another recent study mentioned autoimmune thyroid diseases among a group of autoimmune diseases that were found at increased rates in same - sex married danish individuals . in the present study , it was aimed to investigate the association between history of thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the offspring . this study was a retrospective chart review of patients evaluated by the author - clinician between 2005 to 2013 in 4 outpatient settings in istanbul ; 2 of them attached to marmara university and the remaining ones as private clinics . , all diagnoses had been based on dsm - iv criteria . considering the average age of the onset of same - sex attractions , the charts of the children and adolescents aged 8 to 17 years patients with mental retardation , autism and psychosis were excluded from the review . the excluded children with mental retardation charts with missing perinatal information were not included in the study . although the study was a retrospective one , the approval of local ethics committee of marmara university medical school was obtained . parents consent for treatment involved consent for the use of their data for audit and research purposes provided that their identity not to be disclosed . after the cases with overlapping conditions had been identified ( n=12 ) , a second - line consent for the use of their data in a study correlating complaints of the child with pregnancy history , was asked in telephone interviews and none of the mothers refused to participate . more detailed and updated data about the course of their thyroid dysfunction was collected in those interviews . a respectful approach to the subjects has been adopted because of the sensitivity of the topic . in accordance with current clinical practice parameters , the term same - sex attraction was reserved for the youngsters who display such behavior whereas the term childhood gender nonconformity was rather indicated for preadolescent cases who had behavior that fell outside gender norms . patient records , kept as electronic office files were reviewed consecutively beginning from the earliest admission until the last one , at the end of october 2013 . besides this , since all files are electronically searchable , all files were explored using relevant search terms in turkish and english , relating to pregnancy thyroid dysfunction in the mothers and same - sex attraction / gender nonconformity in the children . in order to prevent possible biases while searching for relevant cases , another clinician who had had no responsibility for any of the patients between 2005 to 2013 also reviewed the files . based on this criteria , the statistical package for the social sciences version 15 was used for statistical analysis ( spss 15 , chicago , il , usa ) . fisher s exact test and t - tests were conducted for the analyses of the data . the prevalence of same - sex attraction / gender nonconformity in the group of children having positive maternal history in pregnancy was compared with the total sample . we also used logistic regression analysis to assess variables predicting same - sex attraction / gender nonconformity . the level p<0.05 was taken as the cut - off value for statistical significance of results . the statistical package for the social sciences version 15 was used for statistical analysis ( spss 15 , chicago , il , usa ) . fisher s exact test and t - tests were conducted for the analyses of the data . the prevalence of same - sex attraction / gender nonconformity in the group of children having positive maternal history in pregnancy was compared with the total sample . we also used logistic regression analysis to assess variables predicting same - sex attraction / gender nonconformity . the level p<0.05 was taken as the cut - off value for statistical significance of results . a total of 790 charts were identified as eligible for further search in this study . this group consisted 520 male children ( 65% ) and 270 ( 35% ) female children , with a mean age of 12.182.81 . the review of this archive revealed 39 mothers with thyroid dysfunction either in pregnancy , or currently . from this sample , twenty - four ( 61% ) mothers reported current , 5 ( 12% ) mothers reported only in pregnancy and 10 ( 25% ) mothers reported both in pregnancy and current thyroid problem . in sum , 15 ( 38% ) no statistically significant difference existed between the mean delivery age of mothers who had thyroid problem in pregnancy and those who did not . any thyroid problem was found to affect 4.9% of the entire population ( n=790 ) , whereas the rate was 1.8% for pregnancy thyroid problem . the most common principal diagnoses given to the children of mothers in this group were attention deficit hyperactivity disorder ( n=14 ; 35% ) and depression ( n=10 ; 25% ) . the mothers who had pregnancy thyroid dysfunction ( n=15 ) reported their diagnoses as either hypothyroidism , goiter or hashimoto disease whereas three mothers could only recall the presence of a thyroid disorder . among the mothers interviewed , only three reported medication use in pregnancy which were thyroid hormones . we have identified 16 children and adolescents in the total population who had behavior indicative of same - sex attraction / gender nonconformity . of these children , 13 were males and 3 were females , having a mean age of 13.181.83 . different diagnoses were given to these children , except for three , for whom no diagnosis was applicable . the percentage comparison of same - sex attraction / gender nonconformity in the focus group with other control groups are presented in figure 1 . for each group compared , there was a significant association between the history of thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the children born to these women ( p<0.0001 , by fisher s exact test ) . the association was also significant for each sex ( p<0.0001 , by fisher s exact test ) . in logistic regression analysis , no other factor has been found to influence same - sex attraction / gender nonconformity . homophobic bullying , which targets children with same - sex attraction / gender nonconformity appeared to be a common problem ( n=7 ; 58% ) . in table 1 no child was identified as having both mental retardation and perinatal history of thyroid dysfunction . in excluded cases , in this study , a significant association between thyroid dysfunction in pregnancy and same - sex attraction / gender nonconformity in the offspring was found . , this study is the only one that explored this association with a focused objective and found positive results . an initial point of note is that the prevalence of thyroid disorders in the study population , both currently and in pregnancy , is far less than the reported prevalence rates that may be expected . it is possible to hypothesize that only certain patients had received medical attention in pregnancy . current case - finding procedures are estimated to fail to identify many pregnant women with subclinical and overt hypothyroidism . also , a significant rate of children and adolescents with same - sex attraction may remain to be identified . first of all , same - sex interest may manifest later than the mean age we found in this population . third , as can be seen in table i , most subjects were male children who assume a feminine role . active or manly same - sex attraction was not a reason for referral . similarly , behavioral manifestations of same - sex attraction in girls were lower than population estimates in this study . this finding of sex difference in referral rates is in line with previous reports of gender issues . despite these difficulties , there existed sufficient cases to show the magnitude of underlying pathology . the assumption that same - sex interest is caused by child sexual abuse is no longer supported . situational homosexuality , which occurs when heterosexual partner is inaccessible , like in prisons , constitutes only a small group . therefore , it is likely that the index group in our study represents general characteristics of individuals with same - sex attraction in a population . as 3 children had not received any psychiatric diagnosis and the remaining children s disorders were either temporary or common ones seen in child psychiatry practice , this may call for further research about the generalizability of the present findings to general population . on the other hand , although the statistical association we found in this study is such strong , there were 3 cases who had no sex attraction / gender nonconformity despite maternal thyroid history dysfunction in pregnancy . depending on the timing , duration , severity and type of maternal thyroid dysfunction , there may be a group of children who could survive the negative impact on behavior . as described in the methods section in accordance with current clinical practice parameters , although two different terms , gender nonconformity and same - sex attraction were used to identify the explored behavior , all cases have been merged in a single group as can be seen in table 1 . there is agreement that gender nonconformity often is a developmental precursor of homosexuality in males and it is also associated with negative health outcomes . the present study provides further evidence that both conditions are developmentally linked . on the other hand , in all youngsters in table 1 , the extent of same - sex attraction was beyond the limits of sexual experimentation which is a feature of adolescent development . a particular point is that , no case had been diagnosed as having gender identity disorder , which is a different developmental pathway . however , gender identity disorder in the offspring should also be considered among the behavioral consequences of prenatal thyroid dysfunction and may be explored in further research . in contrast to what may be expected , no case was identified as having low iq because of early thyroid dysfunction , which is a well - established consequence . it is evident that the impact of early thyroid dysfunction on developing neurons produces more common but not less serious results which manifest later in life at a behavioral level . first of all , as different specialties are involved in this model , namely endocrinology , obstetrics , pediatrics , child and adult psychiatry , grasping the whole picture may be elusive . however , unlike other disciplines , child psychiatry has a unique advantage of being able to collect data for both parents and offspring . secondly , the impact of thyroid dysfunction on sexual behavior unfolds after so many years , even after a couple of decades . thirdly , because of understandable sexual privacy and secrecy in the context of strong moral , cultural and religious values , even very close relatives of an individual with same - sex attraction may be unaware of the condition . fourth , a significant rate of pregnant women with thyroid dysfunction may go undiagnosed , or may have subclinical forms of thyroid dysfunction . all possible explanations of the findings will be based on thyroid dysfunction during pregnancy , as it is the most likely predictor of same - sex attraction in the majority of cases . either present before pregnancy or developed afterwards , thyroid diseases may result in diminished supply of thyroid hormones which are crucial for optimal brain development . developing brain areas , which will be responsible for determining heterosexual orientation remain undifferentiated without the influence of essential thyroid hormones . as the timing , duration , severity and type of insult during development determine the specific consequence , the characteristics of same - sex attraction ( i.e. , bisexual or entirely homosexual ) in the offspring are expected to be shaped by these factors . once the timely effects of specific hormones are missed , these functions may have been never recovered in a lifetime . besides , these areas may also be overly sensitive to the availability of the thyroid hormones . we should also consider whether there is such a mechanism in the brain that in case thyroid hormones are low , less vital neurons are sacrificed to save more crucial ones to preserve optimal intelligence . further to these explanations , we need to think about the possible role of autoimmunity in the generation of deviant sexual orientation , as hashimoto thyroiditis was quite common in the index group . it is known that , in hashimoto thyroiditis , tsh receptor blocking antibodies are capable of crossing the placenta and affecting the fetal thyroid . it is possible to hypothesize that besides lowering thyroid hormone levels , various antibodies may attack sensitive areas in the developing fetal brain and may produce permanent effects . finally , there is also possibility that maternal thyroid dysfunction may impact prenatal androgen system and generate permanent consequences . , there are cases having same - sex attraction / gender nonconformity despite medication history addressing thyroid dysfunction during pregnancy . although more detailed medication histories were not available , thyroid hormone use during pregnancy in the index cases will be discussed in two ways that i ) medication use rate is low and ii ) simple supply of thyroid medication may be ineffective in altering the pathogenesis if basic underlying factor is autoimmunity . at this point , i am going to refer to the study by ellis and hellberg , cited above . the most significant finding in that study was increased prenatal use of thyroid hormone and diet pills in the mothers of young females with homosexual / bisexual orientation . according to the authors , thyroid and dieting medications may trigger immune reactions that may result in homosexual / bisexual orientation in the female offspring . however , correction of hypothyroidism with synthetic levothyroxine is known to be safe , and in the present study there was no history of medication use in the majority of the cases . thus , it appears that it is not the medication use that leads to same - sex attraction , but the basic thyroid dysfunction itself . definitely , as there may be several cases who never come into medical attention , the subjects with medication use represent only a sub - group . i will also argue that a broad spectrum of thyroid disorders should be considered rather than only immune conditions . finally , failure to find significant results in male subjects may stem from methodology : both sexes are affected in the present study as might be expected . finally , a recent study that reported increased rates of certain autoimmune diseases in homosexual individuals but no increased overall risk can best be explained with the mechanism presented in our study . from a child psychiatry point of view , it is clearly visible that there is a new kid in the playground . what was found in the present study is obviously a challenge to pre - existing theories about homosexuality and there s sufficient ground to introduce a new model . the prenatal androgen model , which refers to rare disorders such as congenital adrenal hyperplasia and testicular feminization for explanation , is going to be affected by the decisive role of thyroid hormones in sexual orientation . it is not surprising to find significant results for homosexuality in genetic studies as prevalent thyroid disorders ( i.e. , hashimoto thyroiditis ) have a very strong genetic background . in the search for possible factors for the persistence of homosexuality in human populations , thyroid disorders provide a reasonable explanation as they are prevalent and more common in women . fraternal birth order effect , which refers to increasing odds of homosexuality in having older brothers , may simply reflect continuously decreasing capacity in thyroid disorders in subsequent pregnancies . in sum , the findings of this study and review of the preexisting views indicate the birth of prenatal thyroid theory to explain the underlying dynamics of same - sex attraction in human beings . there is a need to add relevant statements for the ethical position of this study and manuscript , as it is known that there exist views that oppose scientific study on the causes of same - sex attraction . we believe that clinicians as researchers do not have an option to suppress data , experience or insight gained in their practice . if there is a medical risk factor that may highly predict certain behaviors , clinical researchers can not remain silent and can not keep it hidden , which apparently constitutes an ethical violation . individuals with same - sex attraction have the right to know the possible origins of their behavior which can not be denied by making decisions on behalf of them . apparently , in this age of information , this right extends to any individual who would like to be interested . people at childbearing age , for instance , may not be accepting homosexuality , because they do nt want offspring that is likely to be persecuted and susceptible to negative health sequelae . on the other hand , we are well aware that abuse of scientific knowledge is always a potential risk and we must be prepared to struggle against it at all times . first , the prenatal medical data relied on parental recall . however , as thyroid disorders are chronic in nature , the mothers may be pretty bright in reporting about this . also , the parents and the children had not been questioned systematically about issues of sexual behavior . however , such limitations are inevitable in investigating an issue in which cause and effect are apart in time . based on the insight gained from this work , is expected to focus on confirmation of present findings . then , neurobiology of fetal impact and improvement of endocrinological care during pregnancy may be studied . using current case - finding procedures , many pregnant women with subclinical and overt hypothyroidism may remain undetected . on the other hand , neither targeted case finding nor universal screening have been shown to lead to improved outcomes in population - based studies . in light of this study , screening for thyroid dysfunction in pregnancy should be rethought , which is controversial in current guidelines . outcome criteria for intervention studies should be redefined considering same - sex attraction unfolding much later . newer intervention strategies may be needed as same - sex attraction is a special kind of consequence . psychological effects and therapeutic management of awareness of thyroid impact in affected individuals , either as mother or as offspring , may become a field of highest priority . a final note is that medical implications should always be developed with multi - disciplinary consensus . more convincing neurobiological evidence will be a positive step towards social acceptance and greater adaptation of young and adult people with same - sex orientation . violence and prejudice against individuals with same - sex attraction will inevitably lose part of its ground . with further medical understanding and awareness , all medical specialties have to do their best to help all affected people in their potential problems . there is a real need to review all relevant cultural , legal , political and even historical aspects of the topic from the medical standpoint explained in this article . it is only with the rule of science and knowledge that certain misconceptions and misperceptions will be cleared up and humankind will benefit .

both youngsters and adults with same - sex attraction are at greater risk for negative health outcomes . despite mounting efforts to determine the biological background , a satisfactory conclusion has not been reached and there is a need to explore alternate factors like functioning of thyroid system during pregnancy . a retrospective chart review was undertaken of 790 eligible children and adolescents who had been admitted to child psychiatry between 2005 and 2013 . this population consisted of 520 ( 65% ) males and 270 ( 35% ) females , aged 8 to 17 years . fifteen mothers ( 1.8% ) were found to have a history of thyroid dysfunction during pregnancy . sixteen youngsters ( 2% ) had a history of same - sex attraction . twelve overlapping cases with both same - sex attraction and maternal thyroid dysfunction during pregnancy were identified , which was extremely significant ( p<0.0001 , by fisher s exact test ) . the association was also significant for each sex ( p<0.0001 , by fisher s exact test ) . there is evidence that thyroid gland plays a crucial and decisive role in determining sexual orientation in people . maternal thyroid dysfunctions during pregnancy may result in homosexual orientation in the offspring .

women with psychiatric diseases are treated with antipsychotic medication(s ) with limited evidence to support the safety of their use during pregnancy.olanzapine , a second - generation antipsychotic , is a us food and drug administration pregnancy category c drug with no unequivocal evidence of harm to the fetus.we report the first case of tracheo - esophageal fistula as a possible teratogenic effect of olanzapine exposure . women with psychiatric diseases may become pregnant and are treated with antipsychotics without any proven evidence of safety . due to ethical issues , pregnant women are rarely included in clinical trials , leading to a dearth of data available on the safety of antipsychotic drugs in this population . there have been reports of congenital anomalies in newborns of mothers exposed to antipsychotic medications during pregnancy [ 14 ] . olanzapine , a second - generation antipsychotic , is a us food and drug administration ( fda ) pregnancy category c drug [ 3 , 5 ] with no unequivocal evidence of harm to the fetus . a literature search revealed that maternal exposure to olanzapine appears to be associated with lumbar meningomyelocele , dysplastic kidney , hip dysplasia , atrioventricular canal defect , club foot , microcephaly , ventricular septal defect , absent fingers , craniosynostosis , cleft lip , encephalocele , aqueductal stenosis , etc . the possibility of further teratogenic effects of olanzapine can not be ruled out . in this case report , we report the first case of tracheo - esophageal fistula ( tef ) as a possible teratogenic effect of olanzapine exposure . we report the case of a 29-year - old married woman with bipolar disorder with mixed episodes who had been treated with olanzapine in the psychiatric outpatient department of our institute ( lady hardinge medical college , new delhi , india ) for the last 7 years who delivered a full - term female baby with tef in september 2013 . one week after her marriage in february 2008 , the patient developed psychotic symptoms such as excessive talking , episodes of crying and laughing , and forgetting things frequently . following initial unsuccessful local treatment , she was taken to the psychiatry department of a tertiary care hospital . after initial management of acute symptoms there , she continued her treatment from our institute , where she was prescribed olanzapine ( oleanz 10 mg tablet orally once daily ) . in december 2008 , she had a miscarriage at 4 months gestation , following which she stopped taking the medication of her own choice . a careful history / enquiry from the parents failed to reveal any data available regarding congenital malformation of the aborted fetus . in 2009 , she conceived again ( it is noteworthy that the patient was not taking olanzapine during this pregnancy ) and delivered a full - term healthy female child in january 2010 . eight months after delivery of this baby the patient had a recurrence of psychotic symptoms for which she was prescribed the same treatment . in december 2012 , she conceived a third time but due to persistent psychiatric symptoms , olanzapine was continued throughout the pregnancy by the treating psychiatrist , who considered there to be a favorable benefit : risk ratio for the patient at that time . she received regular antenatal care and was compliant with all prescriptions and instructions , including prophylactic folic acid and two doses of tetanus toxoid . there was no history of maternal alcohol intake , smoking , or exposure to other potential teratogenic drugs or exogenous hormones throughout the pregnancy . all baseline hematological ( hemoglobin 13.8 g / dl , total leukocyte count 6000/l , platelets 2.3 lac/l , etc . ) and biochemical investigations ( random blood sugar 98 mg / dl , blood urea 27 mg / dl , etc . ) tests for trisomy 21 and 18 ( nuchal translucency scan and dual marker tests ) and rubella were negative . , she delivered a full - term female baby by vaginal delivery . immediately after delivery , the baby experienced respiratory distress and excessive salivation , for which she was admitted to the neonatal intensive care unit . on examination , a red rubber catheter could not be negotiated beyond 10 cm in the esophagus . other concomitant anomalies including vertebral , anal , renal , limb ( components of vacterl [ vertebral defects , anal atresia , cardiac defects , tracheo - esophageal fistula , renal anomalies , and limb abnormalities ] ) , eye , ear , and nasal defects were ruled out . the tef was repaired using esophageal esophageal anastomosis with a drainage tube in the fifth intercostal space . on the second post - operative day for this reason , cervical esophagotomy was performed and a gastrostomy was inserted for feeding purposes . the baby was discharged in a stable condition with advice for full breast milk feeding through gastrostomy , some required medications ( multivitamin syrup , calcium , anti - emetic and antibiotics ) for an appropriate duration , and regular vaccination . however , although the baby was well until this point , she was re - hospitalized at 11 months of age because of diarrhea and unfortunately died on 19 august 2014 , probably because of widespread infection ( no definitive record of the last hospitalization is available and a post - mortem was not performed).fig . 1chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula a number of drugs are clearly known to be associated with teratogenicity , such as thalidomide and isotretinoin , but the majority of drugs do not have conclusive data concerning their effect on fetal development and growth . due to strict ethical constraints , at - risk populations ( including pregnant women ) are not included in most clinical trials , limiting the availability of data on the use of drugs ( including antipsychotics ) in these populations , and thus spontaneous reporting of suspected adverse drug effects constitutes an essential tool for generating data on the safety of drugs , especially in the population of pregnant women . according to a retrospective analysis of data from the lilly safety database , of the 610 prospectively identified pregnancies exposed to olanzapine with an available outcome , there were 401 ( 66% ) normal births , 60 ( 9.8% ) premature births , 57 ( 9.3% ) spontaneous abortions , 49 ( 8% ) perinatal complications , 27 ( 4.4% ) congenital anomalies ( cleft lip , encephalocele , and aqueductal stenosis ) , and 16 ( 2.6% ) other outcomes ( post - perinatal condition , ectopic pregnancy , post - term birth , and still birth ) . other reported congenital anomalies with olanzapine included meningocele , ankyloblepharon , hip dysplasia , acheiria , atrioventricular canal defect , and unilateral club foot [ 1 , 68 ] . a link between teratogenicity and antipsychotics is also supported by the fact that these drugs readily cross the placental barrier [ 1 , 4 , 9 ] , with 23.8% placental passage for quetiapine and 72.1% for olanzapine [ 2 , 4 , 9 ] . newport et al . measured placental passage of medication from mother to fetus by measuring levels in the umbilical cord serum and found that olanzapine has the highest rate of placental passage , compared with haloperidol , risperidone , and quetiapine [ 3 , 9 ] . the overall incidence of esophageal atresia / tef ranges from one in every 2500 to 4500 live births . however , a careful literature search revealed a paucity of data on the incidence of tef in children born to women with psychiatric disease and the problem of confounding by indication can not be ruled out . it has been proposed that antipsychotics interfere with the action of calmodulin , which has an important role in organogenesis . esophageal atresia and abnormalities of pharyngeal glands have also been proposed as being related to neural crest cells . the drugs ( olanzapine in this case ) associated with such anomalies may be hypothesized to cause neural crest cell damage during early fetal development as a causative factor . although the neural crest cell damage - related esophageal abnormalities in many cases are also associated with cardiovascular anomalies , these were absent in the present case . we tried to establish the causality between the adverse outcome ( tef ) and use of olanzapine during pregnancy according to the who - umc ( world health organization uppsala monitoring centre ) adverse drug reaction causality assessment scale and the naranjo causality assessment scale . causality is probable ( score + 5 ) as per the naranjo scale , whereas it is one of the important reasons for lower scores of causality is the non - applicability of many questions in these scales . the current case report of tef associated with antenatal olanzapine exposure demonstrates the need for large clinical studies to generate more conclusive data concerning use of this drug during pregnancy . on the basis of the present case and the literature , it is suggested that antenatal olanzapine exposure may be related to the development of tef in newborns . however , the present case report can only be considered as preliminary evidence and further observational prospective studies of women with antenatal exposure to olanzapine should be conducted to explore the strength of association between tef and olanzapine . knowledge of the teratogenic potential of a drug is essential to prevent / reduce the morbidity and mortality associated with a congenital anomaly ( tef in this case ) . such post - marketing reports are effective tools to disclose the adverse effects of drugs , which are difficult to detect in clinical trials and may factually change the safety status of a drug . written informed consent was obtained from the infant s father for publication of this case report .

there is a dearth of evidence on the safety of the use of antipsychotics during pregnancy . olanzapine , a pregnancy category c drug , has no unequivocal evidence of harm to the fetus . against this backdrop , we report the first case of a tracheo - esophageal fistula ( tef ) in a newborn following maternal antenatal exposure to olanzapine . a 29-year - old woman with acute psychotic disorder had been treated with olanzapine for the last 7 years . her first pregnancy , while taking olanzapine , resulted in a miscarriage at 4 months gestation , following which she discontinued olanzapine . she reconceived after a few months and delivered a full - term normal child . however , due to the recurrence of psychiatric illness after her second pregnancy , she was prescribed olanzapine again , which was continued throughout her third pregnancy . the outcome of the third pregnancy was a full - term female baby with a tef . the baby was managed surgically and discharged with satisfactory vital signs . unfortunately , however , the baby did not survive beyond 11 months of age . causality between antenatal maternal olanzapine exposure and tef in the newborn was determined to be probable ( score + 5 ) as per the naranjo causality assessment scale . greater knowledge of this potential teratogenicity caused by olanzapine is needed to reduce morbidity and mortality in newborns .

viruses are divided into two similar - sized groups depending on whether the virus particle contains dna or rna , and , as causes of human fatality , rna viruses are by far the more important ( 1 ) . new viral diseases continue to appear as a result of several changes in human activity : travel , population growth , interaction with wild habitats etc . well - known novel , or emergent , rna diseases include severe acute respiratory syndrome ( sars ) ( 2 ) , human immunodeficiency virus 1 ( hiv-1 ) ( 3 ) , and may come to include avian influenza h5n1 virus ( 4 ) . these emergent diseases are an important factor behind the increase in the number of genome sequences that ncbi treats as representing new species ( figure 1 ) . in 2005 , more than 200 new virus species were submitted to genbank ( more recent dates are less reliable because there is typically a delay between submission and public availability ) . as more emergent viruses appear , it is important to have a site that allows their genomes to be compared to those of known viruses . the origin of most major infectious diseases is unknown because of our ignorance of the diversity of pathogens in wild animals . this restricts our ability to both predict risks and develop treatments ( 5 ) . dates are the earliest given in the accession ( either of submission or publication ) . submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated . dates are the earliest given in the accession ( either of submission or publication ) . submissions after 2006 are excluded because accessions are made public typically only following publication and thus the frequency of submissions is more recent time periods is underestimated . despite some advances ( 6,7 ) , the evolutionary history of rna viruses is in general poorly known , especially the deep phylogenetic relationships between virus families ( 8,9 ) . we believe that one of the reasons for this is a lack of easily available translated genes and genomes for all species , and the lack of aligned genome sequences representing different isolates of the same species . in addition to the need to facilitate greater comparative analysis of rna viruses is the need to link together the existing virus web sites and their underlying databases . there are many web sites that provide genomic data , tools for genetic analysis and/or biological information for some viruses ( see links on our site home page ) . the rna virus database is intended to complement these other sites by providing basic genomic information and tools for all rna viruses and linking the user to more specialist sites , where they exist , e.g. for hiv-1 and hepatitis c virus ( hcv ) , we provide links to sites such as the los alamos laboratory on the main page for each of these viruses ( find by typing hiv-1 or hcv into the search window in the top toolbar ) . for such viruses , we do not duplicate the work of other groups by attempting to display the available diversity of genomes . we intend that the rna virus database should develop further as a hub for other sites and we therefore encourage other workers to contact us with details of their sites that they wish linked to ours . also , we encourage workers to adopt a virus and improve and/or expand the information that we provide for individual species . this can be done by emailing us or getting involved directly in developing the database , which is an open source project available at our googlecode site ( http://code.google.com/p/rnavirusdb ) . some of the data and tools on the rna virus database can be found elsewhere , but not all of them can , e.g. ncbi 's genome site provides genomic overviews of virus species and pairwise alignments of other isolates to the reference sequence , but it does not provide multiple alignments or complete translated genomes as we do . similarly , its general entrez site provides pair - wise alignments of the query sequence and similar sequences in the database , plus a phylogenetic tree calculated from those distances ; however , no multiple alignment is built . we also corrected the ( few ) errors in the genbank entries , and our database records features such as rna editing ( 10 ) that make genome translation problematic . we have , therefore , created the rna virus database as a user - friendly site devoted to rna viruses , providing essential genomic data and tools ( discussed in more detail below ) and links to the other virus web sites . provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus speciesidentification and taxonomic searching facilityguidance to other web resources . provide multiple whole - genome alignments , gene and whole - genome translations for all rna virus species identification and taxonomic searching facility guidance to other web resources . we provide multiple alignments of whole genomes ( as nucleotides ) for all species where genbank contains multiple representatives . our database , thus , currently has multiple alignments for approximately half the species ( available from the main page of any virus species under these alignments were made by downloading from genbank all complete ( or near - complete ) genomes using the bioperl genbank modules ( 11 ) . accidental mismatches were excluded by performing a preliminary alignment using blastalign ( 12 ) , which is designed to cope with non- or poorly homologous sequences and reports such matches . the sequences that showed clear homology to the ncbi reference sequences ( we used a cutoff of a maximum of 40% of positions being represented by gaps in the blastalign alignment ) , up to a maximum of 50 , were then aligned using clustalw ( default parameter values ) ( 13 ) . for species for which we have at least three sequences , a neighbor - joining tree was then constructed using paup ( with hky - adjusted genetic distances ) ( 14 ) , and this tree is displayed both as a pdf [ via the treegraph program ( 15 ) ] and using figtree , which is a new java - based tree - drawing application created by one of us ( rambaut , a . , unpublished data ) . our site allows virus nucleotide or amino acid sequences submitted by the user to be identified or , if the query is a new species , its closest relative to be found . in addition , the genomic location of any matched region of the library sequences is shown . for this we use ncbi 's suite of blast programs ( 16 ) ( go to the blast link on the toolbar of the home page ) . once the most closely related reference species has been located , the query sequence can then be placed into a whole - genome multiple alignment for that species ( where such an alignment is present ) in order to show the query 's phylogenetic relationships to the genomes in our database ( go to ( i ) a blast of the query to the reference species sequence provides coordinates from the resulting pair - wise alignment . these coordinates are then used to select homologous regions from the reference multiple alignment , and a new multiple alignment is then built using clustalw along with a phylogenetic tree using paup as described above . ( ii ) blastalign ( described above ) is used to generate a new multiple alignment using the query sequence plus the sequences from the reference multiple alignment . figure 2.screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence . screenshots illustrating use of the rna virus database to investigate a submitted virus nucleotide sequence . isolates , includes the biological data of the isolates used in the whole - genome multiple alignments [ except for hiv-1 , hcv and hepatitis b virus ( hbv ) , where we used manually built multiple alignments ; for these three species , accession numbers for the isolates are given in the supplementary data as accessionhivhcvhbv.xls ] . we provide amino acid sequences for all virus genes ( or , more strictly , for all open reading frames ) plus complete translated genomes for each virus species ( go to the translated genomes are intended to facilitate phylogenetic analysis of more distantly related viruses ( 9 ) . one feature that makes annotation of rna viruses difficult is that most species have some gene overlap ( 17 ) , i.e. where the same nucleotides code for two different genes by being read in two different frames . we , therefore , allow the user to select from three possible options for dealing with this feature : ( i ) have overlapped regions excised from the translated genome , ( ii ) have one only of any overlapped amino acid sequences represented or ( iii ) have all the amino acids sequences present , with overlapped sequences placed sequentially ( and thus the nucleotides represented twice ) . using a key word search of the genbank entries , and a standard reference work ( 18 ) where this did not reveal a match , the rna virus database is a php web application on top of a mysql database . php is available at http://www.php.net , but should come pre - installed on unix machines . all data have been taken from the ncbi 's genome and nucleotide sites at the following two urls . table names are in large bold font and interlinking column names are in small regular font . relationships of tables in the underlying mysql database . table names are in large bold font and interlinking column names are in small regular font . species names , nucleotide sequences and accession numbers were downloaded directly from genbank using bioperl modules , while further details of the virus gene coordinates , taxonomic affinities etc.were subsequently extracted from the flatfile of all genbank entries that can be downloaded from the ncbi genome site ( 19 ) . our approach was to treat all entries in the ncbi virus genome sites as species and to follow their taxonomic classification , although we only give virus type ( e.g. single - stranded positive - sense , retrotranscribing ) , family and genus at our site . we , therefore , follow ncbi 's inclusion of hepadnaviruses , which include hbv , and caulimoviruses among the rna viruses despite their mature virion containing dna rather than rna ( their possession of reverse transcriptase clearly places them biologically and evolutionarily among the reverse - transcribing group of rna viruses ) . the php scripts can be accessed using subversion ( http://subversion.tigris.org/ ) from our googlecode site at http://code.google.com/p/rnavirusdb . the mysql database ( a gzipped 16 mb dump ) may also be downloaded from the same site for installation on the user 's computer if required . if required , the database can subsequently be updated by other users following instructions and perl scripts given in the supplementary material ( perl_scripts.tar ) . this updating involves a series of short intervening manual steps ( we find that complete automation of such processes is inefficient ) . we intend to update the database on at least a 6-monthly basis in order to include newly discovered viruses , and are currently working to incorporate biological and epidemiological data . as discussed in introduction , we are keen to collaborate with other groups over further developments of our database . wellcome trust ( to r.b . ) ; eu marie curie fellowship scheme ( to t.d.o . ) ; the royal society ( to a.r . )

the rna virus database is a database and web application describing the genome organization and providing analytical tools for the 938 known species of rna virus . it can identify submitted nucleotide sequences , can place them into multiple whole - genome alignments ( in species where more than one isolate has been fully sequenced ) and contains translated genome sequences for all species . it has been created for two main purposes : to facilitate the comparative analysis of rna viruses and to become a hub for other , more specialised virus web sites . it is available at the following four mirrored sites . http://virus.zoo.ox.ac.uk/rnavirusdbhttp://hivweb.sanbi.ac.za/rnavirusdbhttp://bioinf.cs.auckland.ac.nz/rnavirusdbhttp://tree.bio.ed.ac.uk/rnavirusdb

adult - onset still 's disease ( aosd ) is a rare inflammatory disease of unknown etiology , which commonly affects young adults . it is usually characterized by high spiking fevers , arthritis , and an evanescent , nonpruritic , macular and salmon coloured rash , appearing on the trunk and the extremities . important laboratory findings include leukocytosis , with predominance of neutrophils , negative testing for rheumatoid factor ( rf ) , and antinuclear antibodies ( ana ) as well as high serum ferritin levels and low serum glycosylated ferritin levels [ 13 ] . severe disease complications include pericarditis , endocarditis , haemolytic anaemia , and macrophage activation syndrome ( mas ) . the latter is characterized by thrombocytopenia , markedly elevated ferritin levels , hypofibrinogenemia , and elevated aspartate amino - transferase ( ast ) . aosd diagnosis can be safely established , after important mimickers including infections , malignancies , and autoimmune diseases are excluded . treatment of patients with aosd includes nonsteroidal anti - inflammatory drugs ( nsaids ) , corticosteroids , and disease - modifying antirheumatic drugs ( dmards ) , while our better understanding of disease pathophysiology allowed the identification of biological agents as important targeted therapies [ 1 , 4 ] . recent studies have added valuable information in regard to the underlying pathogenetic mechanisms of aosd . besides , the exact pathogenesis remains largely elusive , with genetic , environmental , and immunologic contributors being implicated . in the present paper , we aimed to summarize recent advances in pathophysiology and potential therapeutic strategies in the setting of aosd . we also used rheumatology textbooks with chapters relevant to aosd and abstract database from acr and eular meetings at 2010 and 2011 . aosd or still 's disease pathophysiology , aosd or still 's disease therapy , still 's disease or aosd treatment , still 's disease or aosd and inflammasome . several small case studies have previously reported associations with distinct hla alleles in patients with aosd , with often conflicting results . in an early small study of 25 aosd patients , wouters et al . reported an increased frequency of the hla - dr4 allele in 29 patients with aosd compared to normal controls , with the presence of hla - drw6 being linked to root joint involvement . in a subsequent study , a strong disease association with hla - b17 , b18 , b35 , and dr2 has been documented . in japanese populations , an association between a chronic articular form of aosd and hla - drb1 * 1501 ( dr2 ) , drb1 * 1201 ( dr5 ) , and dqb1 * 0602 ( dq1 ) was previously reported , while hla- and dqb1 * 0602 ( dq1 ) have been also associated with the systemic form of the disease . data from a korean report , supported an association between hla - drb1 * 12 and drb1 * 15 and aosd , while hla - drb1 * 04 seemed to be protective . on the other hand , hla - drb1 * 14 alleles were more commonly present in patients with the monocyclic systemic type of aosd . the shared clinical and laboratory findings observed in aosd and infections are highly suggestive of a putative role of infectious agents in disease pathogenesis . several anecdotal reports so far indicate a temporal relationship between bacterial and viral triggers prior to disease onset . several viruses such as rubella , echovirus 7 , mumps , cytomegalovirus ( cmv ) , and others , as well as bacterial pathogens including yersinia enterocolitica , chlamydophila pneumoniae , brucella abortus , and borrelia burgdorferi , have been so far implicated in disease pathogenesis [ 1013 ] . a hallmark of aosd is neutrophil and macrophage activation possibly under the effects of the proinflammatory interleukin-18 ( il-18 ) signalling . neutrophil ( pmn ) cd64 a marker of neutrophil activation has been recently found to be upregulated in patients with active aosd . a calcium - binding protein named calprotectin , secreted by activated neutrophils and macrophages , as well as macrophage migration inhibitory factor ( mif ) , is useful markers of disease activity and severity [ 15 , 16 ] . intercellular adhesion molecule-1 ( icam-1 ) upregulated by il-18- has been also proposed as a potential clinical marker , as its expression typically reflects the level of disease activity . furthermore , activation and differentiation of macrophages appears to be orchestrated by macrophage - colony stimulating factor ( m - csf ) , a cytokine which is substantially elevated in acutely ill aosd patients . the role of cd4 t helper ( th ) cells in the pathogenesis of aosd has been recently appreciated , with th1 subset predominating over that of th2 cd4 cells and being associated with disease activity . accordingly , interferon - gamma ( ifn- ) mrna expression was found to be significantly higher than that of interleukin-4 ( il-4 ) in skin and synovial tissue biopsies . the role of th17 lineage in aosd pathogenesis is also emerging , as evidenced by increased number of peripheral th17 cells in 24 patients with untreated and active aosd compared to healthy controls . th17 cells are a subset of t helper cells , named after their ability to produce interleukin-17 ( il-17 ) . this subset of cells is derived from the differentiation of naive cd4 t cells , under the influence of transforming growth factor ( tgf ) , interleukin-1 ( il-1 ) , and interleukin-6 ( il-6 ) [ 20 , 21 ] . interestingly , heightened levels of t - cell receptor -positive ( tcr+ ) t cells , mostly of the v9/v2 subset have been previously associated with active disease and correlated with inflammatory markers . since , it has been recently appreciated that t/ cells are also represent an important source of il-17 production , the role of these cells in the pathogenesis of aosd requires further attention . interleukin-18 ( il-18 ) , interleukin-23 ( il-23 ) , and inteleukin-21 ( il-21)found to be elevated in active aosd patients seem also to ensure the proliferation / maintenance of th17 cells . circulating th17 cells correlated with disease activity and ferritin as well as il-1 , il-6 , il-17 , il-18 , il-21 , and il-23 levels . of interest , additional t cell populations actively involved in aosd pathogenesis include the cd4 cd25 ( high ) t regulatory ( treg ) cells found to be low in these patients compared to healthy controls and inversely associated with disease activity . furthermore , higher levels of cd4 cd25 ( high ) treg cells have been associated with a more favourable prognosis , as patients with monocyclic disease , a mild form of aosd , typically have higher concentrations of circulating cd4 cd25 ( high ) treg cells than those with polycyclic or chronic articular form . it should be , however , pointed out that according to recent findings , cytokine profile has not been proven useful in differentiating patients with aosd from those with sepsis , limiting their potential use in clinical practice . increased tnf- levels were detected in sera and tissues from aosd patients compared to healthy controls independently of disease activity . on the other hand , serum levels of soluble tumor necrosis factor - receptor-2 ( stnf - r2 ) correlated with serum crp levels , implying its potential use as a disease activity marker [ 8 , 26 , 30 ] . il-1 appears to be implicated in aosd pathogenesis as its serum concentration is elevated in these patients compared to healthy controls . further evidence for the contribution of il-1 in aosd pathophysiology came from the pioneering work by pascual et al . reporting that incubated peripheral blood mononuclear cells ( pbmcs ) with serum from patients with systemic form of juvenile idiopathic arthritis ( sjia ) , led to increased expression of innate immunity genes and release of large amounts of il-1 . however , plymorphisms in the il-1 and il-1 receptor ( il-1r ) genes have not been associated with aosd susceptibility , at least in a korean population . recent findings suggest activation of the protein complex nucleotide - binding oligomerization - domain-(nod- ) like receptor family , pyrin domain containing 3 ( nlrp3 ) inflammasome , as an important source of il-1 ; this activation can occur by recognition of pathogen - associated molecular patterns ( pamps ) and danger - associated molecular patterns ( damps ) . although it seems to contribute at least in one subset of sjia the pediatric counterpart of aosd with a favorable response to il-1 blockade , further studies are required to fully explore its exact role in the pathogenesis of aosd [ 3840 ] . taken together , these observations may suggest that susceptibility to sjia and aosd might be conferred by an interplay with exogenous pathogens - triggers of inflammasome with genetically determined inflammasome responsiveness resulting in dysregulation of il-1 production . heightened sil-2r levels , a marker of t - cell activation , were also reported in two distinct studies of aosd patients , serving as a potential marker of disease activity [ 8 , 29 ] . il-6 levels have been found to be elevated in aosd patients compared to their healthy counterparts in association with disease activity , fever spikes , and crp levels . of interest , skin lesional biopsies from individuals presenting with the characteristic salmon coloured rash revealed heightened il-6 levels [ 2931 ] . in addition , il-6 may contribute to the increased levels of ferritin as it stimulates its production along with crp and other acute - phase proteins by the liver . finally , prolonged exposure to high levels of il-6 may be associated with severe growth impairment , especially in patients with sjia . il-8 , a proinflammatory cytokine , which mobilizes , activates , and degranulates neutrophils at the site of inflammation has been also found to be raised in aosd patients compared to healthy controls , independently of activity status . given that elevated levels of serum il-8 typically characterize the chronic articular form of aosd , they can be used as a marker to predict the persistence of arthritic complaints . as previously mentioned and in line with previous observations in other autoimmune diseases , serum il-17- proinflammatory cytokine derived by th17 cells was higher in patients with aosd and correlated with th17-circulating cells . the fact that th17 cells and il-17 levels were both abated upon therapy administration implies a potential therapeutic role of th17 targeted therapies in the management of those diseases . il-18- member of the il-1 family , which induces th1 cytokine production- , has been shown to be higher in the serum synovial tissue and lymph nodes in patients with aosd than in healthy individuals , serving as a marker of disease severity , possible response to corticosteroids and of aosd - related hepatitis [ 29 , 32 , 33 , 45 ] . the latter is evidenced by the demonstrated association of il-18 serum levels with active liver disease . locally rather than systematically produced il-18 by liver activated macrophages ( cd68 ) seems to contribute to this complication [ 30 , 34 ] . associations of il-18 with serum ferritin , c - reactive protein ( crp ) , and neutrophil count have been also demonstrated [ 8 , 34 , 35 ] . several polymorphisms of the il-18 gene have been associated with aosd in japanese and chinese populations [ 4648 ] . another function attributed to il-18 is that of lymphocyte apoptosis possibly through induction of fas ligand ( fasl ) and p53 pathways , both implicated in the programmed cell death . this hypothesis is also supported by raised fas and fasl levels in untreated aosd patients compared to healthy controls . finally , in a more recent report , il-18 levels were found to be significantly elevated in patients of aosd complicated by mas compared to m - csf levels ; an opposite observation was made in patients with lupus - associated mas . although ifn- levels were also found to be raised in aosd patients compared to healthy individuals , no study so far demonstrated association of this cytokine with disease activity [ 26 , 29 ] . the contributory role of chemokines in the pathophysiology of aosd was supported by a recent study reporting elevated levels of cx3cl1 , cxcl8 , cxcl10 , ccl2 , and ccl3 in serum of aosd patients compared to healthy controls . of interest , only cx3cl could be used as a marker of disease activity as it was correlated well with serum crp , ferritin , il-18 , and sil-2r levels . furthermore , markedly elevated concentration of cx3cl1 and ferritin was able to predict the onset of mas , indicating its value in predicting aosd - related complications . treatment of patients with aosd has been empirical for a long time , given the lack of solid data from well - designed double - blinded randomized clinical trials with the majority of evidence deriving from small case series and retrospective studies . recent advances in better understanding of disease pathophysiology allowed the designation of targeted therapies leading to effective disease control . conventional immunosuppressants and new biologics are the main agents included in our therapeutic armamentarium against aosd . while nonsteroidal anti - inflammatory drugs ( nsaids ) have been previously considered as a first - line medication for the treatment of aosd , they have been replaced by corticosteroids , as they are effective as monotherapy only in 715% of patients [ 66 , 68 ] . the steroid therapy is efficacious in approximately two thirds of patients and more pronounced among those without chronic articular disease . disease - modifying antirheumatic drugs ( dmards ) such as methotrexate ( mtx ) , cyclosporine , hydroxychloroquine , gold , penicillamine , and azathioprine , [ 7072 ] have been proven efficacious in steroid - resistant or -dependent aosd cases , with methotrexate being the most commonly used dmard in clinical practice with response rates up to 60% . in regard to sulfasalazine , reduced efficacy along with some previously raised safety issues given the reported associations with mas development discourages its use in aosd cases [ 7375 ] . in patients refractory to treatment with steroids and/or dmards , biological agents seem to achieve a better control of disease activity . despite the lack of solid evidence of tnf implication in the pathogenesis of aosd as opposed to rheumatoid arthritis , anti - tnf agents have been used in aosd refractory cases with modest success , particularly in the chronic articular form of the disease lagging in efficacy behind il-1 and il-6 inhibitors . in a small case series of twelve aosd cases refractory to dmards , administration of etanercept , a soluble tnf receptor , led to arthritis improvement in 7 patients with nonsignificant adverse events . infliximab , a monoclonal antibody against tnf , as a treatment of eight multidrug - resistant aosd cases led to full response in 87.5% ( 7/8 ) of patients . five of these patients remained in remission even after the discontinuation of infliximab and one of them switched to etanercept due to infusion reactions . only one of the responders required chronic therapy to control its arthritis and only one patient did not respond to these biological agents . in two additional cases series , infliximab was administrated along with corticosteroids and dmards in a small number of patients with remission of systemic features , normalization of inflammatory markers , and without serious adverse reactions [ 52 , 55 ] . further information regarding the safety and the efficacy of anti - tnf agents derives from a study published by fautrel et al . in which infliximab or etanercept was administrated to twenty aosd patients , five with systemic and fifteen with polyarticular form , whose response to mtx and corticosteroids was considered inadequate . the majority of patients responded partially to therapy ( 64% , or 16 of 25 patients ) and only five in twenty patients achieved complete remission . anti - tnf--induced cutaneous adverse effects in the setting of sjia have been reported including cutaneous vasculitis and lichen planus , as well as psoriatic palmoplantar pustulosis accompanied by plaque - type psoriasis localized to the scalp . in view of the central role of il-1 in pathogenesis of aosd as previously reported , administration of interleukin-1 receptor antagonist ( anakinra ) in these patients , it has been shown that patients ( 84% ) receiving anakinra either as monotherapy or as adjunct therapy responded completely within a few days and only one of them had its disease relapsed during the subsequent followup . the remaining patients experience a partial clinical ( 12% ) and laboratory ( 16% ) response and only three patients discontinued the drug because of adverse effects . in general , the need for corticosteroids during treatment with anakinra greatly diminished in every patient . the corticosteroid - sparing effect of anakinra along with its effectiveness was also noted in a case series reported by kalliolias et al . in 2007 . furthermore , fitzgerald et al . demonstrated that anakinra is an effective agent to treat aosd patients refractory to corticosteroids , mtx and etanercept , as this drug rapidly resolves the inflammatory response and leads to normalization of laboratory markers . , in a study including both aosd and sjia patients , suggested anakinra as an effective alternative in the treatment of patients with aosd , with somewhat limited efficacy in sjia population . in contrast , in a retrospective chart review of 46 sjia patients , receiving initially anakinra either as monotherapy or together with additional disease - modifying antirheumatic drugs revealed that in 60% of these patients the clinical activity resolved completely and laboratory markers were normalized . the authors concluded that anakinra should be considered a safe and an effective way not only to treat systemic sjia but also to prevent the emergence of intractable arthritis . in addition , according to a case report published by raffeiner et al . , anakinra could be successfully used in the treatment of a patient with aosd and myocarditis . on the other hand , ruiz et al . reported that anakinra could not prevent the progression of aosd - associated cardiac disease despite the excellent control of noncardiac symptoms of a patient , without excluding the possibility that anakinra may be implicated in cardiac events of this patient . although anakinra seems to be an effective treatment of aosd patients regarding the rapid resolution of their clinical and laboratory markers , a recent case report published by lahiri and teng has shown that joint damage may progress despite the administration of this drug . a second generation il-1 inhibitor , the il-1 trap rilonacept , has been used in 3 patients who had failed treatment with glucocorticoids , immunosuppressors , and biologics , including anakinra with promising results . canakinumab , a fully human monoclonal antibody against il-1 with a long half - life , successfully controlled disease flares in aosd patients refractory to dmards , anakinra ( short - acting il-1 blockade ) , and rilonacept ( moderate - acting il-1 blockade ) . in addition , the efficacy and safety of canakinumab in the treatment of sjia , the pediatric counterpart of aosd , has been demonstrated in a phase ii , multicenter , open - label study , with 60% of patients achieving an acr pediatric 50 response . given the emerging role of t - cells in the pathogenesis of aosd , administration of abatacept , a t - cell costimulation modulator , in these patients seems to be a logical approach . abatacept ( ctla4igfc ) , a fusion protein which consists of the extracellular domain of the cytotoxic t - lymphocyte antigen 4 ( ctla-4 ) and the fc portion of immunoglobulin g1 ( igg1 ) , inhibits t - cell activation by binding to cd80 and cd86 receptors on antigen - presenting cells ( apcs ) and preventing their interaction with cd28 receptor on t cells . recent findings support a potential role of the latter in aosd cases refractory to conventional dmards , anti - tnf- agents , and even to il-1 receptor antagonists [ 81 , 82 ] . given that il-6 shares an important pathogenetic role in aosd , as mentioned above , the interleukin-6 ( il-6 ) antagonist , tocilizumab ( toc ) , has recently been proposed as a potential treatment for these patients . indeed , it seems to be an effective drug even against aosd cases refractory to anakinra and tnf- antagonists in anecdotal cases , even as monotherapy [ 8391 ] . tocilizumab was also able to control disease activity in a patient with diffuse intravascular coagulation ( dic ) and aosd , refractory to cyclosporine and high - dose glucocorticoids . in addition , the dose of corticosteroids was greatly reduced as toc was added on maintenance therapy . on the other hand , mas seemed to follow toc administration in a patient with intractable aosd , implying that caution should be taken in very active forms of the disease . in the first case series of tocilizumab in fourteen patients with intractable aosd at a dose 58 mg / kg every two or four weeks , eleven patients completed the 6-month followup and the remaining three discontinued the drug due to adverse effects , including necrotizing angiodermatitis , infusion - related chest pain , and systemic flare . over the course of 6 months , the clinical activity resolved completely in 57% of patients ( 8/14 ) and corticosteroid maintenance dose was dramatically reduced , suggesting that toc may be an effective alternative treatment , when dealing with multidrug - resistant cases of aosd . toc has also been approved for the treatment of sjia patients , as it is associated with substantial clinical and laboratory responses . of interest , administration of this drug in sjia patients led to improvement of reduced serum cartilage oligomeric matrix protein ( comp ) , further supporting the concept of contribution that high levels of il-6 in the suppression of growth cartilage turnover [ 42 , 94 ] . taken together , these findings support the contributory role of several immune mediators in aosd pathogenesis allowing the determination of rational treatment approaches . while current evidence identifies il-1 blockade as a major therapeutic strategy in patients with refractory aosd , inhibition of il-6 , il-17 , or il-18 molecules holds significant promises . given the complex and multifaceted nature of aosd , carefully designed clinical studies aimed to associate distinct clinical phenotypes with specific pathogenetic pathways would allow the designation of tailored therapies for distinct disease aspects .

adult - onset still 's disease ( aosd ) is a systemic inflammatory disorder affecting primarily young individuals . the diagnosis is primarily clinical and necessitates the exclusion of a wide range of mimicking disorders . given the lack of solid data in regard to the underlying pathogenetic mechanisms , treatment of aosd has been for years largely empirical . recent advances have revealed a pivotal role of several proinflammatory cytokines such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , and interleukin-18 ( il-18 ) in disease pathogenesis , giving rise to the development of new targeted therapies aiming at optimal disease control .

a healthy 35-year - old man was scheduled for laparoscopic cholecystectomy under general anesthesia for acute cholecystitis . he had a history of asthma and allergy to animal hair and was treated with a salbutamol inhaler 3 years prior , but showed no symptoms recently . physical and pre - anesthetic examinations , including a chest x - ray , electrocardiography ( ecg ) , laboratory results , and a pulmonary function test were unremarkable . the patient received tazobactam intravenously for preventive antibiotics and glycopyrrolate ( 0.2 mg ) intramuscularly for premedication 30 min before the operation . at arrival to the operating room , standard monitoring included non - invasive blood pressure , ecg , and peripheral oxygen saturation ( spo2 ) . the patient 's initial blood pressure was 155/85 mmhg , heart rate was 70 beats / min , and spo2 level was 99% . general anesthesia was induced with 1% propofol ( 150 mg ) with lidocaine ( 40 mg ) pretreatment and continuous infusion of remifentanil ( 0.10.5 g / kg / min ) . after the patient was asleep , rocuronium ( 80 mg ) was administered intravenously and , after 1 min , his trachea was intubated successively . next , anesthesia was maintained with sevoflurane , and his vital signs , including blood pressure , heart rate , and spo2 , were maintained stably during the operation . fifty minutes after starting anesthesia , efforts of self - ventilation were shown , and we administered additive rocuronium ( 10 mg ) . ten minutes before the end of the surgery , we administered fentanyl ( 100 g ) and discontinued remifentanil . the operation was finished within 90 min without complication . at the end of surgery , at a train of four ( tof ) count of 3 , we administered sugammadex ( 200 mg ; about 1.8 mg / kg ) to antagonize neuromuscular blockade intravenously . two minutes after sugammadex administration , his tof ratio was recovered to 0.9 , and he could breathe spontaneously . at that time , we discovered an erythematous wheal in his anterior thorax that was considered strange but not serious , and decided to extubate . soon after , he complained of dyspnea , his blood pressure decreased to 85/40 mmhg , his heart rate was 130 beats / min , spo2 level was 83% , and bilateral wheezing was demonstrated on chest auscultation . we administered dexamethasone ( 15 mg ) and dexchlor - pheniramin ( 4 mg ) intravenously , and a salbutamol nebulizer ( 5 mg ) with 6 l / min of 100% oxygen via face mask was inhaled . despite these interventions , his symptoms did not improve , his blood pressure decreased further to 65/38 mmhg , his heart rate was 135 beats / min , and spo2 level was 83% . our suspicion of an anaphylactic reaction increased ; thus , epinephrine infusion ( 0.06 g / kg / min ) was started after an intravenous epinephrine bolus ( 20 g ) , and 100 ml of fluids was administered over 10 min . at that time , arterial blood gas analysis ( ph 7.277 ; pco2 , 28.7 ; po2 , 63.3 ; hco3 , 13.1 ; be , 12.1 ) and chest radiography , which showed no active lesion on the chest radiographic image , were performed . within 10 min , his vital signs gradually increased to 105/55 mmhg , his heart rate was 105 beats / min , spo2 level was 95% , and his generalized erythema and tachypnea disappeared . after an additional 10 min , his blood pressure increased to 138/59 , his heart rate was 90 beats / min , and spo2 level was 98% . the patient 's vital signs continued to remain stable , and we discontinued the epinephrine infusion and transferred the patient to the intensive care unit . after half of the day , he was transferred to the general ward without any complication , and was discharged satisfactorily 5 days after surgery . ku / l ( reference , < 100 ku / l ) and specific ige for antibiotics were all negative , but he refused the serum tryptase test . after 7 weeks , skin prick tests for sugammadex , rocuronium , and fentanyl were performed , and the result was weakly positive for only 1 : 1 sugammadex ( 3 2 mm ) compared with the positive ( histamine ; 5 5 mm ) and negative ( normal saline ; negative ) controls , but negative in 1 : 10 , 1 : 100 , 1 : 1000 sugammadex and all concentrations of fentanyl and rocuronium . based on the time course of the event , anaphylactic reaction associated with sugammadex was strongly suspected . anaphylaxis is defined as " a serious , life - threatening , generalized or systemic hypersensitivity reaction " and " a serious allergic reaction that is rapid in onset and might cause death " . the diagnosis of anaphylaxis is mainly based on the clinical course because the course rapidly deteriorates , as mentioned in the definition . the most common clinical manifestations are cardiovascular symptoms such as hypotension , tachycardia or bradycardia , cardiovascular collapse , bronchospasm , and mucocutaneous symptoms such as erythema , edema , urticaria , and angioedema . cardiovascular shock in anaphylaxis was reported as 41% . in our case , after 2 min of sugammadex administration , the patient had generalized erythematous wheals , dyspnea , and progressive cardiovascular shock . this case met world anaphylaxis organization criteria for anaphylaxis , and the severity grade using the severity scale was grade iii . it is difficult to identify the cause of anaphylaxis during anesthesia because various drugs are used . the most common cause of perioperative anaphylaxis is neuromuscular blocking agents ( 69.2% ) , latex ( 12.1% ) and antibiotics ( 8% ) . sedatives , analgesics , local anesthetics , and other drugs are less frequent causes . in our patient , antibiotics were administered without complication 30 min before surgery , and other drugs such as neuromuscular drugs and opioids were administered 20 min before the anaphylactic reaction . elevation of serum tryptase and histamine levels is useful to confirm the diagnosis of anaphylaxis . the serum tryptase test has a positive predictive value of 93% and a negative predictive value of 54% , and should be performed within 6 h because of its short half - life . however , in our case , the serum tryptase test was not performed because of the patient 's refusal . the detection of specific ige antibodies using the in vivo ige test ( skin prick test or intradermal test ) and in vitro ige test is useful to identify the cause of the allergic reaction . the skin prick test is more sensitive than in vitro tests and is generally used but remains unvalidated . there is no absolute agreement on the concentration of sugammadex to be used . in british society or allergy and clinical immunology , the use of 1 : 10 diluted or undiluted agent is recommended for anaphylaxis associated with perioperative agents . a positive decision is made when the wheal diameter with a 1 : 10 dilution is at least 2 mm greater than the negative control . however , a positive wheal to only undiluted agent may be considered a positive decision when the clinical course is fitted to the agent and other causes are ruled out . fourteen cases of anaphylaxis associated with sugammadex reported a positive result of the skin prick test or intradermal test . soria et al . reported a positive intradermal test with 1 : 1000 sugammadex , although the patient showed a positive skin prick test only with undiluted sugammadex at 30 min . in our case , we had a weakly positive skin prick test to undiluted sugammadex , but an intradermal test was not recommended because of concern regarding the potential risk of anaphylaxis . in vitro - specific ige for antibiotics were also examined , and the result was negative . when anaphylaxis is suspected , turning off the responsible agent and administering appropriate treatment promptly are important . epinephrine is the drug of choice for anaphylaxis because it maintains blood pressure with an 1 adrenergic effect and relaxes the bronchial smooth muscle with a 2 adrenergic effect . in our case , sugammadex is a modified gamma - cyclodextrin that is used as an antagonist to aminosteroid neuromuscular blockade , and has been used since february 2013 in korea . sugammadex is an innovative agent that has a rapid recovery time and rapid renal clearance . it provides several advantages , including avoidance of cardiovascular side effects associated with neostigmine , avoidance of postoperative residual curarization , and reversal of deep neuromuscular blockade . however , in the united states , the food and drug administration has not yet approved sugammadex due to the possibility of anaphylactic reactions . several cases of hypersensitivity reactions associated with sugammadex have been reported in countries that frequently use the drug . these reactions appear to be more frequent at higher clinical doses . however , godai et al . reported three cases , although they used 1.92.2 mg / kg low - dose sugammadex . cases of sugammadex - induced anaphylaxis have been reported more frequently in japan because sugammadex is widely used . to our knowledge , thirteen cases of anaphylaxis associated with sugammadex have been reported until now in japan . however , to our knowledge , there is no reported case of anaphylaxis to date in our country . . reported that two patients with an asthma history showed bronchospasm associated with sugammadex . five cases of anaphylactic reaction associated with sugammadex had reported an asthma or allergic history . allergic patients may have been predisposed to anaphylaxis to sugammadex . however , most patients with anaphylaxis associated with sugammadex have no history of sugammadex exposure . it has been suggested that the use of cyclodextrins in food may result in sensitization to sugammadex . we suggest that strict caution should be applied when considering sugammadex administration in asthmatic and allergic patients . second , the use of sugammadex is likely dangerous because it 's associated anaphylactic reaction appears at the time of extubation and movement to the postanesthetic care unit or intensive care unit when the patients are less monitored . therefore , we suggest that vigilance after sugammadex administration is necessary . in summary , we present a case of a patient who presented with generalized erythema , dyspnea , and cardiovascular shock 2 min after sugammadex administration ; the case was confirmed to be anaphylaxis associated with sugammadex by a positive skin test . we ruled out other causes using specific ige for antibiotics and a negative skin prick test for rocuronium and fentanyl , as well as the clinical course of the event ( they were administered 20 min before the event ) . this is a suspected case of hypersensitivity reaction associated with sugammadex reported for the first time in korea . we need to be aware that the use of sugammadex is associated with a serious risk of anaphylaxis .

we describe a case of a 35-year - old male patient who was scheduled for laparoscopic cholecystectomy and developed a life - threatening anaphylactic reaction 2 min after the administration of sugammadex . he manifested erythematous wheals on the entire body , dyspnea , hypotension , and tachycardia . these symptoms disappeared after the administration of epinephrine . the patient recovered and was discharged at postoperative day 5 without any complications . after 7 weeks , we performed a skin prick test , and there was a weakly positive reaction for sugammadex . this case is suspected anaphylaxis associated with sugammadex , and we need to be aware that the use of sugammadex is associated with a serious risk of anaphylaxis .

amyotrophic lateral sclerosis / parkinsonism - dementia complex ( als / pdc ) is a rare disorder endemic to guam island and the kii peninsula of japan . it shows a unique combination of parkinsonism , amyotrophy , and dementia , and the form of dementia , which shows a phenotype similar to alzheimer 's disease ( ad ) , is becoming predominant in the kii peninsula . although kii als / pdc shows several unique clinical features , including severe atrophy of the frontal and temporal lobes on magnetic resonance imaging ( mri ) , decreased cerebral blood flow in the frontal and temporal lobes on single - photon emission computed tomography ( spect ) , pigmentary retinopathy , and decreased cardiac i - meta - iodobenzylguanidine uptake , a postmortem examination is required for a definitive diagnosis . since biomarkers for als / pdc have not yet been identified , we analyzed cerebrospinal fluid ( csf ) biomarkers for kii als / pdc to discriminate it from other neurodegenerative disorders . we collected csf samples from 12 patients with kii als / pdc ( 6 men , 6 women , mean age 67.9 3.7 years , mean illness duration 5.63 years ) , nine patients with ad ( 2 men , 7 women , mean age 61.1 8.7 years , mean illness duration 1.92 years ) , 11 patients with als ( 8 men , 3 women , mean age 60.6 12.6 years , mean illness duration 1.1 years ) , nine patients with parkinson 's disease ( pd ; 7 men , 2 women , mean age 71.3 2.2 years , mean illness duration 4.42 years ) , and five disease control patients ( c ; 4 men , 1 woman , mean age 36.2 20.3 years ) . all of the patients with kii als / pdc were natives of hohara village , which is an area of high als / pdc prevalence on the kii peninsula . we collected csf samples over 10 years ; therefore , the period between csf collection and analysis was not standardized . the diagnosis of kii als was made according to the airlie house criteria , since the clinical symptoms of kii als are essentially the same as those of classical als . the diagnosis of kii pdc was made by a unique combination of levodopa - unresponsive parkinsonism and dementia , which are frequently accompanied by amyotrophy of the extremities and/or pyramidal tract signs . mini - mental state examination ( mmse ) was used for the evaluation of dementia and cut - off point was 23 ( data not shown ) . the frontal lobes and/or temporal lobes of als / pdc patients showed atrophy on mri and/or a decrease of cerebral blood flow on spect . csf samples were immediately centrifuged at 1000 g for 15 min and stored at 80c with polypropylene tube . the total tau ( t - tau ) , phosphorylated tau ( p - tau ) , and amyloid beta ( a ) concentrations were measured with an enzyme - linked immunosorbent assay ( elisa ) kit using a monoclonal antibody specific for t - tau , p - tau , and a142 ( innotest htau ag , phosphor tau(181p ) , and -amyloid ( 142 ) , innogenetics , ghent , belgium ) . elisa assays were carried out using several samples from each group on the same plate in a randomized manner and were repeated using randomized samples in the same manner in plural times . a factorial anova was performed with csf - t - tau , csf - p - tau , and csf - a42 , as dependent variables , with the diagnostic category ( ad , als , c , kii als / pdc , and pd ) using jmp 9.0 . the ethics committee of mie university graduate school of medicine approved this study and the declaration of helsinki was followed . csf - a42 , csf - t - tau , and csf - p - tau were compared between ad , als , c , kii als / pdc , and pd . the concentrations of csf - t - tau and csf - p - tau were significantly higher in ad ( t - tau ; 378.0 41.76 pg / ml ; p < 0.001 , p - tau ; 42.4 6.78 pg / ml ; p < 0.028 ) than in the other groups . however , the concentrations of csf - t - tau and csf - p - tau did not differ significantly between kii als / pdc , als , c , and pd ( figures 1(a ) and 1(b ) ) . the concentration of csf - a42 was significantly reduced in ad ( 402.2 56.6 pg / ml ; p < 0.03 ) compared to als and c and relatively reduced in kii als / pdc ( 465.4 53.69 pg / ml ; p < 0.018 ) compared to als . most of the als / pdc patients had csf concentration values that fell below the cutoff based on c ( figure 1(c ) ) . the ratios of csf - p - tau to csf - a42 were significantly increased in ad ( 0.125 0.02 ) compared with kii als / pdc ( 0.043 0.02 ; p < 0.008 ) , als ( 0.035 0.019 ; p < 0.003 ) , pd ( 0.025 0.02 ; p < 0.002 ) , and c ( 0.027 0.09 ; p the concentrations of csf - t - tau , csf - p - tau , or csf - a42 were not related to the clinical parameters ( age , sex , illness duration , or dementia ) in the kii als / pdc patients . the number of c samples was small and the average age of control patients was low . generally , csf tau level gradually increase according to age and csf a is not affected by age . csf tau level of c samples was relatively low , but it was not significant . thus , the csf values of c were not comparable to those of other groups . nevertheless , the optimal cut - off values that discriminate c from ad were similar to those in previous reports [ 6 , 7 ] in which a larger number of control samples were analyzed . csf tau level of kii als / pdc samples did not increase , although the average age of kii als / pdc group was older than that of ad group . the present study showed that csf - t - tau and csf - p - tau concentrations from patients with kii als / pdc were not increased compared to those in the other disease groups , and a42 concentration in the csf was relatively decreased . the ratio of csf - p - tau to csf - a42 segregates kii als / pdc from ad . because als / pdc is associated with tau pathology in the absence of amyloid plaques , the expectation was that als / pdc patients would not show the alzheimer 's disease ( ad ) profile of decreased a42 but might show increased t - tau and/or p - tau in the csf . in general , decreased csf - a42 indicates plaque pathology , and increased csf - t - tau and csf - p - tau indicate axonal degeneration and tangle pathology , respectively . recently , the average age of onset of kii als / pdc is increasing and a deposition is conspicuous in autopsied patients . therefore , decreased csf - a42 may reflect a pathology in the most recent patients . we analyzed the precise tau isoform of over 10 patients with autopsy - proven als / pdc recently and identified a 3r + 4r type , 4r > 3r type , and a 4r predominant type . the glial tau pathology is particularly related to the 4r isoform , and we consider kii als / pdc to be a 4r - dominant tauopathy ( unpublished data ) . noguchi et al . examined the concentrations of csf - t - tau , csf - p - tau , and csf - a42 in patients with progressive supranuclear palsy ( psp ) and corticobasal degeneration ( cbd ) ; the concentrations of csf - t - tau and csf - p - tau did not significantly differ between psp , cbd , and controls , and the concentration of csf - a42 was significantly lower in psp and cbd than in controls . the authors speculated that the absence of an increase of csf - t - tau and csf - p - tau concentrations might reflect 4r tau predominance and a reduction of csf - a42 might suggest deposition or mismetabolism of a . taken together , csf biomarkers of kii als / pdc might have similar properties to those of 4r tauopathy , psp , and cbd ; however the relationship between tau isoform and csf tau level remains to be resolved . finally , the present findings , in which csf - t - tau and csf - p - tau concentrations were not increased and csf - a42 concentration was relatively decreased , suggest that csf analysis may be useful to differentiate als / pdc from ad , als , and pd . nevertheless there is a major limitation of the interpretation of the data . the size of each group is small , the age of the control group is much younger , and there were two populations in the ad group regarding the levels tau , p - tau , and a/p - tau . further study using groups with larger size of subjects is needed to confirm the proposed utility of the csf biomarkers .

objective . amyotrophic lateral sclerosis / parkinsonism - dementia complex is classified as one of the tauopathies . methods . the total tau , phosphorylated tau , and amyloid 42 levels were assayed in cerebrospinal fluid from patients with kii amyotrophic lateral sclerosis / parkinsonism - dementia complex ( n = 12 ) , alzheimer 's disease ( n = 9 ) , parkinson 's disease ( n = 9 ) , amyotrophic lateral sclerosis ( n = 11 ) , and controls ( n = 5 ) using specific enzyme - linked immunosorbent assay methods . results . total tau and phosphorylated tau did not increase and amyloid 42 was relatively reduced in kii amyotrophic lateral sclerosis / parkinsonism - dementia complex . relatively reduced amyloid 42 might discriminate kii amyotrophic lateral sclerosis / parkinsonism - dementia complex from amyotrophic lateral sclerosis and parkinson 's disease , and the ratios of phosphorylated - tau to amyloid 42 could discriminate kii amyotrophic lateral sclerosis / parkinsonism - dementia complex from alzheimer 's disease . conclusions . cerebrospinal fluid analysis may be useful to differentiate amyotrophic lateral sclerosis / parkinsonism - dementia complex from alzheimer 's disease , amyotrophic lateral sclerosis , and parkinson 's disease .

in their companion article , tononi and cirelli argue that i have missed the big picture by conflating questions of sleep function with the underlying mechanisms . as i have discussed elsewhere [ 2 , 3 ] , understanding sleep function is of central importance to biology . any theory of sleep function must also grapple with universal traits of sleep , some of which were enumerated in their response . there are other theories of sleep function and many of the theoretical arguments made in support of shy ( e.g. , a need for offline states , sleep homeostasis , and brain metabolism ) apply to them . my position is that this evaluation must always include a discussion of mechanisms , because they can not be disentangled from functional questions . the underlying message from tononi and cirelli is that what really matters is the the end result [ 1 , page 4 ] rather than how you get there . i find this an odd position to take and a backward step in our pursuit of sleep function . the goal of science is to understand how nature works . that includes an empirical pursuit of physical mechanisms . scientists should therefore be skeptical of any theory of sleep function that fails to elucidate the underlying mechanisms that govern the proposed function . in this regard , the proponents of shy are in an indefensible position when they argue that the mechanisms have no bearing on whether the core claim of shy is true or false [ 1 , page 3 ] . they have bearing because if the underlying mechanisms are not sleep dependent then the theory is wrong . incidentally , the theory is also wrong if the proposed mechanisms do not exist . while it may be true that these mechanisms are complex and perhaps different in different species , that is not a reason to ignore them . on the contrary , that is a reason to explore them . . indeed , i do find shy confusing because the proposed mechanisms ( to date ) seem inconsistent with what is actually known about synaptic plasticity . moreover , their imprecise terminology fogs the issue . they claim that they never meant that the potentiation in wakefulness is hebbian ( i.e. , some form of ltp ) , yet it is difficult to draw any other conclusion based on their own words . their liberal and interchangeable use of the words long - term potentiation , ltp , or this is because ltp is not short - hand for stronger synapse ; it has a precise meaning and commonly refers to hebbian processes . in their rebuttal , they back peddle from their past specific language regarding the significance of neuromodulator release and the expression of plasticity molecules in shy . there is , however , little ambiguity in their original description of how these events purportedly promote ltp ( or ltp - like events ) during wake versus sleep [ 4 , 5 ] . the concepts and terminology of synaptic homeostasis and scaling predate shy and there is simply no mistaking the broad similarities between synaptic scaling and what is described in shy . for example , here is a more complete excerpt from tononi and cirelli . like activity - dependent synaptic scaling ( emphasis added ) , however , sleep - dependent downscaling would affect most or all of a neuron 's synapses . in this respect , downscaling is conceptually different from long - term depression , which affects select groups of synapses , or depotentiation , which affects only recently potentiated ones therefore , their use of these terms , if in fact they mean something else , is perplexing . the distinctions they make between their version of downscaling and scaling proper are welcome , but quite subtle when compared to the overall similarities . for example , synaptic homeostasis is not only manifested at the level of firing rates as implied by tononi and cirelli . that is one outcome of a homeostatic adjustment of synaptic weights in a network , as measured , for example , by mepscs [ 7 , 8 ] . this point seems moot , since tononi and cirelli cite changes in firing rates and mepscs as evidence of shy [ 9 , 10 ] . in the end , it really does not matter what new name they give to their process ( or set of processes ) that weakens synapses . the point i was making , and which they missed , is that any form of plasticity ( especially if possibly novel ) should be examined through the prism of known facts about hebbian and non - hebbian plasticity . case in point : i never stated that the downscaling of shy is identical to synaptic scaling as currently understood in the field . i instead argued that since the concepts of scaling figure prominently in shy , and because scaling is an accepted means of globally adjusting synaptic weight , it is logical to examine the claims of shy based on what is known about synaptic scaling . synaptic renormalization from synaptic scaling , but this name change does not inoculate shy against scrutiny . the veracity and validity of a scientific theory is not solely determined by the amount of data piled on one side of a scale . it is also determined by a careful examination of each piece of evidence , pro and con . upon close examination , threads of supportive findings may unravel , and when contrary evidence is properly considered , an otherwise impressive mass of findings may collapse . a large part of their response is a long catalog of supportive data some of which i included in my review . however , as i pointed out , careful examination reveals alternative explanations for some of their results . they also largely ignore evidence consistent with net increases in synaptic strength after sleep , or findings that question their hypothesized relationship between synaptic potentiation and slow - wave electroencephalographic activity ( swa ) [ 11 , 12 ] . these findings are consistent with my original discussion and completely in opposition to predictions of shy . when these are also considered the universality of shy , and its utility as an explanation for why we sleep , becomes questionable . in my original review , i considered biological processes other than sleep that might explain some findings ascribed to shy . these included the cumulative effects of natural patterns in brain temperature ( and in mammals , glucocorticoid release ) across the 24-hour day or after sleep deprivation . tononi and cirelli present a number of arguments against these ideas , but i have a better idea for example , why not examine the effects of cooling or warming on drosophila synaptic proteins ( which are altered by changes in temperature as small as 8c ; not as they state 20c ) ? tononi and cirelli argue strenuously that shy is a theory of function , yet they support their argument with cellular and molecular findings that have no demonstrable function . rather than elaborate upon already dense theoretical arguments about the functional importance of shy , why not demonstrate it empirically ? in science , no argument , no matter how beautifully crafted , trumps a good experiment . in his discussion of sleep theories 50 years ago , nathaniel kleitman reminded his readers of the cautionary tale of ptolemy of alexandria . ptolemy was a revered scholar in the 2nd century ad who codified hundreds of years of prior greek astronomy and mathematics into what came to be known as the ptolemaic universe . the ptolemaic universe held that the stars , planets and sun revolved around the earth , embedded in overlapping crystalline spheres . this theory of the natural world was elegant , mathematical , and even highly predictive of astronomical events . the lesson from ptolemy is that scientific theories are ultimately judged not only by their power to explain , but by the validity of their physical mechanisms . tononi and cirelli argue that for shy to be valid , it does not matter how synapses are weaker after a sleep period , just that they are . but ptolemy reminds us that this is not enough ; scientific truth comes from asking hard questions about how things actually work . i do not question their results , showing for example , changes in evoked responses or mrnas consistent with shy . first , they have not identified how sleep actually alters synaptic strength in any species . second , they have not experimentally excluded the role of other biological processes that coincide with sleep . a final consideration is that if shy is a scientific hypothesis , then tononi and cirelli should propose an experimental outcome that would force a rejection of shy .

in a recent article i reviewed an influential theory of sleep function , the synaptic homeostasis hypothesis ( shy . ) according to shy , sleep renormalizes synapses that are potentiated during prior wakefulness . i concluded that while shy is a seminal theory with important implications about sleep function and the brain , its underlying mechanisms are poorly defined . in an accompanying article , the authors of shy responded at length . their reply is thoughtful and provocative , but unfortunately many of the points i raised were not accurately represented or addressed . in this brief commentary , i attempt to clarify some points of confusion . i also explain why any theory of sleep function is incomplete without an understanding of the underlying cellular mechanisms .

disporopsis pernyi ( hua ) diels , which belongs to disporopsis genus of liliaceae family , mainly grows in south asia such as vietnam , laos , thailand , and yangtze river basin of china . it was a well - known traditional chinese medicine which has been widely used for the treatment of abnormal sweating , chronic cough , women postpartum weakness and irregular menstrual cycle , and so forth . the medical value of disporopsis genus plants has not got much attention until the beginning of 21st century . the roots of the plant are rich in bioactive substances which are possible to have considerable medicinal value and can be used for hypertension cough , inflammation , and tumor . study suggests that a total of 4 polyphenolic compounds are found in extract including rutin , luteolin , quercetin , and betulinic acid , and the phenolic compounds contribute significantly to the antioxidant and antimicrobial activities [ 2 , 3 ] . rutin has both antihypertensive effect and antidiabetes effect [ 4 , 5 ] . luteolin which has anti - inflammatory , antibiosis , and anticancer properties has been used for relieving cough and eliminating phlegm . in addition , it has potential anti - hiv effect [ 6 , 7 ] . quercetin can be used for relieving cough and eliminating phlegm and for hypertension and hyperlipemia ; also , it has neuroprotective and antiproliferative activities [ 8 , 9 ] . acid can kill human melanoma cell without hurting healthy cell and inhibit the hiv-1 infection . additionally , recent study shows that it also has the inhibition effects of cerebroma and leukocythemia [ 10 , 11 ] . the four compounds are major bioactive constitutes in the extract of disporopsis pernyi ( hua ) diels roots . so far , there is no report on the content of the 4 polyphenolic compounds in disporopsis pernyi ( hua ) diels . uplc is a simple and quick tool for the quantitative determination of the bioactive constituents in pharmaceutical industry [ 1214 ] . as rutin , luteolin , quercetin , and betulinic acid the hplc - grade methanol and acetonitrile used were purchased from caledon ( canada ) and formic acid was obtained from dima company ( beijing , china ) . the rutin , luteolin , quercetin , and betulinic acid were purchased from the national institute for the control of pharmaceutical and biological products in china . the purity of the standard compounds was 98% ; their chemical structures are shown in figure 1 . disporopsis pernyi ( hua ) diels were collected from songtao which is in guizhou province of china . standard stock solutions of rutin , luteolin , quercetin , and betulinic acid were dissolved in methanol , at concentration of 1.0 mg / ml . all standard solutions were filtered through 0.22 m syringe filter . the extraction was carried out using 5.0 g of powdered roots . it was dissolved in 50 ml of 70% ethanol solution and back - flow for 60 min . after filter and rotary evaporation to no ethanol smell , 50 ml acetic ether was added and extraction was done three times . the extract and washing liquid were combined and filtered and then evaporated to dryness under reduced pressure in a rotary evaporator . the dried extract was dissolved in methanol and diluted to a 5 ml volumetric flask . the hplc system used was a shimadzu nexera x2 uplc ( kyoto , japan ) chromatograph equipped with a solvent delivery unit ( lc-30ad ) , an autosampler ( sil-30ac ) , a column oven ( cto-20a ) , a degasser ( dgu-20a5r ) , and a photodiode array detector ( spd - m20a ) . separation was conducted on a shim - pack xr - ods column ( 2.0 75 mm , 1.6 m ; shimadzu cooperation , japan ) . the column temperature was set at 30c . the mobile phase consisted of water containing 0.1% formic acid ( a ) and acetonitrile ( b ) . the composition of the mobile phase was 5% ( b ) for 02 min , 5%10% ( b ) for 24.5 min , 10%40% ( b ) for 4.511 min , 40%60% ( b ) for 1113 min , 60%70% ( b ) for 13 - 14 min , 70%80% ( b ) for 1416 min , 80%90% ( b ) for 16 - 17 min , 90% ( b ) for 1720 min , and it was held for 3 min and then reequilibrated to 5% ( b ) until the end of the analysis . the flow rate was 0.2 ml / min and the injection volume was 5 l . the detection wavelengths of all standards and samples were in the uv at 210 , 254 , and 280 nm . the 4 standard compounds were accurately weighed and dissolved in methanol to prepare stock solutions at a concentration of 1.0 mg / ml . stock solutions of the compounds were serially diluted to construct calibration curves . the diluted concentrations of compounds were plotted against the peak area on the calibration curves and the linearity was measured from the correlation coefficient . blank samples were analyzed in triplicate and the area of the noise peak was calculated as the response . the lod and loq were calculated as lod = 3.3 sd / s and loq = 10 sd / s , where sd is the standard deviation of the response and s is the slope of the calibration curve . the precision was calculated by analyzing sample extracts containing low and high concentrations of the compounds . the precision was represented by the relative standard deviation ( rsd ) , which was calculated using the equation rsd = ( standard deviation / mean ) 100 . the precision was measured three times in a single day ( intraday precision ) and over three consecutive days ( interday precision ) . the recovery was calculated as follows : recovery ( % ) = ( ( detected concentration initial concentration)/spiked concentration ) 100 . we used pda detector in this experiment , which could select each wavelength of chromatogram . in our study , we took into account the question that most of the components we studied also have good absorption at wavelength of 350 nm and 370 nm [ 15 , 17 ] . by comparing the resolution and response at different wavelength , the results showed that the resolution and response of the four components at 350 nm and 370 nm are not as good as the wavelengths 210 , 254 , and 280 nm we chose . also , we chose 210 nm to detect betulinic acid for its good response . combined with the literature reports , methanol - water , acetonitrile - water , and methanol - acetonitrile - water were examined as mobile phase as well as the type ( formic acid and glacial acetic acid ) and concentration ( 0.01% , 0.05% , and 0.1% ) of the acid . a shim - pack xr - ods column was employed for the simultaneous determination of the 4 compounds , as it has been the most frequently used technique in the chemical analysis of herbal medicines by uplc . peak resolution and shape of the compounds were considered better indicators when 0.1% formic acid was used as a modifier . taking peak shape , degree of separation , the symmetrical factor , and other factors into consideration , acetonitrile-0.1% formic acid water solution was determined as gradient elution process . the linearity of the calibration curve was measured by the correlation coefficient ( r ) , which ranged in value from 0.9992 to 0.9997 for each compound . the lod and loq were 0.1370.264 g / ml and 0.4560.881 g / ml , respectively ( table 1 ) . the intra- and interday precision , which were represented by the rsd values , were rsd < 2.0% . the recoveries of the 4 compounds were in the range 96.2%102.6% , with rsd < 2.0% ( table 2 ) . the results indicate that the developed analytical method was accurate and precise for the analysis of the 4 compounds in disporopsis pernyi ( hua ) diels . the method we developed was applied to determine the 4 compounds in disporopsis pernyi ( hua ) diels successfully . the calculated contents of the four compounds were 5.63 g / g for rutin , 2.51 g / g for luteolin , 3.87 g / g for quercetin , and 2.41 g / g for betulinic acid . the uplc method mentioned here represented an excellent technique for simultaneous determination of rutin , luteolin , quercetin , and betulinic acid in the extract of disporopsis pernyi ( hua ) diels roots , with good sensitivity , precision , and reproducibility . the method gives a good resolution among the four components with the analysis time ( 25 min ) . furthermore , the method can be used as quality control of polyphenolic compounds in disporopsis pernyi ( hua ) diels roots and will play a reference role on the determination of polyphenolic compounds in other medicinal plants or pharmaceutical preparations .

disporopsis pernyi ( hua ) diels , which belongs to genus disporopsis , has been widely used for the treatment of abnormal sweating , chronic cough , and so forth . an ultra - performance liquid chromatography ( uplc ) analysis was developed for the determination of rutin , luteolin , quercetin , and betulinic acid in disporopsis pernyi ( hua ) diels roots . uplc analysis was conducted by using a shim - pack xr - ods column with gradient elution with the mobile phase of acetonitrile and water containing 0.1% formic acid and with a flow rate of 0.2 ml / min , detected at 210 , 254 , and 280 nm . the method was precise , with relative standard deviation < 2.0% . the recoveries for the four components in disporopsis pernyi ( hua ) diels were between 98.5 and 100.9% . the average contents of rutin , luteolin , quercetin , and betulinic acid in roots were 5.63 , 2.51 , 3.87 , and 2.41 g / g , respectively . the method was accurate and reproducible and it can provide a quantitative basis for quality control of disporopsis pernyi ( hua ) diels .

according to numerous estimations , 15% to 29% of patients with cataract have 1.5 diopters ( d ) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive ( spherical and astigmatic ) disorders at the time of the surgery . corneal incision ( astigmatic keratotomy ) and peripheral corneal relaxing incisions ( pcris ) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age , the depth and length of the incision , complications related to wound healing , epithelial defects , or induction of dry - eye symptoms . these parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction . toric intraocular lens ( iols ) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients . initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs ( acrysof toric iol ; alcon , fort worth , tx ) , approved by the us food and drug administration ( fda ) at the end of 2005 , have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof iols are available in three options : t3 , t4 , and t5 of astigmatic correction 1.5 , 2.25 , 3.00 d , respectively ( at the iol plane ) . the aim of this study is to report the clinical results of acrysof toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively . this prospective study included eyes that had cataract surgery at the papageorgiou general hospital , thessaloniki , greece , between may 2008 and december 2008 . the study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them . inclusion criteria were : cataract , age 70 years or younger , and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were : glaucoma , corneal disease , previous corneal or intraocular surgery , macular degeneration or retinopathy , and history of ocular inflammation . each patient had a complete ophthalmologic examination , including visual acuity ( va ) , slit - lamp examination , intraocular pressure ( iop ) measurement , and dilated fundus examination . topography ( magellan mapper ; nidek technologies , padova , italy ) , automated refractometry ( rk600 ; reichert , inc . , depew , ny ) , and ultrasonic immersion biometry ( ocuscan rxp ; alcon ) were performed to determine the appropriate iol spherical power . cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program ( available from http://www.acrysoforiccalculator.com/ ) . the 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts / lane preoperative toric reference corneal marker ( ae-2791tbl ; asico , westmont , il ) ( figure 1 ) . intraoperatively , the desired implantation axis was marked using an intra - op toric axis marker ii ( ae-2794 ; asico ) ( figure 2 ) . a foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch - ii injector ( alcon ) and placed around 1015 off axis before the ophthalmic viscosurgical device ( sodium hyaluronate 1% ) was removed . after ophthalmic viscosurgical device removal , the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks . all surgeries were performed by the same experienced surgeon ( it ) using topical anesthesia and a standard divide - and - conquer phacoemulsification technique . because of the intraoperative marker design and the pen mark fading during the operation in several cases , it was difficult to assess the proper alignment of the iol after its placement . because of this difficulty , an image was captured from the real - time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software ( screen protractor ; iconico , ny , usa ) ( unpublished data ) . to overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge ( ae-1590 ; asico ) and maloney astigmatism axis marker ( ae-2741 ; asico ) is more appropriate for the whole procedure because it allows maximum visibility . the on - screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location ( figure 3 ) . measurements of visual acuity ( using the snellen opto - type at a six - meter distance ) , iop , and comprehensive slit lamp examination were performed at one - day and one- and six - month postoperative visits . at the one- and six - month follow - up , a digital photograph and corneal topography ( only at the one - month visit ) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above ( figure 1 ) . outcomes of interest included uncorrected va , cylindrical astigmatism power before and after iol implantation , and the possible rotation of the iol one and six months after the operation ( when the initial desired place was at 0 ) . absolute values of the rotation used for the analysis after detecting rotation was not under investigation . completed data forms were analyzed with microsoft excel 2007 ( microsoft corporation , redmond , wa ) and spss software ( version 16.0 ; spss inc . , chicago , il ) . twenty - nine eyes of 19 patients ( mean age 63.03 5.42 years ) were enrolled in this study . uncorrected va was found to be 5/10 or more in 26 of 29 eyes ( 89.7% ) and 8/10 or more in 19 of 29 patients ( 65.5% ) . preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery . mean power of the astigmatism was 2.38 0.91 d ( range 1.55 d ) preoperatively and 0.64 0.61 d ( range 02.5 d ) postoperatively ( figure 4 ) . one month postoperation , the mean toric iol axis rotation was 2.2 1.5 ( range 0.67.8 ) . six months postoperation , the mean toric iol axis rotation was 2.7 1.5 ( range 0.98.4 ) . the later rotation occurred between one and six months , and was found to be more than one degree ( 1.1 ) only in one eye ( 3% ) and in all other cases was less than this value ( figure 5 ) . it is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power . calculated reduction of the desired correction ( in diopters ) because of the observed rotation ( mean value , one and two standard deviations ) for the three iol models is shown in table 1 . no eye had secondary surgery to reposition the iol axis within the six - month postoperative period . the results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one - piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome . in particular , the postoperative rotation of toric iols appears to occur in the early postoperative period ( 1 month ) and remains constant later than six months . the lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule , which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va . acrysof one - piece hydrophobic acrylic toric iols implantation shows satisfactory stability , acceptable clinical results , and is an exceptional option for correction of refractive astigmatism .

purpose : to present clinical results of toric intraocular lens ( iol ) implantation for preexisting astigmatism correction and determine the time of any postoperative rotation.patients and methods : twenty - nine eyes of 19 patients underwent uncomplicated phacoemulsification and were implanted with an acrysof toric iol . uncorrected visual acuity , residual astigmatism , and postoperative rotation of the iol were estimated one and six months after the operation.results:uncorrected visual acuity was 0.5 in 26 of 29 eyes ( 89.7% ) and 0.8 in 19 of 29 patients ( 65.5% ) . the mean toric iol axis rotation was 2.2 1.5 ( range 0.67.8 ) one month postoperation and 2.7 1.5 ( range 0.98.4 ) six months postoperation.conclusion:implantation of one - piece hydrophobic acrylic toric iols appears to have acceptable stability , which encourages visual outcome and emerges as an attractive alternative for correction of refractive astigmatism .

non - st - segment elevation acute coronary syndromes ( nste - acs ) are the most frequent manifestation of acute coronary syndromes ( acs ) . its morbidity and mortality stays high and even equal to those of patients with st - segment elevation myocardial infarction ( stemi ) during long - term follow - up.1)2 ) current guidelines2)3 ) propose risk stratification for tailoring treatment in patients with nste - acs . in patients with intermediate- to very high - risk nste - acs , routine invasive diagnostics and treatments are recommended . multivessel coronary disease ( mvd ) , a leading pathological foundation of intermediate to very high risk clinical manifestation , accounts for approximately 3040% nste - acs cohorts overall and is usually treated with invasive interventions.4 ) the american college of cardiology / american heart association guidelines provide a class iib recommendation that multivessel percutaneous coronary intervention ( pci ) , in contrast to culprit - only pci , might be reasonable in nste - acs patients undergoing pci ( level of evidence : b).3 ) this recommendation is based on reports of studies suggesting that multivessel pci is superior to culprit vessel only pci in terms of repeat revascularization.5)6)7)8 ) however , there is still uncertainty as to whether non - culprit lesions should be treated at the time of culprit - lesion pci for nste - acs.3 ) in the present study , we sought to examine clinical outcomes of " one - time " versus staged multivessel stenting in intermediate to very high - risk nste - acs patients with mvd . between november 2008 and november 2012 , a total of 12047 unselected patients who had undergone a pci were prospectively registered in the pci database of general hospital of shenyang military region . the database contained comprehensive information including clinical and angiographic characteristics , treatment strategies and clinical outcomes . eligible patients had at least one intermediate to very high risk criteria with indication for invasive management defined by the 2015 european society of cardiology ( esc ) guidelines for the management of nste - acs2 ) ; 3 types of very high - risk criteria include recurrent or ongoing chest pain refractory to medical treatment , and recurrent dynamic st - t wave changes particularly with intermittent st - elevation and acute heart failure ; 3 types of high - risk criteria include rise or fall in cardiac troponin compatible with myocardial infarction ( mi ) , dynamic st- or t - wave changes and global registry of acute coronary events ( grace ) score > 140 ; 7 intermediate - risk criteria include diabetes mellitus , renal insufficiency ( estimated glomerular filtration rate [ egfr ] < 60 ml / min/1.73 m ) , left ventricular ejection fraction ( lvef ) < 40% or congestive heart failure , early post - infarction angina , prior pci , prior coronary artery bypass graft ( cabg ) and grace risk score>109 and < 140 . exclusion criteria were patients who had chronic total occlusion ; patients who had procedural failure ( i.e. , technical failure ) , including staged pci patients who had procedural failure during the index pci and scheduled for staging ; patients who had cardiac shock , haemodynamic instability , mechanical complications of mi or malignant ventricular arrhythmia ; patients who had renal dialysis or a egfr < 30 ml / min/1.73 m ; staged pci patients who had any major complication during the index procedure ; patients who had a planned staged pci > 60 days . our final population included 1531 patients from the database having nste - acs with multivessel pci ( fig . all patients were given oral loading doses of aspirin ( 300 mg ) and clopidogrel ( 300 - 600 mg ) prior to pci , unless they had already received antiplatelet medication . interventional procedures were performed according to standard techniques and interventional strategies rested on the operators . the culprit lesion was identified by operators usually based on each patient 's electrocardiogram , angiographic imaging , echocardiogram and , if available , intravascular ultrasound ( ivus ) and optical coherence tomography ( oct ) . a lesion was considered a culprit on angiography if at least two of the following morphological features suggestive of acute plaque rupture were present : intraluminal filling defects consistent with thrombus , plaque ulceration , plaque irregularity , dissection or impaired flow.2)9)10)11 ) after the procedure , the use of aspirin lifelong was advised and clopidogrel was prescribed for 12 months . the study was approved by the hospital ethics committee and all patients gave written informed consent . clinical follow - up was performed via telephone or at an outpatient visit at 30 days , 6 months , and 12 months after the index procedure and annually until 3 years after the index procedure . follow - up angiography was recommended to all patients 6 to 12 months after the index procedure , and repeat revascularization was performed , if clinically indicated . the primary outcome was the incidence of major adverse cardiac events ( mace ) , defined as the composite of cardiac death , mi and target vessel revascularization ( tvr ) during 3-year follow - up . secondary outcomes included mace components , the composite of cardiac death or mi , definite / probable stent thrombosis , and any repeat revascularization . mvd was defined as the presence of a significant atherosclerotic coronary artery stenosis ( 70% diameter stenosis ) or a 50% stenosis of the left main coronary artery ( left main disease ) with additional significant stenosis ( 70% diameter stenosis ) of at least one other coronary artery assessed visually during coronary angiography.12 ) staged pci is defined as the planned pci of non - infarct vessel(s ) within 60 days of the index pci . egfr was calculated from serum creatinine ( scr ) concentrations using the modified glomerular filtration rate estimating the equation for chinese patients with chronic kidney disease : egfr ( ml / min/1.73 m)=175(scr)(age)(0.79 if patient is female).13 ) anemia was defined as hemoglobin less than 12 g / dl for men or less than 11 g / dl for women.14 ) early invasive intervention was defined as a coronary angiography performed within 24 h of hospital admission.2 ) delayed invasive intervention was defined as a coronary angiography performed more than 24 h of hospital admission . technical success was defined as the ability to cross an occluded or stenosed segment and successfully open the artery ( restoration of thrombolysis in myocardial infarction [ timi ] flow grade 3 ) with a residual stenosis 30% by visual analysis . cardiac death was defined as death that could not be attributed to a non - cardiac etiology . mi was defined as third universal definition of mi presented by the third global mi task force.15 ) tvr was determined as any repeated pci or cabg to treat a previously stented vessel . complete revascularization was defined when no visually estimated stenosis 50% for the left main and no stenosis 70% for other major arteries and/or their major branches at discharge . stent thrombosis was classified as definite and probable according to definitions proposed by the academic research consortium.16 ) continuous variables were expressed as meanstandard deviation ( sd ) , and categorical variables were expressed as number and/or percentages . for group comparisons , pearson chi - square test or fisher 's exact test was used for categorical variables . student 's unpaired t - test or the mann - whitney rank - sum test , as appropriate , was used for continuous variables . to minimize the influence of confounders on outcome , we used propensity score matching analysis . patients who underwent staged pci were matched in a 1:1 ratio with patients who underwent a " one - time " pci using the nearest neighbor matching , which were based on all available variables listed in tables 1 and 2 except grace score , ivus used , oct used , contrast volume , length of hospital , medication at discharge and dual antiplatelet therapy ( dapt ) duration . a difference of < 10% was regarded as acceptable . time to event data with estimated event rates calculated by kaplan - meier method were compared with the log - rank test . in addition , we performed kaplan - meier analyses at the landmark periods of 0 to 90 days and 90 days to 3 years to evaluate the effect of revascularization strategy on clinical outcomes at different time periods . between november 2008 and november 2012 , a total of 12047 unselected patients who had undergone a pci were prospectively registered in the pci database of general hospital of shenyang military region . the database contained comprehensive information including clinical and angiographic characteristics , treatment strategies and clinical outcomes . eligible patients had at least one intermediate to very high risk criteria with indication for invasive management defined by the 2015 european society of cardiology ( esc ) guidelines for the management of nste - acs2 ) ; 3 types of very high - risk criteria include recurrent or ongoing chest pain refractory to medical treatment , and recurrent dynamic st - t wave changes particularly with intermittent st - elevation and acute heart failure ; 3 types of high - risk criteria include rise or fall in cardiac troponin compatible with myocardial infarction ( mi ) , dynamic st- or t - wave changes and global registry of acute coronary events ( grace ) score > 140 ; 7 intermediate - risk criteria include diabetes mellitus , renal insufficiency ( estimated glomerular filtration rate [ egfr ] < 60 ml / min/1.73 m ) , left ventricular ejection fraction ( lvef ) < 40% or congestive heart failure , early post - infarction angina , prior pci , prior coronary artery bypass graft ( cabg ) and grace risk score>109 and < 140 . exclusion criteria were patients who had chronic total occlusion ; patients who had procedural failure ( i.e. , technical failure ) , including staged pci patients who had procedural failure during the index pci and scheduled for staging ; patients who had cardiac shock , haemodynamic instability , mechanical complications of mi or malignant ventricular arrhythmia ; patients who had renal dialysis or a egfr < 30 ml / min/1.73 m ; staged pci patients who had any major complication during the index procedure ; patients who had a planned staged pci > 60 days . our final population included 1531 patients from the database having nste - acs with multivessel pci ( fig . all patients were given oral loading doses of aspirin ( 300 mg ) and clopidogrel ( 300 - 600 mg ) prior to pci , unless they had already received antiplatelet medication . interventional procedures were performed according to standard techniques and interventional strategies rested on the operators . the culprit lesion was identified by operators usually based on each patient 's electrocardiogram , angiographic imaging , echocardiogram and , if available , intravascular ultrasound ( ivus ) and optical coherence tomography ( oct ) . a lesion was considered a culprit on angiography if at least two of the following morphological features suggestive of acute plaque rupture were present : intraluminal filling defects consistent with thrombus , plaque ulceration , plaque irregularity , dissection or impaired flow.2)9)10)11 ) after the procedure , the use of aspirin lifelong was advised and clopidogrel was prescribed for 12 months . the study was approved by the hospital ethics committee and all patients gave written informed consent . clinical follow - up was performed via telephone or at an outpatient visit at 30 days , 6 months , and 12 months after the index procedure and annually until 3 years after the index procedure . follow - up angiography was recommended to all patients 6 to 12 months after the index procedure , and repeat revascularization was performed , if clinically indicated . the primary outcome was the incidence of major adverse cardiac events ( mace ) , defined as the composite of cardiac death , mi and target vessel revascularization ( tvr ) during 3-year follow - up . secondary outcomes included mace components , the composite of cardiac death or mi , definite / probable stent thrombosis , and any repeat revascularization . mvd was defined as the presence of a significant atherosclerotic coronary artery stenosis ( 70% diameter stenosis ) or a 50% stenosis of the left main coronary artery ( left main disease ) with additional significant stenosis ( 70% diameter stenosis ) of at least one other coronary artery assessed visually during coronary angiography.12 ) staged pci is defined as the planned pci of non - infarct vessel(s ) within 60 days of the index pci . egfr was calculated from serum creatinine ( scr ) concentrations using the modified glomerular filtration rate estimating the equation for chinese patients with chronic kidney disease : egfr ( ml / min/1.73 m)=175(scr)(age)(0.79 if patient is female).13 ) anemia was defined as hemoglobin less than 12 g / dl for men or less than 11 g / dl for women.14 ) early invasive intervention was defined as a coronary angiography performed within 24 h of hospital admission.2 ) delayed invasive intervention was defined as a coronary angiography performed more than 24 h of hospital admission . technical success was defined as the ability to cross an occluded or stenosed segment and successfully open the artery ( restoration of thrombolysis in myocardial infarction [ timi ] flow grade 3 ) with a residual stenosis 30% by visual analysis . cardiac death was defined as death that could not be attributed to a non - cardiac etiology . mi was defined as third universal definition of mi presented by the third global mi task force.15 ) tvr was determined as any repeated pci or cabg to treat a previously stented vessel . complete revascularization was defined when no visually estimated stenosis 50% for the left main and no stenosis 70% for other major arteries and/or their major branches at discharge . stent thrombosis was classified as definite and probable according to definitions proposed by the academic research consortium.16 ) continuous variables were expressed as meanstandard deviation ( sd ) , and categorical variables were expressed as number and/or percentages . for group comparisons , pearson chi - square test or fisher 's exact test was used for categorical variables . student 's unpaired t - test or the mann - whitney rank - sum test , as appropriate , was used for continuous variables . to minimize the influence of confounders on outcome , we used propensity score matching analysis . patients who underwent staged pci were matched in a 1:1 ratio with patients who underwent a " one - time " pci using the nearest neighbor matching , which were based on all available variables listed in tables 1 and 2 except grace score , ivus used , oct used , contrast volume , length of hospital , medication at discharge and dual antiplatelet therapy ( dapt ) duration . a difference of < time to event data with estimated event rates calculated by kaplan - meier method were compared with the log - rank test . in addition , we performed kaplan - meier analyses at the landmark periods of 0 to 90 days and 90 days to 3 years to evaluate the effect of revascularization strategy on clinical outcomes at different time periods . " one - time " pci was performed in 56.1% ( 859/1531 ) , and the remaining 43.9% ( 672/1531 ) had staged pci ( of these , 80.7% [ 542/672 ] had staged non - culprit intervention during the same hospitalization and 19.3% [ 130/672 ] had planned staged non - culprit procedures after hospital discharge ) . the median delay of the staged pci was 5 days ( interquartile rang [ iqr ] , 3 - 9 days ) . as noted in tables 1 and 2 , male , previous mi , triple - vessel disease , and current smoking were associated with more staging . the volume of contrast media utilized during the index procedure in the staged pci group was smaller ( 200 ml [ iqr , 150 - 220 ml ] vs. 200 ml [ iqr , 200 - 300 ml ] , p<0.001 ) though the total volume utilized in the initial procedure plus staged procedure was greater ( 400 ml [ iqr , 310 - 500 ml ] vs. 200 ml [ iqr , 200 - 300 ml ] , p<0.001 ) . most patients took dual antiplatelet therapy ( dapt ) , which was consistent with the standard recommendation . after propensity matching , there were no statistically significant differences in preselected variables between the two groups . 3a , mace occurred in 132 patients ( 20.8% ) in the staged group and 162 patients ( 19.7% ) in the " one - time " group during 3-year follow - up ( p=0.608 ) . rates of each component of 3-year cumulative mace were similar between both groups . the estimated 3-year composite rate of cardiac death or mi was 7.1% in the staged pci group compared with 9.1% in the " one - time " pci group ( p=0.129 ) . in addition , no significant differences in the 3-year rates of any revascularization ( 22.3% vs. 18.6% , p= 0.083 ) and definite / probable stent thrombosis ( 1.3% vs. 1.9% , p=0.295 ) were observed . at the 90-day landmark analysis ( tables 3 and 4 , fig . 2c and fig . 3c ) , no significant differences in terms of mace , its individual components , any revascularization and definite / probable stent thrombosis were observed at the landmark period of 0 to 90 days and 90 days to 3 years . after generating a propensity score , 420 of the 672 patients who underwent staged pci were matched with a patient , respectively , who underwent a " one - time " pci . there were no differences in preselected variables for the propensity matched subjects ( table 1 , 2 ) . 3b , at 3 years , there were no differences in mace ( 18.9% vs. 21.8% , p=0.249 ) ; whereas there was a significantly lower incidence of cardiac death or mi ( 7.0% vs. 11.1% , p=0.033 ) . the risk for cardiac death in the staged pci group tended to be lower ( 2.5% vs. 4.8% , p=0.067 ) . other clinical outcomes including mi ( 5.0% vs. 7.7% , p=0.162 ) , tvr ( 14.9% vs. 14.5% , p=0.971 ) , any revascularization ( 20.3% vs. 20.4% , p=0.888 ) , and definite / probable stent thrombosis ( 1.2% vs. 2.2% , p=0.280 ) were not significantly different between the two study groups . the results of the 90-day landmark analysis for propensity score matched patients were shown in tables 3 and 4 , fig . the staged pci group showed a lower 90-day incidence of mace ( 1.2% vs. 3.3% , p=0.037 ) , mainly due to a lower composite rate of cardiac death or mi ( 0.7% vs. 2.6% , p=0.031 ) . the 90-day rates of mi did not differ significantly between the 2 study groups , but it presented a trend in favor of staged pci ( 0.7% vs. 2.1% , p=0.081 ) . for the 90-day to 3-year follow - up period , both the incidences of mace ( 17.9% vs. 19.1% , p=0.641 ) and the composite of cardiac death or mi ( 6.3% vs. 8.7% , p=0.191 ) were similar between the two groups . cardiac death , tvr , any revascularization and definite / probable stent thrombosis did not differ significangtly between the two groups at the landmark period of 0 to 90 days and 90 days to 3 years . " one - time " pci was performed in 56.1% ( 859/1531 ) , and the remaining 43.9% ( 672/1531 ) had staged pci ( of these , 80.7% [ 542/672 ] had staged non - culprit intervention during the same hospitalization and 19.3% [ 130/672 ] had planned staged non - culprit procedures after hospital discharge ) . the median delay of the staged pci was 5 days ( interquartile rang [ iqr ] , 3 - 9 days ) . as noted in tables 1 and 2 , male , previous mi , triple - vessel disease , and current smoking were associated with more staging . the volume of contrast media utilized during the index procedure in the staged pci group was smaller ( 200 ml [ iqr , 150 - 220 ml ] vs. 200 ml [ iqr , 200 - 300 ml ] , p<0.001 ) though the total volume utilized in the initial procedure plus staged procedure was greater ( 400 ml [ iqr , 310 - 500 ml ] vs. 200 ml [ iqr , 200 - 300 ml ] , p<0.001 ) . most patients took dual antiplatelet therapy ( dapt ) , which was consistent with the standard recommendation . after propensity matching , there were no statistically significant differences in preselected variables between the two groups . 2a and fig . 3a , mace occurred in 132 patients ( 20.8% ) in the staged group and 162 patients ( 19.7% ) in the " one - time " group during 3-year follow - up ( p=0.608 ) . rates of each component of 3-year cumulative mace were similar between both groups . the estimated 3-year composite rate of cardiac death or mi was 7.1% in the staged pci group compared with 9.1% in the " one - time " pci group ( p=0.129 ) . in addition , no significant differences in the 3-year rates of any revascularization ( 22.3% vs. 18.6% , p= 0.083 ) and definite / probable stent thrombosis ( 1.3% vs. 1.9% , p=0.295 ) were observed . at the 90-day landmark analysis ( tables 3 and 4 , fig . 2c and fig . 3c ) , no significant differences in terms of mace , its individual components , any revascularization and definite / probable stent thrombosis were observed at the landmark period of 0 to 90 days and 90 days to 3 years . after generating a propensity score , 420 of the 672 patients who underwent staged pci were matched with a patient , respectively , who underwent a " one - time " pci . there were no differences in preselected variables for the propensity matched subjects ( table 1 , 2 ) . 3b , at 3 years , there were no differences in mace ( 18.9% vs. 21.8% , p=0.249 ) ; whereas there was a significantly lower incidence of cardiac death or mi ( 7.0% vs. 11.1% , p=0.033 ) . the risk for cardiac death in the staged pci group tended to be lower ( 2.5% vs. 4.8% , p=0.067 ) . other clinical outcomes including mi ( 5.0% vs. 7.7% , p=0.162 ) , tvr ( 14.9% vs. 14.5% , p=0.971 ) , any revascularization ( 20.3% vs. 20.4% , p=0.888 ) , and definite / probable stent thrombosis ( 1.2% vs. 2.2% , p=0.280 ) were not significantly different between the two study groups . the results of the 90-day landmark analysis for propensity score matched patients were shown in tables 3 and 4 , fig . the staged pci group showed a lower 90-day incidence of mace ( 1.2% vs. 3.3% , p=0.037 ) , mainly due to a lower composite rate of cardiac death or mi ( 0.7% vs. 2.6% , p=0.031 ) . the 90-day rates of mi did not differ significantly between the 2 study groups , but it presented a trend in favor of staged pci ( 0.7% vs. 2.1% , p=0.081 ) . for the 90-day to 3-year follow - up period , both the incidences of mace ( 17.9% vs. 19.1% , p=0.641 ) and the composite of cardiac death or mi ( 6.3% vs. 8.7% , p=0.191 ) were similar between the two groups . cardiac death , tvr , any revascularization and definite / probable stent thrombosis did not differ significangtly between the two groups at the landmark period of 0 to 90 days and 90 days to 3 years . to our knowledge , this is the first study to examine the efficacy of staged versus " one - time " approach in intermediate to very high - risk nste - acs patients with multivessel pci . main findings of this study were as follow : in intermediate to very high - risk nste - acs patients with multivessel disease , staged pci was associated with a lower composite rate of cardiac death or mi compared with " one - time " pci strategy . this benefit of staged pci was more apparent in the early period after procedures , rather than middle - late period . clinicians are often faced with the decision of whether to treat the nonculprit lesions during the index procedure or to treat it at a later time.17 ) hannan et al . compared the " one - time " complete revascularization in the index admission versus pci of the culprit lesion only with staged non - culprit pci for complete revascularization in a subsequent admission among patients with nste - acs and mvd.18 ) at 3 years , no significant difference in all - cause mortality was observed between the two groups . however , the staged pci group did not include the patients who underwent a staged intervention during the same hospitalization . furthermore , it is yet unknown whether it was different in all - cause mortality between the two groups after exclusions of low risk patients . in the current observational study , the superiority of staged pci strategy over " one - time " pci strategy in terms of the composite of cardiac death or mi was presented , especially in the early period after pci . first , nste - acs patients are in a heightened thrombotic and inflammatory state.19)20)21)22 ) " one - time " multivessel pci can lead to prolongation of procedure time , exposure to a higher radiation dose23 ) and larger volume of contrast agents during the index procedure compared to staged revascularization . this may result in an increased number of complications such as periprocedural mi or acute kidney injury as well as stent thrombosis.24)25 ) moreover , patients with high / intermediate risks of nste - acs are often in a poor clinical state or have concomitant comorbidities . pci on the culprit lesion first and staged non - culprit pci at a later date with optimal medical therapy usually results in the patient being stable and also allows clinicians to reassess the patient 's clinical and angiographic states . risk stratification is essential for the clinical decision - making process in nste - acs patients . with respect to outcomes , periprocedural complications of intervention , as well as the long - term ischemic risk , remain higher in high / intermediate risk nste - acs than in low - risk nste - acs patients.26)27)28)29)30 ) when choosing the optimal treatment strategy in the individual patient with nste - acs and mvd , general patient condition and concomitant comorbidities have to be taken into account . however , the optimal strategy for the management of nste - acs patients with poor clinical presentation and multivessel disease has not been well established . therefore , our patient inclusion criteria , which were based on esc guidelines , selected nste - acs patients with intermediate- to very high - risk features . the decisions for one - time vs. staged pci were not based on a randomization but at physicians ' discretion , which resulted in obvious confounding and selection bias . although we analyzed by adjusting many possible confounding factors , unmeasured confounding or selection bias might have influenced our findings . second , in this study , all patients underwent pci in the setting of antiplatelet therapy with clopidogrel . third , the relatively high frequency of angiography during the period between 6 to 12 months after the procedure could bias the mace , especially tvr . however , this bias was limited , because the two groups had similar frequencies of follow - up angiography and because repeat intervention was guided by either recurrent angina or signs of ischemia that had progressed angiographically , even without angina based on invasive or noninvasive testing . fourth , our data were collected before current practice guidelines2 ) were published . fifth , given the low absolute number of events at 90-day follow up in the propensity score matched cohort , our study was not powered to detect significant differences in cardiac death , mi , tvr and stent thrombosis at 90 days . last , considering follow - up angiography and routine cardiac biomarkers surveillance was not mandatory , therefore the incidence of mi might have been underestimated . in intermediate to very high - risk nste - acs patients with multivessel disease , staged pci is superior to " one - time " pci in terms of the composite of cardiac death or mi . our findings require further confirmation by randomized trial . in intermediate to very high - risk nste - acs patients with multivessel disease , staged pci is superior to " one - time " pci in terms of the composite of cardiac death or mi . our findings require further confirmation by randomized trial .

background and objectivesto compare clinical outcomes of staged versus " one - time " percutaneous coronary intervention ( pci ) in intermediate to very high - risk patients with non st - segment elevation acute coronary syndromes ( nste - acs ) and multivessel coronary disease ( mvd).subjects and methods1531 nste - acs patients with multivessel pci and meeting the criteria of intermediate to very high risk were screened from a prospectively registered database obtained from general hospital of shenyang military region between 2008 and 2012 . they were categorized into " one - time " pci ( n=859 ) and staged pci ( n=672 ) according to intervention strategy . the primary outcomes included a 3-year major adverse cardiac event ( mace ) , a composite of cardiac death , myocardial infarction ( mi ) , and target vessel revascularization.resultsat 3 years , no significant differences in mace ( 20.8% vs. 19.7% , p=0.608 ) and cardiac death / mi ( 7.1% vs. 9.1% , p=0.129 ) were observed between the two groups . after propensity score matching , there was no statistical significance in mace ( 18.9% vs. 21.8% , p=0.249 ) ; whereas cardiac death / mi was significantly lower in the staged pci group ( 7.0% vs.11.1% , p=0.033 ) . ninety - day landmark analysis showed that the staged pci group had a lower 90-day incidence of mace ( 1.2% vs. 3.3% , p= 0.037 ) and cardiac death / mi ( 0.7% vs. 2.6% , p=0.031 ) . for the 90-day to 3-year follow - up period , the incidences of mace ( 17.9% vs. 19.1% , p=0.641 ) and cardiac death / mi ( 6.3% vs. 8.7% , p=0.191 ) were similar in both groups.conclusionin intermediate- to very high - risk nste - acs patients with mvd , staged pci is superior to " one - time " pci in terms of cardiac death / mi .

plant traits such as leaf shape , trichomes , leaf surface waxbloom , and chemical compounds can affect the animals associated with the plant ( hare 2002 ; whitham et al . plant traits may affect both herbivores and predators , and interactions between predators and their prey may be altered to produce indirect ecological effects ( hare 2002 ) . studies of hybrid plants and their parent species indicate that traits affecting community structure can have a genetic basis ( whitham et al . species associated with hybrid plants can have idiosyncratic responses and may increase or decrease in abundance ( fritz et al . 1994 ; messina et al . the responses of each associated species to hybrid genotypes can change the species richness and evenness of the associated community . for example , leaf - galling aphid survival on individual poplar trees within a hybrid zone varies 75-fold ( whitham 1989 ) . the subsequent decrease in aphid density reduces associated arthropod species richness by 31% ( dickson and whitham 1996 ) . the pattern of community structures of associated arthropods on two eucalyptus species and their f1 and f2 hybrids is comparable to patterns expected from inherited quantitative traits ( dungey et al . 2000 ) . however , many traits differed between the hybrids and their parents ( dungey et al . studies of isoline or near - isoline plants that vary in only one trait may help pinpoint genetic mechanisms that affect community structure . knowledge of genetic mechanisms that affect community structure in a crop - based system may improve pest management ( bottrell et al . all else being equal , a crop variety with a trait that reduced pest density would be clearly preferable to a variety without that trait . however , even recognizing traits that increase one pest but decrease another may aid in developing integrated pest management strategies ( smith and van den bosch 1967 ; pedigo 2002 ) . in peas , pisum sativum l. , single gene mutations can change the amount and composition of waxy bloom that the plant produces ( marx 1969 ; holloway et al . 1977 ) . the other line is homozygous for the allele wel and has reduced wax crystals over its stems , leaves , stipules , and pods ( eigenbrode et al . 1998b ) . foraging predators can walk more effectively on peas with reduced wax , thereby increasing predation on the pea aphid ( acyrthosiphon pisum harris ) ( eigenbrode et al . 1998a ; white and eigenbrode 2000 ) . the convergent lady beetle ( hippodamia convergens guerin - meneville ) , a green lacewing ( chrysoperla plorabunda fitch ) , and a parasitoid wasp ( aphidius ervi haliday ) all cause greater pea aphid mortality on peas with reduced wax than on normal wax peas ( eigenbrode et al . such enhanced predation is partly responsible for consistently lower field densities of the pea aphid on peas with reduced wax than on normal peas ( white 1998 ; white and eigenbrode 2000 ; rutledge et al . for example , interactions between a carabid ( poecilus scitulus leconte ) and the pea aphid and convergent lady beetle do not appear to be altered on peas with reduced wax ( chang and eigenbrode 2004 ) . visual canopy sampling of arthropods found 12 unambiguous morphospecies that responded differently to peas with reduced wax ( rutledge et al . for example , predatory syrphids are more abundant on normal peas , while coccinellids are more abundant on peas with reduced wax ( rutledge et al . . however , information on the effects of wax on the broader community of arthropods is lacking . we used pitfall sampling to complement and extend the information on community effects of peas with reduced wax . one advantage is that many more morphospecies , over 200 taxa at our site , could be counted and identified from pitfalls than from visual canopy sampling . however , a limitation of pitfalls is that they will only capture individuals that walk or fall into them . thus , mostly ground - dwelling species are collected , although some foliage - dwelling and flying insects may fall into the traps . we used pitfall trap data to address two related questions : 1 ) does wax expression of peas alter the structure of the arthropod community , and 2 ) which taxa show the greatest disparity in abundance between normal and peas with reduced wax ? the arthropods associated with peas were assessed at the university of idaho ( u.s.a . ) plant science research farm ( 4643 n , 11657 w ) in 1998 and 1999 . the peas are near isolines differing in expression of the mutation wel ( marx 1969 ) , which reduces wax crystals over the stems , leaves , stipules and pods ( eigenbrode et al . four pairs in 1998 , and five pairs in 1999 , of 5 5 m plots were located on the farm . one plot in each pair was planted with normal peas , while the other was planted with peas with reduced wax . the assignment of normal and reduced - wax pea plots within a pair was random . with the exception of the line of peas planted , identical cultivation practices were used on all of the plots . one pitfall trap ( 8-cm diameter plastic cups ) was placed in the center of each plot buried flush with the soil surface . collected arthropods were identified in the laboratory to order , and when possible , to family , genus , or species . therefore , a morphospecies in this study refers to the finest taxonomic level at which individuals could be classified and is our best estimate of a single species . the number of individuals collected in the pitfall traps is our best estimate of the abundance of a morphospecies , although pitfall trap captures are influenced by density and activity level ( thomas et al . 1998 ) . the individuals collected in pitfalls from each of the two types of peas were the effective arthropod communities for our analyses of the diversity of community structure . the number of individuals was plotted versus abundance rank for the total samples ranked within plots with normal peas and plots with reduced - wax peas . a randomization test for difference in community structure was performed according to solow ( 1993 ) . for the randomization test , the shannon and simpson diversity indices were calculated for both normal and reduced - wax peas . the shannon index is more sensitive to species richness , while the simpson index is more sensitive to species evenness ( magurran 1988 ) . simulated datasets were generated by randomly partitioning the total number of observed individuals into two sets equal in size to the observed numbers of individuals in normal and reduced - wax plots . for each simulation , shannon and simpson diversity indices were calculated for both types of peas , and the difference between the diversity indices from normal peas and peas with reduced wax were calculated . the number of simulations producing a difference in the diversity indices greater than or equal to that observed from the pitfall traps estimates the probability that any observed difference was due to chance . the advantage of using a randomization test on the pooled data is that it retains information from species that occurred only once in our samples . the number of morphospecies collected per plot over each sampling period was tallied as the measure of species richness . a 2-way anova was performed with species richness as the dependant variable and wax level and year as factors and plots as replicates . wardle 's ( 1995 ) index v was modified to calculate the disparity between captures in normal and reduced - wax pea plots for each taxon : mr being captures in peas with reduced wax , mn being captures in normal pea plots . as we have defined it , the value of v increases when relatively more individuals of a morphospecies were collected from normal peas , and decreases when relatively more individuals were collected from peas with reduced wax . the observed values of v were tested for whether morphospecies with 20 or more individuals in our total collection differed from what would be expected from chance . an expected distribution of v was generated based on chance by calculating v for each morphospecies with 20 or more individuals in each of 10 simulated datasets ( the random partitions of the observed individuals , as described above ) . the fit of the observed distribution of v to the simulated distribution of v was then assessed . the observed absolute values of v were grouped into categories of 0.05 increments , with v > 0.2 lumped into a single category to eliminate sparse cells for a g - test ( sokal and rohlf 1981 ) . the number of captures of the selected morphospecies from 1998 and 1999 was tested using manovas with wax level as a factor . manova can detect a difference in situations where species have traded places in terms of relative abundance , without changing the species richness or evenness of the community . such a change in community structure can not be detected by a randomization test based on a diversity index ( solow and costello 2001 ) . furthermore , inspection of univariate anovas within a manova can reveal which species changed in response to the difference in wax . first , the statistical power of manova decreases with the inclusion of more dependant variables ( scheiner 2001 ) . second , the number captured of many of the morphospecies in our data set deviate too much from the normal distribution to meet the assumptions of manova . deviation from normality is particularly problematic in our data because several morphospecies occur in one year but not the other ( or were extremely abundant in one year and not the other ) . therefore , two separate manovas were applied , one to data from 1998 , and one to data from 1999 . the 3 most abundant morphospecies in both 1998 and 1999 were analyzed ( appendix 1 ) . a total of 12,737 individual arthropods were recovered in the pitfall traps , 6,657 in the reduced - wax and 6,080 in the normal peas . of those individuals , 12,113 were identified to one of 229 morphospecies , with 171 taxa in reduced wax and 186 in normal plots . eleven taxa were identified to species and 21 others were identified to genus ( appendix 1 ) . of the remaining morphospecies , the randomization test suggested an effect of wax reduction on the overall community structure ( table 1 ) . in particular , the simpson index was lower in normal pea plots than in peas with reduced wax . inspection of the rank abundance curves revealed greater species evenness in the reduced - wax plots than in the normal pea plots , particularly among the eight most abundant taxa ( figure 1 ) . the most captured taxon on both normal and reduced - wax peas was the pea aphid , which constituted 27% of all individuals collected in normal peas and 19% in peas with reduced wax . the next 7 highest ranked taxa collectively constituted 35% of all individuals in normal peas but made up 46% in peas with reduced wax . the difference between the shannon index obtained from plots of the two pea varieties was not significantly greater than what was expected from chance . the distribution of wardle 's index v for morphospecies represented by 20 individuals in the total sample indicated that arthropod taxa in peas responded idiosyncratically to the peas with reduced wax . several taxa illustrate apparent preference for normal or reduced - wax peas , as well as species that were about evenly divided between the two lines ( appendix 1 ) . the observed and simulated distributions of v ( figure 2 ) were significantly different ( = 43.556 , df = 16 , p < 0.001 , n = 53 ) . in particular , the observed distribution of v had a higher frequency of morphospecies with large disparities in captures in one pea variety versus the other ( a higher absolute value of v ; note the flatter distribution of observed frequencies of v compared to the simulated frequencies ) . the mean ( se ) number of morphospecies per plot in 1998 was 74.3 3.8 and 73.8 3.6 in reduced - wax and normal peas , respectively ; in 1999 , 55.8 2.3 and 54.4 3.2 morphospecies were captured in reduced - wax and normal peas . the number of morphospecies captured in 1998 was significantly greater than in 1999 ( f1 , 14 = 34.76 , p < 0.0001 ) , but wax type and its interaction with year were not significant ( p > 0.05 ) . manova of each year 's 3 most abundant taxa did not find a significant overall effect of pea variety in 1998 ( hotelling - lawley trace = 3.083 , f3,1 = 1.03 , p = 0.6033 ; figure 3 ) . pea variety did have a significant effect on the 3 most abundant taxa in 1999 ( hotelling - lawley trace = 38.871 , f3,2 = 25.91 , p = 0.0374 ; figure 4 ) . univariate f - tests within the 1999 manova revealed that pea leaf weevil ( sitona lineatus ) were strongly influenced by wax reduction . the pea leaf weevil was three times more abundant in peas with reduced wax than in normal peas in 1999 ( figure 4 ) . . communities of arthropods often differ depending on the plant species they are associated with ( ehrlich and raven 1964 ; price 1984 ) . for example , changes in pubescence among species in the genus arctostaphylos correspond to changes in their associated arthropod communities ( andres and connor 2003 ) . other similar plants such as hybrids , their parental species , and backcrosses between those hybrids and parental species , can also harbor different communities of arthropods ( whitham et al . peas with the wel homozygous genotype have a reduced - wax phenotype , which increased the evenness of the associated arthropod community . species richness , another component of diversity , was not significantly different between normal and peas with reduced wax . the change in evenness was driven by changes in the capture frequency of particular species , and responses of insects to peas with reduced wax were idiosyncratic ( appendix 1 ) . for example , two herbivores of agronomic importance had contrasting responses to peas with reduced wax . pea aphid capture frequency was greater from normal peas whereas the pea leaf weevil was much more frequently captured from peas with reduced wax . the pitfall data are consistent with data on pea aphid densities and pea leaf weevil damage obtained from visual surveys of the canopy of peas ( white 1998 ; white and eigenbrode 2000 ; rutledge et al . 2003 for example , in 1996 and 1997 , normal wax peas were associated with higher populations of pea aphids , but peas with reduced wax suffered greater damage from adult pea leaf weevils ( white and eigenbrode 2000 ) . predators decrease pea aphid densities on peas with reduced wax relative to normal peas because they are able to walk and thus forage more effectively when less wax is on the plant surface ( white and eigenbrode 2000 ) . all of the pea leaf weevils captured were adults , which are highly mobile and probably able to choose to forage upon peas with reduced wax . apparent preferences of predatory arthropods for either normal or reduced - wax peas may be a function of both prey abundance and prey accessibility in each habitat ( stephens and krebs 1986 ) . although pea aphids are more abundant on normal peas than on peas with reduced wax , greenhouse experiments in which pea aphids were presented in equal densities on the two types of peas have found that aphids on peas with reduced wax are more accessible to certain predators ( eigenbrode et al . therefore , all else being equal , it can be predicted that the ratio of predators to pea aphids will be higher on peas with reduced wax than on peas with normal wax , although no clear prediction can be made regarding the absolute densities of predators . indeed , several predators had higher ratios to pea aphids on peas with reduced wax . this group includes h. convergens , coccinellid larvae which were probably mostly h. convergens , geocoris sp . , spiders ( as a group ) , and green lacewings ( two morphospecies combined ; appendix 1 ) . other predators had higher ratios to pea aphids in peas with normal wax ; namely , adult coccinella ( two species combined ) , nabidae ( two morphospecies combined ) , and syrphidae ( six morphospecies combined ) . some of the apparent preferences of predators for peas with normal wax may be an artifact of low capture rates of the taxa involved , but ecological factors may also explain some of the apparent paradoxes . for example , some syrphids prefer to oviposit on normal brassicas to those with reduced wax ( chandler 1968 ) . the syrphids in our study may have had a similar preference for peas with normal wax . finally , the greater slipperiness of peas with normal wax may have increased the number of active insects such as coccinellids captured in the pitfall traps located in those plots . the relative abundances of arthropods might be affected by changes in the environment that extend beyond the foliar surfaces of the plant but are ultimately due to differences in plant wax . for example , peas with normal wax typically grow faster than peas with reduced wax ( chang and eigenbrode 2004 ) , probably because greater amounts of cuticular wax improve the ability of plants to cope with water shortages ( fitter and hay 2002 ) . the amount of vegetative cover can affect abiotic conditions such as soil temperature and soil moisture ( daubenmire 1974 ) . certain taxa may be responding to habitat differences that are an indirect consequence of the difference in wax , while predators or competitors may mediate the responses of other species . scitulus was over three times more frequently captured on peas with reduced wax ( v = 0.52 ) , while pt . melenarius was nearly three times more frequently captured on normal peas ( v = 0.44 ) . future work might determine whether their contrasting responses are caused by different preferences in abiotic conditions or some degree of competitive exclusion . we also suggest that differences in the arthropod communities captured in 1998 versus 1999 are also largely due to differences in abiotic conditions . during the 1998 sampling period , mean monthly temperatures were higher and more rain fell than in 1999 ( idaho state climate services 2004 ) . the comparison between normal peas and peas with reduced wax illustrates considerations for rational crop design . the greater evenness of the arthropod community in peas with reduced wax may also be desirable if a crop is being managed for biodiversity in addition to yield ( hails 2002 ) . furthermore , the greater growth rate of peas with normal wax under water - limited conditions ( chang and eigenbrode 2004 ) will be an important practical consideration in many environments . knowledge of the relative advantages and disadvantages of different crop varieties may improve the match between specific cultivars and particular geographic regions . rank abundance curves for arthropod morphospecies found in pitfall traps within plots of normal and reduced wax peas . species rank is determined within a variety ; in other words , the rank order of a morphospecies in reduced wax peas is different from that in normal peas . histogram of observed and simulated values of wardle 's ( 1995 ) index v. observed v 's were calculated for 53 morphospecies that were represented by 20 or more individuals in our total sample . v = 0 for morphospecies with equal numbers of individuals collected from reduced wax and normal pea plots . positive values of v indicate that more individuals were collected from normal wax peas , while negative values indicate that more individuals were collected from reduced wax peas . greater absolute values indicate a greater disparity in collections from the two types of peas . mean abundances of the 3 most abundant morphospecies in pitfall collections from peas , 1998 . the taxa on the x - axis are listed in descending order of abundance in all plots . bars represent the standard error of the mean abundance of each morphospecies per plot ( n = 4 ) . mean abundances of the 3 most abundant morphospecies in pitfall collections from peas , 1999 . the taxa on the x - axis are listed in descending order of abundance in all plots . bars represent the standard error of the mean abundance of each morphospecies per plot ( n = 5 ) .

crop traits can alter economically important interactions between plants , pests , and biological control agents . for example , a reduced waxy bloom on the surface of pea plants alters interactions between pea aphids and their natural enemies . in this study , we assess whether the effect of wax reduction extends beyond the 2 or 3 arthropod species closely associated with the plants and into the structure of the broader arthropod community of over 200 taxa at our site . we sampled arthropods on lines of peas with normal and reduced wax in latah co. , idaho using pitfall traps within randomly assigned pairs of 5 5 meter plots . during the 1998 and 1999 growing seasons , we collected 12,113 individual arthropods from 221 unambiguously identified morphospecies . the number of individuals collected from each morphospecies responded idiosyncratically to the reduced wax peas . to test whether arthropod community structure differed between the collections from plots having peas with normal or reduced wax , we performed a randomization test . the collection from peas with reduced wax had higher species evenness and thus higher community diversity despite having lower species richness . our results demonstrate the potential of a single plant trait , epicuticular wax , to affect a community of arthropods . two pests of peas had opposite responses to peas with reduced wax . the number of pea aphids collected was greater from peas with normal wax peas than those with reduced wax . in contrast , the number of pea leaf weevils collected was greater from peas with reduced wax .

while there is a large body of evidence in favor of a clustering of representations of body effectors in the posterior parietal cortex , recent advances have revealed a hierarchical organization beyond a flat intentional map composed of functionally distinct subdivisions operating in parallel at same level . in particular , the parietal reach region ( prr ) and dorsal area 5 ( area 5d ) have been found to play distinct roles in visually - guided reach . based on three lines of neurophysiological studies on nonhuman primates that utilize sophisticated behavioral tasks , including reference frame , effector choice , and sequential reach , this essay proposes that the prr and area 5d are involved in translation of general motor intentions into detailed motor programs at different stages . the posterior parietal cortex ( ppc ) has historically been considered a typical association cortex , important for spatial attention and multisensory integration in the generation of a unitary map whose output is relayed to the frontal lobe to guide behavior ( critchley , 1953 ; ungerleider and mishkin , 1982 ; colby and goldberg , 1999 ) . since the 1970s , tremendous progress in neurophysiology , neuroanatomical tracing , functional imaging , and experimental intervention has yielded evidence of a variety of distinct functional subareas in the ppc , as opposed to a homogeneous area that constructs a single unified perceptual representation ( rizzolatti et al . , 1997 ; mountcastle , 1998 ; andersen and buneo , 2002 ) . furthermore , the ppc has been shown to be involved in movement planning in a number of different contexts ( andersen and cui , 2009 ; rizzolatti and kalaska , 2013 ) . the ppc seems to be composed of a mosaic of intentional maps , each of which is thought to be specialized for a different kind of movement for a particular body part ( andersen and buneo , 2002 ; scherberger et al . , 2013 ) . recent advances have further suggested that the individual effector - specific regions are heterogeneous ( heed et al . , 2011 ; leone et al . , 2014 ) , with a hierarchical organization among different subdivisions preferring the same effector ( cui and andersen , 2011 ; verhagen et al . , 2013 ) , indicating that some subareas in ppc are involved in sensorimotor integration at multiple levels , instead of working in parallel within a flat intentional map . this essay will focus on two reach - related areas in brodmann s area 5 ( fig . 1a ) , the parietal reach region ( prr ) , and dorsal area 5 ( area 5d ) , and discuss the functional relationship between them based on recent neurophysiological studies using a variety of sensorimotor contingencies . b , three paradigms to distinguish general motor intention from detailed motor programs . in the reference frame task , general motor intention is represented in extrinsic space , while physical movement is prepared in intrinsic space . in the effector - choice task , multiple potential action plans can be formed prior to specification of the concrete motor parameters controlling the end - effector . in the sequential reach task , an action sequence encompassing all motor components at a cognitive level is formed before it is unfolded into a series of detailed programs of element movements . the prr was originally defined as an area medial and posterior to the lateral intraparietal area ( lip ) , including more than one cytotectonic area , whose cells are more active for reaching arm movements than for saccadic eye movements ( snyder et al . , 1997 ; quian quiroga et al . , 2006 ; cui and andersen , 2007 ) . many subsequent neurophysiological studies of the prr have targeted the medial bank of the ips ( mip ) ( baldauf et al . , 2008 ; cui and andersen , 2007 , 2011 ; pesaran et al . , 2006 , 2008 ; reversibly inactivating the prr in monkeys causes miss - reach , similar to the optic ataxia reported in human patients ( hwang et al . in addition , pre - movement activity is not simply related to the cue , because the prr has been found to encode the desired movement goal during an anti - reach task ( gail and andersen , 2006 ) . moreover , the reaching plan embedded in the prr includes not only target locations or end - points , but also high - level kinematics such as movement trajectories ( torres et al . , 2013 ) , supporting the view that movement trajectories are geometrically formed , independently of physical motor commands ( torres and zipser , 2002 ; torres and andersen , 2006 ) . area 5d , which is located caudal to the primary somatosensory cortex ( si ) and medial to the ips , is involved in representing spatial information for limbs and control of reaching arm movements ( lacquaniti et al . 1995 ; kalaska et al . 1978 ; pearson and powell 1985 ) , and is reciprocally interconnected with primary and premotor cortex ( strick and kim , 1978 ) . it was previously thought of as a higher somatosensory area that codes posture and joint positions ( sakata et al . however , studies on behaving monkeys demonstrate that cells in area 5 became much more active during arm movements ( mountcastle et al . , 1975 ) , with strong directional tuning ( kalaska et al . , 1983 ) . during delayed reach , many area 5 cells exhibit sustained activity far before the earliest increase in electromyographic activity ( kalaska and crammond , 1995 ) . compared with primary and premotor cortex , area 5 seems to be less directly involved in control of musculoskeletal dynamics : it appears to be insensitive to external force load , and to encode movement kinematics instead of dynamics ( kalaska et al . , also , many area 5 neurons show strong firing patterns that continuously covary with hand trajectory ( archambault et al . , 2011 ; hauschild et al . , 2012 ) , a manually / brain controlled cursor ( mulliken et al . , 2008 ) , or even the seen position of a realistic fake arm ( graziano et al . , 2000 although both the prr and area 5d have been found to be intimately involved in planning of reach , several recent studies of reference frame , decision making , and sequential movement suggest that they might play distinct roles in planning and control of reaching arm movement at different ( abstract vs concrete ) levels of complexity ( fig . 1b ) , indicating a hierarchical circuitry that is involved in translation from cognitive intention into detailed motor programs to implement a key step of the complex sensorimotor transformations from receptors to muscle activation patterns . the first findings in favor of a hierarchical prrarea 5d circuitry emerged from experiments characterizing sensorimotor coordinates with a variety of combinations of target , eye , and hand positions ( andersen at al . , 1997 ) . unlike an oculomotor plan in eyecentered coordinates that is always registered to its sensory target in a retinotopic map , goal - directed reach initially planned in visual space ( flanagan and rao , 1995 ; wolpert et al . , 1995 ) requires a transformation from extrapersonal to joint / muscle coordinates . recent studies demonstrate that different coordinate systems are employed in the prr and area 5d ( batista et al . , 1999 ; bremner and andersen , 2012 ) , suggesting that these areas are involved in reach planning in extrinsic and intrinsic spaces , respectively . neurophysiological recordings during a delayed - reach task revealed a gradient distribution in the medial wall of the ips , with deep ( superficial ) neurons tending to be more modulated in the cue ( movement ) epoch ( johnson et al . , 1996 ) , suggesting that they are involved in different stages of the sensorimotor transformation ( battaglia - mayer et al . , 2003 ) . reaching goals have been found to be represented in retinal coordinates ( batista et al . , 1999 ; pesaran et al . 2006 ) in the prr , indicating a cognitive plan at a more conceptual level than at the subsequent stages of motor planning . that is , the prr only conveys abstract information ( i.e. , the spatial goal ) about upcoming movements , without specifying physical movement parameters ( i.e. , joint angles ) . nonetheless , accurate motor control must take into account intrinsic variables , such as posture , hand position , the dynamics and structure of muscles , etc . therefore , abstract motor intentions ultimately must be converted into detailed physical motor plans , with movement parameters specified in muscle / joint coordinates to activate musculoskeletal system ( kalaska et al . , 1997 ) . the first step in converting target location in gaze - centered coordinates into limb motor commands for reaching is to incorporate proprioceptive and efference copy information about starting hand position and intrinsic arm posture . it seems likely that area 5d plays a key role in this stage by providing target information with respect to initial hand position to specify action parameters in musculoskeletal coordinates ( buneo et al . , 2002 ) . from the prr to area 5d , neuronal representations of the reaching goal progressively shift from eye - centered to hand - centered along the ventraldorsal axis of the medial bank of the ips ( buneo and andersen , 2006 ) , so that pre - movement activity in area 5d predominantly encodes the reach vector ( bremner and andersen , 2012 ) . however , this hand - centered coding is not static , but gradually emerges during the reach planning period ( bremner and andersen , 2014 ) , supporting the view that area 5d integrates the intrinsic biomechanics of the musculoskeletal system by dynamically incorporating information about intrinsic arm posture . another approach to elucidating parietal sensorimotor circuitry is to identify distinct functional areas for decision making and motor planning . decision making initially was assumed to be a neural process separate from action planning ( miller et al . , 1960 ; keele , 1968 ) . nevertheless , attempts to distinguish decision making from motor planning with target selection tasks have proven frustrating , because virtually all motor - related areas seem to convey potential movements to candidate targets , and multiple potential plans exist concurrently before the final action is chosen ( riehle and requin , 1989 ; platt and glimcher , 1997 ; basso and wurtz , 1998 ; cisek and kalaska , 2005 ) . consequentially , it is commonly believed that target selection and movement preparation involve overlapping brain circuits , and are performed in an integrated manner , as opposed to a serial model in which decision making occurs before action planning ( shadlen and newsome , 2001 ; wang , 2008 ; cisek and kalaska , 2010 ) . from the evolutionary point of view , forming multiple potential action plans prior to choosing among them might not only benefit reward prediction , but might also reduce reaction time ( andersen and cui , 2009 ; cisek and kalaska , 2010 ) . nevertheless , the idea of integrated circuitry for decision making and motor planning has only been tested for spatial target selection ( cisek , 2007 ) , which involves spatial attention , and in turn engages numerous cortical areas ( desimone and duncan , 1995 ) . it is unclear if plan selection and action preparation are embedded in segregated areas for other kinds of decision making , such as nonspatial effector choice . to examine anatomical overlap for decision making and motor planning in nonspatial action selection , an effector - choice task was designed in which monkeys autonomously chose between a saccade and a hand reach to the same visual stimulus ( cui and andersen , 2007 ) . monkeys were required to play a mixed - strategy game against a computer ( barraclough et al . , 2004 ) to compensate for a potential bias due to movement costs . the effector - choice trials were pseudo - randomly interleaved with effector - instructed trials in which the monkeys were explicitly cued to make either a saccade or a reach to the target in the middle of a trial to discourage premature decisions . the effector - choice task allows potential motor plans to be formed without immediate specification of concrete motor parameters controlling a particular end - effector , providing an ideal paradigm to determine whether action selection and motor preparation are encoded in overlapping or in distinct areas in nonspatial decision making . if a brain region is involved in effector decision formation , then it should encode potential motor plans prior to effector selection . conversely , if an area only carries selected motor plans , then it should only reflect the decision outcome after the effector is unambiguously specified . interestingly , recent studies demonstrate remarkable qualitative differences between lip / prr and area 5d ( cui and andersen , 2007 , 2011 ) . whereas the lip and ppr encode potential motor plans , area 5d encodes only selected reach plans after the arm is chosen as the effector , suggesting that it is downstream to the prr in a serial visuomotor cortical circuit ( cui and andersen , 2011 ) . while the prr and area 5d presumably work in concert with premotor and primary motor areas , respectively ( wise et al . , 1997 ) , the functional relationship between the parietal and frontal areas in effector choice remains unclear . in contrast to the prefrontal cortex ( tanji , 2001 ) , the ppc has historically received little attention in studies of sequential planning . nonetheless , neurological studies have suggested that the ppc is crucial for serial behaviour ( buxbaum , 1998 ; zadikoff and lang , 2005 ) . patients with damage to the left parietal lobe appear normal in performing elementary movements , but are impaired in generating complex action sequences , indicating a profound role of the ppc in integrating multiple spatial goals into a motor sequence . to reveal the functional role of ppc in sequential arm movements , single - neuron activity from the prr ( baldauf et al . , 2008 ) and area 5d ( li and cui , 2013 ) has been recorded from monkeys performing a double - reach task . when the monkeys prepared to reach two simultaneously presented targets with sequential arm movements , most prr neurons carried information about both the immediate and the subsequent goals ( baldauf et al . , 2008 ) . situated at the early stage of the visuomotor transformation , the prr presumably is involved in the first step to generate a coherent sequence , integrating information about all component movements into a high - level movement plan at an abstract level . nevertheless , a conceptual sequential plan encompassing multiple goals in parallel ultimately must be decomposed into serial motor commands to drive the musculoskeletal system . in contrast to the prr , area 5d has been found to only encode the immediate upcoming reach , and not the subsequent movement ( li and cui , 2013 ) . area 5d activity is tightly coupled only to the next upcoming movement , suggesting it might play a key role in integrating the visual goals from the prr with physical limb information to form a state estimation ( mulliken et al . , 2008 ; moreover , the component reach encoded in area 5d , as opposed to the sequential information being conveyed in the prr and other cortical sensorimotor areas , suggests that unfolding of the movement sequence begins in the parietal - frontal cortex , including the ppc , prefrontal cortex , premotor cortex , primary motor cortex , and supplemental motor cortex , instead of being exclusively conducted by downstream subcortical and spinal circuits . again , decomposition of the motor sequence appears to engage a larger sensorimotor network via mutual communication between parietal and frontal cortices ( pesaran et al . , 2008 ) . a series of recent experiments have been conducted on monkeys performing a variety of sensorimotor tasks to elucidate distinct roles of the prr and area 5d at different stages of the sensorimotor transformation . first , the reference frame task allowed us to isolate general motor intentions in extrinsic space and physical movement preparation in intrinsic space . second , the effector - choice task allowed potential action plans to be formed without immediate specification of the concrete motor parameters controlling the end - effector . third , the sequential reach task enabled us to isolate the cognitive action sequence from the detailed motor program of element movements . three lines of evidence obtained with these tasks suggest that the prr and area 5d form a hierarchical sensorimotor circuitry that translates abstract intentions into detailed motor plans . despite the markedly different functional roles between prr and area 5d , we should keep in mind that they are not sequentially involved in converting extrinsic stimuli into intrinsic motor plans in a hardwired fashion . first , although a serial neural process was observed in the sensorimotor tasks mentioned above , effector specificity and information flow might be different in behavioral contexts other than visually guided reach ( e.g. , swaminathan et al . , 2013 ; woloszyn and shadlen , 2013 ) . secondly , behavioral and computational studies have suggested that the sensorimotor transformation emerges through an intimate interplay between sensory inflow and motor outflow through paired forward and inverse internal models ( wolpert and kawato , 1998 ; shadmehr and wise , 2005 ) : the forward model translates motor commands into anticipated sensory outcomes , whereas the inverse model converts desired sensory consequences into motor commands ( franklin and wolpert , 2011 ; shadmehr and mussa - ivaldi , 2012 ) . although the prevalent view posits that sensorimotor control largely relies on forward prediction of sensory consequences ( wolpert et al . , 2011 ) , direct neurophysiological evidence that persuasively links neural firing to predicted sensory consequences is still lacking , because the internal prediction of sensory consequences is inherently different from actual sensorimotor variables , and seems not to be behaviorally measurable . since most previous studies have used reactive movements to stationary goals predefined by sensory cues , it is difficult to determine whether the neural activity observed reflects sensory stimuli or predicts impending movements . although corollary discharges have been found in diverse species ( crapse and sommer , 2008 ) for gating sensory inputs ( e.g. , lee and malpeli , 1998 ) and updating perception ( e.g. , duhamel et al . , 1992 ; sommer and wurtz , 2002 ; synofzik et al . , 2008 ) , as well as for distinguishing sensory signals between active and passive motion ( e.g. , roy and cullen , 2004 ) , it remains unclear how they are integrated with sensory inflow in recipient structures to form internal models for directing movement . further elucidation of the ppc sensorimotor circuitry calls for novel behavioral tasks that are highly dependent on predictive spatiotemporal transformations . the author did not consent to publish the author response letter the decision was a result of the reviewing editor ranulfo romo , universidad nacional autonoma de mexico and the peer reviewers coming together and discussing their recommendations until a consensus was reached . a fact - based synthesis statement explaining their decision and outlining what is needed to prepare a revision is listed below . the following reviewers agreed to reveal their identity : christos constandtinidis and w. pieter medendorp the reviewers found your review paper of potential interest for publication in eneuro , provided you address their comments . in general , the reviewers think that you must lay down the scope of your review in the introduction section : input out - put organization of ppc and 5d . also , there are many issues regarding what you mean by sensory - motor hierarchy , sensory - to - motor transformation , and integrated circuits for decision making , planning , and the link with frontal cortex . in the absence of a clear lay - out of these terms . it would be useful to place the prr-5d circuit in a broader context , at least in the introduction . it would be worth considering the inputs to the prr area from other parietal areas and outputs of area 5d to the premotor cortex . line 77 : please , discuss the effector specificity could change over time , and that the network is not hardwired . line 88 : be more specific about the involvements of ppc and 5d in the different stages of the sensory - motor transforms . here and again , what is it a hierarchy and should refer also to caminiti and colleagues . but , there are so many examples of hierarchy and sensory - to - motor transformation and their roles in planning , and decision making , etc . lines 123 - 125 : it is unclear what is meant by integrated circuitry for decision making and motor planning for spatial target selection . the source being cited is a review ( and a 20-year old one , as such ) . line 145 : what is the implication of this finding and the link with frontal cortices ? line 170 : clarify the term parietal - frontal cortex , by spelling out the relevant areas . line 171 : expand communication via synchronization and de - synchronization between ppc and frontal cortices . line 174 : it is unnecessary to start the discussion with in summary . line 195 : could you please explain or consider that sensory consequences are not behaviorally measurable ? the reviewers found your review paper of potential interest for publication in eneuro , provided you address their comments . in general , the reviewers think that you must lay down the scope of your review in the introduction section : input out - put organization of ppc and 5d . also , there are many issues regarding what you mean by sensory - motor hierarchy , sensory - to - motor transformation , and integrated circuits for decision making , planning , and the link with frontal cortex . in the absence of a clear lay - out of these terms . it would be useful to place the prr-5d circuit in a broader context , at least in the introduction . it would be worth considering the inputs to the prr area from other parietal areas and outputs of area 5d to the premotor cortex . line 77 : please , discuss the effector specificity could change over time , and that the network is not hardwired . line 88 : be more specific about the involvements of ppc and 5d in the different stages of the sensory - motor transforms . here and again , what is it a hierarchy and should refer also to caminiti and colleagues . but , there are so many examples of hierarchy and sensory - to - motor transformation and their roles in planning , and decision making , etc . lines 123 - 125 : it is unclear what is meant by integrated circuitry for decision making and motor planning for spatial target selection . the source being cited is a review ( and a 20-year old one , as such ) . line 145 : what is the implication of this finding and the link with frontal cortices ? line 170 : clarify the term parietal - frontal cortex , by spelling out the relevant areas . line 171 : expand communication via synchronization and de - synchronization between ppc and frontal cortices . line 174 : it is unnecessary to start the discussion with in summary . line 195 : could you please explain or consider that sensory consequences are not behaviorally measurable ?

while there is a large body of evidence in favor of a clustering of representations of body effectors in the posterior parietal cortex , recent advances have revealed a hierarchical organization beyond a flat intentional map composed of functionally distinct subdivisions operating in parallel at same level . in particular , the parietal reach region ( prr ) and dorsal area 5 ( area 5d ) have been found to play distinct roles in visually - guided reach .

these include human population density and behaviour , housing type and location , water supply , sewage and waste management systems , land use and irrigation systems , availability and use of vector control programmes , access to health care , and general environmental hygiene . meteorological factors that influence transmission intensity of infectious diseases include temperature , humidity , and rainfall patterns . social and demographic factors such as population growth , urbanization , immigration , changes in land use and agricultural practices , deforestation , international travel , and breakdown in public health services have been mainly responsible for the recent resurgence of infectious diseases1 . the intergovernmental panel on climate change noted in its 2007 report that climate change may contribute to expanding risk areas for infectious diseases such as dengue and may increase the burden of diarrhoeal diseases , putting more people at risk2 . global climate change is a phenomenon that is now considered strongly associated with human activities . atmospheric carbon dioxide levels , which have remained steady at 180 - 220 ppm for the past 420,000 years , are now close to 370 ppm and rising3 . due to improvements in meteorology , we are now able to better understand long - term changes in climate . such understanding might enable the prediction of where and when infectious disease outbreaks may occur . box 1 outlines some of the consequences of climate change that clinicians in india could expect to see over the coming years . we describe in this paper these links between climate and health , future implications , and recommendations for clinician - patient interaction . what clinicians might expect due to climate change the effects of climate change on human health in india is a broad topic , covering areas from extreme weather events to shifts in vector - borne diseases . in south asia , scientists predict an increased frequency of floods due to greater intensity of rainfall events and to glacier lake outburst floods ( glofs ) in mountainous regions5 . these trends are already being seen . in 2007 , floods resulting from monsoon rains killed more than 2,000 persons and displaced more than 20 million persons in bangladesh , india , and in nepal6 . in the himalaya region of south asia , the frequency of glofs rose during the second half of the 20 century , and glofs have occurred recently in nepal , india , pakistan , and bhutan78 . for example , if floodwaters become contaminated with human or animal waste , the rate of faecal - oral disease transmission might increase , allowing diarrhoeal disease and other bacterial and viral illnesses to flourish . faecal - oral transmission of diseases is of particular concern in regions such as south asia because of limited access to clean water and sanitation . in developed countries , flood control efforts , sanitation infrastructure , and surveillance activities to detect and control outbreaks minimize disease risks caused from flooding9 . in developing countries , increase in diarrhoeal disease , cholera , dysentery , and typhoid is of specific concern10 . for example , after flooding in west bengal in 1988 , cholera was thought to be the cause of an outbreak of diarrhoea that resulted in 276 deaths11 . numerous studies have linked previous floods in bangladesh and parts of india with outbreaks of diarrhoea as well as respiratory infections12131415 . flooding can also contribute to increased vector - and rodent - borne and other infectious diseases . for example , collections of stagnant water provide breeding grounds for mosquitoes , potentially aiding in the spread of malaria16 . other studies have linked flooding in bangladesh and parts of india with outbreaks of rotavirus and leptospirosis1718192021 . rising sea - surface temperatures although cyclones originating in the bay of bengal and the arabian sea have decreased in frequency since 1970 , these have increased in intensity , causing significant damage in india and bangladesh222324 . public health effects of cyclones include diseases and illnesses associated with the loss of clean water , hygiene , and sanitation , loss of shelter and belongings , population displacement , toxic exposures , and hunger and malnutrition risk due to food scarcity25 . a warmer climate could cause water - borne diseases to become more frequent , including cholera and diarrhoeal diseases such as giardiasis , salmonellosis , and cryptosporidiosis9 . diarrhoeal diseases are already a major cause of morbidity and mortality in south asia , particularly among children . it is estimated that one - quarter of childhood deaths in south asia are due to diarrhoeal diseases26 . as rising ambient temperatures increase , bacterial survival time and proliferation and thus the incidence of diarrhoeal diseases might further increase27 . diarrhoeal diseases are largely attributable to unsafe drinking water and lack of basic sanitation ; thus , reductions in the availability of freshwater are likely to increase the incidence of such diseases28 . rapid urbanization and industrialization , population growth , and inefficient water use are already causing water shortages in india , pakistan , nepal , and bangladesh6 . climate change will exacerbate the lack of available fresh water as annual mean rainfall decreases in many areas . cholera is a well - known water - borne diarrhoeal disease that has afflicted humankind since ancient times . outbreaks of cholera have occurred in india , bangladesh , and more recently , latin america and africa29 . molecular techniques have shown that bacteria are now recognized as naturally occurring in aquatic environments , with bacterial population peaks in spring and fall in association with plankton blooms29 . the discovery of vibrio cholerae in the natural environment , with a dormant state between epidemics , changed the understanding that this disease had only a human reservoir . a relationship has been observed between increase in sea - surface temperature and the onset of cholera epidemics , with the cholera outbreaks following the seasonal rise and fall in sea - surface height and temperature29 . increases in cholera bacterial populations associated with plankton blooms in spring and summer have been noted off the coasts of several latin american countries and in bangladesh29 . about 400 - 500 million cases of malaria and more than 1 million malaria - related deaths occur globally each year3 . several factors have caused the global resurgence of malaria , including the emergence of insecticide and drug resistance , human population growth and movement , land - use change , and deteriorating public health infrastructure . changes in temperature , rainfall , humidity , and immunity levels also affect malaria transmission . all these factors can interact to affect adult mosquito densities and the development of the plasmodium parasite within the mosquito . both the parasite and the mosquito that houses the parasite are susceptible to temperature changes . at temperatures close to the physiological tolerance limit of the parasite , a small increase in temperature can kill the parasite , thereby decreasing malaria transmission . at lower temperatures , however , a small increase in temperature can greatly increase the risk of malaria transmission due to increased numbers of mosquitoes3 . for example , deforestation might elevate local temperatures and changes in housing types might change indoor temperatures where vectors spend most of the time resting . deforestation , vegetation clearance , and irrigation can form open pools preferred by malaria vectors and thus increase transmission . in south asia , malaria is one of the most pertinent examples of increased occurrence of a vector - borne disease . malaria is already one of the most important vector - borne diseases in india , bangladesh , and sri lanka ; changes in temperature and precipitation patterns have the potential to expand the geographical range of malaria into temperate and arid parts of south asia9 . for example , in india malaria distribution is expected to expand to higher latitudes and altitudes . because the relationship between climate and disease distribution is complex , in some areas reductions in transmission intensity in endemic areas might lead to greater proportions of the population losing immunity , resulting in epidemics in later years30 . currently , all of india 's population is at risk for contracting malaria except for those in the areas above 1700 m above sea surface . more than 973 million persons are exposed to vector - borne malarial parasites in india , and in 1998 an estimated 577,000 disability adjusted life years ( dalys ) were lost due to malaria31 . the multifactorial nature of malaria causation does not lend itself easily to a simple model of disease prediction . to determine the role of climate change in malaria transmission , research efforts will be required that incorporate a disease surveillance system combining trend analyses from multiple sites to account for local factors . the system combined human and meteorologic factors to provide about a month 's lead - time of epidemic risk . rainfall alone accounted for about 45 per cent of the variation in malaria transmission32 . by the 2050s , the geographic range of malaria vectors the duration of the transmission window is likely to widen in northern and western states and shorten in southern states . malaria is likely to persist in orissa , west bengal , and southern parts of assam . it might shift from central india to the southwestern coastal states ( maharashtra , kerala , and karnataka ) . the northern states might also become prone . the duration of exposure is likely to widen in north and west india , and shorten in south india33 . these include mosquito - borne diseases such as chikungunya fever and dengue , parasitic diseases such as leishmaniasis , lymphatic filariasis and onchocerciasis , and tick - borne diseases , which may exhibit changes in transmission intensity or shifts in their geographical ranges due to the impact of climate on the relevant vector populations . climatic factors might also influence human plague , a bacterial disease carried by rodents and transmitted by fleas . temperature and rainfall are important determinants of rodent population abundance and distribution . combined with the influence of temperature and humidity on flea survival and development , changes in any of these climatic components may result in changes in plague incidence30 . murine typhus , a rickettsial disease , is also transmitted by fleas and thus may exhibit similar climate sensitivity30 . the arboviral diseases chikungunya and dengue may also be influenced by climate , as both are transmitted by the common vector aedes aegypti . the first reported outbreak of chikungunya in india was in 1963 in calcutta ( now kolkata ) , with transmission continuing until 197334 . the virus reemerged in 2005 , and has since spread rapidly , with more than one million cases reported - despite no standardized surveillance system for the disease3435 . dengue has also been a significant problem , with more than 50 dengue outbreaks reported in india since 196036 . the activity , abundance , distribution , and ability to transmit viruses is influenced by temperature and precipitation30 . as a developing country with high population density , india might experience myriad human health effects because of climate change . these effects could include infectious diseases such as malaria , chikungunya , and water - borne illnesses . monitoring the spread of infectious diseases will require early warning systems , which have both health and economic benefits . displacements due to the loss of housing , hunger , and injuries are some of the adverse outcomes to the population . health care providers will need to address at the primary level the negative health outcomes associated with climate change in india . there will likely be a rise in demand for emergency medicine services such as urgent and ambulatory care37 . expanded surveillance activities could detect shifting patterns of disease distribution so that emergency department personnel would be aware of emerging threats . many other fields of health care in india could be impacted by climate change , including family practice , internal medicine , paediatrics , geriatrics , and psychiatry37 . the climate and health council of the general medical council of the united kingdom38 has outlined several actions that health care providers can take , many of which are relevant to providers in india39 . these include informing professional colleagues and the wider community about the health consequences of climate change , and advocating for carbon reductions to promote human health . several recommendations have been made for health system preparedness , some of which are relevant for indian hospitals39 . these include undertaking energy audits , pursuing energy and water conservation and energy efficient construction . a review of overall emissions from petroleum - based energy sources such as diesel generators and hospital transportation systems is a key first step . contingency plans for alternative methods of energy generation during electricity blackouts , particularly during the summer months , will facilitate preparedness for these events . to reduce waste , health care without harm has recommended several options : recycle and buy recycled products , collect and recycle nitrous oxide and anaesthetic gases , prevent waste , and dispose of waste locally40 . using native vegetation and planting trees on site can mitigate the heat - island effect . vegetation lowers ground level temperatures near buildings , reducing the buildings cooling load , energy requirements , and greenhouse gas emissions . those of low socio - economic status will likely to be the most affected by the health impacts of climate change , as they have the least adaptive capacity4 . as india 's economy continues to expand , the growing middle class presents a unique situation . while rising out of poverty will improve sanitation levels and living conditions , thus increasing resilience to infectious diseases , it will also lead to higher consumption patterns that can initiate new health problems while leading to more carbon pollution . box 2 outlines some of the topics health care providers can discuss with their patients to help maximize the benefits . general practitioners as well as specialists both can play a role in anticipating the health effects of climate change and improving the health through discussing effective preventive health care with their patients . discussing climate change effects with patients

climate change has the potential to influence the earth 's biological systems , however , its effects on human health are not well defined . developing nations with limited resources are expected to face a host of health effects due to climate change , including vector - borne and water - borne diseases such as malaria , cholera , and dengue . this article reviews common and prevalent infectious diseases in india , their links to climate change , and how health care providers might discuss preventive health care strategies with their patients .

periodontal disease is a multifactorial infectious disease triggered by the development of dental biofilm , which can harm periodontal tissues . there are several factors which influence periodontitis : personal and social characteristics , behavioral , systemic , genetic and dental factors , and the microbial composition of the dental biofilm [ 1 , 2 ] . stress may be defined as the combination of physical and mental responses caused by certain external stimuli ( stressors ) which allow an individual ( human or animal ) to overcome certain environmental demands , and the physical and mental weariness caused by the stress process itself . stress has been related not only to a higher occurrence of periodontal disease but also to its severity . other social and behavioral factors that could be related to periodontal status are tobacco use , social economic status , nutritional status , psychological aspects , and abusive alcohol intake . among these factors , alcohol has become relevant because it is one of the few socially accepted psychotropic drugs . this social acceptance has contributed to a rise in alcoholism , which has become a serious public health problem . according to silva furtado amaral et al . the world health organization has reported that the rates of death and functional limitations associated with abusive alcohol intake have surpassed those associated with tobacco smoking . alcoholism per se imposes a state of stress because , besides its physiological demand , it acts as a chemical aggressor to the body . all the alcoholic drinks consumed by an individual do not remain stored in the body and , before being eliminated , it is first metabolized in the liver , which results in high levels of reactive oxygen species [ 7 , 8 ] . several cross - sectional studies have verified an association between the abusive consumption of alcohol and poor periodontal health [ 911 ] . abusive consumption of alcohol has also been associated with the severity of periodontitis . however , many questions on this subject are still not answered and laboratorial research could be helpful to clarify these doubts . animal model studies may more specifically help elucidate questions related to alcohol intake , stress , and periodontal disease [ 12 , 13 ] . as a lifestyle factor drinking , and stress seem to contribute to poorer periodontal status . in the present study it was hypothesized that alcohol consumption by stressed animals could be accompanied by an increase in the severity of periodontitis . therefore , aiming to confirm or reject this hypothesis we evaluated whether forced alcohol intake by stressed animals affects histometrical parameters of periodontal breakdown . thirty - two animals of the rattus norvegicus species lewis breed ( two - month old ) , weighing average 250 g , were selected for the present study . the animals were divided in groups of four and each group was kept in a cage ( polyethylene 16 40 30 ) [ 14 , 15 ] . they all received standard rat feed and water or alcohol ( ethanol 20% ) ad libitum . they were kept in light / dark cycles of 12 hours ; the temperature was controlled at 24c and humidity at > 50% . the experiment took place at the university center of vrzea grande univag and it was approved and registered by the ethics in animal research committee ( cep / unic-2009 no . initially , a research assistant randomly divided the animals in four experimental groups : group gal : alcohol + ligature ( n = 8);group gasl : alcohol + stress + ligature ( n = 8);group gpc : stress + ligature , positive control ( n = 8);group gcn : negative control ( n = 8) . group gal : alcohol + ligature ( n = 8) ; group gasl : alcohol + stress + ligature ( n = 8) ; group gpc : stress + ligature , positive control ( n = 8) ; group gcn : negative control ( n = 8) . after the division , the animals in the gal and gasl groups were offered alcohol ( ethanol 20% ) ad libitum for 60 days [ 11 , 16 ] while negative control ( gcn ) received water ad libitum for the same period of time . because alcohol intake is very limited on the first day , ligatures were inserted only in the second experimental day ( gal , gasl , and gpc ) . the animals received general anesthesia through the intramuscular administration of 0.05 ml xylazine hydrochloride ( rompun , bayer . animal health , so paulo , sp . brazil ) and 0.1 ml ketamine ( dopalen , agribrands . animal health , paulnia , sp , brazil ) per 100 grams of body weight . after anesthesia , a sterile silk suture number 4 ( ethicon , johnson and johnson , so paulo , sp , brazil ) was placed in the gingival crevice of the second right upper molar . the animals in the positive control group were kept in the same environment in the first experimental day , drinking water , but without suffering any kind of intervention . the animals in the negative control group ( gnc ) did not receive any type of intervention but were kept in their cages in the same environment during all the study . the model of physical stress induction chosen for this study was immobilization done during 60 days , six times a week , in different times of the day as previously described [ 18 , 19 ] . at room temperature , the animals in the gasl group were placed in polyvinyl chloride tubes compatible to their body sizes . the tubes were closed on both ends with metallic wire , which enabled the animals to breathe while they were immobilized for four consecutive hours . after that process , the jaw segment was decalcified in edta for approximately five weeks ( edta was renewed six times ) ; following dehydration in graded alcohol , the samples were embedded in paraffin . the tissue blocks included in paraffin allowed 6 m histological slices that followed the long axis of the teeth in the mesio - distal plane and were stained with hematoxylin and eosin . for the histometric analysis , ten serial sections containing the 1st and 2nd upper molars and the following structures ( a ) dental pulp , ( b ) the mesial cementoenamel junction of the 2nd molar , ( c ) proximal bone crest , and ( d ) connective attachment had to be observed . histometry was performed in the mesial aspect from first molars , by the same blinded examiner , through the capturing of images in the microscope and measured in millimeters ( software imagelab 2000-diracon bio informtica ltda . previous to the study , the error was calculated by double measurements of 10% of the specimens with a one - week interval . during the study , paired t - test statistics were run and no differences were observed in the mean values for comparison ( p > 0.5 ) . additionally , pearson 's correlation coefficient was obtained between the 2 measurements in each time point and revealed a very high correlation ( 0.98 and 0.99 ; p < 0.01 ) . the distance between the cementoenamel junction and the bottom of the junctional epithelium ( cej - je ) in the mesial site of the first molars was defined as the loss of histological attachment . in addition , the distance between the cementoenamel junction and the alveolar bone crest was also analyzed and it was considered as the indicator for bone loss ( cej - bc ) . analysis of variance ( anova ) and tukey ( p < 0.05 ) tests were applied . there were no statistically significant differences among the body weights of the groups at any of the evaluation periods ( anova , p < 0.05 ) . loss of histological attachment ( cej - je ) and bone loss ( cej - bc ) ( p < 0.05 ) were statistically different in the animals exposed to alcohol ( gal ) and the ones exposed to alcohol and stress ( gasl ) . the most severe periodontal breakdown was observed among the nonstressed animals exposed to alcohol , followed by the group of stressed animals exposed to alcohol . this last group ( gasl ) did not differ from the positive control group ( gpc ) . figures 1 and 2 illustrate results from gasl group at two different experimental times . finally , the negative control group showed the lowest values of cej - je and cej - bc when compared to the animals in the other groups ( table 1 ) . the severity and rate of the progression of periodontitis are influenced by a great variety of determinants and risk factors . oral hygiene , tobacco smoking , diabetes , age , and the presence of specific microorganisms can be considered as risk factors , since individuals under these conditions seem to be more likely to develop periodontitis . however , the combination of these factors is not enough to explain the difference in the progression rate and severity of the disease . other systemic conditions such as stress , osteoporosis , obesity , and abusive alcohol drinking have been considered as partially responsible for this variability as well . the effects of alcohol to the body and the functional imbalances associated with its consume have been constantly investigated . such interest is justified by the wide range of injuries and toxic effects that alcohol drinking causes the organs and tissues , which can reflect in different systemic diseases . besides , the world health organization estimates that two billion people around the world are alcohol consumers , and that 76 million show some kind of systemic adverse event related to that . in brazil , it is estimated that 68% of the population consumes alcohol and that 11.2% are alcoholics . de souza et al . demonstrated that alcohol consumption directly framed the progression of bone loss in an induced periodontitis model . on the other hand , instead of a direct action of alcohol over the periodontal tissues , these effects are based on poor oral hygiene , which could better explain a higher occurrence and severity of periodontal disease in people who drink alcohol . in this context , animal studies are important as they may help understand the biological events once they allow the elimination of certain bias by the control of some variables . therefore , animal studies have observed that alcohol affected the periodontal tissues through different mechanisms . reduced host responses , due to deficiency and functional changes in neutrophils , as well as changes in the protein metabolism and in the healing of tissues have been previously reported . in the present study alcohol drinking negatively impacted the progression of periodontitis . similarly de souza et al . also observed a direct effect of alcohol drinking in the bone loss progression in a ligature - induced periodontitis model . one common question is in which level the alcohol as a water substitute under experimental conditions reflects human alcohol drinking . the answer to that is not simple , especially considering that the metabolism of rats is faster than that of humans . according to the world health organization mild drinkers are men who drink 21 alcohol units ( 1 unit = 10 grams ) per week . for women , for example , a glass of red wine ( 90 ml ) has 12% or 1.7 unit of alcohol . a beer has 5% ( 1.1 unit ) while distilled drinks such as vodka and whisky have 40% or 2.0 alcohol units . supporting dose dependence , de souza et al . reported a significantly higher alveolar bone loss in rats receiving 20% ethanol in comparison to rats receiving 10% ethanol or water . . carried an interesting cross - sectional study to evaluate the effect of the consumption of different alcoholic drinks ( wine , beer , and liquor ) on the severity of periodontal disease . they observed that the effects over the periodontal tissues did not change depending on the kind of drink taken . these same authors also verified contrary activity of alcohol over some periodontal pathogens such as actinobacillus actinomycetemcomitans and porphyromonas gingivalis . clinical results suggested that alcohol seems to affect more severely the gums , followed by the periodontal ligament and the alveolar bone and that the effect of alcohol on periodontal disease may depend on the dose , frequency , and time of drinking . . also demonstrated that individuals that consumed more than 15 grams of alcohol a day had a significant increase in the progression rate of periodontal disease and a higher inflammatory infiltrate , besides a higher number of periodontal pockets when compared to those who did not consume alcohol . although , dental biofilm acts besides some recognized risk factors such as tobacco use , as previously mentioned , more recently , researchers are considering whether and in what degree lifestyle could affect the periodontium . in this context , stress seems to affect not only periodontal status but also personal lifestyle . individuals exposed to stressful situations tended to smoke a higher number of cigarettes and become alcohol consumers . also , a decrease in oral hygiene was observed in a group of stressed individuals . furthermore , direct tissue damage could be attributed to the stress process . due to the frequent combined occurrence of the studied factors , this study evaluated the effect of alcohol intake by stressed animals . the fact that the stressed animals exposed to alcohol showed better periodontal conditions than the nonstressed animals was only a partially unexpected result of this study . yaroslavsky and tejani - butt evaluated the relation between stress and alcohol consumption and observed that rats exposed to chronic stress consumed a greater amount of alcohol . in addition , changes in central dopamine type-2 receptor sites were found indicating an altered dopamine neurotransmission following stress and alcohol exposure . since stressed animals consumed more alcohol , the authors concluded that it is possible that the consumption of alcohol reverses these alterations related to dopamine , suggesting a self - medicating phenotype . compared the immunological effect of the discontinuous feeding of a liquid diet containing a moderate amount of ethanol to that of continuous ethanol administration or a control diet . the discontinuous alcohol group received the ethanol diet for 3 days and the control liquid diet for the remaining 4 days of each week ( for a total of 4 weeks ) . the authors concluded that discontinuous drinking of a moderate amount of ethanol can be more harmful for the immune system than a continuous ethanol intake , as the model applied in the present study . those authors suggested that probably the discontinuous alcohol intake induces a greater stress as indicated by the searched immune indicators . therefore , future studies must be carried as to elucidate the isolate and combined effects of alcohol and stress over periodontal tissues . our data suggest that although stress and alcohol may be harmful separately , their association probably triggered different processes . the evaluation of these factors is relevant because the combination of stress and alcohol drinking is a frequent condition in human beings with periodontitis . although the association did not result as expected , the effects of alcohol drinking confirmed to be an important factor for the development of a periodontal disease . however , nonstressed animals who also consumed alcohol for the same period showed a pattern of periodontal breakdown even more severe . therefore , the factors of alcohol drinking and stress do not seem to show any synergic effect .

this study histometrically evaluated the effect of forced alcohol intake by stressed animals on the severity of ligature - induced periodontitis in rats . thirty - two rats were randomly divided in four groups : group gal alcohol and ligature ; group gasl alcohol , chronic physical stress , and ligature ; gnc negative control ; gpc positive control . gal and gasl received 20% ethanol ad libitum , and gnc received water ad libitum for 60 days . after 24 hours of exposition to alcohol intake by gal and gasl immobilization was applied as a chronic stressor in the gasl group for a two - month period , six times a week , in random hours . the means of the respective groups were statistically compared ( analysis of variance and tukey tests , p < 0.05 ) . the most severe periodontal breakdown was observed in nonstressed animals which drank alcohol ( gal ) , followed by stressed animals exposed to alcohol ( gasl ) . gasl did not differ from the positive control group ( gpc ) . the negative control group showed the lowest values of periodontal breakdown ( p < 0.05 ) . conclusions . non - stressed alcohol consumer animals showed the most severe pattern of periodontal breakdown . although stressed animals which were forced to drink alcohol showed poorer periodontal status than the negative controls , their results were similar to those of positive controls .

prostate cancer is the second most common cause of cancer death in men in the united states . in the united states , the age - adjusted number of new cases of prostate cancer was 147.8 per 100,000 men per year based on 2007~2011 data and the age - adjusted number of deaths was 23.0 per 100,000 men per year based on 2006~2010 data . according to 2008~2010 data , approximately 15.3% of men will be diagnosed with prostate cancer at some point during their lifetime , and in 2011 , an estimated 2,707,821 men were living with prostate cancer in the united states.1 the most common sites of prostate cancer metastasis include the bone , lymph nodes , lungs , liver , pleura , and adrenal glands.2 rarely has metastatic prostate cancer been reported to involve the gastrointestinal tract.3,4,5,6,7,8,9,10,11 here , we report the case of a 64-year - old man with prostate cancer that had metastasized to the rectum and stomach . a 64-year - old african - american man presented with anemia ( hemoglobin level of 6.8 g / dl ) in september 2012 . he had a medical history of prostate cancer ( gleason score of 5 + 4=9 ) diagnosed in june 2011 , hypertension , coronary artery disease with stent placement , and insertion of bilateral percutaneous nephrostomy tubes due to hydronephrosis . the patient was being treated with flutamide and leuprolide acetate for prostate cancer since june 2011 . in september 2012 , he presented with the complaint of dark - colored stools for a week . at that time , he did not experience any chest pain , shortness of breath , palpitations , dizziness , or headache , and no history of hemoptysis or hematemesis was reported . physical examination of the patient was unremarkable except for the findings of a cachectic elderly man with pale conjunctiva and bilateral nephrostomy tubes . laboratory data on admission revealed a hemoglobin level of 6.8 g / dl , hematocrit of 21% , platelet count of 281,000/mm , white cell count of 4,500/mm , and normal prothrombin time and activated partial thromboplastin time . iron studies revealed a total iron level of 80 mg / dl , total iron binding capacity of 227 mg / dl , ferritin level of 357 ng / ml , and reticulocyte count of 0.9% . a complete metabolic profile showed a blood urea nitrogen level of 54 mg / dl and a creatinine level of 3.3 mg / dl . the patient had a computed tomography ( ct ) scan performed without contrast on his abdomen and pelvis . the ct scan revealed multiple cystic lesions in the liver , marked thickening of the wall of the stomach ( fig . the patient also underwent a colonoscopy , which revealed circumferential nodularity and poor distensibility of the rectal lumen ( fig . biopsy of the rectum indicated diffuse infiltration of high - grade neoplastic cells that were positive for human prostatic acid phosphatase and prostate - specific membrane antigen . these findings were consistent with a diagnosis of metastatic , poorly differentiated carcinoma of the prostate . since it was known that the patient had hormone - refractory prostate cancer , he was started on docetaxel and received 3 cycles until february 2013 , at which point he was lost to follow - up . in may 2013 , microscopic examination revealed sheets of atypical cells with prominent nucleoli and no glandular pattern within the gastric lamina propria ( fig . immunohistochemical studies demonstrated that the tumor cells were positive for prostate specific antigen ( psa ) and alpha - methylacyl - coenzyme a racemase ( amacr , p504s ) ( fig . the patient 's serum psa level was more than 1,000 mg / dl at that time . the bone scan , as well as the ct scan of the abdomen and pelvis , showed no metastases but showed retroperitoneal lymphadenopathy . at that time , the patient had an eastern cooperative oncology group performance status of 3 . the patient and family refused further chemotherapy for metastatic prostate cancer and opted for palliative care instead . the patient was admitted multiple times for similar complaints and was managed with supportive care . prostate cancer is the most common noncutaneous cancer in men in the united states and is among the most commonly diagnosed cancers in many developed countries . classic risk factors for this cancer include older age , african - american race / ethnicity , and a family history of prostate cancer . the increase in prostate cancer incidence rates in groups that have migrated from countries with low rates to countries with high rates strongly suggests the importance of environmental factors in its etiology . kolonel12 reported that the the incidence rates of prostate cancer steadily increased in the japanese group with migration from mainland japan to hawaii , and the united states mainland in that order based on the ethnic studies . the majority of men are diagnosed with prostate cancer at an age older than 65 years , and the vast majority of prostate cancer deaths occur in this older age group . the median age at prostate cancer diagnosis is 71 years in caucasians and 69 years in african - americans in the united states . metastatic prostate cancer has a poor prognosis and median survival time ranges from 1 to 3 years.3 prostate cancer preferentially spreads to the skeleton . more than 80% of men who die from prostate cancer are identified with bone metastases at autopsy.13 in contrast to most other cancers , prostate cancer predominantly forms osteoblastic metastases . the vertebral column , pelvis , ribs , and proximal long bones are the most common sites of skeletal metastases . hematogenous , lymphatic , and direct infiltrations are the typical routes of spread.14 patients with prostate cancer can be anemic due to bone marrow involvement . metastasis of prostatic carcinoma to the gastrointestinal tract is a very rare occurrence and presents a diagnostic challenge.6,7,8,9,10,11,13,14 because the prostate is richly supplied with lymphatic channels , metastasis to the gastrointestinal tract may occur via the lymphatic route . this should be taken into consideration during the work up for anemia in a patient with advanced prostate cancer , as supportive management will depend on the etiology of the anemia . in patients with suspected gastrointestinal bleeding or signs of iron deficiency anemia , upper and lower endoscopy two postmortem studies reported gastric metastasis from primary prostate cancer in 1% to 4% of cases.15,16 histologically , primary adenocarcinoma of the stomach is composed of atypical glands with cribriform ( back to back ) formation and a mucin - producing infiltrating growth pattern . carcinoma in situ or high - grade dysplasia is usually found adjacent to the carcinoma . in contrast , tumor cells of metastatic carcinoma from prostate cancer usually have prominent nucleoli and no glandular formation . the prostate biomarker amacr has been used in conjunction with morphology with very high sensitivity and specificity in diagnostically challenging cases . amacr , also known as racemase or p504s , is an enzyme identified by cdna subtraction and microarray technology . it is a sensitive and specific immunohistochemical marker that has been found to be consistently up - regulated in prostate carcinoma.17,18 treatment for metastatic prostate cancer is palliative . several new agents have been introduced for the treatment of metastatic prostate cancer in the past two decades , with excellent disease control and good patient tolerability . the management of advanced prostate cancer with metastases to the gastrointestinal tract includes local control measures , supportive care , and treatment of the underlying cancer . if the disease progresses and hormone - refractory metastatic prostate cancer is diagnosed , alternative treatments include chemotherapy , immunotherapy with sipuleucel - t , androgen receptor antagonist drugs such as enzalutamide , and androgen synthesis inhibitors such as abiraterone . although rare , it is important to consider the possibility of prostate carcinoma metastasizing to the gastrointestinal tract in patients presenting with gastrointestinal bleeding and a history of prostatic adenocarcinoma . it is crucial to distinguish primary gastrointestinal cancer from metastatic lesions , especially in cases of a previous history of cancer at another site , for appropriate management . this can be achieved by determining the histopathologic classification of the tumor and by immunohistochemical staining for psa .

prostate cancer is the second most common cause of cancer death in men in the united states . the most common sites of metastasis include the bone , lymph nodes , lung , liver , pleura , and adrenal glands , whereas metastatic prostate cancer involving the gastrointestinal tract has been rarely reported . a 64-year - old african - american man with a history of prostate cancer presented with anemia . he reported the passing of dark colored stools but denied hematemesis or hematochezia . colonoscopy revealed circumferential nodularity , and histology demonstrated metastatic carcinoma of the prostate . esophagogastroduodenoscopy showed hypertrophic folds in the gastric fundus , and microscopic examination revealed tumor cells positive for prostate - specific antigen . bone scanning and computed tomography of the abdomen and pelvis did not show metastasis . it is crucial to distinguish primary gastrointestinal cancer from metastatic lesions , especially in patients with a history of cancer at another site , for appropriate management .

the incidence of acute pancreatitis is increasing around the world , where it is associated with morbidity and even the risk of mortality . it may presents with various skin manifestations as rash and rarely , with erythematous tender subcutaneous nodules , known as pancreatic panniculitis . alcohol and gallstones are the most important causes , while routinely prescribed drugs have been linked to the causation of acute pancreatitis . metformin , one of the most widely prescribed oral hypoglycemic agents , was linked to pancreatitis , secondary to overdose or in case of impaired renal function . here , we report a case of metformin induce acute pancreatitis in young healthy man with normal renal function . nineteen year - old - man , known case of type 2 diabetes mellitus for 4 y on 1 g metformin twice daily since diagnosis of his diabetes . he was in his usual state of health till he presented to the emergency department reporting nausea , vomiting and epigastric pain for 3 d. on physical examination , his height was 170 cm and body weight 99 kg ; body mass index ( bmi ) 34.3 kg / m2 , looked mildly dehydrated . laboratory investigations showed hba1c 7.7% , creatinine 58 mol / l , amylase 462 units / l ( normal range < 100 ) , lipase 1378 units / l ( 060 ) , white blood cells 16.8/mm3 ( 411 ) 80% of which was neutrophils , crp 258 mg / l ( 05 ) , mg 0.76 mmol / l ( 0.71.05 ) , ca 2.17 mmol / l ( 2.22.6 ) , ast 18 units / l ( < 39 ) , alt 34 units / l ( < 41 ) , tg 0.95 mmol / l ( < 2.3 ) , lactate 1.4 mmol / l ( 0.51.6 ) . abdominal ultrasound and ercp were done for the patient , results showed no gallstones and clear biliary tract , respectively . normalization of amylase and lipase was reached after metformin cessation , and supportive treatment in the form of iv insulin and iv fluids . few days later , after re - exposure to metformin , he presented with recurrence of his previous symptoms , and elevation of amylase and lipase was documented . as a result , metformin was suspended with improvement of his symptoms and biochemical profile . metformin is one of the most effective and valuable oral hypoglycemic agents in the biguanide class . it has been selected as the drug of choice in management of majority of patients with type 2 diabetes mellitus , as it works by several mechanisms ; decreasing hepatic gluconeogenesis , increasing glucose transport into glucose utilizing cells , decreasing appetite and caloric intake and reducing intestinal glucose absorption . as any other medication , metformin is associated with undesirable side effects and it has been reported in few cases to cause lactic acidosis and pancreatitis . acute pancreatitis is attributed to many etiological factors ; one of them is medication , where a large number of single case reports on drug - induced pancreatitis were published . diuretics , antimicrobial agents , hiv therapy and neuropsychiatric agents as well as metformin are known medication to induce pancreatitis . the underlying pathogenesis in drug induced pancreatitis in some medications as codeine was due to spasm of sphincter of oddi , in others as azathioprine , immune- mediated mechanism or a hypersensitivity to the drug is the postulated pathogenesis . in metformin , the exact mechanism is not known , but toxicity is probably secondary to acinar cell injury leading to intercellular leakage of digestive enzymes from ductules . fortunately , drug - induced pancreatitis is an acute edematous pancreatitis of a benign course and good prognosis , but fatal outcome may ensue if proper management is not initiated . among the published case reports of metformin induced pancreatitis , one was attributed to metformin overdose , other was caused by metformin accumulation , resulting from combination of drug overdose and acute renal failure triggered by vomiting in patient with concealed renal insufficiency . additionally , two case reports were found to be associated with drug induced ( angiotensin converting enzyme inhibitor , angiotensin receptor blocker , non - steroidal anti - inflammatory drug as ibuprofen and celecoxib and diuretics ) renal failure , which reduced excretion of therapeutic dose of metformin causing toxicity , leading to sever lactic acidosis and pancreatitis . lastly , in a case report of patient presented with hyperglycemia after an ingested dose between 64- 85 g of metformin in a suicide attempt , the potential mechanism of hyperglycemia is not clear and nothing among metformin s known mechanisms would logically explain the progressive and severe hyperglycemia , pancreatitis remains a potential mechanism , as the patient s clinical presentation with reportts vomiting and abdominal pain is consistent with pancreatitis . in our case , several signs can guide to and support the diagnosis of metformin induced pancreatitis ; first is the clinical presentation of abdominal pain , nausea , vomiting and dehydration . second , high levels of lipase and amylase , which correlate positively with ct findings . the lack of other known causes of acute pancreatitis such as gall stones , alcohol abuse , hypercalcaemia , hypertriglyceridaemia , and trauma , added more evidence to the diagnosis . apart from the therapeutic dose of metformin ( 2 g daily ) , our patient has never consumed any other medications known to cause pancreatitis . finally , resolution of his symptoms upon metformin cessation and relapse upon re - exposure would strongly suggest that metformin is deemed responsible of our patient s clinical presentation . available evidence suggests that acute pancreatitis in our patient was probably precipitated by therapeutic dose of metformin with normal renal function , with unknown exact mechanism . in summary , our case demonstrates the possibility of metformin to induce pancreatitis in a healthy patient without preexisting renal disease . accordingly , every diabetic patient on metformin should be counseled about symptoms of acute pancreatitis and the urgency to discontinue metformin and visit the emergency department to receive the appropriate treatment . and for us as physicians , metformin should be kept in back of our minds as a cause that would contribute to acute pancreatitis even in a healthy patient .

acute pancreatitis frequently presents with abdomen pain but may presents with various skin manifestations as rash and rarely , pancreatic panniculitis . metformin , one of the most effective and valuable oral hypoglycemic agents in the biguanide class was linked to acute pancreatitis in few cases . here , we report a case of metformin induce acute pancreatitis in young healthy man with normal renal function .

several decades ago , treatments for benign prostatic hyperplasia ( bph ) were almost entirely surgical . during the 1990s , various minimally invasive surgical procedures ( mis ) were developed , including transurethral microwave thermotherapy , transurethral needle ablation , and transurethral laser vaporization . although techniques have advanced , transurethral resection of the prostate ( turp ) remains the gold standard surgical intervention for bph . the introduction of -antagonists in the early 1990s resulted in significant improvements in the medical treatment of bph . after the early 2000s , combination therapy with -antagonists and 5--reductase inhibitors has resulted in significant changes in the management of lower urinary tract symptoms ( luts ) secondary to bph . in the proscar long - term efficacy and safety and medical therapy of prostate symptoms ( mtops ) studies , some patients can not or will not tolerate medical therapy , whereas others experience symptomatic deterioration despite long - term medical therapy . despite the advances in medical and surgical intervention , many men continue to suffer from bph . the incidence of bph is 50% in men between 51 and 60 years of age and as high as 88% in men up to 80 years of age . in an aging population , it would be advantageous to determine the roles of medical and surgical intervention for bph and the effect of changes in medical therapy trends on the indications and outcomes of surgical intervention for bph . the aim of this study was to compare the outcomes in patients who underwent surgery in 1985 to 1989 ( before the widespread use of medical therapy for bph ) , in 1995 to 1999 ( when medical therapy was developed and began to be prescribed as alternative treatment to surgery ) , and in 2005 to 2009 ( when medical therapy superseded surgical intervention to become first - line treatment and when combination therapy became widely adopted ) . medical records were retrospectively reviewed for all patients who underwent surgery for bph from 1 january 1985 to 31 december 1989 ( group 1 ) , from 1 january 1995 to 31 december 1999 ( group 2 ) , and from 1 january 2005 to 31 december 2009 ( group 3 ) . university staff urologists .with more than 1 year of surgical experience with bph performed all the operations . preoperatively , we evaluated age , body mass index ( bmi ) , prostate - specific antigen ( psa ) level , prostate volume , prostate transitional zone volume , maximum urinary flow rate , postvoid residual urine volume , international prostate symptom score ( ipss ) , qol score , bph medication use , and chief complaints . bph medication history included -blocker or 5--reductase inhibitor therapy for bph more than 3 months before surgery . perioperative and postoperative parameters were investigated representatively in patients who underwent turp , who were divided into 3 subgroups according to time period : 1985 to 1989 ( subgroup 1 ) , 1995 to 1999 ( subgroup 2 ) , and 2005 to 2009 ( subgroup 3 ) . transfusion and the weight of the resected tissue were evaluated as operative parameters . the postoperative course was evaluated on the basis of length of hospital stay , the postoperative day on which the catheter was removed , and postoperative complications . a postoperative complication was defined as any event requiring additional treatment within 1 year of bph surgery : secondary hemorrhage that required transfusion or hospital admission within 4 weeks postoperatively , impaired detrusor contractility that required suprapubic cystostomy as a result of recurrent acute urinary retention ( aur ) or failure to void , repeat surgery for bph , urethral stricture that required surgical intervention , long - term stress incontinence , and irritative luts that required medication use until 1 year postoperatively . for the statistical analysis , one - way analysis of variance was used for continuous variables and a chi - square test was used for categorical variables to assess differences between means . a p value < 0.05 was considered to be statistically significant . spss ver . 18.0 ( ibm , new york , ny , usa ) was used for the statistical analysis . a total of 190 men underwent surgery for bph in group 1 , 484 in group 2 , and 278 in group 3 . the mean ages of the men were 65.4 , 65.9 , and 69.3 years in group 1 , group 2 , and group 3 , respectively . over time , the mean age increased and was significantly different between group 2 and group 3 ( p<0.001 ) . the prevalences of hypertension , a history of surgery , and " others " ( e.g. , stroke , cancer , and cardiac conditions ) increased significantly over time . the mean bmis were 21.6 , 22.9 , and 24.0 in group 1 , group 2 , and group 3 , respectively ( p<0.001 ) . the most common indication for surgery in all groups was worsening symptoms , followed by aur . the incidence of symptomatic deterioration continued to increase , but the rate of aur kept decreasing . , only one psa level was found in the chart review ; therefore , group 1 was excluded from the psa comparison . the mean psa levels of group 2 and group 3 were 6.8 and 5.8 , respectively ; however , they were not significantly different . there was a slight increasing trend from 34.4 ml to 38.4 ml between group 1 and group 2 . however , there was a sharp increase between group 2 and group 3 ( p<0.001 ) . we could not elucidate sufficient data to analyze prostate transitional zone volume , maximum urinary flow rate , residual urine volume , total ipss score , and qol score in group 1 . therefore , we compared groups 2 and 3 , and a significant difference was detected in prostate transitional zone volume ( p<0.001 ) ( table 1 ) . only two patients ( 1.1% ) in group 1 required bph medication ; the remaining patients in this group did not receive medical therapy . however , 33.5% of the patients in group 2 used -blockers . a marked improvement was observed in group 3 . -blockers were taken by 47.8% of patients , and 25.9% of patients required combined therapy with an -blocker and a 5--reductase inhibitor ( fig . on the other hand , mis had only begun to be performed with group 2 ( fig . the basic characteristics of these patients were similar to those of patients who underwent surgery for bph ( table 1 ) ; however , one difference was that the mean prostate volume gradually and significantly increased over the 20-year study period ( p<0.001 ) . we analyzed perioperative and postoperative parameters in subgroups who underwent turp for direct comparison in one type of surgery : 131 patients in subgroup 1,339 patients in subgroup 2 , and 194 patients in subgroup 3 underwent turp for bph . perioperative parameters and postoperative variables for the three patient subgroups are illustrated in table 2 . the mean weight of resected tissue was significantly greater in subgroup 3 than in subgroup 1 and subgroup 2 . the number of patients who required transfusion because of perioperative hemorrhage continuously deceased over time . over time secondary hemorrhage within four postoperative weeks and the need for repeat turp within 1 year after surgery decreased significantly ( p=0.03 and p=0.003 , respectively ) . the number of patients who required suprapubic cystostomy gradually increased over time , albeit not significantly so ( p=0.523 ) . no significant changes in any of the other complications were found in the three studied periods . over the past two decades , medical therapy has been the standard first - line therapy for bph . long - term combination therapy reduced the incidence of aur and bph surgery . in our study , aur decreased significantly , but worsening symptoms increased significantly . our results confirmed an increasing trend in -blocker monotherapy and combination therapy with 5--reductase inhibitors after the 1990s , when medical therapy became widespread . however , surgery for bph is still required and turp became the mainstay of surgical intervention for bph . although medical therapy is appropriate for patients with symptomatic bph , it has obvious limitations . failure of medical therapy results in worsening luts and recurrent aur . in this study , the most common chief complaint was worsening symptoms followed by aur . in a study by izard and nickel , patients who finally required surgery were inevitably older , had worse health conditions , and had more advanced disease . therefore , late surgical intervention could result in poorer outcomes associated with worse postoperative complications . in this study , the mean age of patients who underwent surgery was greater in the 1990s than in the 1980s , and the increase was larger after 10 years . this trend was also shown in another study . according to vela - navarrete et al . , patients who underwent surgery for bph in 2002 were significantly older than those who underwent surgery in 1992 , by a mean of 3.1 years . a considerable number of patients had hypertension and previous surgical and other medical histories in the 2000s . we concluded that the increasing comorbidity was due to aging , advancement in diagnostic techniques , and increasing health concerns . the decreased aur and the increased frequency of symptomatic deterioration were due to combination therapy that reduced aur . according to our results , the mean prostate volume increased during the 20 year period in patients who underwent surgical intervention for bph . a review by han et al . showed that the mean prostate volume in surgically managed patients over 5 successive years increased significantly , from 37.1 ml to 53.6 ml , between 1998 and 2002 . they attributed these findings to the priority given to medical therapy by patients who required surgery in the past and to development of the endoscopic technique , which extended the indication of turp . because of larger prostate volumes , it is natural that the weight of the resected tissue increased correspondingly . this study showed that the mean prostate volume was significantly greater in group 3 than in group 1 ; simultaneously , the weight of the resected tissue was also significantly greater in subgroup 3 than in subgroup 1 . shin and park reported that the mean resection volume increased with both turp and open prostatectomy between 1999 and 2010 . reich et al . evaluated 10,654 patients who underwent turp and showed that mortality and morbidity were closely associated with the weight of tissue resected . according to mebust et al . , increased morbidity was seen in patients with a gland size greater than 45 g and an age greater than 80 years . according to our survey of perioperative and postoperative variables , the length of hospital stay , the number of days to catheter removal , and the number of transfusions , secondary hemorrhages , and repeat turps deceased with time . these findings were due to advancements in surgical techniques , experience , and improved instruments . we believed that critical outcomes such as impaired detrusor contractility would be poor and hypothesized that long - term bladder outlet obstruction would impair bladder detrusor contractility . experimented with rat bladders and found that whole - bladder pressure was significantly lower in rats subjected to long - term outflow obstruction than in controls . however , the number of suprapubic cystostomies required after turp did not increase significantly , because new techniques and equipment reduced the number of complications and overcame the risks associated with aging . in addition , combination therapy resolved bladder outlet obstruction and protected progress to impaired detrusor contractility . the results showed that surgical intervention conferred benefits to patients , despite their older ages and larger prostate volumes at the time of surgery . following the widespread adoption of medical therapy for the treatment of bph , the rate of medical therapy increased and the number of surgeries decreased . although this study did not represent all bph patients , it analyzed data spanning two decades to determine the changing trends in surgery for bph . however , because of its retrospective nature , ipss and transitional zone volume data were not available in the 1980s . an additional limitation was the lack of pre- and postoperative data , such as ipss data and maximum urinary flow rates , with which to compare the direct results of surgery . furthermore , we were unable to determine the length of time required to complete the surgeries - a variable that is useful in determining the skill level of surgeons when resection weights are identical . instead , we used transfusion history in the perioperative period to indirectly determine the level of surgical skill and care . the length of hospital stay appeared to be longer in the current study than in other studies , which was due to korean health insurance policies and patient tendencies . the mean number of hospitalizations in subgroup 3 in the current study was similar to that in other korean publications . several studies reported that these technologies have led to shorter hospital stays , shorter catheterization periods , and fewer complications . even in patients with large prostate volumes , in whom turp was inappropriate in the past , these procedures have shown excellent results , especially concerning perioperative blood loss . continued advancement in surgical intervention the ages and comorbidities of patients who underwent surgery for bph increased over the two decades studied . our study showed that the mean prostate volume at the time of surgery was significantly greater than it was 20 years ago , as was the weight of the resected tissue . although these conditions can increase the risk of complications , the prevalence of secondary hemorrhage within four postoperative weeks and the need for repeat turp within 1 year after surgery decreased significantly . although patients who underwent surgery were older after widespread use of medical therapy for bph , advancements in surgical techniques have benefitted these patients .

purposefollowing the introduction of medical therapy for benign prostatic hyperplasia ( bph ) , we determined the effect of the change in trends in medical therapy on the indication and outcome of surgical intervention for bph.materials and methodswe compared the basic characteristics of , weight of resected tissue of , transfusions in , and postoperative complications of patients who underwent surgery between 1985 and 1989 ( before the advent of medical therapy for bph ) , between 1995 and 1999 ( when medical therapy was developed and became widely used as alternative treatment ) , and between 2005 and 2009 ( when medical therapy superseded surgical intervention to become first - line treatment and when combination therapy became widely adopted).resultsat our institution , the mean age and bmi of patients increased over the past two decades ( p<0.001 ) . hypertension , operation history , and other comorbidities also increased significantly ( p<0.001 , p=0.005 , and p<0.001 , respectively ) . the indications for surgery in 1985 to 1989 , 1995 to 1999 , and 2005 to 2009 were as follows : acute urinary retention in 34.7% , 20.2% , and 15.1% of patients and symptomatic deterioration in 61.1% , 72.3% , and 73.0% of patients , respectively . prostate volume and the weight of resected tissue increased from 34.414.5 ml to 61.332.4 ml and from 7.26.4 g to 10.87.6 g , respectively , over two decades . patients who underwent surgery in 2005 to 2009 had their catheters removed earlier ( p<0.001 ) . secondary hemorrhage within four postoperative weeks and repeat transurethral resection of the prostate within 1 year decreased significantly ( p=0.03 and p=0.003 , respectively ) . no statistically significant change in impaired detrusor contractility was found ( p=0.523).conclusionsalthough patients who underwent surgery were older after widespread use of medical therapy for bph , advancements in surgical techniques have benefitted these patients .

spinal fusion and pedicle screw fixation techniques are usually used in cases of vertebral fractures , dislocation , scoliosis , kyphosis , spinal tumor and for severe back pain that does not respond to other therapies.123 the pedicular screw fixation offers a stable and safe possibility for stabilization during correction of malalignment.123 the pedicle is surrounded by many sensitive structures such as nerve root , dura , cord which are not visible during pedicle screw insertion . screw malposition pedicle wall perforation , nerve roots and cord impingement and very rarely , damage to vascular structures.456 therefore , the exact location of entry points and screw orientation is of great importance . in case of conventional transpedicular fixation especially the minimally invasive pedicle screws insertion , the surgeon is only provided with intraoperative two dimensional x - ray images for the alignment and positioning of the pedicle screws and in up to 40% of the cases a perforation of the pedicle occurrs , depending on both the surgeon 's performance and the definition of error.456 there was also much more radiation exposure to the surgeons during minimally invasive pedicle screws placement.78 in order to ease the surgeon 's hand - eye coordination and to reduce the iatrogenic radiation injury to the surgeons , a robot assisted surgery is expected to increase the quality of percutaneous pedicle screw placement . this study to feasibility and clinical value of robot - assisted navigated drilling in percutaneous pedicle screw placement . additionally , the results would form the basis for the development of a new robot for pedicle screw fixation surgery . the spine robot system [ figure 1 ] was domestically developed by and the department of science and department of orthopedics at our university . the robot includes three main parts : robot arm , base of the robot arm and console [ figure 2 ] . the robot , composed of six revolute joints , is a serial manipulator with 6 of freedom . the motion patterns of the robot consist of manual traction mode , longitudinal shift mode , angular deflection mode and horizontal shift mode . therefore , the 6 of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . the end of the robot arm equipped with bone drill holder which can hold the pneumatic drill . the pneumatic drill can be conveniently sterilized by separation from the robot and used for drilling the pedicle screw trajectory during operation . the bone drill holder integrated six dimensional force / torque sensor , surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process . ( 1 ) robot arm , ( 2 ) base of the robot arm , ( 3 ) controller of the drill , ( 4 ) console operation interface of the console . ( 1 ) power button ( 2 ) touch screen operator interface ( 3 ) operating lever longitudinal shift ( 4 ) operating lever angular deflection ( 5 ) operating lever horizontal shift preoperative computed tomography ( ct ) of eight bovine lumbar spines ( l1l5 ) in axial plane was captured for each vertebra , the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement . during preoperative planning this process needs to be done for each of the vertebrae involved in the procedure . l. distance between the posterior median line of the spinous process and the entry point ; ( a ) angle between the posterior median line of the spinous process and the insertion line bovine is a tetrapod , its anatomical characteristics and common fracture site is different from the human and bovine spine segments are presumed to have higher bone mineral density than human spines and the pedicles of the bovine spine were much more thin than human spines , all pedicle screws will make cortical perforation , so that we did nt insert pedicle screw into the pedicle to avoid the misjudgement about cortical perforation . the purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placemen . in each screw insertion , full procedures from preoperative tasks to postoperative tasks were tested and evaluated to determine whether they are proper to apply to clinical fields . we checked preoperative planning , robot movement and surgical procedure in every pedicle screw insertion case . engineers and orthopedic surgeons participated in these experiments and they agreed that this system had proper roles for percutaneous pedicle screw insertion procedures and that those results were applicable to clinical applications . we positioned the relative positions of the drill and the bovine lumbar spines , drilled the bovine lumbar spines according to preoperative plans and then placed k - wires in the holes [ figure 4 ] . the bovine spine used in our study were devoid of skin - soft tissue and muscles , it saved a lot of time . we noted surgical time and intraoperative fluoroscopy times and then we assessed the position of the k - wires through postoperative ct [ figure 5 ] . eight bovine lumbar spines ( l1l5 ) were inserted 80 k - wires using the spine robot system . the most important characteristic of the spine robot system is that the angle of the drill can be deflected to keep drill tip in centre . this function is helpful for us to deflect the angle of the drill according to preoperative plan after the tip of the drill touch the entry point of the bony surface and then insert the drill to the bone ; the whole operation process is smooth . ( a ) adjustment the pneumatic drill parallel to the upper vertebral body end plate , ( b ) adjustment the distance between the tip of the drill and the posterior median line according to preoperative planned distance , ( c ) longitudinal shift of the drill to the entry point on the bone surface , ( d ) adjusting the entry angle of the drill according to preoperative planned angle preoperative and postoperative computed tomography ( ct ) scan of the bovine lumbar spine . ( a ) preoperative plan through the preoperative ct scan , ( b ) evaluating the position of k - wire through the postoperative ct scan the spine robot system [ figure 1 ] was domestically developed by and the department of science and department of orthopedics at our university . the robot includes three main parts : robot arm , base of the robot arm and console [ figure 2 ] . the robot , composed of six revolute joints , is a serial manipulator with 6 of freedom . the motion patterns of the robot consist of manual traction mode , longitudinal shift mode , angular deflection mode and horizontal shift mode . therefore , the 6 of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . the end of the robot arm equipped with bone drill holder which can hold the pneumatic drill . the pneumatic drill can be conveniently sterilized by separation from the robot and used for drilling the pedicle screw trajectory during operation . the bone drill holder integrated six dimensional force / torque sensor , surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process . ( 1 ) robot arm , ( 2 ) base of the robot arm , ( 3 ) controller of the drill , ( 4 ) console operation interface of the console . ( 1 ) power button ( 2 ) touch screen operator interface ( 3 ) operating lever longitudinal shift ( 4 ) operating lever angular deflection ( 5 ) operating lever horizontal shift preoperative computed tomography ( ct ) of eight bovine lumbar spines ( l1l5 ) in axial plane was captured for each vertebra , the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement . during preoperative planning , we measured angle a and distance l [ figure 3 ] . this process needs to be done for each of the vertebrae involved in the procedure . l. distance between the posterior median line of the spinous process and the entry point ; ( a ) angle between the posterior median line of the spinous process and the insertion line bovine is a tetrapod , its anatomical characteristics and common fracture site is different from the human and bovine spine segments are presumed to have higher bone mineral density than human spines and the pedicles of the bovine spine were much more thin than human spines , all pedicle screws will make cortical perforation , so that we did nt insert pedicle screw into the pedicle to avoid the misjudgement about cortical perforation . the purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placemen . in each screw insertion , full procedures from preoperative tasks to postoperative tasks were tested and evaluated to determine whether they are proper to apply to clinical fields . we checked preoperative planning , robot movement and surgical procedure in every pedicle screw insertion case . engineers and orthopedic surgeons participated in these experiments and they agreed that this system had proper roles for percutaneous pedicle screw insertion procedures and that those results were applicable to clinical applications . we positioned the relative positions of the drill and the bovine lumbar spines , drilled the bovine lumbar spines according to preoperative plans and then placed k - wires in the holes [ figure 4 ] . the bovine spine used in our study were devoid of skin - soft tissue and muscles , it saved a lot of time . we noted surgical time and intraoperative fluoroscopy times and then we assessed the position of the k - wires through postoperative ct [ figure 5 ] . eight bovine lumbar spines ( l1l5 ) were inserted 80 k - wires using the spine robot system . the most important characteristic of the spine robot system is that the angle of the drill can be deflected to keep drill tip in centre . this function is helpful for us to deflect the angle of the drill according to preoperative plan after the tip of the drill touch the entry point of the bony surface and then insert the drill to the bone ; the whole operation process is smooth . ( a ) adjustment the pneumatic drill parallel to the upper vertebral body end plate , ( b ) adjustment the distance between the tip of the drill and the posterior median line according to preoperative planned distance , ( c ) longitudinal shift of the drill to the entry point on the bone surface , ( d ) adjusting the entry angle of the drill according to preoperative planned angle preoperative and postoperative computed tomography ( ct ) scan of the bovine lumbar spine . ( a ) preoperative plan through the preoperative ct scan , ( b ) evaluating the position of k - wire through the postoperative ct scan preoperative ct of eight bovine lumbar spines ( l1l5 ) in axial plane was taken for each vertebra , the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement [ table 1 ] . assisted by spine robot system , the average time for system registration was ( 343.4 18.4 ) s , the time for procedure of drilling one k - wire was ( 89.5 6.1 ) s , times of fluoroscopy for procedure of drilling one k - wire were ( 2.9 0.8 ) s. overall , 12 ( 15.0% ) of the 80 k - wires violated the pedicle wall . four screws ( 5.0% ) were medial to the pedicle , and 8 ( 10.5% ) were lateral . the rate of the k - wire wholly within the pedicle was 85% [ table 2 ] . preoperative measurement index of the experimental group according to different vertebrae surgical results of the spine robot system for predrilled pedicle screw trajectory percutaneous pedicle screw placements with conventional and image guidance techniques have demonstrated acceptable results,9101112 but there were so much radiation exposure to the surgeons during minimally invasive pedicle screws placement.78 with the development of computer assisted surgery , spine robot system had been developed for pedicle screws insertion and even some spine robot system has already been used in clinic.131415161718 a biplane fluoroscopy guided robot system ( bfrs ) was developed by kim et al.15 for surgical robotic systems , minimally invasive surgeries and cooperative robotic systems , as well as enhanced surgical planning and navigation with preoperative and intraoperative image data . they pointed out that the bfrs might be helpful in improving the accuracy of percutaneous pedicular screw insertion procedures . in the future , they will attempt to improve the accuracy and reliability of the bfrs and to determine new clinical applications for the bfrs . the spine robot system in our study has motion patterns of the robot consist of manual traction mode , longitudinal shift mode , angular deflection mode and horizontal shift mode . therefore , the 6of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . the pneumatic drill can be conveniently sterilized by separation from the robot to ensure that sterility is maintained throughout the entire operation procedure . the bone drill holder integrated six - dimensional force / torque sensor , surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process to ensure more safety during the whole drilling process . the spine robot system , which has already been used in the clinic , is spineassist.161718 kantelhardt et al.17 reported a retrospective cohort analysis comparing conventional open to open robotic - guided and percutaneous robotic - guided pedicle screw placement . use of robotic guidance significantly increased the accuracy of screw position while reducing the x - ray exposure . lieberman et al.18 pointed out that the robotic guidance group had fewer screw placement deviations , less surgeon radiation exposure , lower fluoroscopy time per screw and shorter procedure time compared to the no robotic guidance group . to our knowledge , the spineassist robot system only provided the optimized trajectory , the pedicle screws insertion was performed only by the surgeon but not the spineassist system itself . in the current study , the surgeons can perform the pedicle screws insertion technique behind the radiation protection screen using the tele - manipulation function of the spine robot system so that the radiation exposure to the surgeons can be decreased and the spine robot system can insert the pedicle screws itself . bovine lumbar spine were used for robot - assisted navigated drilling because the bovine lumbar spines can be more easily available than human cadaver specimens . due to traditional concept in china , a very few people accept body donation , so human cadaver specimens were hard to get . the purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placement , so we think that the bovine spine were acceptable for the study.19 the function of the spine robot system is to pre - drill pedicle screw trajectory , the system ca nt offer help for rod placement , when we have inserted the pedicle screws , we can insert the rod using some special instrument such as instrument in sextant system to place the rod through minimally invasive technique . the rate of the k - wire wholly within the pedicle in the current study was 85% . the reasons can be divided into the following two points : firstly , the pedicle of the bovine lumbar spine was too thin , little deviation of the insertion angle can cause the k - wires violated the pedicle wall . secondly , we ca nt accurately determine the relative position of the drill and the bovine lumbar spine . so , the accuracy and reliability of spine robot system should be improved . in order to improve the accuracy and reliability of the spine robot system for clinical use , further research such as building the virtual surgery system and intraoperative electrophysiological monitoring system will be performed . in recent years , many researchers developed simulators for pedicle screw insertion ; the simulators offer many helpful features to the surgeon with respect to complex cases and to the surgical trainee learning the basic technique of pedicle screw insertion.202122 this technology has also begun to be used in preoperative planning for selected cases , the surgeons can make the surgical plan , practice , and visualize pedicle screw surgery on a particular patient before operation through the simulator.2324 however , when the screws are being inserted , there is no projection fluoroscopy image provided to the surgeon . next , we will develop a ct based patient specific pedicle screw insertion simulator to better prepare surgeons to perform pedicle screw insertion using free - hand technique under the projection fluoroscopy and help reduce the risk of pedicle screw misplacement.25 the second main research direction is to build intraoperative electrophysiological monitoring system . based on strong evidence that multimodality intraoperative neuromonitoring ( miom ) is sensitive and specific for detecting intraoperative neurologic injury during spine surgery , it is recommended that the use of miom be considered in spine surgery where the spinal cord or nerve roots are deemed to be at risk , including procedures involving deformity correction and procedures that require the placement of instrumentation.2627 the basic function of the spine robot system can satisfy spine surgeons for percutaneous pedicle screw placement . using the spine robot system , the operation time and intraoperative fluoroscopy times per pedicle screw was less , but we should improve the accuracy and reliability of spine robot system such as building the preoperative planning simulator and intraoperative electromyography monitoring system for clinical use . we think the spine robot system will be used in clinical practice with the development of preoperative planning simulator and intraoperative electromyography monitoring system in the near future .

background : there is much more radiation exposure to the surgeons during minimally invasive pedicle screws placement . in order to ease the surgeon 's hand - eye coordination and to reduce the iatrogenic radiation injury to the surgeons , a robot assisted percutaneous pedicle screw placement is useful . this study assesses the feasibility and clinical value of robot assisted navigated drilling for pedicle screw placement and the results thus achieved formed the basis for the development of a new robot for pedicle screw fixation surgery.materials and methods : preoperative computed tomography ( ct ) of eight bovine lumbar spines ( l1l5 ) in axial plane were captured for each vertebra , the entry points and trajectories of the screws were preoperatively planned . on the basis of preoperative ct scans and intraoperative fluoroscopy , we aligned the robot drill to the desired entry point and trajectory , as dictated by the surgeon 's preoperative plan . eight bovine lumbar spines were inserted 80 k - wires using the spine robot system . the time for system registration and pedicle drilling , fluoroscopy times were measured and recorded . postoperative ct scans were used to assess the position of the k-wires.results:assisted by spine robot system , the average time for system registration was ( 343.4 18.4 ) s , the average time for procedure of drilling one pedicle screw trajectory was ( 89.5 6.1 ) s , times of fluoroscopy for drilling one pedicle screw were ( 2.9 0.8 ) times . overall , 12 ( 15.0% ) of the 80 k - wires violated the pedicle wall . four screws ( 5.0% ) were medial to the pedicle and 8 ( 10.5% ) were lateral . the number of k - wires wholly within the pedicle were 68 ( 85%).conclusions : the preliminary study supports the view that computer assisted pedicle screw fixation using spinal robot is feasible and the robot can decrease the intraoperative fluoroscopy time during the minimally invasive pedicle screw fixation surgery . as spine robotic surgery is still in its infancy , further research in this field is worthwhile especially the accuracy of spine robot system should be improved .

beyond its role of cellular energy currency and phosphate donor , atp plays a potent signaling role through its extracellular release and activation of cell surface purinergic receptors . fast responses to atp are mediated through activation of p2x receptors , a family ( p2x1p2x7 ) of atp - activated ligand - gated ion channels and metabotropic effects through the activation of p2y receptors ( p2y12 , p2y4 , p2y6 , p2y1114 ) which couple to heterotrimeric g proteins . human leukocytes including monocytes , mast cells , neutrophils and central microglia express a diverse repertoire of p2y receptors though p2x1 , p2x4 and p2x7 are the dominant p2x subtypes present . activation of purinergic receptors in leukocytes is coupled to the production and secretion of cytokines and other pro - inflammatory molecules including prostaglandin e2 . purinergic receptors are associated with inflammation , with some receptors inhibited either directly ( p2y12 ) or indirectly through the activity of anti - thrombotic , in the case of platelet p2y12 , or anti - inflammatory agents , in the case of the action of statins on monocyte p2x4 . atp can act as a non - peptide damage - associated molecular pattern ( damp ) release from injured cells and tissues . in this fashion the release of cellular atp is unregulated and released due to cell lysis or puncture . cell surface purinergic receptors are activated by this atp damp signal which serves to initiate an inflammatory response and promote wound healing . however , atp can be released from cells physiologically and act as a critical signal for painful , inflammatory processes . do release other nucleotides including utp and udp - sugars but our focus here is atp release . mechanisms of atp release during physiological processes remain diverse and controversial . investigation into how cellular stress stimulates atp release in non - leukocytes suggests roles for connexin and pannexin hemichannels , maxi- and volume - regulated anion channels and efflux through the p2x7 receptor , though release routes and signal transduction mechanism underlying atp release in leukocytes remain poorly defined . in neutrophils atp is released in response to activation of fmlp receptor by bacterially derived n - formylmethionine . the precise release mechanism is unclear but occurs at the leading edge of migrating neutrophils . released atp and its subsequent metabolism to adenosine at the cell surface activate p2y2 and a3 receptors serving to direct cell orientation and promote migration in response to chemotactic signals . bacterially derived lipopolysaccharide can stimulate central microglia to secrete atp which in - turn activates neighboring astrocytes and modulates excitatory neurotransmission . this raises the possibility that atp can act in a feed forward loop possibly amplifying responses to itself or other external cues which couple to atp secretion . furthermore , such constitutive secretion leads to activation of cell surface gq - coupled p2y receptors which elevate intracellular calcium levels through release of calcium . this mode of constitutive secretion generates a constant pericellular atp cloud or halo which appears to be important in regulating intracellular calcium homeostasis following p2y receptor activation . in cells of hematopoietic lineage such as monocyte / macrophage , nk killer and mast cells , secretory lysosomes have evolved as bifunctional organelles which combine classical degradative properties with secretion . the mechanism of lysosomal atp transport remains undefined though a nucleotide transporter ( v - nut ) has been characterized for other atp containing vesicles . the signal transduction coupling external cues such as cytokines , chemokines and bacterially derived molecules to atp release is poorly defined . what are the release machines in leukocytes and do they differ from other cell types ? it is expected that therapeutic intervention in agonist stimulated atp release is a potentially novel route to pharmacological modulation of innate immune responses but also in chronic inflammatory disease where normal inflammatory responses are heighten and act deleteriously .

release and reception of extracellular atp by leukocytes plays a critical role in immune responses to infection , injury and cardiovascular disease . leukocytes of both the innate , adaptive immune and central nervous system express a repertoire of cell surface receptors for atp ( p2x and p2y receptors ) and its metabolites . atp acts as a damage - associated molecule pattern ( damp ) released by injured or dying cells . detection of released atp by neighboring leukocytes initiates inflammation and wound healing . however , recent evidence from our group and others suggests atp release by leukocytes themselves serves to regulate homeostatic mechanisms and coordinate responses to external pro - inflammatory cues . examples include the homeostatic control of intracellular calcium and regulation of migratory guidance during chemotactic response to external cues . though there has been some progress in elucidating atp release mechanisms of some mammalian cells types , release conduits and coupling signal transduction machinery remain larger elusive for leukocytes . our recent studies suggest a role for secretory lysosomes in releasing atp in monocytes . though poorly defined , targeting atp release mechanisms in leukocytes have great anti - inflammatory potential .

an increased incidence of hashimoto thyroiditis has been reported in patients with turner syndrome . in view of its pathogenic relationship with autoimmune thyroiditis we report the case of a 9-yr - old girl with turner syndrome who developed graves disease during recombinant human gh therapy , and we discuss previous reports of this association . the patient was the first child of healthy unrelated parents of normal stature , father 175.0 cm and mother 159.0 cm . the patient was born after an uncomplicated pregnancy at a gestational age of 37 wks and 1 d. her birth weight was 2490 g. on examination , lymphedema and webbed neck were detected . chromosomal analysis of peripheral blood lymphocytes ( n=26 ) showed complete deletion of the short arm of the second x chromosome ( karyotype : 45 , x ) . at the age of 5 yr and 2 mo , her height was 98.2 cm ( 2.25 sd ) and her weight was 14.4 kg ( 1.48 sd ) . a gh stimulation test was performed , and the peak gh values after stimulation with clonidine and arginine were 8.39 ng / ml and 5.56 ng / ml , respectively . of note is that diagnosis of complete gh deficiency is less than 5 ng / ml in this assay . recombinant human ( rh ) gh therapy ( 0.175 mg / kg / wk ) was initiated with success ( fig . thyroid function tests showed a tsh level of 0.01 u / ml ( normal , 0.50 to 5.50 u / ml ) , a free ( f ) t4 value of 6.00 ng / dl ( normal , 0.85 to 1.80 ng / dl ) , and a free t3 value of 21.76 pg / ml ( normal , 2.50 to 5.50 pg / ml ) . anti - thyroglobulin antibody was 3.4 u / ml ( normal , < 0.3 u / ml ) . anti - thyroid peroxidase antibody was 5.5 u / ml ( normal , < 0.3 u / ml ) . tsh receptor antibody ( trab ) was 52% ( normal , < 15% ) . these results are compatible with a diagnosis of graves disease and treatment with thiamazole was started . growth curve of the patient at the latest follow up ( 9 y old ) , the development of the patient s breast was tanner stage 1 . treatment with thiamazole was continued and the thyroid function has stayed within normal ranges . the association of turner syndrome with graves disease was reported in english about 1020 years ago ( 1,2,3,4,5,6,7,8,9,10 ) . table 1table 1 graves disease in turner syndrome summarizes the historical thirteen known cases of graves disease with turner syndrome , including our case . this table reveals that the clinical characteristics of graves disease with turner syndrome are similar to those known generally in patients without turner syndrome in terms of ages of the onset , symptoms and prognosis . treatment with rhgh was given to 4 patients in table 1 , and it was continued in 3 out of the 4 patients including our case . this continuation of gh treatment , together with the successful treatment of hyperthyroidism , suggests that a direct link between gh treatment and graves disease is unlikely . consistent with this hypothesis , the incidence of hyperthyroidism in japanese patients with turner syndrome receiving rhgh therapy is 0.40% , which is similar to overall incidence of hyperthyroidism in patients with turner syndrome , 00.5% ( 11 , 12 ) . it is well known that hyperthyroidism in patients with turner syndrome leads to acceleration of height velocity ( 6 , 13 ) . in our patient , the height velocity was not increased by hyperthyroidism , probably because her growth had been already accelerated by rhgh therapy before the onset of hyperthyroidism . it is important to monitor changes of thyroid function as well as growth parameters in patients with turner syndrome , as is recommended by sybert and mccauley ( 14 ) .

an increased incidence of hashimoto thyroiditis has been reported in patients with turner syndrome , but several cases of graves disease were also described ten to 20 years ago . we report the case of a patient with turner syndrome who developed graves disease , 3 years after successful treatment with recombinant human growth hormone ( gh ) . a diagnosis of graves disease was made and treatment with thiamazole was started , which resulted in normalization of the thyroid function . it is important to monitor thyroid function as well as growth parameters in patients with turner syndrome .

optical coherence tomography ( oct ) is a noninvasive method that evaluates biological tissues by in vivo imaging.1 since its introduction , oct has undergone several improvements and revolutionized the diagnostic , monitoring , and therapeutic approaches to many retinal diseases and glaucoma . computerized algorithms can be used on the high - resolution images obtained by modern oct devices in order to identify and measure the thicknesses of discrete retinal layers , including the retinal nerve fiber layer ( rnfl ) , macular ganglion cell complex ( gcc ) , and choroid.2,3 the choroid is the most vascular tissue in the eye and is known to have an important role in ocular nutrition , volume regulation , and temperature control.4 choroidal abnormalities are critical to the onset and progression of many chorioretinal disorders such as age - related macular degeneration ( amd ) , polypoidal choroidal vasculopathy , vogt - kayanagi - harada disease , and central serous chorioretinopathy.5 with the recent development of enhanced depth imaging ( edi ) , in vivo assessment of the choroid has become an area of interest . edi allows better visualization and quantitative evaluation of the choroid , which was not possible before . information about the choroidal thickness ( ct ) is useful in many clinical situations for decision making regarding the management and monitoring of disease progression.6 neurodegenerative diseases such as glaucoma preferentially affect the gcc , which is the sum of the three innermost layers : the rnfl , the retinal ganglion cell layer , and the inner plexiform layer . these contain the axons , cell bodies , and the dendrites of the retinal ganglion cells , respectively.7,8 recent studies have shown that measurement of macular gcc thickness has the same diagnostic potential for glaucoma as rnfl thickness.911 moreover , it has been reported that measurements of average gcc thickness had better sensitivity in detecting changes in retinal thickness in multiple sclerosis neurodegeneration than rnfl thickness measured by oct.12 both ct and gcc thickness measurements can be affected by several factors such as pupil size , scan quality , and cataract . in this study , we aimed to evaluate the effect of uneventful cataract surgery on subfoveal ct and gcc thickness measured by edi - oct . this prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . the present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus , hypertension , glaucoma , amd , diabetic retinopathy , retinal vein occlusion , uveitis , or other inflammatory and vascular pathologies . patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure ( iop ) or cystoid macular edema were also excluded . all patients had a full ophthalmic examination , including best - corrected visual acuity ( bcva ) assessment , as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation , and iop measurement by goldmann applanation tonometry . axial length ( al ) was measured by immersion echography ( axis ii ; quantel medical inc . , cedex , france ) and central corneal thickness ( cct ) was measured by ultrasonographic pachymetry ( pacline ; optikon 2000 spa , rome , italy ) . prior to the operation , tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . phacoemulsification was performed by one experienced surgeon ( ec ) using infiniti vision system ( alcon laboratories , inc . ) . a clear corneal incision of 2.8 mm , 600 mmhg vacuum , an aspiration flow rate of 30 ml / min , bottle height of 75 cm , and quick - chop technique were used in all surgeries . two different dispersive viscoelastics ( viscoat and bivisc ) and two different cohesive viscoelastics ( healon and bio - hyalur ) were used during cataract surgery . one - piece hydrophobic acrylic intraocular lens ( sensar model : aab00 ; abbott medical optics inc . , santa ana , ca , usa ) was used in all surgeries . viscoelastics were cleaned out with marked attention , even behind the intraocular lenses . the procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . the operative times ( ots ) and effective phaco times ( epts ) were recorded in each case . postoperatively , all patients were treated with topical antibiotics ( ofloxacin 0.3% ) for 1 week and topical steroid drops ( prednisolone acetate 1% ) for 4 weeks . cirrus edi - oct ( carl zeiss meditec ag , jena , germany ; software version 6.5.0.772 ) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis ( macular cube 512128 ) scan protocols , respectively . scans with misalignment , segmentation failure , decentration of the measurement circle , and poor illumination , or those out of focus , were also excluded . the one - line raster is a 6 mm line consisting of 4,096 a - scans . scan 3 of the five scans taken , which passes through the fovea , was used for all measurements . ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . to avoid the effects of mydriatic agents or diurnal changes , the ct was measured from the subfoveal region by using the cirrus linear measurement tool . when there was a discrepancy of > 10% of the mean of the two values between the observers in two eyes in the study , observers discussed and repeated the measurements , and the average results from the two observers were determined as the final ct readings . the cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers ( gcipl ) in six circular sectors centered in the fovea . it also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans , each composed of 512 a - scans . a built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . the annulus has an inner vertical diameter of 1 mm , which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately , and an outer vertical diameter of 4 mm , which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . the ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . the difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . similar to other cirrus printouts , it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . those that are abnormal at the < 5% and at the < 1% level are represented by yellow and red backgrounds , respectively . the primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes ; however , central macular thickness ( cmt ) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols , respectively , using the cirrus oct device . statistical analysis was performed using spss statistics for windows , version 20.0 ( ibm corporation 2011 , armonk , ny , usa ) . paired - samples t - test was used for equality of means , and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . this prospective study included consecutive patients who had undergone uneventful phacoemulsification surgery for senile cataract at sakarya university medical education and research hospital between november 2013 and june 2015 . the present study was approved by the institutional review board of sakarya university medical education and research hospital and adhered to the tenets of the declaration of helsinki and all patients signed informed consent after they received an explanation of the nature and possible consequences of the procedure . exclusion criteria included a history of prior ocular surgery and systemic / ocular diseases such as diabetes mellitus , hypertension , glaucoma , amd , diabetic retinopathy , retinal vein occlusion , uveitis , or other inflammatory and vascular pathologies . patients with unstable fixation or dense cataracts leading to a poor scan quality of oct images and eyes with increase in postoperative intraocular pressure ( iop ) or cystoid macular edema were also excluded . all patients had a full ophthalmic examination , including best - corrected visual acuity ( bcva ) assessment , as well as slit - lamp biomicroscopy and dilated fundoscopy evaluation , and iop measurement by goldmann applanation tonometry . axial length ( al ) was measured by immersion echography ( axis ii ; quantel medical inc . , cedex , france ) and central corneal thickness ( cct ) was measured by ultrasonographic pachymetry ( pacline ; optikon 2000 spa , rome , italy ) . prior to the operation , tropicamide 1% and phenylephrine hydrochloride 2.5% were applied to patients eyes for pupil dilation . phacoemulsification was performed by one experienced surgeon ( ec ) using infiniti vision system ( alcon laboratories , inc . ) . a clear corneal incision of 2.8 mm , 600 mmhg vacuum , an aspiration flow rate of 30 ml / min , bottle height of 75 cm , and quick - chop technique were used in all surgeries . two different dispersive viscoelastics ( viscoat and bivisc ) and two different cohesive viscoelastics ( healon and bio - hyalur ) were used during cataract surgery . one - piece hydrophobic acrylic intraocular lens ( sensar model : aab00 ; abbott medical optics inc . , santa ana , ca , usa ) was used in all surgeries . viscoelastics were cleaned out with marked attention , even behind the intraocular lenses . the procedure ended with intracameral cefuroxime axetil 1 mg/0.1 ml for prophylaxis for endophthalmitis . the operative times ( ots ) and effective phaco times ( epts ) were recorded in each case . postoperatively , all patients were treated with topical antibiotics ( ofloxacin 0.3% ) for 1 week and topical steroid drops ( prednisolone acetate 1% ) for 4 weeks . cirrus edi - oct ( carl zeiss meditec ag , jena , germany ; software version 6.5.0.772 ) was used to obtain subfoveal ct and gcc thickness measurements using high - definition one - line raster and ganglion cell analysis ( macular cube 512128 ) scan protocols , respectively . scans with misalignment , segmentation failure , decentration of the measurement circle , and poor illumination , or those out of focus , were also excluded . the one - line raster is a 6 mm line consisting of 4,096 a - scans . scan 3 of the five scans taken , which passes through the fovea , was used for all measurements . ct was defined as the vertical distance from the outer portion of the hyperreflective line corresponding to bruch s membrane beneath the retinal pigment epithelium to the outermost hyperreflective line of the inner scleral border . to avoid the effects of mydriatic agents or diurnal changes , the ct was measured from the subfoveal region by using the cirrus linear measurement tool . when there was a discrepancy of > 10% of the mean of the two values between the observers in two eyes in the study , observers discussed and repeated the measurements , and the average results from the two observers the cirrus oct device allows a quantitative assessment of the ganglion cell and inner plexiform layers ( gcipl ) in six circular sectors centered in the fovea . it also gives information on the mean and minimum gcipl thickness for each eye and compares these figures with a normative database . macular cube 512128 acquisition protocol generates a cube through a 6 mm square grid of 128 b - scans , each composed of 512 a - scans . a built - in gcipl analysis algorithm detects and measures the thickness of the macular gcipl within a 662 mm elliptical annulus area centered on the fovea . the annulus has an inner vertical diameter of 1 mm , which was chosen to exclude the portions of the fovea where the layers are very thin and difficult to detect accurately , and an outer vertical diameter of 4 mm , which was chosen according to where the ganglion cell layer again becomes thin and difficult to detect . the ganglion cell analysis algorithm identifies the outer boundaries of the rnfl and the inner plexiform layer . the difference between the rnfl and the inner plexiform layer outer boundary segmentations yields the combined thickness of the retinal gcipl . similar to other cirrus printouts , it provides gcipl measurements in six wedge - shaped sectors with a pseudocolor scheme . those that are abnormal at the < 5% and at the < 1% level are represented by yellow and red backgrounds , respectively . the primary objective of this study was to evaluate the subfoveal ct and average gcc thickness changes ; however , central macular thickness ( cmt ) and peripapillary rnfl thickness were also obtained by the macular cube 512128 and optic disc cube 200200 protocols , respectively , using the cirrus oct device . statistical analysis was performed using spss statistics for windows , version 20.0 ( ibm corporation 2011 , armonk , ny , usa ) . paired - samples t - test was used for equality of means , and analysis of variance regression and pearson correlation analysis were used for correlations between parameters . a total of 30 eyes of 30 patients ( 16 male and 14 female ) , with a mean age of 60.24.2 years were included in this study . mean bcva improved in all eyes , and this improvement was statistically significant ( 0.460.15 logarithm of minimum angle of resolution [ logmar ] units preoperatively and 0.060.49 logmar units postoperatively ; p<0.001 ) . mean preoperative al was 22.80.6 mm ( range : 21.524.1 mm ) and mean cct was 551.522.1 m . mean ot was 8.62.7 minutes and mean ept was 6.92.5 seconds ( table 1 ) . the mean subfoveal ct was 294.439.2 m ( range : 201356 m ) preoperatively and 301.439.9 m ( range : 231367 m ) postoperatively . the mean gcc thickness was 85.04.4 m ( range : 7894 m ) preoperatively and 89.25.3 m ( range : 8199 m ) postoperatively . the mean cmt was 247.917.6 m preoperatively ( range : 211280 m ) and 249.017.8 m ( range : 213278 m ) postoperatively . the mean rnfl thickness was 97.45.4 m preoperatively ( range : 87110 m ) and 101.75.6 m ( range : 91113 m ) postoperatively . regression analysis showed that age , sex , al , cct , ot , and ept were not associated with ct changes ( p=0.834 , p=0.129 , p=0.203 , p=0.343 , p=0.547 , and p=0.147 , respectively ) and gcc thickness changes ( p=0.645 , p=0.542 , p=0.152 , p=0.664 , p=0.448 , p=0.268 , respectively ) after cataract surgery . in this study , we investigated the effect of cataract surgery on subfoveal ct and gcc thickness , as measured by edi - oct , and we found that both subfoveal ct and gcc thickness increased slightly after cataract surgery . cmt and peripapillary rnfl thicknesses were also evaluated as a secondary objective , and we observed statistically significant increases in cmt and rnfl thickness . our results indicate that cmt , subfoveal ct , as well as rnfl and gcc thicknesses are slightly affected after phacoemulsification surgery . although our study included a small group ( only 30 eyes ) , we assume that our data reliability is high because of the homogeneous nature of the patients ( similarities of age , cataract type and severity , cct , al , ot , ept and so on ) and the careful / detailed oct examinations performed by two observers , avoiding the effects of diurnal changes and mydriatic agents . zhao et al13 revealed significant diurnal variations of the ct at the macular region in young healthy female individuals . cataract surgery by phacoemulsification is one of the most common intraocular surgeries that improve the quality of vision . however , it is an invasive procedure leading to an inflammatory response mostly induced by the release of prostaglandins in the retina and choroid and , in many patients , can lead to worsening of preexisting retinal diseases such as diabetic macular edema.15,16 recent studies have shown subclinical increase of cmt and peripapillary rnfl thickness after uneventful cataract surgery.1719 however , the morphologic changes that this inflammatory insult produces in the choroid have not been studied well because of the lack of detailed imaging techniques for the choroid . after the introduction of edi , changes in ct in several pathologic ( central serous chorioretinopathy , choroidal neovascularization , and high myopia ) and physiologic ( age , sex , and al ) conditions can be easily assessed.2022 therefore , ct is being measured increasingly often and is becoming an accepted procedure for clinical and research applications . falco et al23 evaluated the effect of uneventful phacoemulsification on the subfoveal ct and cmt , and they reported that phacoemulsification induced nonpathologic increases in cmt ; however , these changes were not accompanied by significant changes in ct . they claimed that the inflammatory insult due to phacoemulsification mainly affects at the retinal level and seems to be independent of ct changes.23 by contrast ; our results support the recent studies by ohsugi et al24 and noda et al,25 which report significant increases in ct after uneventful phacoemulsification . ohsugi et al24 evaluated 100 eyes and emphasized that the al and changes in iop were critical for evaluating the changes in ct after cataract surgery . noda et al25 evaluated 29 eyes and observed that the increase in subfoveal ct did not subside to baseline even at 6 months postoperatively . they also found that male sex and thicker baseline ct predicted a larger magnitude of increase in ct after cataract surgery.25 in our study , interestingly , the parameters of age , sex , al , baseline ct , and cct did not influence the postoperative ct statistically in regression analysis . we also analyzed the effect of ot and ept on postoperative ct and we did not find any relationship . this may be explained by the short / similar results in terms of ots and epts , as well as the experience of the surgeon conducting a quick surgery with less anterior segment manipulation , thus reducing the inflammatory response after cataract surgery . a speculation is that it may be related to postoperative inflammation because proinflammatory prostaglandins and cytokines are considered to explain macular edema after cataract surgery , and inflammatory disorders are also known to increase the ct.26,27 another speculation is that cataract surgery introduces more light into the eyes , leading to greater metabolic activation in the retinal pigment epithelium , causing angiogenesis and inflammation.28,29 in the beaver dam and blue mountains eye studies,3032 it has been suggested that cataract surgery is associated with the onset of amd , resulting in visual impairment due to neovascularization originating from the choroid . noda et al25 report that cataract surgery influences the ct and this influence persists for as long as 6 months postoperatively and may affect the process of amd . choroidal abnormalities are central to chorioretinal disorders such as amd , polypoidal choroidal vasculopathy , and central serous chorioretinopathy . furthermore , it has been claimed that pathologic choroidal blood supply is an important factor in open - angle glaucoma leading to optic nerve damage.33,34 cts of glaucomatous eyes have been reported to be thinner in postmortem histologic studies . in addition , significant increases in choroidal extravascular volume have been confirmed in many primary angle - closure glaucoma eyes and abnormal ct has been hypothesized to be a contributing feature in these eyes.35,36 however , it is unclear whether these findings represent a risk factor or a consequence of these diseases . the hallmark of glaucoma is the loss of the retinal ganglion cell axons , which leads to a typical optic neuropathy . qualitative and quantitative evaluations of the optic nerve head and rnfl have been used to detect evidence of glaucomatous damage.911 in a recent study,8 it has been suggested that the gcc thickness may be the most relevant parameter to measure in glaucoma . nouri - mahdavi et al37 also reported that regional gcc measurements derived from cirrus hd - oct performed as well as regional rnfl outcomes for the detection of early glaucoma . moreover , macular gcc thickness has been found to have better structure function correlations than rnfl thickness with both visual function and central nervous system findings in multiple sclerosis neurodegeneration.12 as oct uses near - infrared light and is based on interferometry , its image quality is influenced by opacities in the optical path . loss of oct image quality is caused by attenuation of the light in the oct scanning spot on the retina.17,18 several studies have shown that lens opacity decreased image quality and postoperative rnfl thickness measurements increased significantly.3840 the more advanced the cataract , the less is the signal quality and the thinner are the recorded rnfl and gcc thicknesses . the coexistence of neurodegenerative diseases such as glaucoma and cataract among elderly patients is not unusual , and cataract may decrease the quality of fundus images , leading to an incorrect evaluation in the diagnosis and follow - up of these patients . nakatani et al7 examined the effect of cataract on the measurements of gcc and rnfl thicknesses , and they reported that all thickness parameters in gcc and rnfl increased slightly after phacoemulsification surgery . therefore , we decided that the presence of cataract may lead to an underestimation of the thicknesses of gcc and rnfl , and this should be taken into account when analyzing progression in glaucoma and/or neurodegenerative diseases such as multiple sclerosis . after cataract surgery , the examiners should consider obtaining new baseline measurements , as suggested in previous studies . this study has many limitations such as short follow - up time , relatively small sample size , and lack of eyes with glaucoma and neurodegenerative diseases . in addition , we analyzed just mean subfoveal ct and mean thickness of gcc and rnfl , leading to lack of segmentation of these parameters . scleral interface and measured ct with cirrus linear measurement tool . although we used scans with signal strength 6 for analysis and excluded poor images , the effect of signal strength index on measurements was not investigated . furthermore , we used postoperative topical steroids after cataract surgery ; thus , the true effect of phacoemulsification on oct parameters could not be evaluated and we could not determine whether and when the increased ct can return to baseline measurements . phacoemulsification surgery increased ct and it should be kept in mind that the small changes in ct may affect the onset of amd , which is a serious disease that results in central visual impairment . although the mechanism is unclear , it has been postulated that ct changes may also play a role in glaucoma . we suggest establishment of new baseline measurements after cataract surgery for monitoring patients with glaucoma and neurodegenerative diseases . further studies with a greater number of patients and longer duration of follow - up and conducted using new computerized oct algorithms are needed .

purposewe aimed to evaluate the effect of cataract surgery on subfoveal choroidal thickness ( ct ) and ganglion cell complex ( gcc ) thickness , as measured by enhanced depth imaging - optical coherence tomography ( oct).methodsthis prospective study included 30 eyes of 30 patients who had undergone uneventful phacoemulsification surgery for senile cataract but had no previous ocular surgery or other ocular abnormality . best - corrected visual acuity , slit - lamp biomicroscopy , intraocular pressure , axial length , and central corneal thickness were measured preoperatively . the operative times ( ots ) and effective phaco times were also recorded in each case . oct measurements were performed at the preoperative visit and 1 month after cataract surgery . study of ct and gcc thickness changes was the primary objective , but central macular thickness ( cmt ) and peripapillary retinal nerve fiber layer ( rnfl ) thicknesses were also obtained by oct.resultsthe mean subfoveal ct was 294.439.2 m preoperatively and 301.439.9 m postoperatively ( p<0.001 ) . the mean gcc thickness was 85.04.4 m preoperatively and 89.25.3 m postoperatively ( p<0.001 ) . the mean cmt was 247.917.6 m preoperatively and 249.017.8 m postoperatively ( p=0.029 ) . the mean rnfl thickness was 97.45.4 m preoperatively and 101.75.6 m postoperatively ( p<0.001 ) . regression analysis showed that age , sex , axial length , central corneal thickness , operative time , and effective phaco time were not associated with ct changes ( p=0.834 , p=0.129 , p=0.203 , p=0.343 , p=0.547 , and p=0.147 , respectively ) and gcc thickness changes ( p=0.645 , p=0.542 , p=0.152 , p=0.664 , p=0.448 , and p=0.268 , respectively ) after cataract surgery.conclusionour results indicate that all subfoveal ct , cmt , as well as rnfl and gcc thicknesses are slightly affected after uneventful phacoemulsification surgery . after cataract surgery , the examiners should consider obtaining new baseline measurements .

defects in many of the molecules that regulate the cell cycle have been implicated in cancer initiation and progression . these include p53 , rb , p107 and prb2/p130 , and cdk inhibitors ( p15 , p16 , p18 , p19 , p21 , p27 ) , all of which act to prevent cell cycle progression until all repairs to damaged dna have been completed . in this section , we will review tumor suppressor genes that regulate cell cycle and apoptosis that have been found to be methylated in gastric cancers . tumor suppressor genes which are most recently found to be methylated in gastric cancers regulate cell cycle progression and apoptosis . kim et al . reported that promoter methylation of early b - cell factor-3 ( ebf3 ) was detected in 40.4% ( 42/104 ) of gastric cancer tissues but not in normal gastric tissues . the ebfs are a group of four highly conserved , dna - binding transcription factors with an atypical zinc - finger domain and a helix - loop - helix domain . functional analysis demonstrates that ebf3 represses gastric cancer cell growth and migration but activates cell cycle arrest and apoptosis . ebf3 up - regulates p21 and p27 in gastric cancer cells but represses cdk2 , cyclin d1 , cyclin d2 , and rb as previously reported in different types of cancers , . interestingly , in addition to their central roles in apoptosis regulation , the bcl-2 family of proteins influences the cell cycle , specifically the transition between quiescence and proliferation . bcl2l10 methylation was detected in 44.5% of gastric cancers and 21.34% of normal gastric mucosae ( p < 0.05 ) by bisulphite sequencing . the pro - apoptotic effect of bcl2l10 and growth promotion by sirna targeting bcl2l10 in gastric cancer cells suggests that bcl2l10 may be a tumor suppressor by inducing apoptosis through mitochondrial pathways . the iroquois homeobox protein 1 ( irx1 ) gene is located on chromosome 5p15.33 , which is a cancer susceptibility locus . irx1 transcription was suppressed by hypermethylation in gastric cancer , and restoring irx1 expression in sgc-7901 and nci - n87 gastric cancer cells inhibited tumor growth , invasion , and tumorigenesis in vitro and in vivo . irx1 directly targets bradykinin receptor b2 ( bdkrb2 ) , an angiogenesis - related gene , as well as histone h2b type 2-e ( hist2h2be ) and fibroblast growth factor 70 ( fgf7 ) , cell proliferation and invasion related genes . hypermethylation of irx1 was detected not only in primary gastric cancer tissues but also in the peripheral blood cells of gastric cancer patients , suggesting that irx1 could potentially serve as a biomarker for gastric cancer . the cklf - like marvel transmembrane domain - containing family ( cmtm ) is a novel family of proteins linking chemokines and the transmembrane-4 superfamily . wang et al . reported that cmtm3 is frequently methylated in many carcinoma cell lines and some primary tumors including gastric cancer , resulting in loss of its expression at both the mrna and protein levels . ectopic restoration of cmtm3 expression in tumor cells leads to the suppression of cell growth and induction of apoptosis with caspase-3 activation , suggesting that cmtm3 may function as a tumor suppressor . hypermethylation signals were also observed in some cultured and primary gastric cancers with little or no expression of transcription factor sox2 , a sox transcription factor . among the 52 patients with advanced gastric cancers who were tested , those having sox2 gene methylation in their cancer tissues had a significantly shorter survival than did those without the methylation ( p = 0.006 ) . exogenous expression of sox2 inhibited cell growth through apoptosis and cell cycle arrest . sox2-overexpressing cells exhibited characteristics of apoptosis , such as dna laddering and caspase-3 activation . cell cycle arrest was associated with decreased levels of cyclin d1 and phosphorylated rb as well as an increased p27 level . reported that zinc - finger transcription factor znf382 functions as a tumor suppressor in many types of carcinomas including gastric cancer . ectopic expression of znf382 in tumor cells in which the gene was silenced significantly reduced clonogenicity and proliferation and induced apoptosis . further found that znf382 inhibited nf-b and ap-1 signaling and down - regulated the expression of multiple oncogenes , including myc , mitf , hmga2 , cdk6 , stat3 , stat5b , id1 , and ikbke , most likely through heterochromatin silencing . a , pyrosequencing ; b , q - msp ; c , high - resolution melting ( hrm ) analysis ; d , methylight ; e , bisulfit sequencing . methylation of the ubiquitin carboxyl - terminal hydrolase l1 ( uchl1 ) was detected in primary digestive tumors , including 77% ( 53/69 ) of gastric carcinomas , but not in or occasionally in paired adjacent non - tumor tissues . restoring uchl1 expression in cell lines where the gene had been silenced significantly inhibited their growth and colony formation ability by inhibiting cell proliferation , causing cell cycle arrest in g2/m phase , and inducing apoptosis through the intrinsic caspase - dependent pathway similarly , sdr - related gene product that binds to c - kinase ( hsrbc ) , another novel methylated tumor suppressor gene in gastric cancer , increased the stability of p53 and expression of p53 target genes , such as p21 , puma , and noxa . hsrbc - mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function . these results suggested that epigenetic inactivation of hsrbc contributes to the malignant progression of gastric tumors , in part , through attenuated p53 response to stress . moreover , opioid binding protein / cell adhesion molecule - like gene ( opcml ) is also a stress- and p53-responsive gene , but this response is epigenetically impaired when the opcml promoter becomes methylated . ecotopic expression of opcml led to significant inhibition of both anchorage - dependent and -independent growth of carcinoma cells in which the gene had been silenced . as reviewed above , many methylated tumor suppressor genes in gastric cancer contribute to the dysregulation of the important aspects of tumorigenesis : cell cycle and apoptosis . the understanding of these molecular mechanisms will provide accesses to novel molecular therapeutic strategies for inhibiting oncogenic activity of signaling pathways . morbidity in most cancer patients is not due to primary cancer but to metastatic disease . the high death rate of gastric cancer strikingly correlates with the high metastatic capacity of most gastric cancers . thus , understanding tumor progression to the metastatic state and changes in highly aggressive cells is important for the development of novel approaches to diagnose and treat aggressive malignancies . detachment of cells with increased motility from a primary tumor is the first step of cancer invasion and metastasis . gastric cancer cells with fibroblastoid morphological changes show increased motility and invasiveness due to decreased cell - cell adhesion , which is reminiscent of epithelial - mesenchymal transition ( emt ) during embryonic development . the dynamic regulation of cell - cell adhesion is crucial for developmental processes including tumorigenesis . with reduced cell - cell adhesiveness , cancer cells can disobey the social order and result in destruction of the histological structure . a common and early requirement for cell motility is actin polymerization , which drives the formation of cell protrusions that are used to adhere to the extracellular matrix , define the direction of migration and initiate cell crawling . animal cells respond to signaling at the plasma membrane by remodeling their actin cytoskeleton . on the other hand , the movement of cells into a tightly woven extracellular matrix also require an active proteolytic system , which can cleave a path for cell migration . so the genes regulating the host cell cytoskeleton rearrangement and proteolytic activity also have important functions in cell morphogenesis and motility . our group reported that epigenetic inactivation of phospholipase c delta 1 ( plcd1 ) is common and tumor - specific in gastric cancer and that plcd1 acts as a functional tumor suppressor in gastric carcinogenesis . located at the important tumor suppressor locus 3p22 , plcd1 encodes an enzyme that regulates energy metabolism , calcium homeostasis , and intracellular movements . ectopic expression of plcd1 in gastric tumor cells with silenced plcd1 dramatically inhibited clonogenicity and migration , possibly through down - regulation of mmp7 expression and hampered cyto - skeletal reorganization via phosphorylation and inactivation of cofilin . deleted in liver cancer 1 ( dlc1 ) is another tumor suppressor gene involved in the regulation of cytoskeletal organization and other functions and is frequently methylated in many cancers , including gastric cancer , . recently , its new isoform , dlc1 isoform 4 ( dlc1-i4 ) , was also found to be silenced epigenetically , to have tumor inhibitory properties in multiple carcinomas , and to be regulated by p53 . . the down - regulation of e - cadherin may favor dissociation of cancer cells from one another , facilitating their invasion through the basal membrane . although mutation and allelic loss have been confirmed as major mechanisms for e - cadherin ( cdh1 ) gene inactivation in many malignancies , cdh1 promoter methylation could be frequently detected in gastric carcinoma . as a co - partner of e - cadherin , -catenin not only is critical for cell adhesion in the membrane and cytoplasm of cells , but also plays a role as a transcription activating protein in nuclei . aberrant activation of the wnt/-catenin signaling pathway is frequently found in many cancers , including gastric cancer , . in addition to genetic deletions and point mutations , a number of negative regulators of wnt signaling , including secreted frizzled - related protein ( sfrp ) , dickkopf homolog 2 ( dkk2 ) , dickkopf homolog 2 ( dkk3 ) and wnt inhibitory factor 1 ( wif1 ) , were frequently methylated in gastric cancer. recently , other genes involving wnt pathway were also reported as methylated tumor suppressor genes in gastric cancer . for example , sry - box containing gene 17 ( sox17 ) was reported to be indispensable for embryonic development and a candidate tumor suppressor gene that antagonizes the canonical wnt/-catenin signaling pathway in colorectal cancer . treatment with a demethylating agent induced sox17 expression in gastric cancer cells , thus indicating the down - regulation of sox17 by methylation . transgenic expression of sox17 suppressed dysplastic tumor development in the stomach of k19-wnt1/c2me mice by suppressing wnt activity . these results suggested that sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis , and down - regulation of sox17 contributes to malignant progression through promotion of wnt activity . the popeye domain - containing ( popdc ) genes bves , popdc2 , and popdc3 encode proteins that regulate cell - cell adhesion and cell migration during development . bves and popdc3 were reported to be hypermethylated in 69% and 64% of the gastric cancer tissues , respectively . promoter hypermethylation is a causal event for long - term repression of bves and popdc3 , whereas egf stimulation is an immediate repression mechanism for both genes in gastric cancer . bves , popdc3 , and e - cadherin mrnas were down - regulated and snail mrna expression was up - regulated in egf - induced emt in snu-216 cells . interestingly , accumulating evidence suggests that a major subfamily within the cadherin superfamily , protocadherins , frequently act as tumor suppressor genes . recent studies have shown that the main structural and functional properties of protocadherins are distinct from those of classical cadherins . they may not have the strong cell - cell adhesion activity but do have other functions , such as mediating specificity of cell - cell interactions and signal transduction . yu et al . reported that protocadherin 10 ( pcdh10 ) was silenced or down - regulated in 94% ( 16 of 17 ) of gastric cancer cell lines . furthermore , pcdh10 methylation was detected in 82% ( 85 of 104 ) of gastric tumors compared with 37% ( 38 of 104 ) of paired non - tumor tissues ( p < 0.001 ) . re - expression of pcdh10 reduced colony formation in vitro and tumor growth in vivo . it also inhibited cell proliferation , induced cell apoptosis , and repressed cell invasion , possibly through up - regulation of the pro - apoptosis genes fas , caspase 8 , jun , and cdkn1a ; the anti - proliferation gene fgfr ; and the anti - invasion gene htatip2 . in cancer cells , glucose is often converted into lactic acid , which is known as the warburg effect . the reason that cancer cells have a higher rate of aerobic glycolysis , but not oxidative phosphorylation , remains largely unclear . recently , fructose-1,6-bisphosphatase-1 ( fbp1 ) , which functions to antagonize glycolysis , was reported to be down - regulated through the nf-b pathway in ras - transformed nih3t3 cells . fbp1 was hypermethylated in 57% ( 4/7 ) of gastric cancer cell lines and 33% ( 33/101 ) of gastric carcinomas . restoration of fbp1 expression suppressed anchorage - independent growth , indicating the relevance of fbp1 down - regulation in carcinogenesis . leucine - rich repeat - containing 3b ( lrrc3b ) is an evolutionary highly conserved leucine - rich repeat - containing protein , but its biological significance is unknown . recently , lrrc3b was identified as a putative tumor suppressor gene and is reportedly silenced in gastric cancers by epigenetic mechanisms . stable transfection of lrrc3b in gastric cancer cell line snu-601 inhibited anchorage - dependent and anchorage - independent colony formation . moreover , lrrc3b expression suppressed tumorigenesis in nude mice . microarray analysis of lrrc3b - expressing xenograft tumors showed induction of immune response - related genes and ifn signaling genes . hematoxylin and eosin ( h&e)-stained sections of lrrc3b - expressing xenograft tumors showed lymphocyte infiltration in the region . these results suggested that lrrc3b silencing in cancer may play an important role in tumor escape from immune surveillance . as reviewed above , aberrant dna methylation of promoter cpg islands is one of the major inactivating mechanisms of tumor suppressor genes and is deeply involved in gastric carcinogenesis . moreover , in gastric cancer , dna methylation from the pre - malignant till the most aggressive stage of cancer can be defined . for instance , many genes such as thbd , lox , hrasls , flnc , and hand1 were found to be infrequently methylated in non - cancerous gastric mucosae , in addition to their frequent methylation in cancers . similar findings were reported for cdh1 , dapk , p14 , thbs1 , and timp-1 . the presence of trace amounts of methylation in non - cancerous gastric mucosae suggested that some gastric carcinogens could have induced the methylation . the most important gastric carcinogenic factor is helicobacter pylori ( h. pylori ) infection , which increases the risk of developing gastric cancers by 2.2- to 21.0-fold , . recent studies to quantify dna methylation changes induced by the h. pylori infection in light of imflammatory reactions have been performed in both animal models and in the clinical setting . the findings suggest that h. pylori infection induces aberrant methylation in gastric mucosae and that levels of accumulated methylation are associated with gastric cancer risk . notably , although h. pylori infection is important to trigger inflammation capable of inducing aberrant dna methylation , some inflammation processes appear to be critical in induction of aberrant dna methylation. indeed , the inflammation induced by h. pylori infection , not h. pylori itself , was critically involved in methylation induction . h. pylori may induce methylation of promoters containing cpg islands by release of reactive oxygen species ( ros ) and nitric oxide ( no ) and by activation of dna methyltransferase , . more recently , sepulveda et al . suggested that permanent dna methylation after h. pylori eradication may occur in stem cells , thus promoting tumorigenesis . considering the significant involvement of aberrant dna methylation of cpg islands in human cancers , further identification of the inducing factors and mechanisms can be expected to provide novel targets for cancer prevention . in addition to regional hypermethylation , global hypomethylation is also purported to be a hallmark of cancer cells and is known to cause chromosomal instability as an early event during gastric carcinogenesis . however , yoshida et al . reported that h. pylori infection potently induces alu and sata hypomethylation in gastric mucosae as an early event during gastric carcinogenesis , whereas global hypomethylation is present only in some individuals . thus , the use of hypomethylation as a risk marker has not been considered realistic . aberrant hypermethylation of promoter regions of specific genes is a key event in the formation and progression of cancer . an accumulating number of studies have reported that gene silencing by dna methylation may be established at an early stage in the multistep process of carcinogenesis . methylations of specific genes or methylation patterns of groups of genes were found to be associated with chemotherapy response and prognosis . thus , aberrant dna methylation can be applied to cancer diagnostics in three ways : as a marker to detect cancer cells or cancer - derived dna , as a marker to predict prognosis , and as a biomarker for the assessment of therapeutic response . methylation analysis has an advantage in that it can be performed using chemically stable dna ( compared to rna ) . moreover , detecting gene methylation is easier than detecting gene mutation because the exact location of a mutation is usually unknown , making it difficult to specifically amplify dna molecules with an embedded mutation in excess of wild - type molecules . detection of aberrant methylation can provide confirmation of the presence of intact cancer cells or cancer - derived dna in bodily fluids , such as blood , urine , sputum , saliva , and stools . recently , the methylations of bnip3 , chfr , cyp1b1 , mint25 , sfrp2 , and rassf2 were analyzed in 107 specimens of peritoneal fluid by quantitative methylation - specific polymerase chain reaction ( msp ) . the results showed that dna methylation in periotoneal fluid is a possible marker for detecting occult neoplastic cells on the peritoneum . methylation analysis along with a cytological examination might , therefore , improve the positive detection of cancer cells in the peritoneal fluid of gastric cancer patients . the development of the bisulfate conversion technique that reproducibly changes unmethylated cytosines to uracil but leaves methylated cytosines unchanged rapidly increased progress in dna methylation detection . bisulfite sequencing , msp , and combined bisulfite restriction analysis ( cobra ) were all developed on the basis of bisulfite conversion among these technologies , msp is a subjective , gel - based assay and can not provide quantitative information . to date , several real - time msp methods , such as bisulfite treatment in combination with methylight , quantitative multiplex - msp(qm - msp ) , , or pyrosequencing , have been developed and used in dna methylation studies . for example , a recent study showed that one pyrosequencing analysis of global and site - specific dna methylation in peripheral blood samples from 105 gastric cancer patients provides quantitative dna methylation values that may serve as important prognostic indicators . however , prior to clinical application , these findings require validation in prospective clinical studies . , an increasing number of drugs targeting dna methylation have been improved to increase efficacy and to decrease toxicity . 5-azacyti - dine , the most successful epigenetic drug to date , is currently recommended as the first - line treatment of high - risk myelodysplastic syndromes ( mds ) . new methods for gene - specific epigenetic modification are being developed and tested in solid tumors . gastric cancer is a biologically heterogeneous disease with various molecular tumor subsets that likely respond distinctly to therapy . methylation of genes involved in dna repair and maintaining genome integrity ( e.g. mgmt , hmlh1 , wrn , and fancf ) , as well as genes involved in cell cycle checkpoints ( e.g. chfr , 14 - 3 - 3 , cdk10 , and p73 ) all reportedly influence sensitivity to chemotherapeutic drugs , suggesting that dna methylation could serve as a molecular marker for predicting tumor responsiveness to chemotherapy . an investigation of dna methylation using specialized high - throughput platforms can potentially be applied to further stratify patients to individualized therapies . methylated tumor suppressor genes are being intensively investigated in gastric cancer , but the underlying functions and mechanisms need to be carefully examined . future investigations of the connections between h. pylori infection , gastric cancer , and epigenetic changes will greatly expand our understanding . we believe that information obtained from studying dna methylation will have an impact on cancer prevention , diagnostics , and treatment , and will contribute to cancer elimination . however , proper selection of methylation markers is crucial for sensitive and specific detection , and further technological advances are necessary in the future .

gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide . the pathogenesis mechanisms of gastric cancer are still not fully clear . inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis . accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes , particularly caused by hypermethylation of cpg islands in promoters , is critical to carcinogenesis and metastasis . here , we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer . we also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications .

pregnancy has a profound physiological impact on the thyroid gland and thyroid function . during pregnancy , the thyroid gland increases in size by 10% in iodine sufficient countries and to a greater extent in iodine deficiency countries . production of thyroid hormones and iodine requirement both increases by approximately 50% during pregnancy as part of physiology . in addition , pregnancy is a stressful condition for the thyroid gland resulting in hypothyroidism in women with limited thyroid reserve or iodine deficiency . data from recently published studies have underscored the association between miscarriage and preterm delivery in women with normal thyroid function who test positive for thyroid peroxidase ( tpo ) antibodies . prenatal and postnatal adverse effects including attention deficit and hyperactivity syndrome have been reported in children born to hypothyroid mothers . during the first trimester , approximately 1 in 10 pregnant women develop antibodies to tpo or thyroglobulin and hypothyroidism develops in roughly 16% of these women . there are a few reports of prevalence of hypothyroidism during pregnancy from india with prevalence rates ranging from 4.8% to 11% . therefore , this study was carried out in a larger cohort of pregnant women during the first trimester from a government hospital setting catering to majority of women from lower socioeconomic status . this was a cross - sectional multicenter study conducted at allahabad , bengaluru , rohtak , chennai , kolkata , hyderabad , nasik , pune , new delhi , srinagar , and vizag to assess the incidence of hypothyroidism in pregnant women . complete blood count ( hemogram ) , total cholesterol , triglycerides , serum creatinine , and blood urea nitrogen were evaluated for all subjects . estimation of thyroid stimulating hormone ( tsh ) , free t4 , and anti - tpo antibodies was carried out using roch modular kit using eclia technology . the intra - assay precision of tsh and free t4 was 3 and 2 , whereas interassay precision was 7.2 and 4.8 , respectively . the mean age of the study population was 25.5 5.6 years with a mean gestational age of 19.3 15.9 weeks . demographic characteristics of the study population ( n=2955 ) keeping upper normal limit of tsh < 4.5 , we found in our study population 13.13% of pregnant women to be hypothyroid ( n = 388 ) . anti - tpo antibodies were positive in 20.74% of all pregnant women ( n = 613 ) , whereas 40% ( n = 155 ) of hypothyroid pregnant women were positive for anti - tpo antibodies . a total of 36.07% ( n = 816 ) of pregnant women were found to be hypothyroid when we used trimester - specific tsh reference ranges as suggested by the american thyroid association ( ata ) [ table 2 ] . thyroid profile of pregnant women the prevalence of hypothyroidism was 15.1% , 12.06% , and 14.36% in the first , second , and third trimester , respectively , whereas anti - tpo antibody positivity was seen in 18.07% , 19.45% , and 22.91% in each trimester [ table 2 ] . miu / l for the second and third trimester as suggested by ata , we found 44.3% , 32.0% , and 34% women were found to have hypothyroidism in the first , second , and third trimester , respectively [ table 2 ] . when we analyzed prevalence of hypothyroidism in individual states / cities , srinagar ( kashmir ) , reported unusually high prevalence of hypothyroidism of ( 39% ) and the lowest prevalence was reported from bengaluru ( karnataka ) ( 7.8% ) [ table 3 ] . this study was aimed to evaluate thyroid function during different trimesters of pregnancy in a large cohort across india . the major findings are that 13.3% women attending secondary and tertiary public hospitals have hypothyroidism majority being subclinical . the prevalence of hypothyroidism has been reported from different countries very recently . on analysis , results of this study are consistent with recently published data from india and other countries . previous studies conducted in delhi reported a 14.3% prevalence of hypothyroidism during the first trimester . there are at least two small - scale published studies from the south , one from chennai , and another from hyderabad . rao et al . included 163 nonpregnant women with recurrent pregnancy loss in a gestational age up to 12 weeks ( 2006 ) in hyderabad . similarly , in a community - based large - scale study involving over 500,000 pregnant women from the usa showed a 15.5% prevalence of hypothyroidism . hypothyroidism was found in 7 ( 4.12% ) women with recurrent pregnancy loss and one in the control group . the study demonstrates that hypothyroidism has a statistically significant relationship with recurrent pregnancy loss in the first trimester . another study examined 500 pregnant women attending two government obstetrics and gynecology hospitals in chennai for a period of 5 months in 2007 for thyroid function . subclinical hypothyroidism was detected in 2.8% , among them , tpo antibodies positivity was seen in 57.1% , whereas euthyroid women had significantly lower positivity ( 7% ) . in another study , sahu et al . have done thyroid function during the second trimester in high - risk pregnant women and reported that prevalence of thyroid disorders , especially overt and subclinical hypothyroidism was 6.47% . further , in this study , significant adverse effects on maternal and fetal outcomes were observed emphasizing the importance of routine antenatal thyroid screening . therefore , findings of our study are consistent with other previously reported data from india and this study also shows a rising trend of hypothyroidism among the indian pregnant women . the impact of thyroid dysfunction on pregnancy outcomes appears to manifest with a tsh threshold of > 2.5 miu / l in the first trimester rather than with a tsh range based on percentiles cutoff derived from apparently normal pregnant women . ata recommends > 2.5 and > 3.0 iu / ml as cutoff range for diagnosis of hypothyroidism during the first and later part of pregnancy , respectively . however , we have taken 4.5 iu / ml as cutoff for diagnosis of hypothyroidism in different trimesters . in addition , marwaha et al . have reported that normal range of thyroid hormones in the indian pregnant women are higher as compared to international cutoffs . the large burden of subclinical hypothyroidism in pregnancy may prove to be a major public health burden in india , once it becomes clear that adverse outcomes can be corrected with screening and early replacement of levothyroxine . children born to hypothyroid mothers have a poor intellectual function during the later part of their life . therefore , majority of the developed countries have the national neonatal screening program in place and routinely screen all newborn for hypothyroidism . the question whether to screen all pregnant women for hypothyroidism ata in its recently published guidelines has stated against the universal screening of pregnant women for hypothyroidism . some of these studies have shown an adverse effect on fetal and maternal health outcomes . however , these studies are too small , and it is advisable that a large - scale study is done in this regard from india . the strong point of this study is that we have included the largest number of subjects in this study from different parts of india . further , the study population belongs to different socioeconomic strata and therefore , represents probably the pregnant population of india . this study also demonstrates a secular trend in prevalence of hypothyroidism in india , when data from other previous studies was analyzed . however , there are few limitations of this study . we have not carried out clinical and radiological thyroid examination including ultrasound , and apart from autoimmunity , we have not evaluated other causes of hypothyroidism in these women . this study concludes that there is a high prevalence of hypothyroidism in pregnancy ( 13.13% ) . further studies are required to evaluate impact of thyroid disorders during pregnancy in the indian population to decide whether universal screening is needed for indian pregnant women .

background : a previous hospital based study from delhi revealed a high prevalence of hypothyroidism in pregnant women . several other studies with small sample size also indicate a rising trend of prevalence of hypothyroidism during pregnancy in india.objective:to assess prevalence of hypothyroidism in pregnant women from various states / cities across india.materials and methods : this was a cross - sectional multicenter study conducted at allahabad ( uttar pradesh ) , bengaluru ( karnataka ) , chennai ( tamil nadu ) , kolkata ( west bengal ) , hyderabad ( telangana ) , nasik ( maharashtra ) , rohtak ( haryana ) , pune ( maharashtra ) , new delhi ( delhi ) , srinagar ( kashmir ) , and vizag ( andhra pradesh ) enrolling 2599 pregnant women . estimation of thyroid stimulating hormone ( tsh ) , free t4 , and antithyroid peroxidase ( tpo ) antibodies was carried out using roche modular kit using eclia technology in a central laboratory.results:we found in our study population that 13.13% of pregnant women have hypothyroidism ( n = 388 ) , using a cutoff tsh level of 4.5 iu / ml . this prevalence was much higher using the american thyroid association criteria . anti - tpo antibodies were positive in 20.74% of all pregnant women ( n = 613 ) , whereas 40% ( n = 155 ) of hypothyroid pregnant women were positive for anti - tpo antibodies.conclusion:this study concludes that there is a high prevalence of hypothyroidism ( 13.13% ) , majority being subclinical in pregnant women during the first trimester from india and universal screening of hypothyroidism may be desirable in our country .

lesch - nyhan syndrome was first recognized and clinically characterized in 1964 by lesch and nyhan , observing two affected brothers . lns is a rare x - linked recessive disorder , resulting from a mutation on the gene encoding for the enzyme hypoxanthine - guanine phosphoribosyltransferase ( hgprt ) , located on the long arm of the x chromosome between xq26 and xq27 . the hgprt is an enzyme involved in purine metabolism , which catalyses the condensation reaction which combines phosphoribosyl pyrophosphate ( prpp ) and the purine bases hypoxanthine and guanine to form the respective nucleotides , inosinic acid , and guanylin acid . the hgprt deficiency causes a backlog of guanine and hypoxanthine that oxidize in uric acid and an increase of intracellular prpp with a consequent major production of new purine . it results in an overproduction and accumulation of uric acid , which , if untreated , usually leads to renal failure and death in early childhood . one of the first symptoms of the disease is the presence of orange sand - like crystals of uric acid in the diapers of the affected infant . overproduction of uric acid may lead to the development of uric acid crystals or stones in the kidneys ( nephrolithiasis ) , ureters , or bladder , causing hematuria and increasing the risk of urinary tract infection , of nephrite , and of renal failure . another potential consequence of untreated hyperuricemia is gouty arthritis caused by uric acid crystal deposition in articular cartilage . the affected children appear to be normal for the first few months of life , but hypotonia and development delay are evident from age three to six months . later extrapyramidal involvement with dystonia , choreoathetosis , opisthotonos , dysarthria , dysphagia , and sometimes ballismus becomes evident . the patients also develop signs of pyramidal involvement as spasticity , hyperreflexia , and extensor plantar reflexes . the motor disability is so extensive that most individuals never walk and are confined to a wheelchair . the cognitive disturbs , attention deficit problems , and mental retardation are also present in patients with lesch - nyhan syndrome . the affected children appear normal for the first months of life ; with the eruption of deciduous teeth , they begin to show self - mutilative behavior , biting their oral / perioral tissue and their fingers . the finger nails may be completely ripped , and the self - destructive process involves the bone . in some cases , extensive lesions the secondary infection complicates and retards the healing of traumatic lesions [ 4 , 5 ] . nyhan ( 1997 ) reported that children with lns feel pain and remorse when they mutilate themselves , but they are unable to control and to stop their action . they are usually relaxed when they have physical restraints , instead when it is removed the patients become agitated and their compulsive actions increase . the relationship between the enzyme deficiency and the neurological manifestations in lesch - nyhan syndrome is not so clear . those disorders may be associated with deficits in dopaminergic activity in the basal ganglia ; neurochemical studies of lns patients ' brains show large reductions in da levels , elevated numbers of da receptors , and decreased levels of da transporters . the therapy with allopurinol , a xanthine oxidase inhibitor which blocks the metabolism of hypoxanthine and xanthine into uric acid , can control the overproduction of uric acid , reducing the risk of nephrolithiasis , gouty arthritis , and correlated diseases . the dose of allopurinol has to maintain the uric acid within normal limits ; an excessive allopurinol therapy results in the accumulation of hypoxanthine and xanthine , which could result in xanthine stones . the pharmacological treatment of self - injurious behavior is addressed to control the deregulation in the dopaminergic , opiatergic , or serotonergic systems , as a possible cause of compulsive self - mutilating behavior . the administration of baclofen or benzodiazepines helps in muscle relaxation and keeps the patient calm . the use of hydroxytryptophan together with decarboxylase inhibitor has been proven effective in reducing self - mutilation for a short time . recent therapeutic options also include gabapentin , botulinum toxin a , injected into bilateral masseters , and deep brain stimulation in globus pallidus . the self - destruction behavior should be managed by a combination of physical restraints , behavior modifications , and pharmaceutical therapy , according to olson and houlihan 2000 . in the literature various therapeutic approaches to manage self - destruction behavior are described , but to date the treatment to solve the source of the problem has not been found yet . pharmacological treatment of dopaminergic dysfunction , the possible opiate , and/or serotonin system dysfunction have shown variable successes , but there are disadvantages to pharmacologic treatment , as it usually requires chronic use of the drug that often places the patients in a state of chronic stupor . the use of botulinum toxin a ( btx - a ) should be considered as a good treatment for self - mutilating behavior in lns , but the action of btx - a in decreasing self - inflicted injury is not clear . btx - a may be acting directly on peripheral nerve endings , inhibiting the release of acetylcholine and indirectly on the basal ganglia , resulting in muscle weakness and in decreased behavior . inquiries on btx - a nowadays are not enough and a further study is necessary to define the mechanism of action and the therapeutic results [ 9 , 10 ] . the deep - brain stimulation has been performed on a few patients with lesch - nyhan syndrome and some patients experienced a decrease in spastic self - injurious symptoms . published literature suggests that the role of dentist is crucial to prevent and control self - mutilation . in several cases , the extraction of deciduous teeth is adopted in young patient , but the extraction of all teeth is necessary also in permanent dentition . the extraction of only anterior permanent teeth ( incisors and canines ) , as recommended by rosenbloom and other authors , is not effective in preventing self - injury and so all permanent teeth are involved . the extraction of all teeth represents an extreme solution that may cause functional problems and requires a treatment under general anesthesia . to avoid the extractions , a series of intraoral appliance budnick studying a lesch - nyhan syndrome patient , unable to chew his lips for lack of anterior occlusion , decided to create an acrylic splint firstly cemented in the lower arch and secondly in the upper one . he experimented a new technique by covering the posterior teeth to create anterior open bite , eliminating wounds caused by incisors . in the most collaborative patients with few self - mutilations he proposed only normal care of preventive odontology ; in patients showing such a grade of collaboration and of spasticity to allow the intraoral alginate impression , he suggested the use of soft splint without cemented retention which caused hygienic and demineralization problems . finally , in patients with insufficient collaboration the only solution was represented by the extractions of all teeth . a hard acrylic chin - cup with lip extension fitted by means of orthodontic headgear was proposed . the appliance was well tolerated by patients and it guaranteed the health of the oral tissue but increased the dribbling habit with a consequent fungal infection of chins dermis . to solve fungal inflammation the appliance had to be removed . a thin soft resin mouth guard was employed to prevent finger and lower lip self - mutilation in combination with an arm restraint . in six days of treatment and after three months the patient self - mutilations disappeared and he stopped biting himself , also without the mouthguard . in order to prevent dermatitis a soft resin mouthguard not covering the lip was built . it was first cemented and then to increase the retention he preferred heat - cured acrylic resin due to adequate rigidity and he added head strap ; it had to rebuild to be adapted to development of teeth and it was not indicated in periodontal patients . in some resistant casese orthognathic surgery was proposed to create open bite and when the conservative options of treatment failed he suggested the amputation of teeth , as an alternative method to extraction of all teeth to preserve the alveolar bone . the upper and the lower incisors , canines , and the first premolars were performed with formocresol pulpotomies , the crowns were amputated on gingival level , and the dental root canals were filled with light curing glass ionomer cement and polished with softlex discs . the patient was sedated using choral hydrate and hydroxane , but in case of poor cooperation the general anesthesia was indicated [ 1719 ] . a removable acrylic dental appliance was made to create anterior open bite , orthodontic acrylic resin covered the palate , and occlusal surface of posterior teeth and adam 's claps on first upper molar increased the retention . a resin mouthguard thick in posterior part was combined with a lip bumper in order to open anterior bite and to protrude lower lip from the teeth , but this treatment had minimal to partial success . the acrylic resin bite plate 's retention was increased by a combination between adam 's clasps on the premolars and ball clasps among incisors . the operators of the orthodontic ward of gaslini hospital realized a new orthodontic device to preserve oral and perioral tissue of lns patients . at first irreversible hydrocolloid impressions of maxillary and mandibular arches these impressions were finally taken in spite of patient 's lack of compliance ; operators ' persuasive capacity , family , and operators ' efforts to change the negative behavior of m.c . were crucial . on these impressions a plaster model was built , the undercuts were eliminated ( figures 1 and 2 ) , and the orthodontic dispositive was realized by thermoforming disc materials ( with a hard copolyster outer layer and a soft polyurethane inner layer ) ( figure 3 ) , using a positive - pressure thermoformer ( ministar ) ( figures 2 and 5 ) . the hard side was placed in contact with teeth to increase friction and retention ; on the contrary the soft one was placed in contact with antagonist dental arch to prevent traumatic occlusion . the first devices were removed by patients ' tongue , so it was decided to change the dispositive , reducing mucosal flange to prevent the mouthguard 's removal ( figure 4 ) . at the beginning , patients used the device only for few hours , but time by time they were persuaded to wear it longer . at first a thick lower bite was made to produce an anterior open bite ; then it was substituted by a thinner maxillary and mandibular mouthguards well tolerated by the patient also during the meals . after these treatments , the patients were able to wear the device for 24 hours and it guaranteed the health of the oral and perioral tissue . the most common disadvantages of preventive intraoral devices are the design that may cause fungal infections , extensive laboratory time , and difficulty in daily oral care . also frequent adjustments are required and heavy biting forces added to poor cooperation may interfere with impression taking . these devices extend to the vestibule area and the splint that creates an open bite increases the drooling habit with a consequent fungal infection of chin dermis . cemented splint requires an extensive laboratory work , daily hygiene care is difficult , and the demineralization of the teeth is frequent . some patients with lns show a degree of collaboration to allow the intraoral alginate impression , in other lns patients is impossible to have a degree of cooperation on making the intraoral impression ; in fact sedation is sometime necessary . the use of putty - type vinyl polysiloxane impression material or quick alginate to overcome the insufficient patient 's collaboration that makes the intraoral impression very difficult is suggested . it is possible to obtain greater patient 's collaboration thanks to behavioral modification and persuasive capacity of family and operators . the simple intraoral device presented by gaslini operators is easy to fabricate ; it is a mouthguard realized by thermoforming disc and characterized by two different matrices : hard and soft . the hard matrix guarantees a good retention , so during the cementation , the head straps , the splint extension to vestibule area , and the metal clasps are not necessary to give stability . the double matrix device is readily removed for everyday cleaning and the placement is also comfortable . the bite is very thin , it permits that the patient can wear the mouthguard all day , also during the meals , and consequently the drooling habit is not accentuated . the device is built on the patient plaster model ; it can be customized for each individual case . heavy biting force and poor patient 's collaboration may interfere with impression taking . in the case described here the first impression to create an individual impression trays was taken and a second one to fabricate the device . also the work was simplified by the use of quick alginate . in a second moment the efficacy of this device was observed during a period of about 7 years . during these years the major difficulties encountered during this therapy are as follows : necessity of modifying the dispositive to adapt it to the mouth changes , risk of small cracks on the occlusal resin bite that must be often repaired . necessity of modifying the dispositive to adapt it to the mouth changes , risk of small cracks on the occlusal resin bite that must be often repaired . in conclusion the literature analysis demonstrates that the intraoral appliances , despite some limitations , can represent a good alternative option to invasive treatment and a good choice of therapy to limit and to prevent self - mutilative behavior in lns patients . the thin resin mouthguard described was successfully employed to prevent self - mutilation of oral and perioral structures . it represented a conservative solution and a good alternative option to invasive treatment of extracting the teeth or of orthognathic surgery . in addition , the device is easy to build and is well accepted by patient . a good choice of treatment for improving the quality of life of these patients can be represented .

lesch - nyhan syndrome , described in 1964 by lesch and nyhan , is a x - linked recessive disorder , occurring in 1 : 100000 to 1 : 380000 live births . lns is characterized by a decrease in activity of hypoxanthine guanine phosphoribosyl transferase , an enzyme involved in purine metabolism , resulting in overproduction of uric acid . hyperuricemia and neurological features including choreoathetoid spasticity , self - mutilation , and mental retardation clinically characterize this syndrome . in lns patients the typical feature is loss of tissue from biting themselves with partial or complete amputation of fingers , lips , and tongue . the self - mutilation compares with the eruption of the deciduous teeth . several drugs trials have been administered to improve self - destructive behavior and invasive treatment approaches , such as extractions of teeth and orthognathic surgery , have been suggested with variable effectiveness . nowadays prevention is , therefore , the standard of care . the role of dentistry is essential in the management of the self - mutilating behavior , because the teeth represent the main self - injury instrument . this report presents a revision of various therapeutic approaches to manage self - destruction , highlighting the effectiveness of a preventive treatment . it describes a new technique : a resin mouthguard , realized at gaslini hospital , to obtain immediate healing of the oral lesions , confirmed in the follow - up period .

the field of small - molecule organocatalysis via noncovalent interactions has seen rampant growth over the past decade . this area , which aims to mimic the mechanisms used by nature in enzyme catalysis , is attractive due to its potential for high catalyst tunability and substrate specificity , as well as obviating the use of metals . while such research has resulted in many catalysts operating through bifunctional mechanisms , the primary interaction responsible for electrophile activation occurs through hydrogen - bonding to an acceptor moiety . the consequent lumo - lowering results in rate enhancement . in comparison to metal - based systems ( i.e. , lewis acids ) , current metrics to estimate the reactivity of hydrogen bonding catalysts are ineffective . although pka values of the donor and acceptor may be used to infer hydrogen - bond strengths , this analysis fails to account for several important secondary interactions , including sterics , dual - activation , and binding geometry . as a result , the discovery of reactions compatible with hydrogen - bond catalysis is far outpacing understanding of catalyst interaction and mechanism . indeed , while certain privileged organocatalyst motifs have been identified to be successful for several reaction types , rational design of these structures remains limited , relying on trial and error to achieve optimal reactivity and selectivity . in a previous communication , we described preliminary results showing the utility of small organic chromophore s for the detection of hydrogen - bonding interactions by uv herein , we assess the general utility of this colorimetric sensor as a predictive gauge for the relative reactivity of a broad range of organocatalysts , including several widely used motifs , with the goal of encompassing many different hydrogen - bonding arrays . an additional goal was to validate the sensor measurements across significantly different reaction profiles , particularly those involving noncarbonyl electrophiles . the lack of comprehensive rate data for a range of catalysts in different reactions is a barrier to understanding the factors controlling catalytic activation . as a consequence , the relative rates have been measured for an array of catalysts in two reactions with different groups that interact with the catalysts . the sensor signal has been analyzed with respect to reaction profile , catalyst structure , acidity , and acceptor preference . our findings establish the sensor as a useful substrate surrogate for probing and gauging catalyst performance . moreover , the data unequivocally establish that catalyst structure and binding mode are far more relevant to catalytic activity than acidity . a key consideration in designing a method for measuring hydrogen - bond strengths is the very broad range of these noncovalent interactions ( 0.240 kcal / mol ) . observing the very weak range of these interactions is a challenge with commonly employed spectroscopic techniques . for example , despite successful application in measuring lewis acid binding effects , preliminary nmr studies proved too insensitive for detecting the interactions of weak hydrogen - bonding catalysts with a carbonyl acceptor . we proposed an alternative approach using the sensitivity of uv vis absorption profiles , in which a change in electronic excitation of an acceptor chromophore occurs upon binding to a hydrogen - bond donor ( scheme 1 ) . specifically , imidazopyrazinone s displays solvatochromism with protic solvents as well as color changes with a small number of lewis acids . we postulated that upon treatment with various hydrogen - bond donors , the carbonyl moiety of s would act as an acceptor moiety . the resulting hydrogen - bonding interaction would alter the electronic transition of the chromophore , detectable by simple uv vis spectroscopy . in line with this reasoning , treatment of sensor s in dichloromethane with various hydrogen - bond donors resulted in visible hypsochromic ( blue ) shifts ( figure 1 ) . importantly , compounds anticipated to be weaker donors , such as diphenylthiourea ( 1 ) , yielded significant changes in sensor signal . variation of binder concentration resulted in titration - like behavior , with a measurable end point upon saturation of sensor with catalyst . an array of catalysts ( chart 1 ) , varying in structure and anticipated strength , was examined with the colorimetric sensor , and the max upon saturation was determined . response in the uv vis spectrum of s upon increasing amounts of 12 . [ s ] = 2.22 10 m in ch2cl2 , = 0 to 1.78 10 m. dft molecular orbital calculations were performed on bound and unbound sensor for selected hydrogen - bonding agents to gauge the orbital perturbation ( table 1 ) . the calculated lowest energy transition accurately predicts the observed absorbance maximum for the free sensor . lumo energy gap was larger for all bound complexes , in accord with the empirically observed hypsochromic shift in figure 1 . an increased shift ( lower max ) is predicted for binders of ostensibly greater strength ( e.g. , proton > benzoic acid > phenol ) . the uv absorption behavior of the sensor with the hydrogen - bonding agent can be represented as shown in figure 2a . the lowest energy electronic transition may be ascribed to the n ( homo ) to * ( lumo ) transition , e1 , corresponding to the measured max . as supported by the above calculations , addition of a hydrogen - bonding agent stabilizes the ground state ( homo ) to a greater extent than the excited state ( lumo ) , i.e. , e3 > e2 . as a consequence , a hypsochromic shift is observed upon interaction of the sensor with the hydrogen - bond donors . for comparison , figure 2b illustrates the energy diagram for a typical reaction with a hydrogen - bonding catalyst , in which catalysis is effected by lumo - lowering of the electrophile ( ea ) . e2 is proportional to ea , i.e. , the wavelength shift of the bound sensorcatalyst is proportional to the rate enhancement afforded in a reaction with the hydrogen - bonding catalyst . ( a ) proposed energy diagram of the lowest energy electronic transition of the sensor upon interaction with catalysts of increasing strength , corresponding to the hypsochromic wavelength shift ( max ) . ( b ) lumo - lowering of reactants via hydrogen - bonding catalysts , corresponding to increased reaction rates ( krel ) . as shown in figure 1 , a continuous wavelength shift was revealed upon saturation of the sensor with the catalyst . the lack of two distinct peaks in intermediate measurements containing both bound and unbound sensor indicates a rapid equilibration . thus , plots of absorbance vs [ catalyst ] ( see figure 3 for an example with bisamidinium 12 ) were used to determine the binding constants ( keq ) for the sensorcatalyst complex . a significant range of blue shifts was observed for the different catalyst donors , ranging from 490 to 465 nm ( max 1030 nm ) . in general , catalysts with larger max values possessed much stronger binding constants . since e1 is proportional to 1/max , the energetics of the interaction of the sensor with the catalysts ( e3 e2 ) is proportional to 1/max(sensorcatalyst ) 1/max(sensor ) . indeed , a good correlation of this inverse wavelength shift with ln(keq ) was found ( figure 4 ) . note that in this plot , both axes are linearly proportional to energy terms : to the e of the sensor electronic absorption , and ln(keq ) to g of sensorcatalyst formation . importantly , this relationship establishes the observed wavelength shift as a reliable gauge for binding affinity of a catalyst to the sensor molecule . uv - titration curve of catalyst 12 in ch2cl2 using [ s ] = 2.22 10 m. inlay : visible color change of sensor before ( red ) and after addition of 12 ( yellow ; = 1.78 10 m ) . all titrations were performed with [ s ] = 2.22 10 m in ch2cl2 . using the sensorcatalyst wavelength shift as predictors of catalyst reactivity yields several noteworthy observations . diol - based 30 ( taddol ) and silanol catalysts 31 and 32 afforded very weak shifts , despite application in numerous transformations , including rawal s seminal report on the asymmetric hetero diels alder reaction . the greater max shift of 32 compared to the related monosilanol 31 mirrors the increased reactivity of this silanediol scaffold , as elegantly reported by mattson and franz . benzoic acids and phenols spanned the intermediate range of sensor shifts , with trends clearly based on the electronic effects of aromatic substitution . although these structures are not as commonly incorporated as hydrogen - bond catalysts , schafmeister and co - workers have recently demonstrated the spiroligozyme catalyst 24 , containing a carefully arranged carboxylic acid and phenol , as an effective ketosteroid isomerase mimic for the aromatic claisen rearrangement.n , n-diaryl thioureas and ureas , particularly those with multiple trifluoromethyl substituents such as schreiner s catalyst 4 , afforded some of the largest sensor shifts , indicative of the immense utility of these structures in various organocatalysts . the internally activated bf2-urea 9 provided the largest shift within this class , in line with experimental reactivity data reported by mattson and co - workers . finally , formally cationic species , including guanidinium , amidinium , and takenaka s azaindolium 14(22 ) were the strongest binders , with wavelength shifts ranging from 26 to 34 nm ( max = 473465 nm ) . interestingly , one of the strongest noncationic binders was thiophosphoramide 16 , possessing a pocket of three potential n h donors . squaramide - containing scaffolds have yielded excellent results as hydrogen - bond activators ; however , these compounds possess limited solubility , and are typically employed as heterogeneous catalysts . representative squaramide 11 , containing the common n-3,5-(cf3)2aryl and n-alkyl array , was synthesized , and gave an apparent sensor end point of 480 nm . due to its relative insolubility , an accurate binding equilibrium value could not be determined . it is worth noting the experimental ease with which the sensor metric can be obtained . compound s itself is easily obtained in 2 steps from commercial materials , and very little sensor or catalyst ( particularly for strong catalysts ) is necessary to obtain the wavelength shift . the titration experiment is largely insensitive to moisture , as illustrated by the poor binding observed in the sensor titration with water . applying the method of continuous variation to the sensor with catalyst 12 revealed a 1:1 binding stoichiometry with the sensor molecule ( figure 5 ) . this observation is significant , since several other binding situations may be postulated , including donation of one catalyst molecule to several sensors ( 4 equivalent n job plot analysis of catalyst 12 with sensor s showing 1:1 binding stoichiometry . benzoic acids and phenols offer useful templates to study electronic effects on sensor signal due to availability and well - understood behavior of aromatic substitution . due to solubility limitations , ortho - substituted benzoic acids were studied rather than the para - substituted analogs . the electronic effects from substitution on the sensor interaction can be illustrated via a hammett - type plot , as shown in figure 6 . as may be anticipated from the brnsted catalysis law ( see section 2.6 for further discussion ) , increasingly electron - withdrawing substituents on these structures correlate with larger hypsochromic shifts of the sensorcatalyst complex . for both catalyst sets , highly linear relationships are evident with substituent parameters indicating that the wavelength shift provides an accurate readout of electronic perturbation on the hydrogen - bonding ability . correlation of hammett parameters for o - benzoic acids ( ortho ) and p - phenols ( para ) with sensorcatalyst wavelength shifts . notably , the wavelength shifts seen with the sensor do not correspond directly with pka either in water ( figure 7a , r = 0.0007 ) or dmso ( figure 7b , r = 0.0950 ) . however , correlations are observed for closely related catalyst structures , wherein electronic perturbations modify the acidity of the donor moiety without introducing significant secondary effects . this observation provides potential for the sensor to estimate pka values within a series of related compounds . persubstituted phenol 29 deviates from other phenolic catalysts , which may be attributed to the increased steric demand around the donating o plot of catalyst acidity in water ( a ) or dmso ( b ) vs sensorcatalyst wavelength shifts . the correlation of the observed blue shift with the binding strength across a large range of hydrogen - bond donors proved the metric to be able to quantitatively detect these interactions . however , this finding does not necessitate a correlation with catalyst reactivity . in order for this correlation to occur , the sensor must be a good facsimile of the substrate that is undergoing reaction . other factors , including alternate binding modes and steric effects , might come into play when a substrate interacts with hydrogen - bonding agent in a catalyzed reaction . to be a useful metric for the community , the sensor signal must correlate to empirically obtained rate enhancement via hydrogen - bond catalysis ( scheme 2 ) . myriad reaction profiles have been reported that are established to proceed via hydrogen - bond activation of the electrophile ( lumo - lowering activation ) . in order to best isolate the reactivity enhancement offered by the catalysts strictly due to hydrogen - bonding , we first targeted a reaction where the electrophile has only one possible point of interaction with the catalyst , and the nucleophile does not contain binding points ( i.e. , no heteroatoms ) . additionally , the reaction should have minimal background rate and a method to easily analyze starting material and/or product concentrations . the reaction of methyl vinyl ketone ( mvk ) with cyclopentadiene ( cp ) offers a useful reaction platform that fulfills these criteria ( scheme 3 ) , and has been used to gauge the relative strength of thiourea and bisphenol catalysts previously . importantly , the binding in the sensorcatalyst complex is very similar to that of the mvkcatalyst intermediate as both interactions arise from a carbonyl acting as a hydrogen - bond acceptor . systematic investigation of the diels alder reaction of mvk and cp with a variety of catalysts was performed under pseudo - first order conditions as described in scheme 3 . kinetic data was acquired via continuous sampling ( 5 min intervals ) by h nmr spectroscopy , and each rate measurement was performed in triplicate . relative rate constants , krel , were calculated as described in eq 1 from the observed pseudo - first order rate constant kobs and background rate kbackground , and were normalized for catalyst concentration n. the resulting values directly provide the rate enhancement afforded by the catalyst.1 as displayed in figure 8 , a plot of ln(krel ) against the inverse sensor wavelength shift of 18 catalysts shows an excellent correlation . catalysts with greater blue shifts when treated with the sensor show greater activity in the diels alder reaction via correspondingly greater lumo lowering of the ketone in the dienophile . more precisely , the change in energy of the sensor upon binding with the catalyst is proportional to the change in activation energy of the hydrogen - bond catalyzed diels alder reaction . correlation between the sensorcatalyst wavelength shift and catalyst rate enhancement in the diels alder reaction between mvk and cp . the observed correlation establishes that , at least in this class of reaction , the sensor signal is a good indicator of lumo - lowering ability of these small molecules as hydrogen - bond catalysts . importantly , the results also indicate that the binding interaction of the sensor with catalysts is similar to that of methyl vinyl ketone with catalyst , i.e. the sensor is a useful gauge of carbonyl activation . the addition of various nucleophiles into nitroalkenes is one of the most widely used reaction motifs in hydrogen - bonding catalysts ; it is often used as a measure of reactivity when developing and comparing novel catalyst structures . to test the effectiveness of our sensor metric beyond the diels alder reaction crafts addition of n - methylindole ( 34 ) into nitrostyrene 35 ( scheme 4 ) . deuterated 35 was easily prepared via henry condensation using d3-nitromethane with the corresponding aldehyde . nucleophile 34 provides a reasonably active coupling partner while minimizing potential catalyst interactions [ pka(h2o ) n - methylindolium = 1.8 ] . thus , the effects of hydrogen bonding catalysts on the activation of the styrene electrophile via binding to the nitro acceptor can be cleanly delineated . significantly , the catalyzed rates of this reaction allow comparison of the effects of hydrogen - bonding catalysts on carbonyl acceptors ( the sensor and mvk ) versus nitro acceptors ( nitrostyrene 35 ) as outlined in scheme 5 . while binding geometries to carbonyl groups are anticipated to be similar , an analogous correlation may not be automatically presumed for a nitro group . in particular , the nitro group contains a formally delocalized negative charge across three atoms , and has been suggested to form -activated intermediates with certain catalyst structural types such as squaramides and thioureas . as illustrated in figure 9 , kinetic reaction data was acquired using h nmr spectroscopy . although initial h nmr spectroscopic studies with proteo-35 provided usable data , employment of a deuterium label provides exceptional signal isolation . moreover , overlap of signals from the catalyst is completely mitigated , since only substrate , product , and internal standard exhibit appreciable resonances . as a result , any catalyst can be readily analyzed . ( a ) stacked h nmr plot and ( b ) kinetic profile for the friedel crafts reaction shown in scheme 4 using catalyst 12 . conditions : = 1.33 m , = 0.133 m , = 0.133 m , = 2.67 10 m. the cdcl3 peak arises from natural abundance in chcl3 . crafts reaction was studied under pseudo - first order conditions using the broad series of hydrogen - bond catalysts shown in chart 1 . the relative catalytic strength of each catalyst was calculated according to eq 1 , using averaged kobs values from duplicate trials . a plot of ln(krel ) and inverse sensor wavelength shift is shown in figure 10a . similar to the diels alder reaction , and predicted based on sensor shifts , cationic binders proved to be the most effective catalysts , followed by electron - deficient ureas and thioureas . squaramide 11 , employed here as a heterogeneous catalyst , provided moderate rate enhancement as predicted by its sensor wavelength shift . the krel values reported for these catalysts and 4 align closely with the values measured in this work . ( a ) correlation observed between the sensorcatalyst wavelength shift and catalyst rate enhancement in the friedel crafts reaction . the overall correlation of the sensor wavelength shift with catalyst strength for the friedel crafts reaction is good ( r2 = 0.84 ) , but not as strong as that found in the diels alder reaction ( r2 = 0.95 ) . analysis of this data suggests that the binding interaction of a hydrogen - bonding catalyst with the sensor carbonyl does not fully mimic that of a hydrogen - bonding catalyst with the activated nitroalkene . closer inspection reveals that the wavelength shift is correlated even more strongly to catalyst strength within an isostructural catalyst series ( figure 10b ) . specifically , the catalysts can be placed in four groups based on their general structure : benzoic acids , phenols , y - type binders , and other n h binders . the y - type group consists of catalysts possessing two n h donor groups separated by a single atom , such as ureas , thioureas , guanidines , bis - sulfonamides , etc . the other n h binders include those catalysts that have more than one atom separating the donor array ( squaramide 11 , bisamidinium 12 ) or can only donate one n interestingly , the silanediol catalyst 32 exhibits reactivity falling nicely in line with benzoic acids , possibly due to a similar o h geometry . this analysis quantitatively shows that y - type structures are superior for nitroalkene activation . in contrast , phenols provided the least activation relative to their binding interaction with the sensor . overall , the sensor provides a good assessment of relative reactivity of hydrogen - bond catalysts in the friedel crafts reaction . the presence of stronger correlations within catalyst structural classes is consistent with some catalysts activating nitroalkenes via a different mode ( e.g. , -binding ) that is not completely captured by the interactions of the catalysts with the carbonyl of the sensor molecule . this analysis underlines the complex nature of hydrogen - bonding , emphasizing that caution must be exercised in generalizing catalyst reactivity or selectivity from one reaction to another . a general equation to describe catalyst strength for reaction r based on sensor response ( ) is presented in eq 2 . parameter rr ( slope ) represents the responsiveness of the rate per unit catalyst strength as determined by the sensor measurement . parameter cr ( y - intercept ) corresponds to inherent complementarity of the catalyst to the electrophilic reaction partner.2 the parameter values ( table 2 ) for the reactions shown in scheme 3 ( diels alder ) and scheme 4 ( friedel crafts ) were obtained using the kinetic data from figures 8 and 10 , respectively . comparison of the rda and rfc values reveals that the friedel crafts is more sensitive to catalyst strength . in other words , the same catalyst produces a greater relative rate enhancement for the friedel crafts reaction than for the diels alder reaction . similarly , a given wavelength shift of the sensor by a catalyst will cause a greater reactivity change in the friedel crafts vs the diels alder reaction . on the other hand , the cda and cfc values indicate the inherent complementarity of the electrophilic substrate with catalysts ; greater complementarity translates to greater reactivity . notably , the c values represent reactivity when there is no wavelength shift ( no perturbation of the sensor by the catalysts ) and represent the lower limit of lumo activation afforded by the catalyst . catalyst group - specific coefficients rr and cr can be introduced ( eq 3 ) to account for variation if the sensor binds the catalyst differently than the reaction electrophile . due to the strong correlation of the sensor shift to relative rates independent of catalyst structure , coefficients are unnecessary for the diels alder reaction ( rda cda 1 ) . as discussed in section 2.4 , the sensor does not completely model catalyst binding to the nitrostyrene acceptor of the friedel crafts reaction . accordingly , the slope and intercept data from figure 10b were combined with the r and c values from table 2 to afford coefficient values for rfc and cfc , respectively , as provided in table 3.3 the coefficient values in table 3 reveal general trends between the different catalyst structural types and rate in the friedel again , rfc values are a measure of responsiveness of a given catalyst architecture to a perturbation in sensor binding . for y - type and phenolic catalysts , rfc is noticeably higher than the other n h binders , and particularly benzoic acids . thus , for the same amount of wavelength shift , the phenol and y - type catalysts provide greater increases in reactivity relative to the other n h binders and benzoic acids . this observation indicates that the sensor can assess electronic effects in a catalyst series . comparison of the hammett effects on ( figure 6 ; acid = 5.8 , phenol = 4.4 ) with those on ln(krel ) ( acid = 0.60 , phenol = 0.33 ) provides support for this assertion ; both measures show a stronger electronic effect for the carboxylic acid series . on the other hand , lower cfc values indicate the inherent complementarity of a given catalyst architecture . for example , the y - type binders activate nitro electrophiles to a greater extent at a given wavelength shift relative to phenols or the other n h binders . interestingly , the cfc value for benzoic acids would predict high catalytic activity relative to y - type binders , but only in the weak binding regime ( left side of plot ) . due to the low rfc value for benzoic acids , the trends invert such that y - type binders are superior in the strong binding regime ( right side of plot ) . considering both terms together , the y - type binders are both more complementary to the nitroalkene and more efficient at lumo lowering , thereby providing superior reactivity . acidity values have widely been used as a guiding principle in hydrogen - bond catalyst design , under the premise that a more acidic donor will form a stronger interaction and stabilize the buildup of anionic charge in the transition state to a greater extent . indeed , several reports have observed increased activity with judicious electronic tuning of the donor hydrogen . however , even ostensibly subtle changes to catalyst structure can cause secondary factors to override the reliability of pka as a predictive measure , as demonstrated by cheng s recent study on thiourea derivatives and even noted in the seminal work by hine on mono- and bis - phenols . having proved the effectiveness of the sensor signal as a gauge for catalyst strength , we undertook a comparison with acidity to determine the similarities and differences between the two metrics . aggregate data for all catalysts spanning 3 orders of magnitude in reactivity for the diels alder reaction and 4 orders in the friedel crafts reaction is organized by increasing sensor wavelength shift in table 4 . recent efforts by schreiner and others provided accurate acidity values of common hydrogen - bond donors . the brnsted catalysis equation ( eq 4 ) , which describes the relationship for the rate of an acid - catalyzed reaction with the pka of the acid , was applied to this data.4 value for 2-naphthol . figures 11 and 12 display the results for selected catalyst series in the diels alder and friedel crafts reactions , respectively . these plots prove the linear free energy relationships ( lfers ) between catalyst acceleration and acidity among catalysts of very similar structure . in general , these lfers exhibited a narrow range of values ( 0.390.47 ; table 5 ) , indicating a similar degree of hydrogen - bonding in the transition states and that these reactions are not proceeding through formal protonation ( = 1 ) . slightly higher values found for benzoic acids in the diels alder ( = 0.57 ) indicate a greater degree of proton transfer in the transition states consistent with the ionic nature of benzoic acids . lower values for ureas in the friedel crafts ( = 0.31 ) indicate a lesser degree of proton transfer in the transition states in line with the covalent nature of the n h bonds . brnsted catalysis plot for the diels alder reaction , demonstrating lfers for closely related catalyst groups . more significantly , these figures clearly demonstrate the inherent limitations of estimating catalyst strength using acidity metrics . based solely on pka measurements , thioureas would be predicted to provide much higher activity . in practice , the opposite is observed where ureas exhibited greater ( 6 , 7 vs 2 , 3 ) or similar ( 8 vs 4 ) activation of the nitro group compared to their thiourea analogues , despite the much greater acidity of the thioureas ( 45 orders of magnitude difference ) . highly reactive catalysts not belonging to a clearly defined series , including common brnsted acids , are included in figure 11 and further highlight the disparity between acidity and activity . interestingly , takenaka s azaindolium catalyst 14 displayed much higher activity than ppts , despite similar acidities and similar pyridinium in conclusion , a sensor is described that provides an assessment of the reactivity over 34 orders of magnitude for 33 hydrogen - bonding catalysts . useful correlations are obtained between the wavelength shifts that catalysts cause to the pyrazinone sensor and the rate - determining steps in diels alder and friedel crafts reactions . as a result , only the wavelength shifts upon saturation of the sensor with catalysts need to be measured vs the more time - consuming titration studies . consequently , the sensor may also find use as a rapid means for measuring pka values in series of related compounds . in contrast to established acidity ( pka ) values , the sensor wavelength shift is a highly predictive metric for the relative reactivity of catalysts encompassing a broad range of structures and strengths . notable acidity - activity disparities include cationic catalysts ( low acidity , high reactivity ) , and benzoic acids ( high acidity , low reactivity ) , the strengths of which are more accurately gauged by their interaction with the sensor . overall , the sensor is a superior surrogate for the diverse electrophiles ( enone and nitroalkene ) used compared to water and is better able to assess secondary interactions . the data collected was used to formulate the relationship described in eq 3 , which provides a direct means of assessing the reactivity of a catalyst in a given reaction using the sensor signal . the resulting parameters also reveal relationships between substratecatalyst binding , catalyst - induced lumo - lowering , and catalyst structure . investigation of additional catalyzed reactions with the sensor , empirically or computationally , has the potential to expand eq 3 to achieve quantitative predictive power across a large range of reaction platforms and catalysts ( hydrogen bonding , brnsted acid , and lewis acid ) .

a spectrophotometric sensor is described that provides a useful assessment of the lumo - lowering provided by catalysts in diels alder and friedel crafts reactions . a broad range of 33 hydrogen - bonding catalysts was assessed with the sensor , and the relative rates in the above reactions spanned 5 orders of magnitude as determined via 1h- and 2h nmr spectroscopic measurements , respectively . the differences between the maximum wavelength shift of the sensor with and without catalyst ( max1 ) were found to correlate linearly with ln(krel ) values for both reactions , even though the substrate feature that interacts with the catalyst differs significantly ( ketone vs nitro ) . the sensor provides an assessment of both the inherent reactivity of a catalyst architecture as well as the sensitivity of the reaction to changes within an architecture . in contrast , catalyst pka values are a poor measure of reactivity , although correlations have been identified within catalyst classes .

streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide , particularlyin lower income countries . pneumococcal diseases are the leading source of vaccine preventable deaths , mostly due to community - acquired pneumonia ( cap ) , accounting for approximately 11% of all deaths in children under 5 years old ( 1 ) . colonization of the nasopharynx is a necessary step along the path to pneumococcal disease ( pd ) ( 2 ) . pneumococcal conjugate vaccines ( pcv ) reduce nasopharyngeal carriage of serotypes which included in the vaccine by conferring capsular - specific immunity . experience from countries where conjugate vaccines have been introduced has shown rapid and sustained carriage reduction of vaccine serotypes ( vt ) following vaccination ( 3 ) . the microorganism produces a plethora of virulence factors , including the polysaccharide capsule , several surface - located proteins , and the toxin pneumolysin ( 5 , 6 ) . the polysaccharide capsule is highly efficient in protecting the bacteria from opsonophagocytosis ( 7 ) . surface proteins of s. pneumoniae ( pneumococcus ) have been investigated for their role in pneumococcal pathogenicity and as candidate antigens for protein based vaccines ( 8) . among the surface - associated proteins , the pneumococcal surface protein a ( pspa ) andc ( pspc ) are the best characterised choline - binding proteins ( 6 , 9 ) . pneumolysin is a cytoplasmic toxin released by autolysis of the cell and it is a very important virulence factor with multiple effects . the major autolytic enzyme of the pneumococcus is lyta ( nacetylmuramoyl - l - alanine - amidase ) , which is responsible for the deoxycholate- and penicillin induced cell lysis in the stationary phase , having a great clinical importance ( 10 ) . the lyta - encoded major autolysin of s. pneumoniae is a member of a widely distributed group of cell wall - degrading enzymes located in the cell envelope and postulated to play roles in avariety of physiological functions ( 11 ) . rrga is a virulence factor in a murine lung infection model and has a varied distribution among serotypes of s.pneumoniae ( 12 ) . s. pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits , rrga , rrgb and rrgc ( 13 ) . some of the pneumococcal virulence factors are potential targets for protein- based pneumococcal vaccine production . thus , in this study the presence of three genes were detected among the isolates of s. pneumoniae . a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory . these samples were collected from children attending day care centers in different geographical areas of mashhad , iran . both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours . the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) . the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 . the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) . the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays . oligonucleotides which were used as primers to amplify particular sequences of s. pneumoniae amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min . amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min . one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) . sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) . a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory . these samples were collected from children attending day care centers in different geographical areas of mashhad , iran . both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours . the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) . the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 . the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) . the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays . amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min . amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min . one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) . sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) . among 260 nasopharanx samples from healthy children under 6 years , 59 isolates were confirmed as s. pneumoniae . distribution of the lyta , rrga and pspc genes among isolates of s.pneumoniae were determined . rrga and pspc were also found in17 ( 28.81% ) , and 2 ( 3.38% ) isolates respectively . five patterns of these genes were seen among s. pneumoniae isolates : lyta ( n=33 , 55.93% ) , rrga ( n=2 , isolates , 3.38% ) , pspc ( n=1 1.69% ) , lyta+rrga ( n=16 , 27.11% ) and lyta+ rrga+ pspc ( n=1 , 1.69% ) . the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) . the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) . it remains unclear , however , why some children develop invasive disease , whereas in the majority of cases , colonization remains asymptomatic , a combination of bacterial virulence and host factors may be responsible ( 18 ) . we studied the s. pneumoniae isolates giving special reference to identification and distribution of virulence markers such as autolysin among the isolates from nasopharynx . this exercise may help in understanding the factors contributing to the pathogenicity of s. pneumoniae . further , there are numerous reports giving us increasing evidences on the role of lyta in pneumococcal pathogenesis suggesting that this might be more appropriate as vaccine antigen against s. pneumoniae infections . the cpsa was used as a novel genetic marker specific for identification of s. pneumoniae and to differentiate it from the closely viridans group streptococci as well as other pneumococcus - like streptococci such as s. pseudopneumoniae ( 19 ) . many virulence genes contribute to the colonization of s. pneumoniae ; however , our study only demonstrates this for pspc , rrga and lyta genes . thus , synergistic effect and correlation of these virulence factors is necessary for our future work . our results showed that most of the isolates had lyta gene ( 84.74% ) which has an important role in colonization . this observation is in concordance with previous report which was showed that all of the unencapsulated isolates of s. pneumoniae were negative for lyta and psaa by pcr . detection of lyta and psaa in six encapsulated isolates for which a serotype could be determined was negative ( 9 ) . false negative results were obtained by the pcr assays for these two genes may be due to mutations or sequence variation . on the other hand , lyta is essentially a rather conserved gene displaying limited genetic variation ( 11 ) . in the study of whatmore , 33 out of 62 isolates of s. pneumoniae were selected to represent a diverse range in terms of serotype , clinical association , and time and place of isolation . autolysin which was found in all strains , might appear to be a suitable target virulence , and apparently highly conserved , for inclusion in a potential vaccine ( 11 ) . the pneumococcal protein lyt a is the major autolysin of s. pneumoniae , it has an important function in pathogenesis by releasing pneumolysin and plays a fundamental biological role in bacterial lysis after exposure to certain antibiotics ( 19 ) . it has been reported that the lyta gene has higher specificity than the pspc for identification of s. pneumoniae ( 15 , 20 ) . in our study , only 2 out of 59 isolates of s. pneumoniae ( 3.38% ) had pspc gene . given the high sequence diversity of pspc , it is unlikely that pspc alone can be a vaccine antigen to provide protection from across different pneumococcal strains ( 16 ) . it was showed that rrga is central in pilus - mediated adherence and disease , even in the absence of polymeric pilus production ( 13 ) . however , it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease ( 21 ) . hence , new vaccine designs are focused on the surface proteins ( e. g. , pspa and pspc ) , cytolysin , and pneumolysin ( 22 ) . we concluded that the gene cpsa was specific and highly conserved among s. pneumoniae isolates which were colonized in nasopharynx . on the other hand , we showed that lyta gene was the most frequent genes among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern.thus this should allow for appropriate screening of adhesin - based vaccines to prevent infections by streptococci .

background and objectives : many surface proteins are implicated in nasopharyngeal colonization and pathogenesis of streptococcus pneumoniae . some of these factors are candidate antigens for protein based vaccines . new vaccine designs focus on the surface proteins ( e. g. , pspa and pspc ) and also cytolysin , and pneumolysin . in this study , 3 key virulence genes , lyta , pspc , and rrga , which encoded surface proteins , were detected among s. pneumoniae isolates.materials and methods : a total of 260 nasopharyngeal swabs were collected from healthy children under 6 years old attending day care centers in mashhad , iran . isolates of s. pneumoniae were confirmed by optochin susceptibility and colony appearance and also by pcr for cpsa gene . the presence of lyta , pspc , and rrga genes were also detected by pcr.results:a total of 59 isolates were confirmed as s. pneumoniae . among these isolates , 50 ( 84.74% ) , 19 ( 32.20% ) , and 2 ( 3.38% ) were positive for lyta , rrga , and pspc genes respectively . the presence of these genes among s.pneumoniae isolates were as follows : 1 ) rrga , lyta , pspc ( 1 isolate ) , 2 ) rrga , lyta(17isolates ) , 3 ) pspc ( 2 isolate ) , 4 ) lyta ( 50 isolates).conclusion : cpsa gene was specific for detection of s. pneumoniae isolates which were colonized in nasopharynx . the lyta gene was the most frequent gene among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern . thus , it is important for future monitoring of vaccine formulation in our country .

we report a 32-year - old iranian male diagnosed with a case of miliary tuberculosis affecting the prostate . computerized tomography increased the clinical suspicion of miliary tuberculosis extending to the prostate where a trans - rectal urethral biopsy was obtained . a strong clinical suspicion and availability of sophisticated tests with confirmation by biopsy , polymerase chain reaction , and culture are needed in order to avoid misdiagnosis of complicated miliary tuberculosis cases . miliary tuberculosis is a rare form of a tuberculosis infection that results from massive lymphohematogenous dissemination of the mycobacterium tuberculosis bacilli.1 this form of infection , which accounts for 2.8% of all tuberculosis infections , usually involves the extrapulmonary organs.2 from extrapulmonary tuberculosis only 22% affects the genitourinary system , while tuberculosis of the prostate gland is seen in only 2.7% of genitourinary tuberculosis.3,4 due to the rare involvement of the prostate , very scarce literature of the subject , and non - specific symptoms , the clinical suspicion of prostate tuberculosis is difficult . here we report a case of a young , previously healthy , immunocompetent patient with miliary tuberculosis involving the prostate . we present a case of a 32-year - old immunocompetent iranian male who presented to the emergency room with a generalized tonic - clonic seizure lasting for 3 minutes . furthermore , he did not have any genitourinary symptoms , including dysuria , hematuria , or penile discharge . a computerized tomography ( ct ) scan of the head failed to show any acute central nervous system insult . prior to this admission , he gave history of an admission 12 months ago to a hospital outside the country , where he complained of fatigue , weight loss , and fever . reports from the hospital showed that he also had chronic moderate ascites and a left pleural effusion . however , several laboratory and imaging tests were done and it was concluded that there was no evidence of infectious disease , chronic liver disease , or malignancy . it was recommended that he may need more investigations or a laparoscopy with peritoneal tissue biopsy , which was not done . upon the latest admission , the respiratory rate was 12 breaths per minute , the body temperature was 36.9c , and the blood pressure was 112/70 mmhg . digital rectal examination was not performed since the patient did not have any genitourinary or gastrointestinal symptoms . during this hospitalization , the laboratory investigations , urine routine and microscopy , cultures , virology screen , including human immunodeficiency virus , as well as the autoimmune markers did not show any significant issues . also , three sets of sputum acid - fast bacilli smears were done , which were all negative . a lumber puncture was also performed with a normal initial pressure , 0 mmcell count , glucose of 4.1 mm per liters , and protein of 764 mg per liter . at the same time a magnetic resonance image of the brain was done concluding multifocal enhancing foci suggestive of an infective process ( figure 1 ) . due to his previous history , ct - chest , the abdominal ct showed mild peritoneal ascites with evidence of mild splenomegaly . the ascetic fluid taken was translucent and yellow in color and the serum - ascites albumin gradient 5 gram per liter ( 0.5 gram per deciliter ) furthermore , the analysis was negative for ziehl neelsen , gram stain , and culture . the pelvis ct also showed an enlargement of the prostate and a right prostate lesion extending to the seminal vesicles ( faint ring enhancing lesion measuring 32.73 cm ) ( figure 2 ) . the case was discussed with the patient and he agreed to start anti - tuberculosis treatment and to have a trans - rectal biopsy of the prostate . the biopsy showed multiple slides of necrotizing granulomata suggestive of tuberculosis ( figure 3 ) . neelsen stain was also performed , and the diagnosis of miliary tuberculosis was confirmed ( figure 4 ) . he was started on anti - tuberculosis therapy with isoniazid , rifampicin , ethambutol , pyrazinamide , and pyridoxine for 12 months . at follow - up the patient was asymptomatic with a normal physical examination and laboratory tests with no drug side effects . this complication is seen in 1%3% of all tuberculosis cases.5 although most cases of miliary tuberculosis are treatable , the mortality rate among young adults with miliary tuberculosis remains 15% 20% and for adults 25%30%.2 one of the main causes for these high mortality rates includes late detection of disease caused by non - specific symptoms . tuberculosis of the prostate is rare and the literature is scarce . most often , the cases are under - reported , and easily missed . patients with prostatic tuberculosis usually present with non - specific symptoms except in rare cases . these rare cases present with irritative voiding symptoms , terminal dysuria or hemospermia.6 however this was not the case in our patient . the patient was asymptomatic and like most cases in the literature , tuberculosis of the prostate was diagnosed incidentally . as in our case , tuberculosis of the prostate is mainly diagnosed by pathologists where a trans - rectal ultrasound guided biopsy of the prostate is the mainstay of the diagnosis.7 as wang and chang have demonstrated , it was the clinical usefulness of the contrast enhanced ct for the diagnosis of tuberculosis of the prostate in which low density multiple and bilateral lesions with irregular borders are seen that prompted us to further investigate the prostate.8 once the disease is diagnosed , the patient should receive a full course of anti - tubercular therapy . often , the presentation can be non - specific and misleading , as in our case . a strong clinical suspicion and availability of sophisticated tests with confirmation by biopsy , polymerase chain reaction , and culture are needed in order to avoid misdiagnosis of complicated miliary tuberculosis cases .

introductiontuberculosis of the prostate is a rare complication of miliary tuberculosis.case presentationwe report a 32-year - old iranian male diagnosed with a case of miliary tuberculosis affecting the prostate . the patient was admitted to the hospital with convulsions . computerized tomography increased the clinical suspicion of miliary tuberculosis extending to the prostate where a trans - rectal urethral biopsy was obtained . the biopsy revealed multiple necrotizing granulomata suggestive of tuberculosis.conclusiona strong clinical suspicion and availability of sophisticated tests with confirmation by biopsy , polymerase chain reaction , and culture are needed in order to avoid misdiagnosis of complicated miliary tuberculosis cases .

transversus abdominis plane ( tap ) block is a widely practiced peripheral nerve block , utilized to anesthetize the somatic nerves supplying the anterior abdominal wall by depositing local anesthetic in the neurovascular plane between internal oblique and transversus abdominis muscle layers . it was introduced in anesthesia practice in 2001 by rafi utilizing the traditional anatomical land marks . tap block has subsequently been used as a component of multimodal analgesia for postoperative pain relief following various surgical procedures such as large bowel resection , open appendectomy , retropubic prostatectomy , nephrectomy , hernia repair , laparoscopic cholecystectomy and cesarean section . although carney et al . and atim et al . have observed analgesic benefit of tap block in total abdominal hysterectomy by landmark based approach and ultrasound guided ( usg ) approach respectively , griffith et al . found that tap block does not confer any definite analgesic benefit in major gynecological procedures over a multimodal analgesic regimen . furthermore , the effect of preincisional tap block on intraoperative as well as postoperative analgesia in patients undergoing total abdominal hysterectomy remains yet to be elucidated . based on these observations , this study was conceptualized to elucidate the efficacy of bilateral preincisional tap block as a component of multimodal analgesia for providing perioperative pain relief in patients undergoing total abdominal hysterectomy . after obtaining approval by the institute ethics committee and written informed consent , 90 adult female patients of american society of anesthesiologists ( asa ) physical status ( ps ) i or ii , scheduled for total abdominal hysterectomy by a lower abdominal transverse incision were recruited in this randomized double - blind controlled clinical trial . unwilling patients and patients with body mass index > 30 kg / m , compromised renal and liver function , uncontrolled diabetes , severe cardiovascular , respiratory disease , having a history of allergy to any of the study drug , and history of abdominal surgery were excluded from the study . secondary outcomes were intraoperative fentanyl requirement and hemodynamic changes , postoperative hemodynamic changes and time to request first postoperative analgesic . a thorough review of related literature was performed from standard textbooks and an internet search of related articles was performed pubmed . sample size was calculated on the basis that a 20 mm difference in the mean vas between the two groups would be clinically useful . we used ps power and sample size calculations software , version 3.0 [ department of biostatistics , vanderbilt school of medicine , nashville , tn ] . we assumed a standard deviation of 30 mm as a standard deviation of vas score in the population . forty one patients in each group would be needed , assuming the probability of alpha ( ) error is 5% and a power of the study is 85% . assuming a probable drop out of 10% , 90 patients were recruited . patients were randomly allocated into two equal groups of 45 patients in each group using a random number generators in microsoft excel 2003 [ microsoft corporation , redmond , wa ] and allocation concealment were maintained by using an opaque sealed envelope technique . baseline parameters such as heart rate , continuous electrocardiogram , noninvasive blood pressure , spo2 were noted down . patients were randomly allocated into two groups , group b or group n to receive one of the following solutions for bilateral tap block . group b : injection bupivacaine , 0.25% ( 0.5 ml / kg body weight on each side).group n : injection normal saline ( 0.5 ml / kg body weight on each side ) . group b : injection bupivacaine , 0.25% ( 0.5 ml / kg body weight on each side ) . group n : injection normal saline ( 0.5 ml / kg body weight on each side ) . the anesthesiologist , who prepared the solution in identical syringes , remained unaware of the nature of the study and was not involved in further data collection . the lumbar triangle of petit , located just anterior to the latissimus dorsi muscle was identified by palpating the iliac crest in an anterior to posterior direction , until the edge of the latissimus dorsi was felt . the skin was pierced just cephalic to the iliac crest over the triangle of petit with a blunt 18 gauge tuhoy needle [ smiths medical international limited , hythe , kent , uk ] after infiltration with 2% lignocaine . the needle was advanced perpendicular to the skin in the coronal plane until the first resistance of external oblique muscle was encountered . gentle advancement of the needle resulted in a pop sensation as the needle entered the plane between the external and internal oblique fascial layers . a second loss of resistance was encountered when the needle reaches the transversus abdominis fascial plane between the internal oblique and transversus abdominis muscle . a test dose of 1 ml was injected to determine resistance to flow and confirm the needle tip placement within the neurovascular plane . after this one of the study solutions was injected on each side following careful aspiration to exclude vascular puncture . ten min after tap block , all patients received a standardized general anesthesia with fentanyl 1 mcg / kg , propofol and atracurium . they were monitored for any signs of inadequate analgesia in the intraoperative period such as sweating , lacrimation , tachycardia ( > 100/min ) and hypertension ( > 20% elevation of baseline mean arterial pressure ) and supplemental doses of injection fentanyl 0.5 mcg / kg were given as needed . an anesthesiologist who was unaware about the patient allocation did recording of intraoperative hemodynamic data and other anesthesia management . intravenous infusion of paracetamol ( 1 g to patients with body weight > 40 kg and 750 mg to patients with body weight < 40 kg ) was given 30 min prior to completion of surgery . fluid deficit arising from preoperative fasting was corrected by maintenance fluid . blood loss and other plasma losses blood losses up to the transfusion threshold were replaced with 3 ml of ringer 's lactate for each ml of blood loss . after the patient had adequately recovered from anesthesia , and was able to assess pain , postoperative analgesia was assessed with vas 0 - 100 mm in the immediate postoperative period ( when the patient was able to communicate in the postanesthesia care unit ) , at 1 , 2 , 3 , 4 , 5 , 6 and 24 h and whenever the patient complained of pain . the time of administration of rescue analgesic in the form of injection tramadol 2 mg / kg intravenous ( iv ) was noted when vas > 40 mm . vas score was assessed in both rest and movement ( knee flexion ) by an independent observer who was unaware about the allocation . after administration of rescue analgesic , patients were shifted to a postoperative analgesic regimen of injection tramadol iv 2 mg / kg 8 hourly and injection paracetamol 6 hourly up to 24 h. the incidence of postoperative nausea and vomiting was noted during the first 24 h. rescue antiemetics were given to any patient who complained of nausea or vomiting . any signs of adverse effects of the technique like local site infection , hematoma formation , local anesthetic toxicity due to intravascular injection of anesthetic ( like dizziness , tinnitus , perioral numbness and tingling , lethargy , seizures , signs of cardiac toxicity like atrio - ventricular conduction block , arrhythmias , myocardial depression and cardiac arrest ) , peritoneal perforation , bowel perforation , difficulty ambulating or fall and injury secondary to spread of local anesthetic to nerves of the buttock , lateral thigh or leg in the distribution of the femoral nerve were sought for . all raw data were entered into a microsoft excel spreadsheet and analyzed using standard statistical software . continuous numerical data were expressed as mean and standard deviation ( for normally distributed data ) , or median and inter - quartile range ( for data that are not normally distributed ) . normally distributed numerical data between groups were analyzed using the student 's t - test . skewed data between groups were analyzed using the mann categorical variables were analyzed using the fisher 's exact test or the pearson 's chi - square test as applicable . ninety patients were recruited for the trial and data from all of them has been analyzed . a consort flow diagram depicting the passage of participants through the trial has been provided in figure 1 . the two groups were comparable in terms of baseline demographic parameters ( age , sex and body weight ) , duration of surgery and anesthesia and preoperative hemodynamic parameters ( pulse rate , systolic and diastolic blood pressure , respiratory rate ) and the volume of the study drug required in tap block . a summary of base line characteristics of the patients has been furnished in table 1 . consort flow diagram for patient selection baseline characteristics of the patients in each group from the analysis of the intraoperative hemodynamic parameters it was found that pulse rate was significantly higher in patients receiving placebo ( 95.9 11.2 bpm vs. 102.9 8.8 bpm , mean the difference 7.0 s , p = 0.001 ) after surgical skin incision . both systolic and diastolic blood pressure after surgical skin incision was also significantly higher in patients receiving placebo , but similar at all other time points . comparison of mean preoperative and intraoperative pulse rate , b = group b , n = group n , pr1 = 10 min after tap block , pr2 = before induction , pr3 = after induction , pr4 = after incision , pr5 = 15 min intraoperative , pr6 = 30 min intraoperative , pr7 = 60 min intraoperative , pr8 = 90 min intraoperative , pr9 = 120 min intraoperative comparison of mean preoperative and intraoperative systolic blood pressure and diastolic blood pressure . bsbp = systolic blood pressure of group b , nsbp = systolic blood pressure of group n , bdbp = diastolic blood pressure of group b , ndbp = diastolic blood pressure of group n , bp1 = 10 min after tap block , bp2 = before induction , bp3=after induction , bp4 = after incision , bp5 = 15 min intraoperative , bp6 = 30 min intraoperative , bp7 = 60 min intraoperative , bp8 = 90 min intraoperative , bp9 = 120 min intraoperative median requirement of intraoperative fentanyl was significantly higher in patients receiving placebo in comparison to bupivacaine in tap block ( 81 mcg vs. 114 mcg , p = 0.000 , mann the difference between the medians is 32.0 mcg with a 95% confidence interval ( ci ) of 22.0 , 42.5 ( hodges lehman median difference ) . there was no statistically significant difference between the two groups in terms of median values of immediate postoperative pulse rate , systolic blood pressure and diastolic blood pressure ( mann whitney u - test ) . postoperative oxygen saturation varied from 97% to 100% in all patients of both groups b and n. vas scores in the immediate postoperative period both at rest ( median vas 3 mm vs. 27 mm ) and with activity ( median 8 mm vs. 35 mm ) were significantly lower in patients who received tap block . median duration of analgesia was significantly higher in patients belonging to group b ( 290 min vs. 16 min , p = 0.000 , mann the difference in median duration of analgesia is 275 min with 95% ci of 250307 min ( hodges lehman median difference ) . a kaplan meier survival analysis for the cumulative duration of analgesia in first 24 h shows a significantly longer duration of analgesia in patients receiving tap block [ figure 4 ] . comparison of quality of analgesia duration of analgesia in either group has been depicted in by kaplan meyer survival analysis . the survival graph shows significant cumulative analgesia in patients receiving transversus abdominis plane block incidence of postoperative nausea - vomiting was also similar in both groups . no opioid related side effects such as respiratory depression , pruritus or urinary retention was noted in any of the patients . none of the patients in either group had any complication that can be attributed to tap block . median value of pulse rate , systolic blood pressure , diastolic blood pressure , vas scores at 1 , 2 , 3 , 4 , 5 , 6 and 24 h were not compared as > 30% patients in group n received rescue analgesic during the immediate postoperative period . the principal finding of our study is that bupivacaine in tap block provides effective intraoperative and immediate postoperative analgesia in patients undergoing total abdominal hysterectomy . our finding of preincisional tap block reducing intraoperative fentanyl requirement was consistent with those of mukhtar and khattak who reported a significant reduction in intraoperative morphine consumption in patients receiving tap block with 0.5% bupivacaine in renal transplant recipients ( 0.4 1.2 mg vs. 9.3 1.4 mg ; p < 0.0001 ) . el - dawlatly et al . reported a similar significant reduction in intraoperative sufentanil consumption in patients undergoing laparoscopic cholecystectomy ( 8.6 3.5 mcg vs. 23.0 4.8 mcg , p < 0.01 ) . similar findings were reported in a study by ra et al . in patients undergoing laparoscopic cholecystectomy where intraoperative remifentanil use was significantly lower in patients receiving either 0.5% or 0.25% bupivacaine in comparison to placebo . however , no other rct , to the best of our knowledge has addressed the efficacy of preincisional tap block in preventing hemodynamic response to surgical stimuli . we have found the superiority of tap block in providing immediate postoperative analgesia reflected by a lower vas score both at rest and with activity . the current literature on tap block is not unanimous in the matter that whether it improves postoperative pain score or not . carney et al . in open appendicectomy . in 2008 , carney et al . found that anatomical tap block in total abdominal hysterectomy patients significantly reduces postoperative pain scores up to 48 h period . postoperative morphine consumption also decreased at 12 h , 36 h and 48 h time period . recently , sharma et al . also found that tap block by landmark technique improves vas score in first 24 h in patients undergoing major abdominal surgery . petersen et al . in 2012 also found that us guided bilateral tap block in patients undergoing laparoscopic cholecystectomy provides superior postoperative pain scores . petersen et al . in 2013 found that tap block does not provide superior analgesia in comparison to placebo after inguinal hernia repair . a previous cochrane review and a meta - analysis in 2012 failed to demonstrate the beneficial effect of tap block on postoperative pain scores . in this context , it is worth mentioning that the meta - analysis found that tap block decreases postoperative opioid consumption , which may be a more important parameter to decide an analgesic regimen . the median duration of effective postoperative analgesia from our study was 290 min in patients receiving tap block , and we did not use any additive in tap block . clonidine in peripheral nerve block has been shown to significantly increase the duration and may be considered here also . time to the requirement of first postoperative analgesic is also significantly increased in patients received tap block ( 290 min vs. 16 min ) . this was consistent with mcdonnell et al . , who demonstrated in their anatomic study that tap block with 0.5% lignocaine may provide analgesia for 4 - 6 h. only three patients in bupivacaine tap group required rescue analgesic in first two postoperative hours whereas 43 patients in the saline group required the same . four patients in bupivacaine group did not require rescue analgesia in first 24 h postoperative period . the cause of prolonged duration of analgesic effect following single shot tap block is not entirely clear . this may be explained by the fact that the tap is relatively poorly vascularized , and therefore drug clearance may be slowed . inadequate analgesia even after tap block may be either due to technical failure or due to visceral pain component , which is not addressed by tap block . as such , until now , all local anesthetic techniques carry an inherent failure rate of 5 - 20% , depending on the skill of the operator . the most important clinical implication of our findings is the significant opioid sparing effects of tap block both in the intraoperative as well as the postoperative period . opioids , though very effective in perioperative pain management , may be associated with nausea - vomiting , pruritus and respiratory depression . moreover , some patients who are morbidly obese or having obstructive sleep apnea will be maximally benefitted from tap block as it provides opioid sparing effects . patients having ischemic heart disease or stenotic valvular lesion like mitral or aortic stenosis , where tachycardia is undesirable , will also be benefitted from preincisional tap block . it may be a relatively safer alternative to neuraxial block for intra and postoperative analgesia in patients having coagulopathy . though we controlled the depth of anesthesia by bis monitoring , ensured adequate muscle relaxation , prevented hypovolemia , indirect assessment of intraoperative pain by hemodynamic parameters may be unreliable . second use of real time usg for tap block is increasing ; we used a landmark based anatomical approach . however , as real time us guidance may increase the efficacy of tap block , it wo nt change the primary finding of our study . third , use of patient controlled analgesia in the postoperative period could have accurately delineated postoperative opioid consumption . preincisional tap block decreases intraoperative fentanyl requirements , prevents hemodynamic responses to surgical stimuli and also provides effective postoperative analgesia .

background and aims : transversus abdominis plane ( tap ) block has been shown to provide postoperative pain relief following various abdominal and inguinal surgeries , but few studies have evaluated its analgesic efficacy for intraoperative analgesia . we evaluated the efficacy of tap block in providing effective perioperative analgesia in total abdominal hysterectomy in a randomized double - blind controlled clinical trial.materials and methods : a total of 90 adult female patients american society of anesthesiologists physical status i or ii were randomized to group b ( n = 45 ) receiving tap block with 0.25% bupivacaine and group n ( n = 45 ) with normal saline followed by general anesthesia . hemodynamic responses to surgical incision and intraoperative fentanyl consumption were noted . visual analog scale ( vas ) scores were assessed on the emergence , at 1 , 2 , 3 , 4 , 5 , 6 and 24 h. time to first rescue analgesic ( when vas 4 cm or on demand ) , duration of postoperative analgesia , incidence of postoperative nausea - vomiting were also noted.results:pulse rate ( 95.9 11.2 bpm vs. 102.9 8.8 bpm , p = 0.001 ) systolic and diastolic bp were significantly higher in group n. median intraoperative fentanyl requirement was significantly higher in group n ( 81 mcg vs. 114 mcg , p = 0.000 ) . vas scores on emergence at rest ( median vas 3 mm vs 27 mm ) , with activity ( median 8 mm vs. 35 mm ) were significantly lower in group b. median duration of analgesia was significantly higher in group b ( 290 min vs. 16 min , p = 0.000 ) . no complication or opioid related side effect attributed to tap block were noted in any patient.conclusion:preincisional tap block decreases intraoperative fentanyl requirements , prevents hemodynamic responses to surgical stimuli and provides effective postoperative analgesia .

innate immunity is the first line of defense of the body , present throughout evolution from lower ( invertebrate ) to more complex organisms ( vertebrate ) ( 1 ) . in order to rapidly protect the host against infectious or stressful events , the innate immune cells initiate triggers an inflammatory response . when not properly regulated or in excess , inflammation may contribute to many and different pathological conditions , from autoimmune and chronic inflammatory diseases to atherosclerosis and cancer ( 234567 ) . they play a pivotal role in the immune response to pathogens , by generating and then resolving the inflammatory reaction . but they also have a central role in tissue development ( by shaping the tissue architecture ) , and in surveillance and monitoring of tissue changes ( by acting as sentinel and effector cells ) . especially , they are important in maintening tissue homeostasis , by clearing apoptotic or senescent cells , and by repairing and remodeling structural and functional integrity of the tissue soon after a damage . in 1893 , elie metchnikoff first described the phagocytosis of pathogens by macrophages ( " the big eaters " in greek ) , observing starfish challenged with rose thorns ( 8) . macrophages were identified as tissue resident cells able to eat and kill infectious agents . in 1924 , macrophages were defined by aeschoff as cells of the reticulo - endothelial system ( res ) . this implied that macrophages originate from , and reside and renew within , that tissue , which comprises the kupffer cells of the liver , the cells lining the sinuses of spleen , lymph nodes , bone marrow , and the endothelial cells of various organs ( 9 ) . in the late 1960s , ralph van furth proposed the existence of the mononuclear phagocyte system . within this system , all macrophages , including tissue macrophages and inflammatory cells ( macrophages recruited during inflammation , also meaning the monocyte - derived macrophages ) , are terminally differentiated cells derived from blood monocytes ( 10 ) . however , in 1984 van furth together with diesselhoff - den dulk ( 11 ) suggested that not all macrophages derive from blood monocytes . in the same years it was observed that macrophages were not terminally differentiated ( 1213 ) and were persistent in tissues ( 14 ) . it has now become clear that tissue - resident macrophages may arise independently of monocyte input ( see below ) , originating prenatally from c - myb - independent hematopoiesis ( 15 ) prior to the establishment of definitive hematopoiesis . accordingly , there is now a consensus that some macrophages may proliferate , although it is not clear whether this takes place through self - renewal or through the proliferation of local progenitors ( 1617 ) . thus , in light of the monocyte - independent origin of macrophages , other aspects of macrophage biology need reassessment , including macrophages heterogeneity , the role of environment in the tissue - specific specialization , and macrophage polarization into different functional phenotypes during inflammation . in this review several questions are still open on differences , similarities , and development lineage relationship between monocyte - derived macrophages and tissue macrophages . these are discussed in depth elsewhere ( 18 ) . finally , we will briefly report on the recently renewed interest for innate immune memory . tissue macrophages are heterogeneous and versatile cells found in virtually all tissues of adult mammals , where they can represent up to 10~15% of the total cell number in quiescent conditions . the functional specialization of macrophages in unique tissue microenvironments explains their heterogeneity ( 1920 ) . thus , macrophages take different names according to their tissue location , such as osteoclasts ( bone ) , alveolar macrophages ( lung ) , microglial cells ( brain ) , histiocytes ( connective tissue ) , kupffer cells ( liver ) , langerhans cells ( lc ) ( skin ) , etc . if on the one hand tissue macrophages acquire specific morphological and functional phenotypes according to the microenvironment in which they reside , on the other hand some functions of macrophages are the same in all tissues , such as the surveillance that maintains tissue homeostasis ( recognizing and removing anomalous and senescent cells ) , and the protective function ( reaction to infections and tissue damage by initiating , developing and resolving an inflammatory response ) . as mentioned above , it recently became evident the existence of a myb - independent hematopoietic stem cell ( hsc)-independent lineage of tissue macrophages . fate - mapping experiments showed that tissue - resident macrophages in multiple organs , including the liver ( kupffer cells ) , epidermis ( lc ) , and brain ( microglia ) , originate from the yolk sac ( ys ) or fetal liver ( fl ) ( 1521 ) . overviews of all the experiments demonstrating myb - independent macrophage origin are critically reviewed in ( 22 ) . primitive hematopoiesis that takes place in ys and gives rise to macrophages without going through a monocytic progenitor ( myb - independent ) ; 2 . definitive hematopoiesis that takes place in the fl ( myb - dependent ) , which is initially seeded by hematopoietic progenitors from the ys and subsequently by hematopoietic stem cells from endothelium of the aorta - gonads - mesonephros ( 23 ) . the relative contribution of ys progenitors to the circulating definitive hematopoietic progenitor cell pool that seeds the fl ( and that then replenishes macrophages of peripheral organs ) remains controversial . ys- and fl - derived macrophages have been considered distinct so far , but a recent work identified an erythro - myeloid progenitor population that originates in the ys and later migrates to the liver ( 24 ) . thus , a common origin in the ys for both ys- and fl - derived macrophages is a reasonable hypothesis . during embryogenesis , the fl subsequently becomes the source of definitive hematopoiesis that generates all major hematopoietic lineages , including circulating monocytes . fl monocytes populate most peripheral tissues ( e.g. , lung , spleen , dermis , liver ) , except the brain , and give rise to tissue macrophages , which generally coexist with , but can progressively displace , ys - derived tissue macrophages . after birth , upon bone formation blood monocyte precursors originate in the bone marrow , and blood monocytes constantly replenish resident macrophages in various organs , such as intestine and dermis ( 252627 ) . together with these monocyte - derived tissue resident macrophages , the tissues may encompass monocyte - derived macrophages that developed from inflammatory monocytes recruited during an infection or damage of the tissue ( 1828 ) . macrophages that prenatally reside the tissues then proliferate to repopulate the expanding tissue with tissue resident macrophages . this proliferative potential or self - renewal capacity has been recently observed in microglia , in peritoneal , pleural and alveolar macrophages , in macrophages of adipose tissue and atherosclerotic plaques , and in cardiac macrophages ( 16 ) . nevertheless , tissue macrophages of embryonic origin can be replaced by monocytes - derived macrophages after severe inflammation ( 18 ) , or in the aging heart ( 29 ) . 30 ) have recently reviewed the current knowledge on resident macrophage development and their functional specialization in the tissue , while dey et al . we refer the reader to these two reviews for deeper and detailed information on these aspects of macrophage biology . the macrophage functional specialization / differentiation defines the presence of morphologically distinct macrophages with tissue - specific functions in homeostatic conditions . on the other hand , macrophage polarization describes the capacity of macrophages to modify their function in a plastic manner during the inflammatory reaction . both tissue macrophages and monocyte - derived macrophages have a high level of plasticity , although their relative contribution to the progression and resolution of inflammation is not fully demonstrated . the relationship between tissue macrophages and monocyte - derived macrophages and their role in steady state conditions and during inflammation has been extensively reviewed in ( 18 ) and , with a focus on brain , adipose tissue and liver , in ( 31 ) . macrophage polarization occurs through different activation programs , by which macrophages carry out their defense functions . in this way , macrophages become able to respond with appropriate functions in distinct contexts , and functional diversity becomes the key feature of these cells . essentially , macrophages can modify their functions from a heal / growth promoting setting ( m2 or alternative macrophages ) , to a killing / inhibitory capacity ( m1 or classical macrophages ) ( 32 ) . in vitro , macrophages are activated towards an m1 functional program by microorganism - related molecules ( e.g. , the gram - negative lipopolysaccharide , lps ) and by the inflammation - related cytokines tnf- or ifn- , alone or in combination . m1 macrophages are efficient producers of toxic effector molecules ( such as reactive oxygen / nitrogen species , and inflammatory cytokines ) , participate as inducers and effector cells in polarized th1 responses , and mediate resistance against intracellular parasites and tumors ( 33 ) . conversely , m2-like polarization has been observed in vitro in response to the th2-related cytokines il-4 or il-13 , to the concomitant triggering of fc receptors and toll - like receptors ( tlr ) , to immune complexes , and to anti - inflammatory molecules such as il-10 , tgf- , and glucocorticoids ( 3435 ) . m2 macrophages take part in polarized th2 responses , allergy , parasite clearance , dampening of inflammation , tissue remodeling , angiogenesis , immunoregulation , and tumor promotion ( 36 ) . m2 is the normal " default " program adopted by resident macrophages ( 37 ) . in addition to functional differences , m1 and m2 macrophages have distinct features in terms of chemokine production profiles ( 38 ) and metabolism ( 39 ) . the main distinction in terms of metabolism is that in m2 macrophages the arginine metabolism is shifted to ornithine and polyamines , which are important in the would healing process . conversely , in m1 cells such metabolism is shifted to no , which plays a key role in the intracellular killing of pathogens ( 40 ) . the m1/m2 classification is useful to understand the plasticity of macrophages but is just a limited attempt to capture the complexity and plasticity of these cells . in fact , this macrophage taxonomy does not fully mirror what really happens in the tissue during inflammation . in vivo the sequence of changing scenarios / signals presumably induces macrophages to adopt a variety of functional phenotypes during the course of an inflammatory reaction . therefore , the m1/m2 polarization of macrophage functions may be taken as a simplified conceptual framework describing a continuum of diverse functional states , of which m1 and m2 activation states are not ontogenically defined subsets but represent the extremes of the functional continuum ( 41 ) . as suggested by martinez and gordon ( 42 ) , recently , murray and coworkers attempted a re - classification of polarized macrophage in response to a range of stimuli ( 43 ) . furthermore , this issue has been recently addressed by network modeling analyses of hundreds of macrophage transcriptomes triggered by a diverse set of stimuli ( 44 ) . this analysis revealed at least nine distinct macrophage activation programs , thus extending the dual m1/m2 macrophage polarization to a spectrum model . moreover , knowing that local environment controls macrophage phenotypes , and given that many experiments with human cells are performed in vitro , we should redefine the in vitro culture conditions to closely match those occurring in a specific tissue microenvironment ( 4546 ) or in the course of an inflammatory reaction ( 47 ) . an important issue of macrophage polarization is whether polarized macrophage populations can switch one to the other in response to different conditions . data from in vitro studies demonstrate that human monocytes can acquire the phenotype of polarized m1 macrophages and then mature into m2 repair macrophages upon exposure in culture to sequential changes in the microenvironmental conditions ( 47 ) . a related open question is whether both tissue - resident macrophages and monocyte - derived macrophages can polarize in m1 or / and m2 functional phenotypes . here we just want to highlight the major role of microenvironmental signals in the functional phenotype switching of monocytes and macrophages by mentioning two severe pathological conditions : obesity and cancer . the metabolic syndrome can lead to a switch in the phenotype of adipose tissue macrophages from m2/wound healing ( as in healthy non - obese humans ) to classically activated macrophages ( 48 ) . in cancer , tumor - infiltrating classically activated macrophages have the potential to contribute to the earliest stages of neoplasia , and then , as the tumor progresses , can progressively differentiate to a regulatory phenotype and eventually become cells that share the characteristics of both regulatory and wound - healing macrophages ( 49 ) . in both cases , signals present in the surrounding environment at a given time drive the changes in the functional phenotype of adipose tissue macrophages and tumor - associated macrophages at given stage of the disease . as previously discussed , innate immune cells have two peculiarities , diversity / heterogeneity and plasticity ( 50 ) . steady state hematopoietic differentiation programs may generate diverse subsets of monocytes ( e.g. , cd14 cd16 or classical monocytes , cd14cd16 or intermediate monocytes , cd14cd16 or non - classical monocytes ) ( 51 ) , and distinct tissue - resident macrophages ( e.g. , kupffer cells , microglia , lc , etc . ) . thus , tissue - resident macrophages may be exposed to endogenous signals from the tissue during development or steady state conditions , in order to develop tissue specialization , whereas both tissue - resident macrophages and monocyte subpopulations ( once in the tissue , monocyte - derived macrophages ) may be subsequently activated by microbial or danger signals during inflammation or tissue damage . thus , in the presence of diverse microenvironmental stimuli , each subset modifies its activation state in a plastic manner depending on the nature of the stimulus . phenotypic , transcriptional and epigenetics mechanisms underlie both diversity and plasticity , inducing functional specialization in different cells and in the same cells in different microenvironmental contexts . we want to underline that heterogeneity , maintenance and specific function of resident macrophages are firmly under tissue control during homeostasis and in inflammatory conditions . it is evident that the expression of different transcription factor is required for specific functions in macrophages ( 5253 ) . several examples of transcription factors that dictate tissue - specific transcription programs in macrophages have been reported . for examples : the heme - induced transcription factor spic is required for the development of red pulp macrophages ( 54 ) , and nr1h3 is needed for the development of all macrophages of the marginal zone ( 55 ) . gata6 is a regulator of a tissue - specific gene expression program in peritoneal macrophages , and retinoic acid released in the tissue is a signal that regulates reversibly the induction of gata6 ( 46 ) . induction of ppar- by gm - csf is critical for the development of alveolar macrophages from fetal monocytes ( 56 ) . transcription factors such as stat1 , stat6 , c / ebpb , irf-4 , irf5 , and ppar- have been shown to regulate transcription programs that control m1/m2 macrophage polarization ( 57 ) . two recent studies that have employed rna - seq have demonstrated the main role of the microenvironment in determining the macrophage phenotype reprogramming ( 5859 ) . authors concluded that tissue macrophages shared with other myeloid cells the epigenetic structure and gene expression ( regulated by the master transcription factor pu.1 ) , and in addition that each tissue has its own unique gene expression profile controlled by changes in enhancer landscapes . moreover , tissue - specific reprogramming was evident by transferring macrophages from a tissue to another , showing that transferred cells lost most of the old tissue programming , acquiring a new one according to their new tissue microenvironment ) . the same was evident upon lethal irradiation of embryonic tissue macrophages and replacement with macrophages derived from transplanted healthy bone marrow . within a tissue , macrophages sense the microenvironmental changes through a series of sensors , such as prr , scavenger receptors , cytokines receptors , and adhesion molecules , and are susceptible to silencing programs dictated by the tissue . indeed , the tissue sets tissue - specific thresholds for the timing and extent of macrophage activation depending on the type and intensity of the stimulus . the exact nature of these silencing circuits differs between tissues and includes innate inherent suppression , as well as acquired deactivation induced activating stimuli ( 34 ) . intestinal gut macrophages express the il-10 receptor , and in order to prevent severe inflammation they must be exposed to homeostatic t regulatory cell - derived il-10 ( 60 ) . on the other hand , microglia is probably silenced by tgf- ( 61 ) and maintained into a down - regulated phenotype by neural - derived cx3cr1 and cd200 ( 6263 ) . local tissue - derived signals are thought to control the development of the tissue - specific phenotypes of resident macrophages , proving the critical role of tissue environment . however , for most tissues the identity of these signals remains largely unknown ( with some exceptions , see microglia , 61 , and bone , 64 ) . in conclusion , the environment may reprogram macrophages in a tissue - specific manner in order to tailor their functions based on the tissue needs , or may polarize macrophages conferring a differently reactive phenotypes ( classical vs. alternative vs. deactivated ) . moreover , it is unknown whether functional heterogeneity and polarization are the result of irreversible lineage - specific differentiation or a consequence of continuous but reversible induction of diverse functional programs . it was recently suggested to classify monocyte / macrophage primarily by their ontogeny and only secondarily by their location , function and phenotype ( 65 ) . however , which is the effect of origin on macrophage function is not yet fully known . considering the new insights in macrophage biology , it is worth mentioning the recent reassessment of the innate immune memory , an old concept that is currently raising a renewed interest in the scientific community . indeed , revisited old knowledge on the repeated stimulation of the innate immune responses ( see for instance 6667 ) has reintroduced the old concept of innate immune memory ( 68 ) . evidence in both plants and invertebrates ( that do not possess adaptive immunity and classical memory ) indicates that phagocytes can respond much better to a challenge if they have been pre - stimulated with the same or with another agent ( 69 ) . thus , innate immunity can have a memory , although different from acquired immune memory . a very interesting notion is that the innate memory is apparently at least in part non - specific , which implies that cells pre - challenged with ( almost ) any kind of agents can attain an improved defensive response to a subsequent challenge . another very interesting concept is that of the exquisitely local dimension of phagocyte memory . in higher vertebrates such as man , however , in the case of external challenges ( e.g. , an infectious agent ) , they can initiate an immediate defensive inflammatory response . it is very interesting to observe that macrophages , after a challenge , can develop a memory and react to a second challenge in different ways : they become less reactive to some challenges to avoid extensive tissue damage or they display an enhanced response to improve tissue surveillance , e.g. , against tumors . these two different responses of innate immune memory are known as " tolerance " and " trained immunity " , respectively . lps activates macrophages into a disruptive inflammatory reaction that also damages the surrounding tissue . upon a second challenge , macrophages react much less because they aim at avoiding an excessive response that would destroy the tissue integrity . on the other hand , challenge with fungal components ( implying a long - term slow infection with tissue debilitation ) induces an innate memory that results in enhanced reactivity to subsequent stimuli , necessary for the adequate defense of a weakened tissue . the molecular mechanisms responsible for shifting macrophages toward a memory status have not yet been elucidated . putative mechanisms may involve different recruitment of different monocyte / macrophage subpopulation ( i.e. , cd14 and cd16 ) ( 68 ) , or changes in the expression of lectin receptors on cell membrane ( 70 ) , or in the functional phenotype ( e.g. , phagocytosis or protein production ) . in any case , all of these changes are probably due to epigenetic reprogramming that regulates gene expression by inducing dynamic alterations in the chromatin structure , through modification of dna , post - translational modifications of histones ( methylation ) , or microrna ( 717273 ) . another interesting aspects of innate memory are changes in metabolic processes , as already observed in macrophage polarization ( 3974 ) . whether monocyte - derived macrophages or tissue macrophages or both retain a memory of past challenge we preliminarily observed ( unpublished observation ) that a mild microbial stimulus could induce different immunological programming in monocyte - derived macrophages vs. tissue macrophages . indeed , in vitro monocyte - derived macrophages and tissue macrophages primed with microbial agents ( e.g. , lps , -glucan ) produce a substantially different profile of inflammatory cytokines upon re - exposure , demonstrating the capacity of adapting their response to an evolving situation ( such as multiple or chronic infections ) . notably , the type of innate memory induced by pre - challenge may be different ( decrease vs. increase vs. no effect ) depending on the inflammatory endpoint ( e.g. , production of tnf- or il-1 or il-8 ) and the type of mononuclear phagocyte , with inflammatory monocytes being more reactive than tissue macrophages . the increased understanding of the properties of innate memory is changing our awareness of host defense and immune memory , and could lead to defining new classes of vaccines and adjuvants . efficacy of many vaccines probably implies the induction of non - specific macrophage memory that contributes to the increased resistance to infections . research in the field of memory macrophages needs a thorough reassessment of a large body of old evidence accumulated in the past decades in the areas of macrophage activation and of adjuvanticity . given the important role of macrophages in acute and chronic inflammation and in other severe diseases ( artherosclerosis , neurodegeneration and cancer ) , it is not surprising the growing interest for their proprieties and for their potential clinical applications . in a recent review , martinez and gordon address the issue of translating the new knowledge of macrophage biology into clinical practice ( 75 ) . they describe a selection of the potential functional targets in macrophages , such as macrophage recruitment , activation , and memory , which are being considered for potential therapeutic / clinical application by using ad hoc inhibitors . moreover they indicate new application areas that are likely to develop in the near future , thanks to our new understanding of macrophage biology , including cell therapy , diagnosis and prognosis . we agree with martinez and gordon especially on the following key needs : more studies on humans to close the gap between the bench and the clinic ; and use of standardized and improved methods to investigate macrophages heterogeneity and to measure genes , proteins and metabolites in situ . the common view is that macrophage diversity and plasticity are driven by cues in the tissue microenvironment , which can include cytokines , growth factors and microorganism - associated molecular patterns . these signals are believed to dictate a transcriptional response that shapes the phenotype and function of macrophages based on the physiological or pathological context . progress has been made in defining the molecular mechanism underlying macrophage biology , but the data are still incomplete and far from being systematic . thus , we need to increase our knowledge of the mechanistic basis of macrophage heterogeneity / diversity and plasticity , and how to pharmacologically manipulate them . considering that various in vitro models currently used do not adequately reflect the heterogeneity and plasticity of tissue macrophages , we conclude by suggesting that future studies should be conducted on macrophages isolated directly from tissues in different homeostatic and pathological conditions ( in steady state condition and in response to a changing environment ) . single - cell transcriptomic and epigenetic analysis ( 76 ) might help to molecularly identify the macrophage functional states and their changes driven by changing tissue conditions .

macrophages are the main effector cells of innate immunity and are involved in inflammatory and anti - infective processes . they also have an essential role in maintaining tissue homeostasis , supporting tissue development , and repairing tissue damage . until few years ago , it was believed that tissue macrophages derived from circulating blood monocytes , which terminally differentiated in the tissue and unable to proliferate . recent evidence in the biology of tissue macrophages has uncovered a series of immune and ontogenic features that had been neglected for long , despite old observations . these include origin , heterogeneity , proliferative potential ( or self - renewal ) , polarization , and memory . in recent years , the number of publications on tissue resident macrophages has grown rapidly , highlighting the renewed interest of the immunologists for these key players of innate immunity . this mini - review aims to summarizing the new current knowledge in macrophage immunobiology , in order to offer a clear and immediate overview of the field .

the term amyotrophic lateral sclerosis ( als ) was first coined by charcot , who postulated the primacy of the upper motor neuron ( umn ) in als pathogenesis.1 assessment of cortical function in als and identification of the characteristic clinical phenotype involving combined upper and lower motor neuron abnormalities remain the key for als diagnosis.24 however , despite charcot 's initial observations , the site of disease onset and mechanisms underlying als pathophysiology remain areas of intense study and debate.5 in this setting , assessment of motor cortical and corticospinal function using non - invasive techniques , such as transcranial magnetic stimulation ( tms ) , has enhanced our understanding of als pathophysiology and resulted in novel diagnostic approaches . single- , paired- and multiple - pulse tms techniques have all been used ( figure 1 ) with the following measures taken to reflect corticomotoneuronal function : motor threshold ( mt ) , motor evoked potential ( mep ) amplitude , central motor conduction time ( cmct ) , cortical silent period ( csp ) , intracortical inhibition and facilitation . the present review will focus on the mechanisms underlying the generation of these tms measures , while at the same time assessing the contributions tms has made in the understanding of als pathophysiology . with an eye towards the future , the review will also consider the potential diagnostic utility of tms in als and incorporation of tms as a disease biomarker in the assessment of neuroprotective medications in a clinical trial setting . transcranial magnetic stimulation excites a network of neurons in the underlying motor cortex with motor evoked potentials recorded over the contralateral abductor pollicis brevis muscle . the motor cortex is preferentially stimulated when the current flows in a posterior anterior direction within the motor cortex . mt reflects the ease with which corticomotoneurons are excited and is proposed to be assessed by the international federation of clinical neurophysiology as the minimum stimulus intensity required to elicit a small ( usually > 50 v ) mep in the target muscle in 50% of trials.6 with the recent adaptation of threshold tracking techniques , mt can also be measured as the stimulus intensity required to elicit and maintain a target mep response of 0.2 mv.79 mt reflects the density of corticomotoneuronal projections onto the spinal motor neuron with the highest density of projections to intrinsic hand muscles having the lowest mts.1012 mts are lower in the dominant hand12 and correlate with the ability to perform fine ( fractionated ) finger tasks,13 so that mt has the potential to map corticomotoneuronal representation and function . as well as reflecting the density of corticomotoneuronal projections , mts may also be a biomarker of cortical neuronal membrane excitability.1416 mts are influenced by the glutamatergic neurotransmitter system , through -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid ( ampa ) receptors , whereby excessive glutamate activity reduces mts.17 in contrast , pharmacological blockade of voltage - gated sodium channels raises mt.18 in als , abnormalities in mt have been inconsistent . while some tms studies reported an increased mt or even an inexcitable motor cortex,1926 others have documented either normal or reduced mt.2732 these discrepancies likely relate to heterogeneity of the als phenotype and the stage of disease at time of testing and rate of progression . longitudinal studies have documented a reduction of mts early in the disease course , increasing to the point of cortical inexcitability with disease progression.29 the early reduction in mt appears most pronounced in als patients with profuse fasciculations , preserved muscle bulk and hyper - reflexia.33 fasciculations may precede other features of als by many months and taken in association with reduced mt suggest a cortical origin of fasciculations in als.34 reduced mt may be modulated by increased glutamate excitation , reduced gamma - aminobutyric acid ( gaba ) inhibition or a combination of both . reduced mt early in als supports an anterograde transsynaptic process , whereby cortical hyperexcitability underlies the development of progressive neurodegeneration . mep amplitude reflects a summation of complex corticospinal volleys consisting of d ( direct)- and i ( indirect)-waves.14 35 at threshold , tms elicits i - waves at intervals of 1.5 ms , which increase in amplitude with increasing stimulus intensity.35 the increase in mep amplitude with increasing stimulus intensity may be used to generate a stimulus response curve that follows a sigmoid function.36 as with mt , the mep amplitude reflects the density of corticomotoneuronal projections onto motor neurons.37 when compared with mt , the meps probably assess the function of neurons that are less excitable or further away from the centre of the tms induced electrical field.38 the mep amplitude should be expressed as a percentage of the maximum compound muscle action potential ( cmap ) evoked by electrical peripheral nerve stimulation.6 doing so takes into account any lower motor neuron pathology and provides insight into the percentage of the motor neurone pool activated in the mep . normative values for the mep to cmap ratio demonstrate a large inter - subject variability thereby reducing the sensitivity and limiting the value of this measure for detecting abnormalities of the corticomotoneurons.38 39 the mep responses are modulated by a variety of neurotransmitter systems within the central nervous system.37 40 specifically , gabaergic neurotransmission via gabaa receptors suppresses while glutamatergic and noradrenergic neurotransmission enhances the mep amplitude.41 of interest , these changes in mep amplitude occur independently of changes in mt , suggesting that physiological mechanisms underlying the generation of the mep amplitude and mt are varied . abnormalities of meps have been extensively documented in als.38 increases in mep amplitude have been reported in sporadic and familial forms of als ( figure 2a ) , most prominently early in the disease course.30 31 42 mep amplitude correlates with surrogate biomarkers of axonal degeneration , such as the strength duration time constant , thereby providing an association between cortical hyperexcitability and motor neuron degeneration.30 43 the increase in mep amplitude in als is not seen in mimic disorders despite a comparable degree of lower motor neuron dysfunction ( figure 2b ) . this suggests that the mep amplitude changes in als are excitotoxic in nature.4447 ( a ) the motor evoked potential ( mep ) amplitude , expressed as a percentage of compound muscle action potential ( cmap ) response , is significantly increased in sporadic amyotrophic lateral sclerosis ( als ) and familial als ( fals ) when compared with healthy controls . ( b ) the mep amplitude is significantly increased in als when compared with pathological and healthy controls , thereby distinguishing als from als mimic disorders . * cmct represents the time from stimulation of the motor cortex to the arrival of corticospinal volley at the spinal motor neuron.6 multiple factors contribute to the cmct including time to activate the corticospinal cells , conduction time of the descending volley down the corticospinal tract , synaptic transmission and activation of spinal motor neurons.48 cmct may be measured using either the f - wave or cervical ( or lumbar ) nerve root stimulation methods;49 50 both methods provide only an estimation of the cmct,48 51 and given that a variety of technical , physiological and pathological factors influence cmct,48 there is a range of normative data . in als , cmct is typically modestly prolonged,21 29 52 probably reflecting axonal degeneration of the fastest conducting corticomotoneuronal fibres and increased desynchronisation of corticomotoneuronal volleys secondary to axonal loss.28 53 54 the d90a - sod1 als mutation is a unique exception ; in this disorder cmct is typically very prolonged.55 the sensitivity of detecting a prolonged cmct may be improved by recording from both upper and lower limb muscles , or from cranial muscles in als patients with bulbar onset disease.26 56 csp refers to the interruption of voluntary electromyography activity in a target muscle induced by stimulation of the contralateral motor cortex.57 the csp duration is measured from the onset of the mep response to resumption of voluntary electromyography activity37 57 and increases with stimulus intensity.5759 the csp is mediated by both spinal mechanisms , in its early part , and cortical inhibitory neurons acting via gabab receptors in the latter part.57 58 6063 since the duration is determined by the latter part , the csp is a measure of cortical inhibition . in addition , the density of the corticomotoneuronal projections onto motor neurons also influences the csp , with the csp duration being the longest for upper limb muscles.38 abnormalities of the csp duration are well established in als.37 absence or reduction in csp duration has been reported in both sporadic and familial als , with the reduction of csp duration being the most prominent early in the disease course.3032 44 46 52 6467 the reduction of csp duration appears to be specific for als among neuromuscular disorders , being normal in x - linked bulbospinal muscular atrophy ( kennedy 's disease ) , acquired neuromyotonia and distal hereditary motor neuronopathy with pyramidal features.4447 although the mechanisms underlying csp duration reduction in als remain to be established , decreased motor drive and reduced gabaergic inhibition , either due to degeneration of inhibitory interneurons or dysfunction of gabab receptors , may underlie the reduction of csp duration in als . an absent or delayed ipsilateral csp has also been reported as an early abnormality in als.67 68 the ipsilateral csp depends on functioning of transcallosal glutamatergic fibres projecting onto inhibitory interneurons in the non - stimulated motor cortex,69 and degeneration of these transcallosal fibres or their targeted inhibitory interneurons may account for abnormalities of the ipsilateral csp in als . the previous section has covered conventional tms parameters that can be assessed through activation of the motor cortex by single impulses . motor cortical excitability may also be assessed using paired - pulse techniques , in which a conditioning stimulus modulates the effect of a second test stimulus . several different paired - pulse paradigms have been developed,37 38 but short interval intracortical inhibition ( sici ) , intracortical facilitation ( icf ) and long interval intracortical inhibition have been most frequently used in als clinical research as methods to determine cortical excitability . to identify sici and icf , a subthreshold conditioning stimulus is typically delivered at predetermined time intervals prior to a suprathreshold test stimulus.8 7072 in the early tms paradigms,70 72 73 the conditioning and test stimuli were kept constant , and changes in the test mep amplitude were evaluated . typically , if the interstimulus interval ( isi ) was between 1 and 5 ms , the test response was inhibited ( sici ) . increasing the isi to between 7 and 30 ms resulted in the facilitation of the test response ( icf).38 by recording the descending corticospinal volleys through epidural electrodes at the level of the cervical spinal cord , it has been deduced that both sici and icf originate at the level of the motor cortex.35 72 specifically , sici is associated with a reduction in the number and amplitude of late i - waves , namely i2 and i3 , with i - wave suppression remaining up to an isi of 20 ms , which is the typical duration of the inhibitory postsynaptic potential mediated through gabaa receptors.71 74 sici and icf appear to be physiologically distinct processes as evident by lower thresholds for activation of sici and sici remains independent of the direction of current flow in the motor cortex induced by a subthreshold conditioning pulse in healthy subjects , while icf appears to be preferentially generated by current flowing in a posterior constant stimulus paired - pulse technique has been the marked variability in mep amplitudes with consecutive stimuli.71 75 to overcome this limitation , a threshold tracking technique was developed whereby a constant target mep response ( 0.2 mv ) was tracked by a test stimulus.7 8 using threshold tracking , two phases of sici were identified,7 8 76 77 a smaller phase at isi 1 ms and a larger phase at isi 3 ms ( figure 3a ) . although synaptic neurotransmission through the gabaa receptor mediates the second phase of sici,74 7880 the precise mechanisms underlying the first phase of sici remain uncertain . it was initially suggested that the first phase of sici reflected local excitability properties , particularly relative refractoriness of cortical axons , with resultant resynchronisation of cortico - cortical and corticomotoneuronal volleys.7 81 subsequently , it has been argued that synaptic processes best explain the development of the initial phase of sici , possibly driven by activation of cortical inhibitory circuits that were distinct to those that mediated the later sici phase.76 77 82 ( a ) short interval intracortical inhibition ( sici ) , defined as the stimulus intensity required to maintain a target motor evoked potential of 0.2 mv , as assessed by the threshold tracking transcranial magnetic stimulation technique . intracortical inhibition is illustrated by an increase in the conditioned test stimulus intensity required to track the target response , while intracortical facilitation is indicated by a reduction in test stimulus intensity . in healthy controls , sici develops between interstimulus intervals ( isi ) of 1 and 7 ms , with two peaks evident at 1 and 3 ms as indicated by the arrows . sici is significantly reduced in both sporadic amyotrophic lateral sclerosis ( sals ) and familial amyotrophic lateral sclerosis ( fals ) . ( b ) averaged sici , between isi 1 and 7 ms , was reduced in two presymptomatic superoxide dismutase-1 ( sod-1 ) mutation carriers 6 months prior to the development of als . ( c ) normalised sici , expressed as a fraction of the sici value measured at the first study , was reduced 8 months prior to development of als in a third presymptomatic sod-1 mutation carrier . a reduction or absence of sici , together with an increase in icf , indicative of cortical hyperexcitability has been documented in cohorts of sporadic and familial als patients ( figure 3a).3032 44 45 8388 of relevance , cortical hyperexcitability appears to be an early feature in sporadic als , correlating with measures of subsequent peripheral neurodegeneration.30 in addition , cortical hyperexcitability appeared as an early feature in familial als due to mutations linked to the superoxide dismutase-1 ( figure 3a ) and fused in sarcoma ( fus ) genes,31 preceding the clinical development of familial als ( figure 3b).31 neuropathological studies in als have identified degeneration of inhibitory cortical interneurons89 and this could account for the reduction in sici . separately , glutamate - mediated excitotoxicity may also contribute to sici reduction , as was suggested by partial correction of sici abnormalities in als patients treated with the glutamate antagonist riluzole.87 a recent study documenting sici reduction at low ( 40% of resting mt ( rmt ) ) , medium ( 70% of rmt ) and high ( 90% of rmt ) conditioning stimulus intensities in als patients provided further support for the notion that abnormalities in sici appeared to be mediated by a combination of glutamate excitotoxicity and degeneration of inhibitory cortical circuits.90 as such , preserving the integrity of intracortical inhibitory circuits , and counteracting excitatory cortical circuits , may serve as potential therapeutic options in als . a peristimulus time histogram technique can assess the function of a select subset of corticomotoneurons by recording the perturbation of voluntarily recruited motor units induced by a threshold cortical stimulation.53 in healthy controls , there is a well synchronised primary peak with a latency of approximately 2030 ms recording from hand or forearm muscles.28 53 analysis of this primary peak in disease states such as als provides information on corticomotoneuronal conduction time , the extent of desynchronisation of corticomotoneuronal descending volleys , the degree of corticomotoneuronal synaptic input onto the anterior horn cell and the timing of excitatory and inhibitory inputs to the motor neuron.33 in als , the primary peak becomes desynchronised , prolonged in duration and delayed.28 91 92 in addition , the amplitude of the primary peak may be increased with additional subcomponents both suggestive of corticomotoneuronal hyperexcitability.53 93 these primary peak abnormalities appear early in als , accompanied by reduced mts . with progression of disease , there is prolongation and increased desynchronisation of the primary peak , findings possibly specific to als when compared with healthy controls and kennedy 's disease.53 94 over recent years , collision techniques such as the triple stimulation technique ( tst ) have been used to reduced the degree of mep desynchronisation which normally occurs following a single cortical stimulus.95 96 this complex technique is performed by first delivering a high - intensity magnetic stimulus to motor cortex followed by supramaximal electrical stimulation of the peripheral nerve supplying the target muscle at the wrist such that the descending corticomotoneuronal volley is collision takes place along the proximal segment of the peripheral nerve at the upper arm . a third stimulus is subsequently delivered to erb 's point ( axilla ) after an appropriate delay , eliciting a highly synchronised motor response in those fibres in which the collision had occurred . the amplitude and area of this test cmap response are compared with the response induced by the conditioned tst paradigm ( erb 's point - wrist erb 's point stimulation ) yielding an amplitude ratio of > 93% and area ratio of > 92% in healthy controls.95 96 in als , the tst is sensitive at detecting subclinical corticomotoneuronal dysfunction.54 97 corticomotoneuronal dysfunction was also reported in kennedy 's disease using the tst technique,98 99 potentially limiting the diagnostic utility of tst in als . recently , however , a combination of tst with single- and paired - pulse tms techniques has reaffirmed the functional integrity of corticomotoneuronal tracts in kennedy 's disease,100 and thereby the diagnostic utility of tst . given the well documented tms abnormalities in als patients , the tms techniques may be of utility in the diagnostic process of als . although umn signs may be clinically evident in als , in some phenotypes such as the flail arm variant , this may not be the case , and detection of subclinical umn dysfunction may facilitate the diagnosis.42 abnormalities of cortical excitability , including an increase in mep amplitude along with reduction of sici and rmts , have been reported in the flail - arm variant of als , underscoring the utility of tms in detecting subclinical umn dysfunction.42 of further relevance , subclinical umn dysfunction has been reported in progressive muscular atrophy ( pma),101103 suggesting that pma may be a phenotype of als . while corticomotoneuronal integrity was recently reported to be intact in pma using a -band intermuscular coherence technique,104 assessment of cortical function with tms techniques may be of diagnostic utility , especially in light of presence of subclinical umn pathology in pma.102 103 importantly , single- and paired - pulse tms techniques reliably distinguish als from the mimic disorders ( table 1 ) , hastening the diagnosis of als by up to 8 months.47 a reduction in averaged sici , between isi 1 and 7 ms , and peak sici at isi 3 ms were the most robust diagnostic tms parameters , with the finding of absent sici exhibiting a sensitivity of 97%.47 of further relevance , tms studies have established the presence of early and subclinical dysfunction of cortico - bulbar and cortico - respiratory tracts in als,26 105107 thereby suggesting a potential diagnostic utility of bulbar and diaphragmatic mep recordings . in addition , combining tms with radiological techniques , such as mr spectroscopy , may further add to the diagnostic yield especially given the sensitivity of mr spectroscopy in detecting subclinical umn dysfunction.108110 consequently , combining tms techniques , in particular the recording of sici as well as bulbar and diaphragmatic meps , together with radiological techniques , such as mr spectroscopy , may enable an earlier diagnosis of als and thereby commencement of neuroprotective therapies and recruitment into clinical trials . transcranial magnetic stimulation ( tms ) techniques in amyotrophic lateral sclerosis ( als ) mimic disorders single - pulse tms studies have established a normal resting motor threshold ( rmt ) and cortical silent period ( csp ) duration in all als mimic disorders . the motor evoked potential ( mep ) amplitude was reported to be increased in spinal muscular atrophy ( sma ) , a finding attributed to greater corticomotoneuronal projections onto the surviving motor neurons . in addition , the central motor conduction time ( cmct ) was reportedly prolonged in distal hereditary motor neuronopathy with pyramidal features ( dhmnp ) . short interval intracortical inhibition ( sici ) and intracortical facilitation ( icf ) , assessed by the paired - pulse tms technique , have been universally normal in als mimic disorders . in contrast , triple stimulation techniques ( tst ) have been reportedly abnormal in kennedy 's disease , suggesting subclinical upper motor neuron dysfunction , although a recent study has reaffirmed functional integrity of corticomotoneuronal tracts in kennedy 's disease ( see utility of peristimulus time histograms section ) . single- and paired - pulse techniques have also been normal in facial onset sensory motor neuronopathy ( fosmn ) syndrome . * neuromuscular disorders include demyelinating neuropathy , myasthenia gravis , lead toxicity and hirayama 's disease . in addition to its diagnostic utility , it has been suggested that tms may exhibit a clinical utility in assessing disease progression in als.111 specifically , longitudinal tms studies in als patients reported a significant reduction in mep amplitude , mt and cmct , and suggested that reduction in mep amplitude may be an objective biomarker of disease progression in als.111 in contrast , others have failed to document any significant longitudinal changes in tms parameters , thereby arguing against tms utility in the monitoring of disease progression in als.52 prospective longitudinal studies are indicated to further clarify the role for tms in monitoring disease progression in als . in his original writings , charcot concluded that als was a disorder of the brain and that the lower motor neuron component resulted from a downstream affect . not all his contemporaries agreed and in particular gowers was adamant that the demise of upper and lower motor neurons were independent events . in the past 2 decades the site of als onset has been revisited , to a large extent precipitated by the advent of tms . three schools of thought have developed pertaining to the role of the umn , and related pathophysiological processes in als : ( i ) the dying forward hypothesis , ( ii ) the dying back hypothesis and ( iii ) the independent degeneration hypothesis ( figure 4 ) . while the site of disease onset in als remains uncertain , tms studies have tended to favour a cortical origin , with excitotoxicity mediating motor neuron degeneration in als.5 112 the dying forward and dying back hypothesis of amyotrophic lateral sclerosis ( als ) . the dying forward hypothesis proposed that als was primarily a disorder of the corticomotoneurons ( highlighted in red ) , with anterior horn cell degeneration mediated via an anterograde glutamate - mediated excitotoxic process . in contrast , the dying back hypothesis proposes that als begins within the muscle or neuromuscular junction , with pathogens retrogradely transported from the neuromuscular junction to the cell body where these pathogens may exert their deleterious effects . the dying forward hypothesis proposes that als is primarily a disorder of the corticomotoneurons , which connect monosynaptically with anterior horn cells.113 corticomotoneuronal hyperexcitability was postulated to induce anterior horn cell degeneration transsynaptically via an anterograde glutamate - mediated excitotoxic process.113 most tms studies have demonstrated that cortical hyperexcitability is an early feature in sporadic and familial als , linked to motor neuron degeneration.27 30 31 43 65 112 114 115 in addition , longitudinal studies in asymptomatic sod-1 mutation carriers revealed that cortical hyperexcitability developed prior to the clinical onset of als,31 also seen in the g93a sod-1 mouse model.116 of relevance , loss of parvalbumin - positive inhibitory interneurons in the motor cortex of als patients would contribute to the development of cortical hyperexcitability.117 in keeping with a cortical origin of als is the now accepted view that als and frontotemporal dementia ( ftd ) represent an overlapping continuum of the same disorder,118 119 an observation underscored by recent genetic findings establishing that increased hexanucleotide repeat expansion in the first intron of c9orf72 gene on chromosome 9p21 is associated with both als and ftd.120 121 of further relevance , accumulation of tdp-43 ubiquitinated inclusions in anterior horn cells appears to be a pathological hallmark of als.119 122 interestingly , identical tdp-43 inclusions may also be evident in cortical neurons within the frontal ( betz cells ) and temporal lobes of als patients,119 122 123 underscoring the link between ftd and als , and thereby a cortical origin of als . of relevance , molecular approaches have provided further corroborating evidence for glutamate excitotoxicity in als . specifically , a significant reduction in the expression and function of the astrocytic glutamate transporter , excitatory amino acid transporter 2 ( eaat2 ) , has been reported in the sod-1 mouse model and the motor cortex and spinal cord of als patients.124128 in addition , dysfunction of eaat2 transporter appeared to be a preclinical feature in the sod-1 mouse model.129 130 further underscoring the importance of astrocytes in als pathophysiology are recent stem cell studies documenting that motor neuron degeneration appears to be initiated by dysfunction of astrocytes.131 on the postsynaptic side , increased expression of glutamate receptors permeable to excessive influx of na and ca ions132 have been reported in als,133137 potentially rendering the motor neurons more susceptible to glutamate excitotoxicity.138 further support for a role for glutamate excitotoxicity has been indirectly provided by the clinical benefit of riluzole , a glutamate antagonist , in als patients.139142 for the glutamate hypothesis to be a plausible mechanism of motor neuron degeneration , the issue of selectivity of motor neuron involvement in als , together with sparing of motor neurons in non - als conditions exhibiting cortical hyperexcitability,38 must be explained . a number of molecular features may render the motor neurons vulnerable to glutamate toxicity in als . first , motor neurons preferentially express glutamate receptors , such as the ampa receptors , which are more permeable to influx of ca ions.133 134 136 137 in addition , motor neurons in als patients lack the intracellular expression of ca binding proteins parvalbumin and calbindin d28k , both required to buffer intracellular ca.143 144 aberrant activity of the inositol 1,4,5-triphosphate receptor type 2 receptor has been reported in als,145 146 thereby resulting in higher intracellular concentrations of ca within the motor neurons . ultimately , an influx of ca ions through the ionotropic glutamate receptors nmda occurs in the motor neurons,147 148 resulting in increased intracellular ca concentration and activation of ca - dependent enzymatic pathways that mediate neuronal death.149151 glutamate excitotoxicity may also result in production of free radicals that can further damage intracellular organelles and thereby cause cell death.152154 it could be argued that the finding of widespread fasciculations in als , an important diagnostic criterion,155 may argue against a dying forward mechanism given that fasciculations are thought to originate from the distal motor axon , are associated with abnormalities of sodium and potassium conductance , and may precede the onset of lower motor neuron dysfunction.156162 it seems unlikely that cortical hyperexcitability could lead to changes in distal axonal excitability that would result in widespread fasciculations . importantly , a supraspinal mechanism for triggering fasciculations in als has been previously reported.34 in agreement with this notion are findings that fasciculations in als may originate at the level of the motor neuron cell body.157 as such , it could be hypothesised that hyperexcitability of descending motor pathways may contribute to generation of fasciculation in als , thereby providing additional support for a dying forward process . in conjunction with glutamate excitotoxicity , there is compelling evidence that mitochondrial dysfunction may exert an important role in the pathophysiology of als.163168 under conditions of excessive ca load , as may be evident with glutamate excitotoxicity,169 mitochondrial production of free radicals increases resulting in injury of critical neuronal cellular proteins and dna . mitochondrial dysfunction may in turn enhance glutamate excitotoxicity by disrupting the normal resting membrane potential , resulting in loss of the voltage - dependent mg - mediated block of nmda receptor channels.170 from a therapeutic perspective , dexpramipexole , a pharmacological agent that enhances mitochondrial function,171 was effective in slowing als progression in a recent phase ii trial.172 a phase iii , multicentre , international trial was commenced in march 2011 to determine the clinical efficacy of dexpramipexole in als ( clinicaltrials.gov-nct01281189 ) . taken further , it is anticipated that tms studies will be used to determine the efficacy of dexpramipexole in the modulation of cortical excitability in an attempt to provide further insight into als pathophysiology . the dying back hypothesis proposes that als is primarily a disorder of the lower motor neurons , with pathogens retrogradely transported from the neuromuscular junction to the cell body where they exert their deleterious effects.173 although some pathological studies have indirectly supported a dying back process,174176 no pathogens of any type have been identified in relation to als . the presence of widespread dysfunction within the frontal cortex , including the primary , supplementary and prefrontal motor cortices in als , remains difficult to reconcile with a dying back process.3 110 177 in addition , the absence of central pathology in other lower motor neuron disorders such as kennedy 's disease or poliomyelitis provides a further argument against a dying back process.33 44 the independent degeneration hypothesis suggests that the upper and lower motor neurons degenerate independently.178 some 100 years after the original gowers publication , neuropathological studies provided support for the independent degeneration hypothesis whereby the degeneration of upper and lower motor neurons appeared to be independent.179 180 these correlative morphological techniques , however , may be confounded by the anatomical and functional complexity of the corticomotoneuronal system.181 in particular , there remains considerable variability in the corticomotoneuronal to anterior horn cell ratio , due to synaptic changes , and as such attempts to correlate upper and lower motor neurons on autopsy studies may not be meaningful.33 in addition to the three competing theories of als pathogenesis , a prion - like propagation hypothesis has also been suggested.182 specifically , the previously documented contiguous spread of als5 183 could be explained by direct neuron - to - neuron transmission of pathogenic proteins via exosomes , defined as small lipid membranous microvesicles.182 the pathogenic exosomes could spread in either a rostral direction , explaining the rostral - to - caudal spread of als , or in a lateral horizontal direction accounting for the lateral - to - medial spread of disease . in addition , non - contiguous propagation of als could also be explained by spread of pathogenic proteins or toxic molecules through the blood or csf via exosomes.182 interestingly , the genes implicated in als pathogenesis , including tdp-43 and fus , possess a putative prion domain.184 although a prion - like propagation mechanism may seem an attractive explanation for the spread of als , at present there is no direct evidence to support such a process in als . although first described by charcot some 150 years ago , the pathophysiological mechanisms underlying als , variability , rate of progression and site of disease onset remain obscure . objective assessment of umn function in als remains a difficult task in clinical neurophysiology.185 while tms is mainly used as a clinical research tool , conducted in specialised neurophysiological laboratories , there is an urgent need to objectively assess umn function in als . this has been underscored by the recent awaji diagnostic criteria.155 186 although needle electromyography is used by the criteria to objectively assess lower motor neuron dysfunction , the detection of umn involvement is based solely on clinical examination . much has recently been learnt about als from mri , especially diffusion tensor mri , functional imaging and network analysis,110 187198 but these tools remain prohibitively expensive , not readily available and may exhibit a modest diagnostic sensitivity.190 commercially available tms systems that will enable an objective assessment of umn function could be readily developed , facilitating the diagnosis of als . such tms systems may result in the development of more functional als biomarkers that could be used in future drug trials for early patient recruitment and monitoring of drug efficacy . although first described by charcot some 150 years ago , the pathophysiological mechanisms underlying als , variability , rate of progression and site of disease onset remain obscure . objective assessment of umn function in als remains a difficult task in clinical neurophysiology.185 while tms is mainly used as a clinical research tool , conducted in specialised neurophysiological laboratories , there is an urgent need to objectively assess umn function in als . this has been underscored by the recent awaji diagnostic criteria.155 186 although needle electromyography is used by the criteria to objectively assess lower motor neuron dysfunction , the detection of umn involvement is based solely on clinical examination . much has recently been learnt about als from mri , especially diffusion tensor mri , functional imaging and network analysis,110 187198 but these tools remain prohibitively expensive , not readily available and may exhibit a modest diagnostic sensitivity.190 commercially available tms systems that will enable an objective assessment of umn function could be readily developed , facilitating the diagnosis of als . such tms systems may result in the development of more functional als biomarkers that could be used in future drug trials for early patient recruitment and monitoring of drug efficacy .

amyotrophic lateral sclerosis ( als ) is a rapidly progressive neurodegenerative disorder of the motor neurons in the motor cortex , brainstem and spinal cord . a combination of upper and lower motor neuron dysfunction comprises the clinical als phenotype . although the als phenotype was first observed by charcot over 100 years ago , the site of als onset and the pathophysiological mechanisms underlying the development of motor neuron degeneration remain to be elucidated . transcranial magnetic stimulation ( tms ) enables non - invasive assessment of the functional integrity of the motor cortex and its corticomotoneuronal projections . to date , tms studies have established motor cortical and corticospinal dysfunction in als , with cortical hyperexcitability being an early feature in sporadic forms of als and preceding the clinical onset of familial als . taken together , a central origin of als is supported by tms studies , with an anterograde transsynaptic mechanism implicated in als pathogenesis . of further relevance , tms techniques reliably distinguish als from mimic disorders , despite a compatible peripheral disease burden , thereby suggesting a potential diagnostic utility of tms in als . this review will focus on the mechanisms underlying the generation of tms measures used in assessment of cortical excitability , the contribution of tms in enhancing the understanding of als pathophysiology and the potential diagnostic utility of tms techniques in als .

slips , trips and falls ( stfs ) lead recurrently to injuries in occupational situations1 . these accidents are triggered by a movement disturbance ( a slip or a trip ) when working , especially when walking . other movement disturbances ( a wrench slipping , an arm colliding with a wall , etc . ) can occur in occupational situations ; moreover , these can arise when performing different types of tasks ( tightening a bolt , moving an item alone or with a colleague , etc . ) . this paper considers ostfs and , more broadly , occupational accidents with movement disturbance ( oamds ) ; the latter composing a set of accidents operationally defined by leclercq et al.2 , 3 , which involve a heavy cost in both human and financial terms4 . the literature often advances workplace design and upkeep5 , 6 , access system configuration7 or , again , human factors8 , 9 for explaining ostfas . implementing actions that neutralise these factors to secure displacements , however , such actions frequently overlook not only task diversity , but also production requirements and they can therefore only offer a partial response to preventing all oamds . research into these accidents shows that , as in all occupational accidents , many accident - causing event configurations stem from arbitration between production and safety , which can not be overlooked if progress is to be achieved in the prevention field . production - safety arbitrations lead to controls applied under working conditions in order to perform the task while maintaining safety . the control most frequently referred to involves walking fast to try to absorb a delay or confront an emergency . these observations provide a partial explanation for worker difficulties in systematically applying certain recommendations based on common sense ( e.g. do nt rush ) that are aimed at preventing oamds . this paper describes initially the need for , and limits involved in , neutralising the environmental factors in play and subsequently the production - safety arbitrations prompted by the so - called organisational oamd factors referred to in the literature . some of these arbitrations imply control implementation by the worker performing the task , which is then reflected in his / her displacements or , more generally , in his / her movements that increase exposure to oamd risk . focusing on organisational factors allows us to integrate these controls into a set highlighted by a general work organisation model . such a model indicates areas of similarity between oamd genesis and other occupational injury geneses . movement disturbance factors may be permanent and visible ( floor in poor condition , congestion , difficult access to parts of a machine , etc . ) in some work situations and may expose many workers over relatively long periods . this is the case of slippery floors in food processing shops , for example . such factors , along with haste , carelessness and awkwardness are frequently advanced when explaining oamd occurrence . neutralising environmental factors often involves taking action on certain working conditions ( installing a slip - resistant floor , reconfiguring an access system , etc . ) . it is commonplace for companies that decide to raise their oamd - related safety level primarily focus of this type of action . however , in common with instructions designed to change behaviour ( e.g. instructions to workers to move carefully from place to place or to adopt a safe , unhurried displacement , avoiding short - cuts ) , this action can not meet the requirements for preventing all oamds . instructions of this type alone in fact disregard other aspects , which are sometimes more difficult to objectify and control , such as urgency of the situation , fatigue or certain task requirements . as in all occupational accidents ( oas ) , an oamd will often be caused by a combination of factors , each of which is of different nature . a clearly visible obstacle is never sufficient to cause a trip : it may simply not be taken into account by a worker , when his / her visual attention is absorbed by his / her task during a displacement . on the other hand , an oamd can occur without involving a permanent , visible anomaly in the environment : a worker , late for his / her appointment , misses a step when running up stairs that are not subject to any design defect . finally , many situations are temporarily more susceptible to oamd occurrence : for example , when performing his / her activity , a worker collides with an element in his / her environment , which obstructs his / her movement ; he / she had intended to move this element on completion of priority work . in the situation illustrated by fig . 1.two images of occupational situations taken from the napo in safe on site / champions of the world video produced by , a first level of prevention would involve disposing of unwanted material or organising storage areas . at a given moment , presence of elements required for performing a task can also represent an accident factor for a worker or his / her colleague . in fig . 1b , this would be the cinder blocks left near a mason , but could also be a batten left lying on the ground when stripping formwork , a wheelchair when transferring a patient or a toolbox when conducting maintenance work . two images of occupational situations taken from the napo in safe on site / champions of the world video produced by neutralising accident factors that are permanent and visible in the work environment is important , but analysing the part played by this factor in a more comprehensive accident genesis is in fact just as important . behaviours adopted in work situations also need to be understood since they often reflect the presence of organisational factors . the literature includes in - depth analyses of occupational slips , trips and/or other movement disturbances conducted at various companies . events leading to injury are integral to the relevant company operation and some are related to production - safety arbitrations referred to long ago in the general occupational accident field . for example , situations described in terms of recovery or momentary co - activity by faverge10 reflect arbitrations revealed during iron mine accident analysis , in particular . monteau11 refers to known organisational risks , when analysing occupational health and safety from an organisational perspective . it should be noted that few of these accidentology studies are considered in relation to understanding and preventing oamds . yet , the contribution of multiple organisational factors has been highlighted during analysis of such accidents . research reported in bentley & haslam12 and in leclercq & thouy13 questions the role of work preparation in stfa occurrence . bentley & haslam12 effectively describe the difficulties encountered in distributing mail on time during periods involving snow and ice . leclercq & thouy13 show that a number of accidents have involved field operators climbing up into and down from trucks , when checking equipment required during the day at various building sites . this phase of their activity called for all the more care since instances of missing equipment were frequent . a specific study of oamds sustained by train drivers14 has revealed problems involving task allocation as well as recovery situations , i.e. situations in which the normal task is interrupted by an incident , from which the worker has to recover , in other words strive to restore the usual course of work10 . these accidents occurred when inspecting a train prior to departure : in the first case , the train started to brake during the operation ; in the second case , an inexperienced driver detected a brake failure he had never before encountered and did not know how to remedy and , in the third case , the driver once again climbed down from the train because he had overlooked an inspection point . in each case , the driver gave his full attention to inspection in order to prevent the train being delayed and , when walking , tripped on a sleeper or a plate creating unevenness in the ground . in the first two cases , the driver was performing a recovery operation at the time of the accident to restore the train braking system operation . all recovery situations introduce or accentuate a time constraint , so resources mobilised to make the braking system operational as quickly as possible were partially lacking in terms of controlling displacement and this effectively caused the driver to trip . bentley et al.15 refer to a concurrent visual task when explaining the occurrence of certain oamds ; these authors also emphasise that , at a given moment , resources dedicated to performing the task may be lacking in terms of controlling displacement . in many cases , existing obstructions to displacement are due to earlier or simultaneous work performed by workers other than the worker , who sustains an oamd ; this reveals the part played by co - activity or a succession of activities in oamd occurrence . for example , a worker has to divert to avoid tools useful to other workers installing new equipment , but left on his / her displacement route . displacement diversion may be considered as a form of recovery activity intended to restore a normal course of work by returning to the initial route . co - activity , historically described by cuny16 , represents task performance by persons pursuing different production objectives and required to share concurrently a common workplace . interim situations or those involving subcontracted work , in particular , can generate co - activity or a succession of activities . finally , bentley & haslam12 have shown that the job and finish policy implemented at the time in the united kingdom s mail distribution company , which allowed workers to go home as soon as the last mail had been distributed , could encourage workers to take risks by hurrying or taking short - cuts . these authors reported that workers explained that the accident risk raised by reading mail addresses while walking was more acceptable than the time wasted in stopping to read the addresses . working conditions ( hence movement performance conditions ) play a part in oamd occurrence since they make it more or less difficult to control displacement and , more generally , movement during task performance . organisational factors highlighted during oamd analysis reveal worker arbitration between production and safety in the work situation , in which he / she is exposed to a risk of movement disturbance . production - safety arbitrations relate particularly to the organisational activity implemented by the company . neutralisation of organisational factors therefore requires local and collective management of the oamd risk to ensure proximity to the company s specific characteristics and to compare existing logics and viewpoints . bentley & haslam12 state that , depending on the workers distributing mail , managers consider performance a priority over safety and that the workers themselves prefer rapid performance to safer performance of their work ; their attitudes reflect those of the management in this respect . as in the presence of any oa risk , controls are implemented to perform the task , while ensuring safety with regard to movement disturbance , in other words while ensuring movement control . , for example in the case of a collision when bolting because the spanner slipped . oamds can also involve more atypical movements , such as picking up an object or walking , and in some cases , being cut by an element in the environment or missing a step when running up stairs . controls implemented in work situations are therefore virtually permanent and the worker manages the available resources to perform his / her task , while controlling his / her movement . the resources required for movement control vary in time and with respect to the work situation . for example , derosier et al.17 report situations , in which metallurgists are sometimes required to move over template elements similar to beams . at these moments , the resources required to control their movements are more extensive than those required when walking on a level floor . likewise , resources required for walking on a floor with variable slip resistance are more extensive than resources required for walking on a surface with uniformly high slip resistance . resources needed to perform the task as a whole are also variable . at certain moments , a worker s visual attention can be taken up by a task and can thus be unavailable for movement control14 , 15 . task characteristics and requirements will therefore condition resources , which could be dedicated to movement control . 2.model of ostfa understanding based on the worker and his / her activity ( adapted from the model developed by vezina ( 2001 ) for musculoskeletal disorders ) . contributes to our understanding of movement disturbance by illustrating a work situation model based on the worker and his / her activity . this has been adapted from the model developed by vzina18 in relation to work - related musculoskeletal disorders ( wrmsds ) . wrmsds and oamds are invariably outcomes of occupational risks , which manifest themselves through worker movements . this is why oamds and wrmsds possess common characteristics with an impact on prevention . oamd prevention has been the subject of little research to date and could therefore benefit from studies in the wrmsd prevention field , at least from a theoretical and methodological standpoint . similarities between wrmsd and oamd and their consequences for prevention have been developed by leclercq et al4 . model of ostfa understanding based on the worker and his / her activity ( adapted from the model developed by vezina ( 2001 ) for musculoskeletal disorders ) . figure 2 shows that controls are implemented in work situations to ensure safety when performing a task . movements performed at work are subject to continuous adjustment with respect to the required task and individual , organisational and environmental constraints , as reported by chassaing19 when studying wrmsds . some of the implemented controls can be easily observed ( rushing , moving round obstructions , etc . ) and the individual strategies , to which these controls contribute , can be examined in detail using personal interviews . sometimes , they can not be visually observed and their detection requires a very fine observation grid : one that accurately describes movements such as heel strike angle when walking , distance provided as a safety margin between the foot and a low - level obstruction during a displacement , supports used , etc . to acquire a best possible understanding of worker controls implemented to perform a task in an occupational situation , while avoiding movement disturbances , we need to combine two levels of analysis : analysis of the activity and analysis of the movement performed within the activity . macroscopic developments such as technological advances or the advent of regulation influence the conditions under which an operator performs his movements and hence the resulting risks present in occupational situations . this model illustrates the outcome of macroscopic developments in the occupational situation through productive organisation choices . illustrates a work organisation model developed by the niosh20 . the nature of the different factors involved in oa occurrence is displayed , along with the boundaries within which these factors are effectively harmful . organisation of the niosh work model taken from sauter et al . , ( 2002 ) . this model illustrates the outcome of macroscopic developments in the occupational situation through productive organisation choices . in general , productive organisation characteristics evolve constantly under the specific effects of technical progress ( automation , introduction of new technologies , etc . ) , subsequent growth in productivity21 , employment market developments ( active population characteristics , etc . ) and reorganisations22 ( outsourcing , etc . ) . these macroscopic developments and choices made by productive organisations affect the work situation ( level of prescription , time and spatial constraints , etc . ) in ways that condition how worker movements are performed . controls are continually implemented not only by a worker performing his / her task , while avoiding injuries , but also at the different levels illustrated in fig . 3 . analysing and combining these controls contributes to our understanding and prevention of occupational accidents , in particular oamds . despite scientific progress in the safety field , oamds are still commonly considered simple accidents resulting from a malfunction in a simple system ; this might suggest that their prevention is also this paper aims to encourage changes in these perceptions by attempting to orient the reader s vision towards organisational factors , which often combine with other accident factors to cause movement disturbance and injury in work situations . these risks manifest themselves in the worker s movement but are none the less an unwanted consequence of productive organisational decisions . oamd organisational factors reveal the need for local and collective management of this risk and the importance of a better understanding of movement / displacement performed under working situations , i.e. in a context integrating specific task requirements and working conditions . while organisational measures implemented by the company represent a lever for oamd prevention , two points should be noted : on the one hand , organisational activity is restricted as illustrated by fig . 3 and , on the other hand , its lever is not unique . maximum possible neutralisation of factors close to the injury in the accident genesis and risk awareness also constitute major lines of prevention . awareness of the oamd risk , in particular , is an essential prerequisite to any progress in preventing these accidents . perception of the oamd risk and its more or less accepted nature are factors , which determine both consideration of this risk at every level of the company and controls implemented by workers .

workplace design and upkeep , or human factors , are frequently advanced for explaining so - called occupational slip , trip and fall accidents ( ostfas ) . despite scientific progress , these accidents , and more broadly occupational accidents with movement disturbance ( oamds ) , are also commonly considered to be simple . this paper aims to stimulate changes in such perceptions by focusing on organisational factors that often combine with other accident factors to cause movement disturbance and injury in work situations . these factors frequently lead to arbitration between production and safety , which involves implementation of controls by workers . these controls can lead to greater worker exposure to oamd risk . we propose a model that focuses on such controls to account specifically for the need to confront production and safety logics within a company and to enhance the potential for appropriate prevention action . these are then integrated into the set of controls highlighted by work organisation model developed by the niosh .

the weissella species are gram - positive , non - spore - forming , heterofermentative , nonmotile , and irregular or coccoid rod - shaped organisms . members of the genus weissella have been isolated from a variety of sources , such as fresh vegetables , fermented silage , meat , or meat products . several lactic acid bacteria belonging to the genera weissella have been introduced to wheat sourdough baking for in situ production of exopolysaccharides . weissella kimchii pl9023 was selected as a prebiotic as it mostly produced hydrogen peroxide which inhibited the growth and adherence of vaginal isolates of candida albicans , escherichia coli , staphylococcus aureus , and streptococcus agalactiae . lactic acid bacteria ( lab ) that produce dextrans include various leuconostoc , streptococcus , and lactobacillus species . patel et al . reported the dextran - production ability of pediococcus pentosaceus for the first time . additionally , dextran production has typically served as a phenotypic test in the identification of bacteria classified in the genus weissella . reported industrially useful dextran - producing weissella confusa e392 which could be a suitable alternative to the widely used l. mesenteroides b512f in the production of linear dextran . first proposed by collins et al . on the basis of the results of 16s rdna phylogenetic analyses , the genus weissella encompasses a phylogenetically coherent group of lactic acid bacteria and includes twelve validated leuconostoc - like species , currently , including w. confusa ( formerly lactobacillus confusus ) , w. minor ( formerly lactobacillus minor ) , w. kandleri ( formerly lactobacillus kandleri ) , w. halotolerans ( formerly lactobacillus halotolerans ) , w. viridescens ( formerly lactobacillus viridescens ) , weissella paramesenteroides ( formerly leuconostoc paramesenteroides ) , w. hellenica , w. thailandensis , w. cibaria , weissella kimchii , weissella soil , and weissella koreensis . alternative molecular biological approaches have been developed in place of classical phenotypic methods for the identification of weissella species . such methods include soluble whole - cell protein pattern analyses , restriction pattern of ribosomal dna fatty acid analyses , random amplified polymorphic dna - pcr ( rapd - pcr ) , denaturing gradient gel electrophoresis ( dgge ) , and pcr targeting 16s/23s rrna gene spacer region . in the present study , a novel weissella species isolated from fermented cabbage was characterised up to genus and species level using 16s rdna - based sequence analyses . its glucan formation capacity has been explored in detail and compared with that of leuconostoc mesenteroides nrrl b-512f . fresh chopped cabbage was subjected to fermentation in 2.5% ( w / v ) nacl solution . one gram of fermented cabbage was ground to paste and mixed in 10 ml of saline ( 0.9% w / v ) homogeneously in test tubes . one hundred microliter from all the dilutions of cabbage from 10 to 10 was taken and spread plated on petri plates of 1.7% ( w / v ) mrs agar with glucose as carbon source . petri plates were incubated at 28c for 24 h. based on the higher - glucansucrase activity and glucan concentration determined ( as described later ) , a microbe cab3 ( named after its colony number ) was isolated . the isolated bacterium was propagated as stab in mrs agar supplemented with sucrose as carbon source at 25c and stored at 4c . for long - term preservation , the colony morphology of isolates grown on mrs medium for overnight at 25c was observed directly and by light microscopy . cell mobility and gliding movement were assessed by phase - contrast microscopy ( 1000x ) using cells of mrs broth . the cell morphology of the selected isolate was examined by scanning electron microscopy ( leo1430 vp , leo electron microscopy ltd . , cambridge , uk ) operated at 10.0 kv . the catalase activity was determined by transferring fresh colonies from mrs agar to a drop of 5% ( v / v ) h2o2 on a glass slide . for the temperature tolerance of the microorganism , the growth of selected isolate was carried out using mrs broth at different temperatures , that is , 4c , 10c , 15c , 20c , 30c , 37c , and 42c for 2 days . for salt tolerance of the isolate , the growth was observed using mrs broth in the presence of 4.0% and 6.5% ( w / v ) of nacl incubated at 25c for 2 d. the production of glucan ( slimy layer ) by isolate cab3 from sucrose was observed on mrs agar in which glucose was replaced by sucrose . the strain cab3 was tested for its ability to ferment various carbohydrates using the method of kandler and weiss . from the overnight - grown mrs broth containing 2% ( w / v ) glucose as carbohydrate source , 50 l was inoculated in 5.0 ml liquid mrs medium lacking glucose but containing phenol red and other test carbohydrates to give a final inoculum to medium ratio of 1% ( v / v ) . the acid production was recorded between 24 and 48 h. the acid production was indicated by a change in the colour of the phenol red indicator dye from red to yellow . the isolated strain cab3 was tested for susceptibility to 30 antibiotics using a standardized filter - paper disc - agar diffusion assay , to determine the drug susceptibility of microorganisms . the antibiotic tests were performed using commercially available antibiotic octadiscs containing amoxyclav ( ac ) , cephalexin ( cp ) , ciprofloxacin ( cf ) , clindamycin ( cd ) , cloxacillin ( cx ) , erythromycin ( e ) , tetracycline ( t ) , ampicillin ( a ) , carbenicillin ( cb ) , cefotaxime ( ce ) , chloramphenicol ( c ) , co - trimazine ( cm ) , gentamicin ( g ) , norfloxacin ( nx ) , oxacillin ( ox ) , amikacin ( ak ) , amoxycillin ( am ) , bacitracin ( b ) , cephalothin ( ch ) , novobiocin ( nv ) , oxytetracycline ( o ) , vancomycin ( v ) , penicillin - g ( p ) , tobramycin ( tb ) , cephaloridine ( cr ) , kanamycin ( k ) , lincomycin ( l ) , methicillin ( m ) , norfloxacin ( nf ) , and oleandomycin ( ol ) from hi - media pvt . ltd . the bacterial cell pellet was lysed using a solution containing guanidium thiocyanate ( a chaotropic agent ) and sds ( a detergent ) , to extract dna . this lysis solution was used to disrupt the cell , remove proteins and polysaccharides , and partial hydrolysis of rna . the genomic dna was extracted by geneitm genomic dna extraction kit ( bangalore genei pvt . ltd . ) . dna was then precipitated using alcohol and washed with 70% ( v / v ) alcohol to remove contaminants . dna pellet was solubilized in sodium acetate buffer at ph 5.5 added to a final concentration of 0.3 m in isopropanol at higher temperature ( 5055c ) , to increase the solubility of genomic dna . the extracted dna was purified using ultrapure prep kit ( kt83b , bangalore genei , india ) . genomic dna was purified in a gravity flow column using elution buffer , followed by treatment with lysozyme , proteinase k , and rnase a supplied with the kit . the quality of purified dna was evaluated on 1.2% agarose gel , a single band of high - molecular - weight dna was observed . the universal 16s rdna primers , forward primer 8f ( 5agttgatcctggctcag3 ) , and reverse primer 1492r ( 5accttgttacgactt3 ) were used for the polymerase chain reaction ( pcr ) . the pcr amplification was carried out in a reaction mixture containing ~10 ng genomic dna as template , 1 l dntp mix ( 2.5 mm each ) , 100 ng/l each of forward and reverse primer , 1x taq dna polymerase assay buffer ( 10x ) , 3u taq dna polymerase enzyme ( bangalore genei ltd.,bangalore , india ) , and distilled water enough to make up the volume to 50 l reaction mixture . amplification conditions were 5 min initial denaturation at 94c , 30 s denaturation at 94c , 30 s of primer annealing at 54c , 1 min elongation at 72c for 35 cycles , and a final extension of 10 min at 72c . the reactions were carried out in a thermal cycler ( applied biosystems , model abi 2720 ) . fifteen l of pcr amplified product was electrophoresed on 1.2% ( w / v ) low - eeo agarose gel in 1x tbe buffer ( 45 mm tris - borate , ph 8.3 , and 1 mm na 2 edta ) at 100 v for 2 h. the gel was stained with ethidium bromide in a final concentration of 0.5 g / ml , visualized , and photographed under uv light . the amplified 16s rrna gene from the isolate was eluted and purified from the gel slice using the geneipure tm gel extraction kit ( qiagen ) . forward and reverse dna sequencing reaction of pcr amplicon was carried out with 8f and 1492r primers using bdt v3.1 cycle sequencing kit on abi 3730xl genetic analyzer . consensus sequence of 1288 bp of 16s rdna gene was generated from forward and reverse sequence data using aligner software . the 16s rdna gene sequence of the isolate cab3 was used to carry out blast with the nr database of ncbi genbank database . based on maximum identity score , first ten sequences were selected and aligned using multiple alignment software program clustalw . distance matrix was generated using rdp database , and the phylogenetic tree was constructed using mega 4 . the enzyme assay was carried out in 1 ml reaction mixture containing 5% ( w / v ) sucrose , 20 mm sodium acetate buffer ( ph 5.4 ) , and 20 l cell - free supernatant . the enzyme activity was determined by estimating the released reducing sugar by nelson and somogyi method . the glucansucrase was produced in the enzyme production medium as described by tsuchiya et al . . fermentations were carried out in triplicate sets of 60 ml enzyme production medium in a 250 ml erlenmeyer flask incubated at 25c under shaking condition at 180 rpm . the samples ( 1 ml ) were withdrawn at indicated time intervals and centrifuged at 8000 g for 10 min at 4c to separate the cells . weissella confusa was grown at various temperatures : 22c , 25c , 28c , 30c , 32c , 37c , and 40c in the flasks as described previously in an orbital shaking incubator at 180 rpm to determine the optimum incubation temperature for the maximum enzyme production . the culture ( 1.0 ml ) was withdrawn at the indicated time intervals and centrifuged at 8000 g for 10 min at 4c to pellet out the cells . the production glucansucrase was compared under shaken - flask condition with the static flask culture at 25c in triplicate sets of 60 ml enzyme production medium in 250 ml erlenmeyer flasks . the samples ( 1.0 ml ) were withdrawn at indicated time intervals and centrifuged at 8000 g for 10 min at 4c to pellet out the cells . the cell - free extract was analysed for enzyme activity and protein concentration as described earlier . the polysaccharide content of the isolated strain ( cab3 ) was determined by phenol - sulphuric acid method in a microtitre plate . the isolated strain was grown in 60 ml liquid medium described by tsuchiya et al . at 25c and 180 rpm up to 32 h. the samples ( 1 ml ) were withdrawn at regular intervals . to 200 l of the culture supernatant , three volumes of the prechilled ethanol were added and centrifuged at 12,000 g. the supernatant was discarded , and the precipitate was resuspended in 200 l distilled water . the crude 50 ml cell - free supernatant with enzyme activity 6 u / ml and specific activity 1 u / mg was subjected to purification by fractionation using peg-400 ( 33% , w / v ) . to confirm the existence of glucansucrase , the pas staining of the sucrose- and raffinose - incubated gel was carried out on 7% nondenaturing sds - page . the electrophoresis was performed on a mini gel unit ( biorad ) using 1.5 mm thick gels , following the method of . after run , the gel was treated thrice with a solution containing 20 mm sodium acetate buffer , ph 5.4 , 0.1% ( w / v ) triton x-100 , and 0.005% ( w / v ) calcium chloride for 20 min to remove sds . then , the gel was incubated with 5% ( w / v ) sucrose / raffinose solution in 20 mm sodium acetate buffer , ph 5.4 for 1012 h. following incubation , the gel was washed twice with 75% ( v / v ) ethanol for 20 min and incubated in solution with 0.7% ( w / v ) periodic acid in 5% ( v / v ) acetic acid for 20 min at room temperature . the gel was then washed thrice with 0.2% ( w / v ) sodium bisulfate in 5% ( v / v ) acetic acid solution and finally stained with schiff 's reagent ( 0.5% w / v basic fuchsin , 1% ( w / v ) sodium bisulfate , and 0.1 n hcl ) until the discrete magenta bands appeared within the gel . the purified enzyme having 10.4 u / mg specific activity ( 1.0 mg / ml ) was used for further optimization of assay conditions . the ionic strength of the buffer was varied from 10 mm to 400 mm . the assay was carried out in 1 ml reaction mixture containing 5% ( w / v ) final sucrose concentration , in sodium acetate buffer ( ph 5.4 ) at 30c for 15 min . to study the effect of varied ph on enzyme activity , the ph of buffer was varied from 4 to 7 in 1 ml reaction mixture containing 5% ( w / v ) final sucrose concentration in 20 mm sodium acetate buffer at 30c for 15 min . the enzyme assay was done in 5% ( w / v ) sucrose solution in 20 mm sodium acetate buffer ( ph 5.4 ) for 15 min . based on higher glucansucrase activity and glucan concentration determined , a microbe cab3 was isolated from the fermented cabbage . the isolated culture was identified according to their morphological , cultural , physiological , and biochemical characteristics [ 37 , 38 ] . the tests used were gram reaction , production of catalase and growth at different temperatures and salt concentrations , and acid production from carbohydrates ( 2% w / v ) . lactococcus spp . , and leuconostoc spp . by its biochemical and physiological properties like arginine deamination , esculin hydrolysis , the microorganism was identified by comparing the results with those from the literature ( table 1 ) . the results of the comparison showed that the isolated strain cab3 has the same characteristics as w. confusa ( table 1 , column 5 , which is , 4 ) . from scanning electron microscopy , the isolate cab3 was found to be phenotypically homogeneous with irregular short rod cells in pairs or chains with a width and length of 0.5 - 0.6 m and 1.21.4 m , respectively ( figure 1 ) . the colony produced slimy layer when grown on mrs agar plate was supplemented with sucrose . the strain could grow in presence of 4.0% , 5.0% , 6.0% , and 6.5% ( w / v ) nacl , but not beyond 8.0% nacl . the strain very efficiently utilised cellobiose , galactose , maltose , ribose , sucrose , and xylose but poorly fermented mellibiose , raffinose , and trehalose which was in accordance with fermentation pattern of weissella confusa reported earlier . the cab3 did not utilize rhamnose which is also characteristic of weissella sp . . the isolate cab3 was sensitive to chloramphenicol , erythromycin , and tetracycline and moderately resistant to gentamicin , kanamycin , norfloxacin , and vancomycin which is characteristic of weissella confusa . 16s rrna gene sequence analyses after pcr amplification was performed for identifying the isolate cab3 . the profiles obtained by pcr amplification allowed the identification of the isolate at both genus and species levels . the amplified product of 16s rrna showed a single band of 1,500 bp ( figure 2 ) . forward and reverse dna sequencing reaction of pcr amplicon was carried out with 8f and 1492r primers using bdt v3.1 cycle sequencing kit on abi 3730xl genetic analyzer . the consensus sequence of 1288 bp of 16s rdna gene was generated from forward and reverse sequence data using aligner software ( figure 3 ) . the 16s rrna sequence was compared with reference bacteria from the national centre for biotechnological information ( ncbi ) genbank ( http://www.ncbi.nlm.nih.gov/ ) and ribosomal database project ( rdp ) . the homology in sequences identified by the above methods was used to find out common ancestry . the bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the taxa analyzed . the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 500 replicates ) is shown next to the branches . the evolutionary distances were computed using the kimura 2-parameter method and are in the units of the number of base substitutions per site . all positions containing gaps and missing data were eliminated from the dataset ( complete - deletion option ) . the isolate cab3 and the strain weissella confusa imau:10190 ( genbank accession number gu 138518.1 ) clustered together ( figure 4 ) which confirmed the isolate to be weissella confusa . hence , the isolate cab3 was identified as weissella confusa and assigned the genbank accession number gu138518.1 . a bioinformatic tool , genedoc software , version 2.7.1 , was used for more 16s rrna gene sequence investigation of the other closely related weissella sp . the partial sequence ( 1288 nucleotides ) of 16s rdna of weissella confusa ( cab3 ) was 100% similar to 16s rrna genes from other related strains of weissella sp . in ncbi ( temperatures ranging from 22c to 40c were studied for the production of enzyme under shaking at 180 rpm ( figure 5 ) . at 25c , the activity achieved was maximum ( 6.2 u / ml ) , at a temperature lower than 25c , the cell growth was slow which might be the cause for lower enzyme activity as shown in figure 5 . at higher temperature , the enzyme activity decreased which might be due to the deactivation of the enzyme at higher temperatures . shaking condition significantly favoured the enzyme production giving 6.1 u / ml enzyme activity which was 1.5 times higher than the enzyme activity observed under static condition ( 4.1 u / ml ) as shown in figure 6 . the maximum enzyme activity was achieved 4 h later in case of static flask culture . figure 7 shows a complete fermentation profile of the new strain of weissella confusa ( cab3 ) . the fermentation profile of the new strain of weissella confusa ( cab3 ) was compared with other lactic acid bacterial strains using the same medium composition and the parameters compared are listed in table 2 . it was reported that 23c temperature under static flask culture condition was optimum for the production of dextransucrase from l. mesenteroides nrrl b-512f , and 25c temperature under shaking condition was found to be optimum for l. mesenteroides nrrl b-640 , whereas , for weissella confuse , the temperature 25c and shaking conditions were optimum . from the results , it was clear that the enzyme activity ( 6.1 u / ml ) from weissella confusa was 50% , 27% , and 65% higher than the enzyme activity from l. mesenteroides nrrl b-512f ( 4.1 u / ml ) , l. mesenteroides nrrl b-640 ( 4.8 u / ml ) , and p. pentosaceus , respectively , as shown in table 2 . the cell growth obtained in all strains after 12 h was similar . there was a sharp decrease in the ph of the broth with weissella confusa after 12 h ( figure 7 ) , when the maximum activity was achieved , whereas the ph drop was much lower from the point where the maximum enzyme activity was observed for l. mesenteroides nrrl b-512f . the ph range for maximum enzyme production for weissella confusa was narrow 5.25.6 , whereas for l. mesenteroides nrrl b-512f , l. mesenteroides nrrl b-640 , and p. pentosaceus , it was 6.06.5 , 4.45.9 , and 4.0 - 5.0 , respectively . the cell - free supernatant of isolated strain weissella confusa ( cab3 ) gave glucan concentration of 34 mg / ml ( figure 7 ) . this was very high as compared to other lactic acid bacteria such as from l. mesenteroides nrrl b-512f ( 6.9 mg / ml ) , l. mesenteroides nrrl b-640 ( 7.0 mg / ml ) , and pediococcus pentosaceus ( 10.2 mg / ml ) . the glucan concentration of the strain can be further increased by optimizing the medium composition and culture conditions . the enzyme was purified from cell - free supernatant with 1.0 u / mg specific activity . the fractionation by 33% ( w / v ) peg-400 gave 10.4 u / mg specific activity with 10-fold purification and 26% overall yield . the purified enzyme showed , approximately , 180 kda molecular size on the nondenaturing sds - page gel when stained with coomassie brilliant blue ( figure 8 ; lane 2 ) . the pas staining of the gel incubated in sucrose ( figure 8 ; lane 3 ) showed that the activity band corresponded to the presence of active form of the purified glucansucrase ( figure 8 ; lane 2 ) . no activity band after pas staining appeared after the incubation of the nondenaturing sds - page gel in raffinose ( figure 8 ; lane 4 ) , thus excluding the presence of fructosyltransferase . the partially purified enzyme was maximally active at the temperature 35c , at ph 5.4 in 0.2 mm sodium acetate buffer ph 5.4 ( figure 9 ) . the results showed that the glucansucrase activity was stable within the range of 1020 mm ( figure 9 ) . the enzyme lost 41% of its activity at 50 mm . beyond 50 mm , the decrease in the enzyme activity was not significant with the increase of ionic strength . at ph below and above 5.4 , a sharp decrease in enzyme activity was observed , which has been observed with other glucansucrases [ 16 , 17 ] . the isolated bacterium ( cab3 ) was identified at species and genus levels based on 16s rdna sequencing to be weissella confusa imau:10190 ( genbank accession number gu 138518.1 ) . the isolated strain could be very promising candidate for glucan production at industrial level as it has high glucan concentration ( 34 mg / ml ) . the physiological and biochemical characterisation was carried out to distinguish the isolate from other closely related lactic acid bacteria . based on biochemical and physiological studies , the strain showed its relation with other weissella sp . the isolate weissella confusa ( cab3 ) could not ferment mellibiose , raffinose , and trehalose , a common trait of weissella confusa . the partially purified enzyme was confirmed as glucansucrase by pas staining of nondenaturing sds - page gel incubated with sucrose . the presence of fructosyltransferase was ruled out based on the absence of the band in raffinose - incubated nondenaturing sds - page gel . the partially purified enzyme showed highest activity at 35c , when assayed in 20 mm sodium acetate buffer ( ph 5.4 ) . the wide applications of glucan in food , pharmaceutical , and cosmetics industries emphasize the importance of exploration of the new strains and characterization of their traits . this study reveals a novel high glucan producing bacterial isolate which could be a good alternative for glucan - production in the future .

a gram - positive , nonmotile , irregular , short , rod - shaped new strain of weissella confusa bacterium was isolated from fermented cabbage . the isolate was physiologically and biochemically characterised . the 16s rdna was amplified by polymerase chain reaction ( pcr ) . the isolate was identified as weissella confusa ( genbank accession number : gu138518.1 ) based on nucleotide homology and phylogenetic analysis . the isolate produces glucansucrase when grown in sucrose - supplemented culture medium which catalyses glucan formation . this novel isolate possesses high capacity of industrial use due to its high productivity of glucan ( 34 mg / ml ) as compared to other strains reported . the optimum temperature for glucansucrase production was 25c . the shaking condition gave an enzyme activity of 6.1 u / ml which was 1.5 times higher than that given by static condition ( 4.1 u / ml ) . the temperature 35c , ph 5.4 , and ionic strength 1020 mm were optimum for enzyme assay . this investigation unraveled the abundance of industrially valuable microflora of the north east india .

a young boy , 16 years old , presented with complaints of poor vision in the left eye since childhood . , he had a vision of 20/200 in the right eye ( re ) and 20/20 in the left eye ( le ) . zonules were lacking around the fissure between the two lobes [ fig . 1 ] . 2 ] revealed a bilobed lens in the anterior segment of the eye with normal posterior segment . partial congenital aphakia is a rare condition in which the lens appears to be partially divided by a deep furrow.1 in the case of congenital duplication of lens reported by thakkar et al.2 two lenses were placed obliquely in a straight line with a clear area in between . richardson3 reported a case in which the two lenses were asymmetrical , one being larger than the other . the mechanism for this anomaly is not known yet . whether it is part of a phylentogenic spectra or represents development from two embryonically separate lens remains a question . duke elder has tried to explain the occurrence of the two entities based on separate embryonic developmental sequences.1 he suggested that the metaplastic changes in the surface ectoderm leads the lens plate to invaginate at two place and forms two lens vesicles which results in double lens.1 this suggests that the lens is divided before the lens starts developing . our case has a deep furrow in the center fig . 1 with localized absence of zonules . we , therefore , suggest that once the deep furrow is formed due to absence of zonules , it may go further ( deep ) enough to divide the lens into two distinct parts . therefore , the absence of zonules is significant and these furrows may be deep enough and progress to duplication . it is a possibility that the lens duplication might be occurring when the lens vesicle is single and it is the laxity and abnormal stretching which causes the deep furrow and possibly duplication . it is interesting to note that both the cases previously reported2 - 3 had lens place in a straight axis at opposite ends . this gives further credence to our theory of the fissure dividing the lens into two and the remaining zonules pulling them away from each other . since lens coloboma occurs frequently such deep furrows ( bilobed lens ) are uncommon . we believe that it represents the missing link between the colobomatous lens and duplicated lens . we understand that a single case report may not be suggestive of any mechanism but nevertheless it does indicate a possible mechanism of lens duplication .

congenital anomalies of the lens include a wide range from lens coloboma to primary aphakia and doubling of lens . there have been few case reports of double lens ; the etiology suggested is metaplastic changes in the surface ectoderm that leads to formation of two lens vesicles and hence resulting in double lens . we report a case with bilobed lens , which raises the possibility of explaining the etiology of double lens .

in every field of dentistry , awareness regarding the importance of preserving tooth tissue is becoming evident . the current odontologic era is characterized by an increasing move toward less invasive treatment and preventive dentistry . the search for a more gentle , comfortable , and conservative caries excavation has led to the development of methods which aim at providing minimal thermal changes , less vibration and pain , and removal of infected dentine only . laser , chemomechanical excavation , and air abrasion are successful in overcoming these problems . the chemomechanical caries removal ( cmcr ) technique stands out among other alternative methods as it is a nonaggresive excavation method which uses a chemical gel that is said to remove only the infected dentine where collagen is degraded , maintaining the demineralized portion that is capable of being remineralized and repaired . this approach is based on principle of minimal invasive dentistry that involves application of substances such as caridex , carisolv , papacarie , and carie - care for the removal of carious dentin . the use of carisolv introduced in the mid 90 's as a chemical agent to remove caries is broadly discussed in the dental literature . although carisolv was quite a success in the field of dentistry , certain drawbacks have also been reported which includes requirement of customized instruments , more time and its high cost - making it available for just a privileged few . in 2003 , a research project in brazil developed a papain - based gel to universalize the use of chemomechanical method , and the new formula was commercially known as papacarie . the union of these three components confers antibiotic , bacteriostatic , and anti - inflammatory properties to this agent . papain is an endoprotein from the proteolytic cysteine family that acts only upon damaged tissue , since plasma antiprotease is not present in the infected tissue , preventing papain 's proteolytic action in tissues considered normal . chloramine is a compound containing chlorine and ammonia with antibiotic and disinfecting properties , used for the irrigation of root canals . papacarie was found to be easy to manipulate , simple and cheap , as well as effective in removing infected tissues . carie - care is a more recent , minimally invasive method for chemo - mechanical dentine caries removal , developed by uni - biotech pharmaceuticals private limited , chennai , india in collaboration with vittal mallya scientific research foundation . even this is a gel - based formulation containing a purified enzyme , derived from the plant carica papaya along with the benefits of clove oil . cys-25 then performs a nucleophilic attack on carbonyl carbon which frees the amino terminal of the peptide , the enzyme is then deacylated by a water molecule and releases the carboxy terminal portion of the peptide . studies have been done using various cmcr agents namely 5% sodium hypochlorite , gk101 , caridex , carisolv and papacarie . however , there is a need to evaluate the efficacy of a more recent material like carie - care. the extent of carious dentine excavation , the time taken by each technique and even the microbiological aspect needs to be evaluated as it plays an important role in the progression of dentinal caries . hence , the present study was designed to evaluate and compare the efficacy of newer enzymatic approaches , papacarie and carie - care as cmcr agents . this in vitro study was aimed to evaluate the efficacy and effectiveness of caries removal by two cmcr agents papacarie and carie - care. the study was carried out in the department of pedodontics and preventive dentistry in association with department of oral and maxillofacial pathology , st . the response or dependent variables were time required to remove dentine measured in seconds ( efficiency ) and the detection of bacteria after carious removal and dentinal tubule destruction ( efficacy ) . materials used in the study a sample size of 30 was determined by statistician by resource equation method . , in which caries distribution is done according to site and stage of progression of the lesion . this concept of sites / stages ( si / sta ) replaced the classification of black and promoted a medical model of conservative dentistry in clinical practice . the extracted teeth having site 2 ( carious lesions at contact area of interproximal surfaces of the crown ) and stage 3 ( large dentin lesion with extended and frank cavitation ) carious lesion were included in the study . the exclusion criteria were deep carious lesion with pulp exposure or potential for pulp exposure [ figure 2 ] . extracted primary molars each tooth was sectioned mesiodistally in the center of the carious lesion using diamond discs mounted in a straight handpiece so that the two halves ( buccal and lingual or palatal ) had equal sized carious lesions . the two specimens obtained from each tooth were alternatively grouped into two allotting thirty specimens each to papacarie and carie - care for caries excavation so as to avoid selection bias . the caries excavation was done according to the manufacturer 's instructions . for both groups , the carious lesion was covered with gel and left undisturbed for 30 s. when the gel was cloudy , it was removed by scraping gently with the spoon excavator without application of any vertical pressure , after which some more gel was applied on the carious lesion and scraped as the gel turned cloudy and the process was continued until the gel was no longer cloudy . the gel was then rinsed and the cavity was then wiped with a moistened cotton pellet . the time taken for procedures was measured from start of caries removal till the cavity was confirmed to be free of caries with the help of a stop watch and was recorded . after caries removal , the tooth samples were decalcified in 10% formic acid at normal room temperature . [ figure 3 ] the decalcified samples were then washed in water , then the teeth were dehydrated in ascending degrees of ethanol ( 70 - 100% ) , they were then cleared in xylene and later embedded in paraffin [ figure 4 ] . during the experiment , the teeth were sectioned into 5 m thickness sections serially and were stained with eosin and hematoxylin to check for the presence of bacterial deposits microscopically and dentinal tubule destruction using conventional light microscope by a single operator to reduce bias [ figures 5 and 6 ] . decalcification of specimens specimens embedded in paraffin visible bacteria in the histological section of carie - care sample no bacteria evident in the histological section of papacarie sample the independent variable investigated in this experiment was the method of carious dentine removal . the response or dependent variables were time required to remove dentine measured in seconds ( efficiency ) and the detection of bacteria after carious removal and dentinal tubule destruction ( efficacy ) . a sample size of 30 was determined by statistician by resource equation method . , in which caries distribution is done according to site and stage of progression of the lesion . this concept of sites / stages ( si / sta ) replaced the classification of black and promoted a medical model of conservative dentistry in clinical practice . the extracted teeth having site 2 ( carious lesions at contact area of interproximal surfaces of the crown ) and stage 3 ( large dentin lesion with extended and frank cavitation ) carious lesion were included in the study . the exclusion criteria were deep carious lesion with pulp exposure or potential for pulp exposure [ figure 2 ] . each tooth was sectioned mesiodistally in the center of the carious lesion using diamond discs mounted in a straight handpiece so that the two halves ( buccal and lingual or palatal ) had equal sized carious lesions . the two specimens obtained from each tooth were alternatively grouped into two allotting thirty specimens each to papacarie and carie - care for caries excavation so as to avoid selection bias . the caries excavation was done according to the manufacturer 's instructions . for both groups , the carious lesion was covered with gel and left undisturbed for 30 s. when the gel was cloudy , it was removed by scraping gently with the spoon excavator without application of any vertical pressure , after which some more gel was applied on the carious lesion and scraped as the gel turned cloudy and the process was continued until the gel was no longer cloudy . the gel was then rinsed and the cavity was then wiped with a moistened cotton pellet . the time taken for procedures was measured from start of caries removal till the cavity was confirmed to be free of caries with the help of a stop watch and was recorded . after caries removal , the tooth samples were decalcified in 10% formic acid at normal room temperature . [ figure 3 ] the decalcified samples were then washed in water , then the teeth were dehydrated in ascending degrees of ethanol ( 70 - 100% ) , they were then cleared in xylene and later embedded in paraffin [ figure 4 ] . during the experiment , the teeth were sectioned into 5 m thickness sections serially and were stained with eosin and hematoxylin to check for the presence of bacterial deposits microscopically and dentinal tubule destruction using conventional light microscope by a single operator to reduce bias [ figures 5 and 6 ] . decalcification of specimens specimens embedded in paraffin visible bacteria in the histological section of carie - care sample no bacteria evident in the histological section of papacarie sample the mean time taken for complete caries removal was 385.8 s for papacarie and 427.13 s for carie - care , which is slightly more . paired t - test showed significant difference between the mean time taken for caries removal for both the groups [ table 1 ] . comparison of time taken in seconds between inter - comparative groups there is no significant dentinal tubule destruction between papacarie and carie - care groups , thus indicating both are conservative [ table 2 ] . comparison of both groups with respect to dentinal tubule destruction carie - care group showed more amount of bacterial emanents when compared with papacarie . fischer 's exact test showed significant results with carie - care group containing more amount of bacterial remnants following caries excavation [ table 3 ] . although the use of burs in both high speed and low - speed handpieces for caries removal conventionally allows faster treatment , they may remove sound tooth structure as well , which may weaken the remaining tooth structure , as well as cause pulpal trauma . philosophies of dental treatment change with time and now there is more than ample evidence provided by research for a reappraisal of the traditional approaches to caries treatment . the chemo - mechanical caries removal technique has generated great interest among dental researchers due to its concept of saving unaffected tooth structure while guaranteeing the removal of the denatured collagen stage of carious dentine . although the two layers of infected and affected dentine can be differentiated by fuschin staining , the removal of infected dentin is operator and technique sensitive method . similarly , the caries detector dyes can not specify correctly dentine removal in the cavity preparations on the pulpal surface of deep cavities and at the amelo - dentinal junction . further , in this present era of esthetic and adhesive dentistry , any remaining color or stain is unacceptable . thus , the best alternative is preserving remineralizable tissue and prevention of overexcavation of the cavity . there is also a need to evaluate and compare the antimicrobial efficacy and efficiency in caries removal of these newer enzymatic approaches available commercially such as papacarie and carie - care. the present in vitro study was conducted with the objective to evaluate the efficacy ( bacterial remnants and dentinal tubule destruction ) and efficiency ( time taken ) of caries removal using papacarie and carie - care. the extracted teeth were selected based on the modified classification of mount and hume , which help determine the type of treatment ( prevention , healing , re - mineralisation , or invasive intervention ) , and assist clinicians in selecting appropriate restorative materials . carious teeth with site 2 and stage 3 lesion were included as cmcr agents can dissolve only dentinal caries structure . the selection bias was avoided by excavating the corresponding cavity halves as this minimizes differences in the excavation results due to variations in the extension , depth , localization , and structure of the caries lesion . as microflora is one of the main etiological factors in caries occurrence , it is essential to reduce the microbial counts in caries lesions . apart from this , the efficacy in caries removal is also of interest . papain , the main ingredient of papacarie , is an enzyme similar to human pepsin and has got bactericidal and bacteriostatic properities . elindt demonstrated that papain acts only on infected tissues since infected tissues lack plasmatic anti - protease called a1 antitrypsin , this is present only in sound tissues which inhibit protein digestion . the infected dentin does not contain a1 antitrypsin enzyme , so this allows partially degraded molecules to be broken by papain . carie - care , a more recent and also economical than papacarie , is a gel based formulation . clove oil is a natural analgesic and is also known to have anesthetic properties . in the present investigation , evaluations for bacterial remnants and dentinal tubule destruction were done histologically by hematoxylin and eosin staining of the sectioned samples which were decalcified in 10% formic acid . formic acid was used as it gives good results with minimal soft tissue shrinkage and minimal loss of tissue when compared with nitric acid which shows crumbling of tissue . bacteria are the most common cause of dental caries and , for this reason , it is important to eliminate the largest possible numbers of bacteria during the removal of carious tissue . in the present study , the manufacturer 's instructions were adopted , and caries removal ceased when the gel attained a clear ( nonturbid ) appearance . papacarie excavation resulted in a highly significant reduction for all tested viable bacteria when compared to carie - care. as found in this in vitro investigation , among the two agents , the time taken for caries removal by carie - care was found to be more which is 427.13 s when compared to papacarie ( 385.8 s ) . papacarie exerts an inhibitory action on cariogenic bacteria and the time taken for removal of caries was about 6 min which is in accordance with the study conducted by motta et al . in addition to papain , both papacarie and carie - care contain chloramines that are used to chemically soften the carious dentin . the chlorination affects the secondary or quarternary structure of collagen , by disrupting hydrogen bonding and thus facilitating caries removal . papacarie was found to have no ability to affect the sound collagen fibers in the inner affected and normal dentin , as papain can digest only dead cells . , who concluded that papacarie is significantly more efficient in reducing the residual cariogenic bacteria in the dentin of primary teeth when compared to carisolv . have shown clinical efficacy of caries removal is best with airotor and the microbiological efficacy ( residual cariogenic bacteria ) of caries removal was almost comparable with airotor and chemo - mechanical methods of caries removal . however , both papacarie and carie - care exhibited no dentinal tubule destruction after caries excavation which shows their minimal invasive method in preserving the underlying affected dentin for remineralization . the drawbacks of this study are this is an in vitro study , so the features such as pain during caries removal and patient 's comfort levels could not be monitored . further large - scale , well - designed randomized controlled trials are needed to substiantiate the clinical outcome of the present study . the following conclusions were drawn from the study : the mean time taken for caries removal was found to be more for carie - care when compared to papacariemore amount of bacterial remnants was present after excavation with carie - careboth papacarie and carie - care were found to be conservative as no dentinal tubule destruction was evident . the mean time taken for caries removal was found to be more for carie - care when compared to papacarie more amount of bacterial remnants was present after excavation with carie - care both papacarie and carie - care were found to be conservative as no dentinal tubule destruction was evident . since this is an in vitro study , the clinical significance of these findings can only be determined with further studies assessing the clinical outcome of these chemo - mechanical methods in caries excavation . within the limitations of the present study , we find the chemo - mechanical caries removal to be an adequate alternative to the conventional rotary instruments . however , the greater time requirement represents a substantial barrier to its wider use by clinicians .

aims and objectives : the chemomechanical caries removal system is made presently available containing a natural proteolytic enzyme for ease in the excavation of infected dentin . the aim of the study was to evaluate the efficacy and efficiency of caries removal using papacarie and carie-care.materials and methods : a total of 30 extracted deciduous molars with proximal caries were collected , and each tooth was sectioned mesiodistally in the center of the carious lesion so that the two halves ( buccal and lingual or palatal ) have similar carious lesions , thus 30 teeth yielded 60 specimens . the specimens from each tooth were divided alternatively into two groups for caries excavation either using papacarie or carie - care so as to avoid selection bias . paired t - test was used to compare mean time taken for caries removal and fischer 's exact test was done for comparing bacterial remnants after caries excavation.results:mean time taken for caries removal was significantly higher for carie - care ( 427.13 s ) when compared to papacarie ( 385.8 s ) . papacarie was found to be significantly more efficient in caries removal with marked reduction in the bacterial remnants following excavation . however , both gels were found to be conservative as dentinal tubule destruction was not evident in either of them.conclusion:both papacarie and carie - care were found to be conservative in caries removal . papacarie was more efficient in removing bacteria in lesser time from the infected carious lesion .

the enclosure of eukaryotic genomes within the nuclear envelope evolutionarily generated the necessity to transport macromolecules selectively between nucleus and cytoplasm . following their synthesis in cytoplasm , histones and nucleic acid polymerases have to reach the nucleus , while specifically cytoplasmic proteins have to be kept out . mature mrna , trna and rrna molecules and their associated proteins have to follow the opposite route , while their immature precursors have to be kept in 1 . , 2 . , 3 . , 4 . , 5 .. eukaryotic mrna is enzymatically metabolized and compacted with proteins within nuclei to generate functional messenger ribonucleoprotein ( mrnp ) particles 6 . , 7 .. the control of protein biosynthesis involves regulation of intranuclear functions with participation of specific proteins , many of which seem to enter from the cytoplasm as a time - specific event 3 . , 4 . , 5 . , 7 .. the heterogeneous nuclear ribonucleoproteins ( hnrnps ) comprise a group of important regulators engaged in these cellular processes 8 . , 9 . , 10 .. extensive research efforts have been devoted to the cloning and molecular characterization of numerous ribonucleoproteins ( rnps ) in the context of their active role in mrna biogenesis and metabolism 1 . , 2 . , 9 . , 10 . , 11 . , as well as in terms of their involvement as autoantigens in human diseases ( 11 ) . hence , emphasis is put on the roles of various rna - binding motifs and their subsequent roles in governing cellular activities , both in health and pathology ( 9 ) . recently , there has been considerable interest in the participation of these rna - binding particles in triggering the immune response in human autoimmune disorders ( 11 ) . substantial research both at cdna and protein level have been performed on immunochemical features of human autoantigens ( http://www.zoo.uni-heidelberg.de/mol_evol/mb/ana_base.html ) . of particular interest is the group of mammalian hnrnps , which constitutes a part of spliceosome , which itself is an autoimmune target ( 12 ) . novel nucleotide sequences are continuously reported , which afterwards are used to delineate homology among various members based on previously determined nucleotides and protein sequences . the mechanism of contribution of numerous hnrnps with a similar structural rnp motif ( 13 ) to the induction of different immune reactions is sequence - specific ( 14 ) . phylogenetic approach is essential to find functionally important genomic sequences based on detection of their high degree of conservation across different species . such approach shows the level of improvement of the prediction of gene - regulatory elements in the human genome . this necessitates the study of the degree of homology of rna recognition motifs ( rrm ) among these proteins , requiring an evolutionary computation . having considered the importance of submitting new sequences for further functional characterization , the newly cloned and expressed cdna of previously unknown member of the hnrnp a / b family of proteins ( figure 1 ) is presented here . its rna - binding properties and tissue - specific gene expression profiles were recently determined ( 15 ) . based on the concept of correlation between sequences and rna - binding modes , a systemic search was performed for nucleic acid association by evaluating sequence conservation using multiple sequence alignments search tools . the aim of the presented work herein was to isolate novel cdna sequences with important functional implications in human pathology . our efforts have been devoted to the cloning and subsequent tissue - specific gene expressions of numerous human rnps from the hnrnp a / b family of proteins ( figure 1 ; ref . the objective was to search for molecular basis of autoimmunity by applying comparative analysis of the sequences of diverse autoantigens . in this context , evolutionary computation approach could give us major clues on how evolutionarily conserved mrna transport machinery fails are linked to development of human autoimmune disorders . we were mainly interested in cdnas , which might encode the yet undescribed hnrnp b2 . the need for this was based on two observations . on the one hand , autoantibodies directed against hnrnp a2 crossreact with hnrnp b1 and hnrnp b2 . since hnrnp b1 is an alternatively spliced variant of hnrnp a2 , this suggests that hnrnp b2 might be an alternatively spliced form of hnrnp a2/b1 . however , no attempts to clone a cdna encoding hnrnp b2 were successful so far . on the other hand , cdnas closely related to hnrnp a1 and hnrnp a2 have been previously isolated from a human fetal brain library and from a xenopus laevis library , respectively . their close relationships with hnrnp a2 suggested that one of these cdnas might actually encode hnrnp b2 ( 15 ) . to isolate the searched cdna , human liver and brain cdna expression libraries were screened by pcr using primers complementary to 5- and 3-untranslated regions of the fbrnp cdna . since the obtained new sequence shared close identity to the xenopus laevis hnrnp a3 cdna sequence ( entrez ; accession number l02956 ) , the protein was termed human hnrnp a3 . nucleotide sequence comparisons between fbrnp , xenopus laevis hnrnp a3 and our newly determined human hnrnp a3 proteins revealed that extensive sequence conservation exist in rna - binding regions . the majority of sequence variations were seen at the gly - rich domain , composed of amino acids at positions 211 - 373 . these sequences were observed more at nucleotide level , as expected , compared to the translated protein sequences ( figure 2 ) . only protein sequences are shown for brevity . identification of various nucleic acid - binding domains of diverse hnrnps was achieved by cloning and sequencing of cdnas encoding these motifs . in general , all known human hnrnp proteins contain at least one rna - binding module and one another auxiliary domain fragment . the rna - binding motifs contain the rnp consensus sequences ( cs - rbd ) , the rna recognition motif ( rrm ; ref . , 13 . , 17 . ) , the rnp-80 motif , the rgg box ( 18 ) , and the kh domain ( 19 ) . this domain is found in hnrnps in various amounts , ranging from 1 ( in hnrnp c ) to 4 ( e. g. in poly a - binding protein ; ref . figure 1 shows the general modular structure of hnrnp a / b type of rnp particles . their general structure is composed of two domains : the first 195 residues comprise the so - called up1 domain , containing two canonical rna - recognition motifs ( rrm 1 and rrm 2 ) , each of which is comprised from the conserved rnp-2 and rnp-1 submotifs . the gly - rich c - terminal domain comprises an rgg box and a nuclear localization motif . this motif contributes to protein - protein interaction patterns , as well as to subcellular localization ( 18 ) . two conserved solvents exposed phe residues at the centre of the -sheet in each rrm - contacted rna . the least conserved 3 loop in u1a is engaged in extensive rna interactions , whose conformation changes upon rna binding 17 . , 20 .. the rnp domain interacts with a flexible single - strand rna and the -sheet provides a large surface for extensive interaction with nucleotides . regions outside of the rrm may also play important roles in rna binding ( 10 ) . identification of these motifs as rna - binding domains has been used for prediction of this activity in various proteins of yet undescribed function possessing these domains . as seen in figure 2 , the newly isolated human hnrnp a3 cdna encodes a protein of 296 amino acids ( a.a . ) . amino acids numbered 198 comprise rbd1 , 112209 comprise rbd2 , 209296 comprise the gly - rich domain , while 99111 contain inter - rrm linker ( irl ) segments . to our surprise , comparison with the previously reported fbrnp cdna sequence , encoding a 269 a.a . therefore , it was assumed that the currently presented cdna encodes a novel and yet undescribed protein . despite the close homologies among the rna - binding regions ( 95% ) , remarkable differences can be seen in the c - terminal domain ( figure 2 ) , where deletions and insertions are apparent . shorter and there are fewer conserved residues in the c - terminal part . both cdnas also show high homologies to the cdna encoding hnrnp a3 from xenopus laevis except for a stretch of 14 a.a . at the rbd1 n - terminal part . the rna - binding regions of the three proteins are almost identical , while the auxiliary domains are less conserved . the xenopus laevis protein shows 75% homology with fbrnp and 83% with the newly cloned human hnrnp a3 protein , respectively ( figure 2 ) . the reduced length of the human hnrnp a3 , as compared to the xenopus laevis homologue ( 296 vs. 373 a.a . ) , is compatible with differences observed between xenopus laevis and rat hnrnp a1 , which are composed of 365 and 320 residues , respectively ( 15 ) . the assumption that the presented clone encodes the human counterpart of frog hnrnp a3 is further reinforced , when conserved residues in the auxiliary domains of the a / b polypeptides are considered . thus , a stricking homology is apparent at the c - terminus , which is well conserved between frog and human hnrnp a3 with 15 of the last 18 a.a . both proteins end with a triplet rrf , which is also present in hnrnp a1 , but neither in fbrnp nor in hnrnp a2/b1 . moreover , a glutamic acid located at the boundary of rbd2 and the auxiliary domains of xenopus laevis and human hnrnp a3 ( a.a . position 207 ) is substituted by valine in fbrnp , a further indication that the novel protein is indeed the human counterpart of xenopus laevis hnrnp a3 . interestingly , this result is reported also for the murine hnrnp mbx protein , which is highly homologous to both human fbrnp and xenopus laevis hnrnp a3 ( 21 ) . most of the conformational differences between the two rrms occur either at the c - terminal end of the b helices , or at the tip of loop 3 , which is tilted and twisted in rrm1 , as opposed to rrm2 ( figure 3 ) . the rnp domain interacts with a flexible single - strand rna and the -sheet provides a large surface for extensive interaction with nucleic acids . the two aromatic side chains of rnp1 and one aromatic side chain of rnp2 provide a convenient template for base stacking of the rna with neighbouring protein side chains , forming hydrogen bonds with the stacked bases . although the major groove of the a - type rna helix is too narrow and deep to provide a site for sequence - specific association , an rna loop with exposed nucleotides provides a large surface for protein binding . however , the rna - binding is not a unique feature of the -sheets . despite the evolutionary conservation of the rrms , which are necessary for both general and sequence - specific nucleic acid binding , regions outside of the rrms may also play important roles in rna binding ( 10 ) . for instance , the flexibility of the linker sequence connecting both rrms , resulting from two pairs of arg and asp involved in irl salt bridges and the ordered residues , creates a position which is highly probable for this sequence to be involved in direct rna binding . figure 3 shows the rrm sequences of 12 hnrnp a / b type of proteins , extending the previous data set of mayeda et al . there is extensive sequence homology between these various proteins , which are conserved throughout evolution from insects to man . each of the rrms forms an ungapped alignment with both rrms of hnrnp a1 . in all of the shown proteins , the evolutionary links between the rrms of human hnrnp a3 and other hnrnp a / b proteins are depicted in a phylogenetic tree ( figure 4 ) . the sequences of rrm1 and rrm2 cluster in two separate groupings , representing insect and vertebrate proteins . the patterns of branching for rrm1 and rrm2 sequences are nearly identical , except for the placement of the branch representing the minor human variant hnrnp a0 . the almost identical pattern of branching for both rrms suggests that these fragments have evolved in parallel . obviously , each of the insect and vertebrate proteins is equally distant from each other . apparently , this fact indicates independent gene duplications of ancestral hnrnp a / b - like protein , in agreement with mayeda et al . the independent evolution of individual tandemly repeated protein domains and their functional relevance has always being an intriguing issue . the rnp domain is commonly encountered scaffold among the nearly 350 different folds , that is , the favourable secondary arrangements of around 10,000 protein structures covered by the currently used databases 22 . , 23 . , 24 . , 25 .. comparisons of various protein structures , in combination with their nucleotide analyses serve as a clue for their evolution . the debate is whether these structures have evolved for achievement of specific functions or for thermodynamic stability and/or for kinetic folding reasons . the same structural topology can determine numerous activities , preserved through evolution . in biological systems , enzymes evolve by acquiring of new thermodynamic or kinetic properties by the already existing protein folds . generally , only the overall folding pattern is conserved in protein groups , and as the sequence diverges , the structures deform . among the widespread catalytic folds , such as the tim barrel and the globins , the rrm domain attracts research efforts into delineating the advancements of evolution starting from all - helical proteins via all -sheet proteins and reaching a group of /-proteins . in this route , the /-barrel is the most frequently seen enzymatic fold and appears to be a selected topology for the directed evolution of new biocatalysts . it is worth studying how the present /-barrels are linked evolutionarily to each other and through which way they have evolved from simpler ancestors . the topic becomes more attractive when these folds comprise a domain involved in a disease state , such as autoimmunity . , 11 .. the rapidly growing information on structures and functions of numerous hnrnps will be invaluable for understanding the pathological mechanisms by which these particles participate as targets of autoantibodies . of particular interest is the determination of the role of rna - protein folding patterns in development of disease - specific epitope formation . in this context , the evolution of selected nucleic acid - binding sequences and how this contributes to disease establishment remain unknown . therefore , this study was devoted to presentation of evolutionary connections between the various hnrnps and their rna - binding domains . a phylogenetic tree is given to depict these motifs in the major group of these particles , including also the recently cloned and sequenced novel human hnrnp a3 ( 15 ) , followed by remarks on the lastly selected sequences , relevant in diseases . figure 1 shows the general 2xrbd - gly type of structure of the a / b type of human hnrnps . the two canonical rrm-1 and rrm-2 , containing the conserved rnp-2 and rnp-1 submoieties , and the rgg c - terminal part constitute the main domains . the translated sequences of the recently characterized human hnrnp a3 particle is presented herein ( figure 2 ) , solely to demonstrate the high degree of sequence homology among the different rrms . its protein sequence is compared to that of other two members of this protein family the selected three members in figure 2 are functionally related quite distantly from each other , each having a buried core tryptophan and there are disulphide bonds on the highly conserved irl - segments ( 16 ) . the sequence comparisons of the three closely related hnrnp a3 members showed that the general need for a large buried hydrophobic moieties is well satisfied . this represents a possible case , where structural constraints make evolution towards a different conformation unlikely . the high level of conservation of these residues is related to the interlocking interactions and restraints of the loop connecting the -sheets , by providing the huge hydrophobic template necessary for core packing . in addition , having large hydrophobic inner part and suitable hydroxyls capable of hydrogen - bond formation , these residues ( e.g. tyrosines ) could be conserved for structural stability needs . performing amino acid mutations , or substitutions at the sites thus removing hydrophobic packaging interactions , would provide further evidence for the core packing of hnrnp globule . these are buried in the compacted core and are expected to participate in overall folding of the scaffold . however , to verify whether this is the case , more mutational analyses , combined with thermodynamic , kinetic , structural and evolutionary computation data are needed . amino acid sequence comparisons of rrm structures ( figure 3 ) depicted the hydrophobic core residues , conserved among different rrms . the inserted sequence of the human hnrnp a3 shared extensive sequence homology ( up to 90% ) existing between rrms of all these numerous proteins , which are conserved throughout evolution . a phylogenetic tree ( figure 4 ) depicted the evolutionary history of the rrms of human hnrnp a3 and other hnrnp a / b proteins . the objective was to deduce whether the observed patterns of conservation resulted due to structural features for this type of fold architecture , or alternatively , were the consequence of divergent evolution . parallel symmetric groupings of rrm1 and rrm2 in two different clusters , which belong to insect and vertebrate members is well seen . with the slight deviation of hnrnp a0 , this branching pattern is an indication of parallel proceeding evolution , in accordance with relevant studies 16 . , 26 .. each of the members of these two subgroups are equally distant from each other . these results suggest that these branching patterns originated from an ancestral hnrnp a / b - like protein probably through independent gene duplications . phylogenetic analyses of this sort provide evidence for evolutionary history and origin of the modular structure of hnrnps and their contemporary functional implications . this approach helps to delineate whether rna - binding ( rbd ) and protein - protein ( auxiliary ) domains have evolved in parallel or follow another evolutionary history . in this context evolutionary computation on every newly characterized hnrnp particle the sequence of human hnrnp a3 was inserted in the data set at figure 4 and becomes a further support of the evolutionary trend of these rnp family of proteins , appearing also in previous studies 16 . , 26 .. these earlier studies proved that the origin of hnrnps is a consequence of independent gene duplication . in their landmark work , fukami - kobayashi et al . ( 26 ) showed that the ancestral gene of the hnrnps had have two rna - binding domains even before the divergence of invertebrates and vertebrates and that it diverged to contemporary hnrnp genes while preserving the tandemly repeated structures . the origin of natural selection of particular nucleotide sequences and their conservation from primitive rna - based catalysis to eukaryotic chromosome evolution remain to be elucidated ( http://manske.virtualave.net/genetik/vorlesung_ws99/teil1-3/rna_und_die_evolution.htm ; ref . , there is now sufficient data to prove the substitution of ancestral rnas by more efficient rna - protein complexes with subsequent replacement of the latter by proteins ( 27 ) . by using molecular modelling approach , it has been recently shown ( 28 ) that in this transition position , the two rna - binding sites of the simulated proto - protein interact with target sites to form a stable rnp complex . since the selected proteins were unable to resolve misfolded rna , in vivo selected protein associated with the two rna - binding sites , indicating that the protein facilitates the correct folding of the ribozyme . this provides evidence that these modelled self - splicing rna - protein complexes can be considered as a primitive form of splicing factors or rna chaperones , which contribute to correct rna folding ( 28 ) . in terms of molecular evolution of primitive catalysts , these self - splicing intron assemblies possibly became splicing rnps , suggesting that an active rnp particles could have originated from a primordial ribozyme ( 28 ) . the evolutionary driving force for this is the small structural cost , through which the protein binding compensates for some folding deficiencies in the rna ( 29 ) . this serves as another proof that the evolution from rna to rnp - determined catalysis represents an evolutionary design against misfolding rather than for the maintenance of a protein binding site . our preliminary biophysical results ( 30 ) suggest that this is the case with human hnrnp a3 , as well . the current study presents the sequence conservation of mainly hydrophobic residues of human hnrnp a3 , which form the hydrophobic packing cores and are essentially and evolutionarily conserved in other rna - binding domains in the hnrnps studied ( fig . this indicates that the rna - binding domains have evolved from a common hnrnp a / b - type proto - protein , supporting further previous models 16 . , 26 .. interestingly , while the majority of models of rbd protein family built up to now are based on divergent evolutionary principles , another view claims that this is open to doubt in cases of similar , but more distantly related functions and structures ( 31 ) . their model suggests that even though the ancient rbds could have developed separately by divergent evolution , these rbds have evolved conserved rnp motifs with a similar structure and function on similar surfaces . the authors emphasize that rnp motifs are conserved both in scaffold architecture and in function , which provides an intriguing case of convergent evolution . further advancements in sequence comparison techniques will help to understand whether these functional sites have arisen multiple times during evolution ( http://online.itp.ucsb.edu/online/infobio01/higgs1/pdf/higgs1.pdf ; ref . 22 . , 23 . , 24 . , 32 . ) . regarding the evolutionary conservation of hnrnp domains , two possibilities exist for explaining their nowadays existence they are either hyperadaptable , or they may have developed features required to perform vital cellular functions ( 32 ) . despite the case with cold shock domain ( 31 ) , hnrnp a / b proteins appear to possess eukaryotic origins , because prokaryotic homologs could not be detected until now . all these rbds have similar dimerization roles leading to similar three - dimensional architectures , suggesting that they may have arisen from a common ancestor , which have diverged in sequence afterwards ( 32 ) . these domains are found also in chloroplast protein sequences , implying the link with endosymbiont hypothesis , and also implys that the multicellularity in plants and animals did not evolved independently 26 . , 32 .. domain evolution of hnrnps rises the question that whether they are consequences of continuous or discontinuous evolution . fukami - kobayashi et al . ( 26 ) showed that the rbd fused with the sr - rich domain proceeded the divergence of splicing factors , and that these two domains have arisen together thus conserving the fused domain organization . the existence of gly - rich domain ( figure 1 ) , also supports the proposal of domain organization of the unique origin of hnrnps , followed by duplication and divergence of rbds and structural transitions of the auxiliary domain ( 18 ) . this indicates that the auxiliary domain shared by functionally related rnps diverged in parallel together with the rbds ( 26 ) . it is thus implied , that these ancestral repeats must have oligomerized afterwards to adopt a similar structure seen in contemporary homologs ( 32 ) . in the light of the fact that hnrnp a / b particles are considered as disease genes , an intriguing issue becomes the use of protein domain data of various hnrnps to delineate the link and etiology of diseases . as stated earlier , the majority of these gene activities related to autoantigenic hnrnps are covered by relevant databases . respect , identification of human paralogous disease genes generates further pathological gene candidates to be sequenced , because paralogous genes are frequently encountered as mutated versions in similar diseases ( 32 ) . the sequence comparison , however , may be insufficient for deduction of its functional role for patients diagnozed with the suspected disease . therefore , we followed the suggestion of ponting , c.s . and russell , r.r . ( 32 ) and combined our sequence determination of human hnrnp a3 with its gene expression profiles , its molecular interaction features ( 30 ) , and its tissue - specific gene expression patterns ( 15 ) . interestingly , our molecular characterization of human hnrnp a3 showed that while the recombinant hnrnp a3 with its 296 a.a . migrates as expected as a 32 kda protein on sds - page analysis , it is recognized by the patients sera as a 50 kda protein . this is attributed to alternative splicing or due to tissue - specific expression of a highly related yet unknown crossreactive protein . surprisingly , neither the 50 kda nor the 32 kda protein was detected in helanuclear extracts , further supporting the assumption that hnrnp a3 is not ubiquitously expressed as hnrnp a1 or hnrnp a2 . northern blotting analysis showed the variable expression patterns of hnrnp a3 mrna in human tissues . thus , it is highly expressed in spleen , ovary , small intestine , lung , liver , skeletal muscle , kidney and pancreas , whereas expression in thymus , testis , colon and peripheral blood was hardly detectable . expression in prostata , heart , brain and placenta was low , but clearly detectable ( 15 ) . in addition , circular dichroism and fluorescence spectroscopic measurements demonstrated that the human hnrnp a3 protein is a stable particle the free energy of unfolding of the full - length hnrnp a3 is 58c , as shown by both urea and temperature denaturation ( 30 ) . this particle increases its apparent stability upon interaction with rna fragments r(uuaggg)4 to higher temperature values . the high binding affinity to this repeat indicates its preference for association with purine - rich consensus sequences and targeting features towards deleterious g - tetrad structures . the latter fact suggests its active participation in alternative splicing a common feature of well - known telomitic repeats interactions of hnrnp a1 . this rna fragment , hnrnp a3 particle recognition , can act by facilitating the splicing of alternative intron of the pre - mrna . since this represents a case of regulation of splice - site selection with further functional implication in human disease 11 . the first one is comprised of both rbd1 and rbd2 , and the second one is composed of rbd2 and certain parts of gly - rich domain 15 . , 30 .. this epitope recognition pattern differs from the epitope determined for the highly related hnrnp a1 and hnrnp a2 , respectively . thus , in hnrnp 2 the rbd2 was found to contain the major epitope , which was recognized by patients suffering from rheumatoid arthritis or systemic lupus erythenathosus . this particular region was also found to be essential for interaction with rna and the patients autoantibodies strongly inhibited rna binding . as oppose to these , autoantibodies derived from patients diagnozed with mixed connective tissue disease recognized an epitope comprising rbd1 and rbd2 . interestingly , the major epitope of hnrnp a1 also comprises both rbds , which were also targeted by patients with rheumatoid arthritis and systemic lupus erythemathosus . taken together , these data confirm that rheumatoid arthritis , systemic lupus erythemathosus and mixed connective tissue disease are immunochemically linked by systemic autoimmunity to the functionally important rna - binding regions of hnrnp a / b proteins . the observed trend brings the question of why so closely related and evolutionarily conserved rbd domains are differentially recognized by the autoantibodies . we proposed recently ( 30 ) the overall folding patterns of these domains , and the protein antigenicity arising from these patterns is the determining factor . the present study combined with previously determined sequences of u1a , hnrnp c , hnrnp a1 and drosophila sex lethal ( sxl ) protein structures reveals that they have the same fold , but have different placing of the second and fourth -strands . individual rrms have preferences towards various rna sequences , due to differences in surface amino acids found outside the conserved rnp submotifs . in our opinion in certain cases , the two rrms somehow act in concerted fashion to give rise to the overall rna- and antibody - binding characteristics , whereas in other cases the presence of only one rrm is sufficient for autoantibody and rna recognition . thus , rbd1 itself bound strongly to rna fragments , however , the joining of rbd2 to rbd1 that increased the overall affinity indicats that rbd2 also strongly affects rna - binding . to isolate the cdna , human liver and brain cdna libraries were screened by pcr using primers complementary to sequences in the 5- and 3-untranslated regions of the fetal brain ( fbrnp ) cdna , as described ( 15 ) . screening of cdna expression library resulted in isolation of a clone , which is a member of a 2xrna - binding domain ( rbd)the glycine family of hnrnp proteins . human hnrnp a3 polypeptide , highly homologous to the fetal brain cdna , as well as to hnrnps a1 and a2 . at the nucleotide level , the cdnas encoding rna - binding fragments of hnrnp a3 sequence were generated by pcr as deletion mutants , starting from the full - length human hnrnp a3 . pcr mix was prepared by adding 660 l water , 220 l 10 mm dntp mix ( pharmacia biotech , uppsala , sweden ) , 220 l 10 cloned pfu polymerase buffer ( promega , madison , usa ) , and 122 l 25 mm mgcl2 for twice . 20 l of each oligonucleotide ( 100 pmol / ml ) was taken and added to 480 l water . pcr reactions were run by taking 172 l of the pcr mix ( i. e. , 86 l per reaction ) , and mixing this amount with 12 l of both primers . denaturation was achieved by incubating at 94 c for 30 sec , while annealing temperature was 52 c for 1 min . the amplified fragments were cloned into ligation independent cloning ( lic ) vector ( novagen , madison , usa ) . all the dna sequencings were performed by the vienna biocenter oligo team ( http://emb1.bcc.univie.ac.at/gem ) . nucleotides and deduced protein sequences of previously determined rna - binding domains of rnps from various species and human hnrnp a3 were aligned employing clustalw programme ( http://www2.ebi.ac.uk/clustalw/ ; ref . to isolate the cdna , human liver and brain cdna libraries were screened by pcr using primers complementary to sequences in the 5- and 3-untranslated regions of the fetal brain ( fbrnp ) cdna , as described ( 15 ) . screening of cdna expression library resulted in isolation of a clone , which is a member of a 2xrna - binding domain ( rbd)the glycine family of hnrnp proteins . human hnrnp a3 polypeptide , highly homologous to the fetal brain cdna , as well as to hnrnps a1 and a2 . at the nucleotide level , the cdnas encoding rna - binding fragments of hnrnp a3 sequence were generated by pcr as deletion mutants , starting from the full - length human hnrnp a3 . pcr mix was prepared by adding 660 l water , 220 l 10 mm dntp mix ( pharmacia biotech , uppsala , sweden ) , 220 l 10 cloned pfu polymerase buffer ( promega , madison , usa ) , and 122 l 25 mm mgcl2 for twice . 20 l of each oligonucleotide ( 100 pmol / ml ) was taken and added to 480 l water . pcr reactions were run by taking 172 l of the pcr mix ( i. e. , 86 l per reaction ) , and mixing this amount with 12 l of both primers . denaturation was achieved by incubating at 94 c for 30 sec , while annealing temperature was 52 c for 1 min . the amplified fragments were cloned into ligation independent cloning ( lic ) vector ( novagen , madison , usa ) . all the dna sequencings were performed by the vienna biocenter oligo team ( http://emb1.bcc.univie.ac.at/gem ) . nucleotides and deduced protein sequences of previously determined rna - binding domains of rnps from various species and human hnrnp a3 were aligned employing clustalw programme ( http://www2.ebi.ac.uk/clustalw/ ; ref .

heterogeneous nuclear ribonucleoproteins ( hnrnps ) are spliceosomal macromolecular assemblages and thus actively participate in pre - mrna metabolism . they are composed of evolutionarily conserved and tandemly repeated motifs , where both rna - binding and protein - protein recognition occur to achieve cellular activities . by yet unknown mechanisms , these ribonucleoprotein ( rnp ) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases . this feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity . since rnp domain is one of the most conserved and widespread scaffolds , evolutionary analyses of these rna - binding domains can provide further clues on disease - specific epitope formation . the study presented herein represents a sequence comparison of rna - recognition regions of recently cloned and characterized human hnrnp a3 with those of other relevant hnrnp a / b - type proteins . their implications in human autoimmunity are particularly emphasized .

the term replication stress describes the slowing or stalling of replication forks by endogenously or exogenously derived impediments to dna polymerization ( zeman and cimprich , 2014 ) . replication stressors can be local factors , such as dna damage or secondary structures that affect forks randomly as they are encountered , or global ones , such as nucleotide pool depletion or imbalance that simultaneously slows all forks ( poli et al . , 2012 , it is now recognized that replication stress induced by nucleotide pool imbalance is an important consequence of the activation of some oncogenes , which drive cells into s phase without upregulation of nucleotide supply ( bester et al . , 2011 ) . the resulting loss of polymerase processivity is thought to lead to localized uncoupling of the replicative helicase and polymerase and formation of tracts of single - stranded dna ( byun et al . , 2005 , pacek and walter , 2004 ) . while this normally induces checkpoint activation and senescence ( bartkova et al . , 2006 , , in cells that can bypass the checkpoint , such replication stress provides a fertile source of genetic instability , particularly in the vicinity of fragile sites and sites capable of forming secondary structures ( de and michor , 2011 , tsantoulis et al . , 2008 ) . in addition to the extensive genetic changes that have been well documented in many types of cancer , there are also extensive local and global alterations in histone and dna modifications . the consequent changes in chromatin structure are accompanied by significant dysregulation of gene expression ( timp and feinberg , 2013 , berdasco and esteller , 2010 ) , which , since it is not accompanied by changes in the dna sequence , may be considered epigenetic ( berger et al . , 2009 ) . these epigenetic changes could act alongside genetic instability to produce clonal variation within a tumor , upon which selective pressure can act , and so may contribute to tumor evolution . mutations in histone and dna - modifying enzymes , and even histone proteins themselves , have been found in several cancers and are likely to explain at least some of the observed epigenetic instability ( timp and feinberg , 2013 ) . however , it is not clear that mutations in histone - modifying enzymes account for all the alterations observed in different cancer types . we recently provided evidence that deficiencies in enzymes responsible for replicating g quadruplex ( g4 ) structures , such as the specialized dna polymerase rev1 and helicases fancj , wrn , and blm , can lead to localized changes in histone modifications and gene expression ( sarkies et al . , 2010 , sarkies et al . , 2012 , schiavone et al . , 2014 ) . g4s can form within motifs comprising four short runs of dg bases , separated by linker sequences . the dg bases in the motif form planar hoogsteen - bonded quartet structures that can stack on top of each other , resulting in an often highly thermodynamically stable secondary structure , the g4 ( reviewed in maizels and gray , 2013 ) . we proposed that persistent replication fork stalling at g4s in mutants such as rev1 or fancj leads to pathologically long daughter strand gap formation , resulting in local uncoupling of dna synthesis from parental histone recycling . this , in turn , leads to loss of the histone modifications present on the parental chromatin , which , if in the vicinity of a gene promoter , results in changes in transcription ( sarkies et al . , 2010 , sarkies et al . , 2012 , schiavone et al . , a prediction of this model is that global replication stressors that lead to loss of processive dna polymerization with uncoupling of the replicative helicase and polymerase also should promote epigenetic instability by dissociating dna synthesis from histone recycling . here we test this hypothesis by examining the effect of hydroxyurea ( hu)-induced nucleotide pool depletion on the epigenetic stability of a sensitive reporter locus , bu-1 , in chicken dt40 cells ( sarkies et al . we show that chronic treatment with low - dose hu induces stochastic instability of bu-1 expression , characterized by loss of the chromatin marks h3k4me3 and h3k9/14ac seen in the normally active locus . this instability depends significantly on the presence of a g4 motif 3 of the promoter , oriented to stall the leading strand of a fork heading toward the transcription start site ( tss ) . the presence of this g4 motif not only increases the rate at which bu-1 expression is lost , but is additionally associated with phosphorylation of h2ax and appearance of the heterochromatic mark h3k9me3 . this is consistent with the g4 acting to focus dna damage induced by the global replication stress imposed by hu , with the damage leading to repression of the locus . further , we show that , across the genome , chronic exposure to hu results in an altered pattern of gene expression similar to that seen in cells lacking the g4-unwinding helicases fancj , wrn , and blm , and that affected genes are enriched in g4 motifs . together , these observations indicate that nucleotide depletion can combine with naturally occurring dna secondary structures to promote epigenetic instability . we first sought conditions in which we could culture dt40 cells in low - dose hu such that replication is slowed but completed ( alvino et al . , 2007 ) . we therefore exposed wild - type dt40 cells to a range of hu concentrations and monitored their doubling time . the cells were able to proliferate for over a week in up to 150 m hu ( figure 1a ) . at this dose , their doubling time increased from 12.3 to 32.7 hr , recovering when the hu was washed out ( figure 1a ) . to determine the effect of low - dose hu on replication dynamics , we performed dna molecular combing after pulse labeling the cells with halogenated nucleotides ( figure s1a ) 3 days after initiating culture in hu . average fork velocity decreased from 1.26 to 0.71 kb / min ( figure 1b ) , with a compensatory decrease in average interorigin distance from 72 to 40 kb ( figure 1b ) . consistent with these perturbed replication dynamics , cell - cycle profiles revealed a significant accumulation of cells in s phase while in hu ( figure s1b ) . we have reported previously that replication - dependent transcriptional instability associated with g4 motifs can be monitored by following expression of a surface marker , bu-1a , in dt40 cells ( sarkies et al . , 2012 , schiavone et al . , 2014 ) . the bu-1 locus contains prominent g4 motifs 3.5 kb downstream of the tss and 3 kb upstream . both are orientated to be g - rich on the feature strand with respect to the bu-1 transcript ( figure 1c ) . epigenetic instability of bu-1 in rev1 cells is entirely dependent on the + 3.5 g4 motif , and it requires the motif to be orientated such that its g - rich strand forms on the leading strand of a replication fork entering the locus from the 3 end ( figure 1c ; schiavone et al . , 2014 ) . we have reported previously that the bu-1 locus is bidirectionally replicated , meaning that during any given s phase there is a 50% probability of the + 3.5 g4 being replicated on the leading strand template ( schiavone et al . , 2014 ) . growth of wild - type dt40 cells in 150 m hu resulted in the appearance of a bu-1a population as cells divided over the course of 7 days ( figure 1d ) . surface expression of bu-1a correlates closely with transcript abundance ( sarkies et al . , 2012 ) , and this held true for bu-1a clones recovered after hu treatment ( figure s1c ) . to estimate the rate at which bu-1a variants are formed in hu , we performed a fluctuation analysis by expanding multiple parallel populations of 10 bu-1a cells in hu for 7 days , after which we monitored the appearance of bu-1a variants . this revealed a striking degree of expression instability despite the small number of cell cycles through which the cells had passed ( figure 1e ) . using our previously described monte carlo simulation of bu-1a loss as a replication - dependent phenomenon ( schiavone et al . , 2014 ) , we estimated a per - division probability of generating of a bu-1a state during culture in hu of c. 0.15 . to obtain additional evidence that this induced transcriptional instability of bu-1 reflected decreased dna polymerase processivity , we asked whether bu-1 variants could be induced by aphidicolin . aphidicolin slows replication by directly inhibiting dna polymerases , particularly pol ( oguro et al . , 1979 ) , and a low dose induces replication stress ( pacek et al . , 2006 ) . dt40 cells were able to proliferate in up to 150 m aphidicolin for 10 days and , as with low - dose hu , this resulted in substantial instability of bu-1a expression ( figure 1f ) . we have shown previously that removal of the + 3.5 g4 motif from both alleles of bu-1 in rev1-deficient cells results in complete stabilization of expression of the locus ( schiavone et al . , 2014 ) . we therefore examined the extent to which this motif also accounted for the observed hu - induced instability of bu-1a expression in wild - type dt40 . we grew wild - type cells lacking the + 3.5 g4 on both alleles , bu-1 ( schiavone et al . , 2014 ) , in hu and assessed the frequency of bu-1a variants after 7 days by fluctuation analysis ( see figure e3 in schiavone et al this revealed that removal of the + 3.5 g4 motif resulted in a significant reduction in the rate at which bu-1a variants were generated . however , it did not result in complete stabilization of the locus ( figures 2a and 2b , i and ii ) . we considered the possibility that the residual instability could be due to the 3.0 g4 upstream of the tss . however , deleting this motif ( figure s2 ) had no impact on hu - induced instability ( figure 2b , iii ) . to confirm the contribution of the + 3.5 g4 motif , this resulted in the return of the high - level hu - induced instability of bu-1 expression observed in wild - type cells ( figure 2c , i ) . however , this was not seen if the motif was mutated to render it incapable of forming an intramolecular g4 ( figure 2c , ii ) or when it was inverted so that the g4 structure would form on the lagging strand template ( figure 2c , iii ) . thus , hu treatment alone can induce instability of bu-1a expression , but its effect is significantly potentiated by the presence of a g4-forming sequence orientated to stall the leading strand replication of a fork heading toward the tss . we next investigated the basis for the hu - induced generation of bu-1a variants . to test whether the bu-1 state is permanent , we isolated five bu-1 clones at the end of 1-week growth in hu and cultured them for a further 3 weeks in hu - free medium . the clones remained stably bu-1 with no evidence of reversion to bu-1 , suggesting that this was a permanent change . we considered the possibility that bu-1 cells resulted from genetic changes in the locus , although the observed rate of mutation would be extraordinarily high for this to be the case . we therefore sequenced the region around the + 3.5g4 to look for mutation of the motif and used pcr with restriction digestion to detect larger deletions ( figure s3 ) . neither assay revealed any evidence of genetic instability consistent with the formation of bu-1 variants being an epigenetic event . we therefore examined the pattern of histone modification at the bu-1 promoter by chromatin immunoprecipitation ( chip ) from bulk populations of cells exposed to hu . bu-1 is a transcriptionally active locus characterized by high levels of h3k4me3 around its tss . after 48-hr treatment with hu , we observed a small but not significant loss of h3k4me3 at the bu-1 promoter ( figure 3a ) , consistent with the size of the population of bu-1a cells generated by this time point ( figure 1d ) . however , after 7-day treatment , we observed a more significant loss of h3k4me3 correlating with the much larger population of bu-1a cells at this time point ( figures 1d and 2a ) . the loss of h3k4me3 was accompanied by a reduction in h3k9/14 acetylation ( figure s4 ) . seven days of hu treatment also induced a marked increase in h3k9me3 at the bu-1 promoter ( figure 3a ) . it has been proposed previously that hu - induced displacement of parental h3/4 and its buffering by the histone chaperone asf1 may lead to unscheduled heterochromatinization by ectopic deposition of pre - marked histones upon their release from asf1 ( jasencakova et al . , 2010 , schwab et al . , 2013 ) . alternatively , the appearance of h3k9me3 may result from dna damage - induced heterochromatinization , which has been observed following double - strand breaks ( ayrapetov et al . breaks can arise from fork collapse in hu ( petermann et al . , 2010 ) , and thus , if unscheduled incorporation of h3 with k9 methylation was responsible , then an increase in h3k9me3 would be observed irrespective of whether the + 3.5 g4 motif was present . however , if localized g4-induced dna damage was responsible , then the appearance of h3k9me3 would be dependent on the + 3.5 g4 motif . we therefore examined h3k4me3 and h3k9me3 at the promoter of bu-1 in cells lacking the + 3.5 g4 motif . after 7 days in hu , h3k4me3 was reduced ( figure 3b ) , but to a lesser extent than in wild - type cells ( figure 3a ) , consistent with the reduced rate at which bu-1a variants are generated in cells lacking the + 3.5 g4 motif ( figure 2b ) . however , we observed no associated increase in h3k9me3 ( figure 3b ) . to monitor the extent to which hu induced dna damage in the two situations , we performed chip for phosphorylated h2ax ( h2ax ) ( rogakou et al . , 1998 ) at the bu-1 promoter . h2ax was enriched 2.5-fold at the bu-1 promoter in wild - type cells after 7 days in hu , but not enriched in cells lacking the + 3.5 g4 motif grown under the same conditions ( figure 3c ) . this favors a model in which heterochromatinization of bu-1 in hu is promoted by dna damage , likely from fork collapse associated with the + 3.5 g4 motif . we next examined the extent to which hu - induced changes in histone modifications were permanent by performing chip at the bu-1 promoter in the five stable bu-1a clones discussed above . this revealed that promoter h3k4me3 remained low , showing that loss of this mark was permanent ( figure 3d ) . thus , while h3k9me3 is induced by hu in cells containing the + 3.5 g4 motif , this mark is not essential to maintain the bu-1a state . this may be because it is installed only transiently during repair of hu - induced dna damage in the locus , or because cells in which h3k9me3 persists are growth disadvantaged and are lost from the population . the data thus far were consistent with a working hypothesis that reduced polymerase processivity increases the probability of g4 formation at the + 3.5 g4 motif through exposure of more single - stranded dna within the replisome , which , in turn , focuses replication stalling at this site . implicit in this model is the idea that the + 3.5 g4 can form during replication but that it is usually rapidly resolved to maintain fork progression . we therefore reasoned that trapping the g4 structure using a g4-binding ligand also might induce instability of bu-1a expression in otherwise wild - type cells further , we predicted that g4 ligands and hu would act synergistically to destabilize expression of the locus . to test these ideas , we treated cells with the g4 ligand n - methyl mesoporphyrin ix ( nmm ) ( nicoludis et al . , 2012 ) . we first identified the maximum dose at which the cells retained normal viability and global replication dynamics . at 2 m nmm , the fork rate , as assessed by molecular combing , was 1.13 kb / min compared with 1.26 kb / min in wild - type cells , with no significant change in the inter - origin distance ( figure s5 ) . nonetheless , fluctuation analysis for bu-1a loss in wild - type and bu-1 cells cultured for 7 days in 2 m nmm revealed instability in bu-1a expression in wild - type cells , but not cells lacking the + 3.5 g4 motif ( figure 4a ) . since 2 m nmm does not in itself significantly reduce global fork rates and the agent will only interact with the formed g4 structure , not with just the linear dna sequence ( ren and chaires , 1999 ) , this observation is consistent with transient formation of g4s during normal replication . we next asked whether combining nmm - induced g4 stabilization with hu - induced reduction in polymerase processivity led to a further destabilization of bu-1 expression . interestingly , use of both drugs together resulted in significant toxicity , meaning that we had to reduce the dose of each drug by 50% in order to carry out the fluctuation analysis . as expected , growth of cells in hu at 75 m or nmm at 1 m individually had little effect on stability of bu-1a expression ( figure 4b ) . however , the combination of nmm and hu at these doses resulted in a significant increase in bu-1a instability , revealing a marked synergy between hu - induced replication stress and g4 stabilization . we replaced the natural + 3.5 g4 motif with a series of four g4 motifs of varying in vitro thermal stabilities ( schiavone et al . , 2014 ) . all four motifs ( g4 14 ) potentiated the formation of bu-1a variants upon treatment with hu ( figure 5a ) . interestingly , we observed no correlation between the degree of potentiation by the motifs and the in vitro melting temperature of the equivalent oligonucleotides ( figure 5b ) . however , there was a significant trend toward greater potentiation of bu-1a loss being associated with longer non - g loops in the range of 1 to 9 bp ( figure 5c , solid line ) . to explore this further , we also tested a single repeat of the g4 motif containing human ceb1 mini - satellite ( piazza et al . , 2012 ) , which has 18 bp between its first three and last run of dgs . this g4 motif , but not a mutated form that is incapable of forming a g4 structure in vitro ( piazza et al . , 2012 ) , also potentiated bu-1a instability after treatment with hu . however , this was not to a greater extent than the natural + 3.5 g4 dna with its central 9-bp loop , suggesting that there may be a limit after which lengthening the loop has no further effect . finally , we asked whether we could detect genome - wide evidence of an interaction between hu and g4s . we therefore performed affymetrix expression microarray analysis on cells before and after culture in 150 m hu . three parallel cultures of dt40 were treated with 150 m hu for 7 days , or mock treated , and then recovered into normal medium for 7 days , after which rna was prepared for array hybridization . despite this relatively short treatment , a total of 2,937 of 12,920 unique genes exhibited a change in expression of > 0.25 log2 units with p < 0.05 , with an approximately equal number of genes being upregulated and downregulated ( figure 6a ) . we previously have observed a similarly large number of dysregulated genes in cells deficient in the 5-3 g4-unwinding helicase fancj and in double mutants for the 3-5 helicases wrn and blm ( sarkies et al . , further , we found a highly significant overlap in the identity of dysregulated genes in the two sets , the direction in which their expression changed , and the association of the dysregulated genes with g4 motifs ( sarkies et al . we anticipated that if transcriptional dysregulation by hu was linked with g4s that there might be a significant similarity in the gene set altered by hu and the sets altered by loss of fancj and wrn / blm . indeed , the overlap in the identities of the genes dysregulated in all three conditions was highly significant ( figure 6b ) , as were the pairwise correlations in the direction of the change in expression ( figure 6c ) . nearly 68% of the 6,061 genes within the venn diagram in figure 6b have a g4 motif within 1 kb upstream of the tss and the end of the body of the gene in comparison with 59% of the 6,859 genes in the remainder of the array ( p < 1 10 ) ( table s1 ) . to ascertain whether the overlaps in the identity of dysregulated genes reflected the perturbation of common pathways in the three datasets , we analyzed the functional annotation terms associated with the genes in each set and in the overlap sets using david ( https://david.ncifcrf.gov ; huang et al . while treatment with hu resulted in dysregulation of genes with gene ontology ( go ) terms associated with cellular stress and nucleotide metabolism , a large number of miscellaneous go terms also were enriched to a similar degree ( table s2 ) . significantly , despite the large number of genes overlapping in the three datasets , there was no evidence of their being members of common pathways ( figure s6 ) . this is consistent with much of the dysregulation of expression resulting from processes that are not related to a coordinated physiological response either to treatment with hu or ablation of fancj or wrn and blm helicases . nonetheless , the degree of overlap in the dysregulated transcriptomes in these three conditions suggest that cells treated with hu and cells lacking fancj and wrn / blm face similar challenges . however , the enrichment of g4s in affected genes , while statistically significant , is still relatively modest , suggesting that other factors , such as secondary effects or other dna secondary structures , may be contributing as well . numerous lines of evidence have linked replication stress with genetic instability ( halazonetis et al . , 2008 , zeman and cimprich , 2014 ) . imbalanced or depleted nucleotide pools during replication are an important cause of such stress and can arise from the expression of oncogenes uncoupling entry into s phase from upregulation of nucleotide supply ( bester et al . , 2011 ) . importantly , the dna damage resulting from replication stressors like hu or aphidicolin that act directly on the replicative dna polymerases is not randomly distributed across the genome , but is instead focused on sites that often have features that make them potentially problematic to replicate even under ideal conditions ( tsantoulis et al . , 2008 ) . many of these hotspots also correspond to classical fragile sites , in which chromosome breaks are observed after replication stress . such sites have been linked to regions depleted in replication origins , meaning that single forks have to traverse long distances ( letessier et al . , 2011 ) . thus , a combination of regions of low fork density and problematic structures may focus sites of fork collapse under conditions of global replication stress ( wickramasinghe et al . , 2015 ) . the mechanisms by which replication stress leads to epigenetic changes are less well explored . alterations in chromatin composition and structure are common features of cancer cells ( berdasco and esteller , 2010 , hansen et al . , 2011 , timp and feinberg , 2013 ) and are particularly associated with g4-dense breakpoint hotspots ( de and michor , 2011 ) . although the cell line we used in this study , dt40 , is itself transformed , we found no evidence of significant stress to the dna replication program under normal growth conditions . however , growth of the cells in low - dose hu recapitulated the key features of the acutely stressed replication observed in oncogene - expressing primary cells ( bester et al . we have been able to explore directly the interaction between global replication stress induced by nucleotide depletion and a dna secondary structure to demonstrate how they conspire to exacerbate replication - dependent epigenetic instability . we have provided evidence that two parallel epigenetic perturbations contribute to permanent and transient epigenetic changes following an episode of replication stress . the first mechanism relates to the uncoupling of the activity of replicative helicase and polymerase ( byun et al . , 2005 , pacek and walter , 2004 ) during hu treatment . this has been shown to lead to interruption of the normal flow of histones from the parental to the nascent daughter strands , with the histone chaperone asf1 buffering the displaced h3/h4 ( jasencakova et al . , 2010 ) . groth and colleagues suggested that their release from asf1 might lead to local alterations in epigenetic state of chromatin due to unscheduled incorporation of inappropriately marked histones ( jasencakova and groth , 2010 , jasencakova et al . , 2010 ) . ( 2013 ) invoked this model to explain an increase in heterochromatin formation in cells deficient in fancj , suggesting that failure to unwind lagging - strand template g4 structures in fancj - deficient cells led to unscheduled deposition of histones bearing marks that would lead to h3k9me3 and heterochromatin formation . however , this model does not adequately explain the bidirectional changes in gene expression changes seen either in fancj cells or in wild - type cells treated with hu ( sarkies et al . in contrast , the model we have developed previously , in which loss of processive replication at g4s leads to localized loss of parental histone mark recycling , could explain both derepression of loci , such as -globin ( sarkies et al . , 2010 ) , and loss of activation , as can be observed in the bu-1 locus ( sarkies et al . however , since the mechanism by which h3k4me3 is maintained during replication is poorly understood , the precise mechanisms by which replication impediments disturb the maintenance of this mark remain to be fully elucidated this mark is induced alongside the loss of h3k4me3 when the + 3.5 g4 motif is present and cells are exposed to hu . importantly , the appearance of this mark of heterochromatin is accompanied by h2ax phosphorylation , a marker of dna damage ( rogakou et al . , 1998 ) . g4 motifs have been linked to hotspots of genetic instability and translocation ( de and michor , 2011 ) . further , the g4 ligand pyridostatin , which acts similarly to nmm , leads to localized h2ax accumulation at g4 motifs across the genome , suggesting the formation of dna breaks ( rodriguez et al . , dna breaks have been shown to induce transcriptional repression ( shanbhag et al . , 2010 ) and to induce h3k9me3 even in a normally euchromatic locus ( ayrapetov et al . , 2014 ) . thus , the appearance of h2ax and h3k9me3 in the bu-1 locus only in hu - treated cells containing the + 3.5 g4 is consistent with collapse or incision of replication forks , already stressed by nucleotide depletion , that have stalled at the g4 . we therefore propose that nucleotide depletion can give rise to loss of parental h3k4me3 and the appearance of h3k9me3 by distinct mechanisms . h3k4me3 is lost stochastically as a result of interruption of parental histone recycling , a mechanism that is locally exacerbated by the presence of a g4 motif . in contrast , we propose that h3k9me3 may reflect protective transient heterochromatinization of the locus during repair of breaks resulting from hu - induced fork collapse at g4 structures ( figure 7 ) . in the case of loci like bu-1 , in which hu - induced epigenetic instability is linked to g4 formation , an interesting question is whether hu results in a greater opportunity for g4 formation during replication or diminished g4 resolution . it is not currently possible to formally distinguish these possibilities and indeed it is likely that elements of both are true . notably , the observation that the g4-binding ligand nmm can induce bu-1a expression instability at a dose that does not significantly impact on global replication dynamics provides strong evidence that g4 structures form readily during normal replication and that they are usually promptly resolved . the synergy between replication stress caused by nucleotide depletion and structured dna is of considerable potential importance to understanding the development of cancer . epigenetic changes are prevalent in many cancer types , although their origin is unclear , and likely complex ( berdasco and esteller , 2010 , timp and feinberg , 2013 ) . recently , several instances of epigenetic instability in cancer have been linked to mutations in histone or dna - modifying enzymes . however , the widespread and often apparently random nature of epigenetic changes in tumors suggests that other processes also may be at work . we have suggested previously that delayed replication of g quadruplex structures could contribute to the epigenetic diversity of cancer ( sarkies and sale , 2012 ) . however , mutations in enzymes that may cause this form of epigenetic instability , for example rev1 , fancj , wrn , and blm ( sarkies et al . , 2010 , sarkies et al . , 2012 ) , are rarely observed in sporadic cancers . replication stress , on the other hand , is emerging as an important feature of cancer cells , particularly in the early stages of their evolution ( bester et al . , 2011 , di micco et al . , 2006 , thus , we suggest that some of the epigenetic changes seen in tumors may be explained by problems managing replication blocks . consistent with this idea , both copy number variations and changes in dna methylation patterns in cancer have been linked to g4 motifs ( de and michor , 2011 ) . finally , it is worth noting that hu is used extensively in treatment of hemoglobinopathies , such as sickle cell disease and thalassaemia , as it can re - induce expression of the fetal -globin gene , ameliorating the effects of the defective adult globins found in these disorders ( platt et al . , 1984 ) . importantly , the effect of hu on -globin expression is unlikely to be specific , since chronic exposure to the drug leads to quite widespread changes in erythroid gene expression ( flanagan et al . , 2012 ) . although the -globin locus in humans has no g4 motifs in the immediate vicinity of its promoter , its key transcriptional regulator , bcl11a ( bauer et al . , 2013 ) , has a high density of g4 motifs on both sides of its tss . it will , therefore , be interesting to explore whether the mechanisms we propose here could help explain the action of hu on fetal globin expression . dt40 cells culture and the strategy for removing and replacing the + 3.5 g4 motif in the bu-1 locus have been described previously ( schiavone et al . , 2014 ) . genetic manipulation of the + 3.5 g4 motif was performed in the bu-1a allele of cells in which the motif had been removed from the bu-1b allele to avoid the transvection - like effect between the alleles ( schiavone et al . , 2014 ) . oligonucleotides are listed in table s3 . for fluctuation analysis , 150 m hu ( sigma - aldrich , h8627 ) after 7 days , cells at a concentration between 0.2 and 1 10 were stained for 20 min at 37c with anti - bu-1a - phycoerythrin ( 1:100 , santa cruz biotechnology clone 5k98 , 70447 ) . bu-1a expression was assessed by flow cytometry using an lsrii cytometer ( becton dickinson ) . experiments with aphidicolin ( sigma - aldrich , a0781 ) and nmm ( frontier scientific , nmm580 ) were conducted in 96-well plates starting with ten cells expanded for 10 days . bu-1a cells were isolated after hu treatment using a moflo sorting cytometer ( dako - cytomation ) . chip was performed as described previously ( nelson et al . , 2006 ) with modifications . following a 10-min incubation at room temperature with 1% ( v / v ) formaldehyde , glycine was added to 0.2 m for 5 min . the extracted nuclei were sonicated at 4c using a bioruptor water bath sonicator ( diagenode ) with 30 cycles of 30 s separated by 30-s intervals . sheared chromatin samples were resuspended in dilution buffer ( 1.1% triton x-100 , 1.2 mm edta , 16.7 mm tris [ ph 8.0 ] , 167 mm nacl supplemented with pmsf , and a protease inhibitor cocktail ) . for immunoprecipitation , lysates were incubated overnight with the following antibodies at 4c : histone h3 ( 1:100 , cell signaling technology , 2650 ) , h3k4me3 ( 1:100 , cell signaling technology , 9727 ) , h3k9/14ac ( 1:200 , millipore , 17 - 615 ) , h3k9me3 ( 1:200 , abcam , ab8898 ) , h2ax ( 1:50 , abcam , ab2893 ) , and the negative control normal rabbit igg ( millipore ) . following overnight incubation at 4c with tumbling and four washing steps , the qpcr was performed with power sybr green master mix ( applied biosystems , 4367659 ) on an abi prism real - time cycler with the following cycle times : 50c for 2 min , 90c for 10 min , 45 cycles of 90c for 15 s plus 60c for 1 min . the cdna was made from 5 mg mrna with super rt ( ht biotechnology ) and oligodt primer in a final volume of 40 l . dna molecular combing was conducted 3 days into culture with 150 m hu . it was performed and analyzed as previously described ( guilbaud et al . , 2011 ) . rna was extracted from three independent wild - type cell populations treated for 7 days with hu and allowed to recover for another 7 days , as well as from three untreated parallel controls . microarray analysis was performed using r ( http://www.r-project.org/ ) and its bioconductor packages ( gentleman et al . , 2004 ) . raw cel files were processed using the robust multichip average ( rma ) algorithm available in the affy package ( gautier et al . , 2004 ) . genes that showed a change of > 0.25 log2 units relative to the mean wild - type intensity , with a p value of < 0.05 ( t test ) , were identified as exhibiting statistically significant transcriptional dysregulation . custom written r scripts were used to identify and plot genes co - dysregulated between different mutants . venn diagrams were generated with the limma package ( smyth , 2004 ) , and significance for the overlaps was calculated using fisher s hypergeometric distribution .

summarynucleotide pool imbalance has been proposed to drive genetic instability in cancer . here , we show that slowing replication forks by depleting nucleotide pools with hydroxyurea ( hu ) can also give rise to both transient and permanent epigenetic instability of a reporter locus , bu-1 , in dt40 cells . hu induces stochastic formation of bu-1low variants in dividing cells , which have lost the h3k4me3 present in untreated cells . this instability is potentiated by an intragenic g quadruplex , which also promotes local h2ax phosphorylation and transient heterochromatinization . genome - wide , gene expression changes induced by hu significantly overlap with those resulting from loss of the g4-helicases fancj , wrn , and blm . thus , the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by g quadruplex formation to induce epigenetic instability and changes in gene expression , a mechanism that may contribute to selectable transcriptional changes in cancer .

gossypiboma refers to a mass resulting from retained cotton , gauze , or sponge accidentally left within the body after surgery . as indicated in its name , gossypiboma is an iatrogenic outcome reported on rare occasions in various parts of the body with diverse clinical findings , often mistaken radiologically as a tumor or abscess . now , we report a literature review and our experience of a case with incidentally found mass , who was first judged as a gastrointestinal stromal tumor ( gist ) of the gastric fundus by abdominal computed tomography ( ct ) and endoscopic ultrasonography ( eus ) , but was later confirmed as gossypiboma resulting from retained surgical sponge . a 78-year - old female visited the hospital for sudden onset epigastric pain which lasted for one day . she received abdominal us at a private clinic indicating multiple gallbladder ( gb ) stones and common bile duct ( cbd ) stones and was transferred to the emergency room of this hospital . she had a history of partial gastrectomy 30 years ago in another hospital for a reason she can not remember precisely . the physical examination revealed blood pressure 110/70 mm hg , pulse rate 76/min , respiration rate 20/min , and body temperature 36.6 without unusual finding except for the epigastric tenderness . peripheral blood test showed hemoglobin 9.2 g / dl , white blood cell ( wbc ) 9,600/mm , and platelet 428,000/mm . iu / l , alt 95 iu / l , total bilirubin 1.5 mg / dl , alkaline phosphatase 404 iu / l , -gtp 58 iu / l , amylase 66 u / l , and lipase 26 u / l . the abdominal ct performed at the emergency room indicated mild dilatation of intrahepatic bile duct and cbd with multiple gb stones , but the finding of cbd stone was not definite . in addition , a 5.5-cm sized well - demarcated round mass with irregular calcification density was found adjacent to the gastric fundus ( fig . 1 ) . esophagogastroduodenoscopy ( egd ) was performed to search the cause of the abdominal pain , where 5-cm sized bulging area covered with normal mucosa was found at the gastric fundus but not any other lesions to cause the abdominal pain ( fig . cbd stone was not detectable on eus performed to exclude the presence of cbd stone . eus also showed an exophytic hypoechoic mass which was connected with the fourth layer of the gastric wall at the area where subepithelial tumor was suspected . it appeared , from the operative field , that the mass was a foreign body covered by the inflammatory membrane rather than a gist of the gastric fundus . the mass was tightly adhered to the gastric fundus , making it difficult to dissect from the gastric wall . the resected mass was 6.54 cm in size and when the mass was cut in half , a surgical sponge was visible with the naked eyes ( fig . the covering material was confirmed as inflammatory pseudomembrane accompanying fibrosis , abnormal calcification , hemorrhage , and necrosis . gossypiboma , also called ' textiloma , ' is a compound of 2 words " gosysspium " meaning ' cotton ' in latin and " boma " meaning ' place of concealment ' in swahili , referring to a mass resulted from the retained cotton or gauze after the operation.1 the occasion of retaining surgical instruments inside the human body is rare but there have been always the possibility from the beginning of the history of surgery . every surgery with incisional wound has the potential to leave a foreign body inside the human body , such as gauze , clamp , retractor , or electrode . surgical sponge , among others , is most frequently left in the human body after surgery , as much as to be named separately as gossypiboma or textiloma . gossypiboma within the abdominal cavity in most patients are asymptomatic and often leads to a medical - malpractice claim only by incidental discovery several years later , further complicating the recognition of precise incidence . the incidence of gossypiboma in south korea is unknown but reported about 1/1,000 - 10,000 surgeries worldwide.2 gossypiboma complication might occur in every form of surgery , ranging from general surgery ( 52% ) to gynecology ( 22% ) , to urology and vascular surgery ( 10% ) , and to ophthalmology and spinal surgery ( 6%).2 a case - control study by gawande et al.3 found emergency operation , unexpected change in operation , and high body mass index of the patient were the risk factors of leaving a foreign material inside the human body after the operation . cotton surgical sponge does not cause any specific biochemical reaction except for the adhesion or granuloma formation inside the body.4 the clinical manifestation of gossypiboma depends on the type of reaction , the location of gauze or sponge , and the degree of chronicity , but could be separated into 2 groups.5 the first group is the exudative inflammatory reaction which could be detected early after the surgery by symptoms caused by the abscess formation , or could be detected later with intestinal obstruction , erosion into the adjacent organs , and fistula formation . the retained gauze inside the abdominal or pelvic cavity could erode into the gastrointestinal tract causing extracorporeal extrusion on rare occasions . the second group is the aseptic fibrinous reaction where the encapsulated sponge does not induce symptoms and would not be detected for a long time . our patient belongs to the latter , since she did not have any symptom for as long as 30 years , and the gauze was encapsulated and adhered to the gastric fundus . there are various ct findings but a cystic lesion with marked rim enhancement is most common.6 an investigator termed the calcium deposition around the rim of the retained surgical sponge appearing in the form of calcified reticulate rind as " calcified reticulate rind sign " and reported it as the characteristic ct finding of gossypiboma . hypoechoic mass with strong posterior acoustic shadowing due to hyperechoic capsule suggests gossypiboma on the us.6 in this patient the mass showed irregular calcification at the center area of the mass . every gauzes recently used for surgery contains radiopaque markers for easier detection by postoperative radiography . gauzes in which these markers are not contained or degraded , or calcified may not reveal the characteristic radiopaque line on the radiography.7 there is no specific eus finding for gossypiboma . eus could be performed for detailed observation when a gastrointestinal subepithelial lesion is suspected . in the patient of this study , it was considered that the 5 cm sized protruded area covered by normal mucosa on egd was the same mass discovered on ct appearing to originate from the stomach . eus was performed for more detailed observation , revealing a hypoechoic mass originating from the fourth gastric wall layer . it is presumed that the inflammatory mass encapsulating the gauze was adhered to the serosal layer due to the fibrosis , and appeared as hypoechoic on eus . subepithelial lesion on eus is diagnosed putatively based on the layer of origination and echogenic findings of the mass . when the pathologic diagnosis and eus impression of subepithelial lesions was compared in the prior studies , the diagnostic accuracy of eus was estimated about 78%.8 the factors contributing to the incorrect eus interpretation include inflammatory change , fibrosis , or tumor microinvasion around the tumor.9 the typical eus finding of gist is hypoechoic tumor originating from the fourth ( proper muscle ) layer , occasionally in multilobulated or pedunculated forms . findings of necrosis , calcification , or ulceration of gist suggests the possibility of malignant transformation.10 our case did not show spongiform air bubbles with marked rim enhancement and cystic lesion on ct as typically found in gossypiboma patients . but the round well - demarcated mass originated from the stomach accompanying radiopaque material was noted and mistaken as a mass with irregular calcified density . this lesion was also found on eus as an exophytic hypoechoic mass connected with the fourth layer of the gastric wall and internal irregular hyperechoic spots with posterior acoustic shadowing . but the resected lesion was found to be a mass resulted from the retained surgical sponge .

gossypiboma refers to a mass resulting from a retained gauze pad accidentally left within the body after surgery . although the clinical features are diverse , it is often found incidentally as a mass having an internal cystic change and adhesion to adjacent organs . abdominal computed tomography ( ct ) is helpful , yet the initial diagnosis can be misleading in cases with atypical findings . we report a case of gossypiboma in a 78-year - old woman that we suspected was a gastrointestinal stromal tumor according to abdominal ct and endoscopic ultrasound , yet was diagnosed as a gossypiboma postoperatively .

while advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species ( lockwood et al . 2005 ) , it is still unclear how introduced populations can successfully establish . as elton ( 1958 ) pointed out , for every successful invasion that occurs , there are enormously more invasions that never happen , or fail quite soon or even after a good many years ( page 109 ) . this modern biological paradox can not readily be reconciled , especially in the characteristic case where the founder population is small , as such populations are definitely in a precarious position ( mayr 1965 ; page 42 ) . introductions of populations at low density and/or small size are often faced with inverse density dependent effects , attributed to demographic stochasticity or reduced cooperative interactions ( courchamp et al . allee ( 1931 ) first proposed that under these conditions , populations may suffer a decrease in the per - capita rate of increase , from here on referred to as the allee effect . upon arrival in a novel environment , individuals need to overcome a series of challenges in order to reduce the population 's risk of extinction . the time period in which this occurs is generally considered the initial establishment phase , and is thought to be a common feature and general pattern of invasion and the process of growth and expansion ( shigesada and kawasaki 1997 ; sakai et al . the occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena ( sakai et al . small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve , leading to the perception of a time lag . where this time lag is more pronounced , populations may be recovering from inverse density dependent effects ( i.e. , allee effects ) . individuals may suffer a reduction in fitness at low densities for many reasons ( reviewed by courchamp et al . even when the initial population is large , rapid dispersal required for expansion could be suicidal as the population density decreases , thereby enhancing inverse density dependent effects ( lewis and kareiva 1993 ; drake et al . component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness , respectively ( stephens et al . 1999 ; gascoigne et al . it is often difficult to decipher the exact mechanism that manifests allee effects ( and it is not always the case that component allee effects lead to demographic allee effects ) . nevertheless , we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . essentially , it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted , and these impacts on individual fitness combine to produce an overall decrease in abundance ( i.e. , demographic allee effects ) . as allee effects impact individual fitness , the underlying traits that influence these effects ( i.e. , component allee effects ) may be subject to natural selection ( courchamp et al . 2008 ) . whereas propagule pressure is an emerging explanation for the establishment of invasive species ( lockwood et al . 2005 ) , it relies on an obvious relationship between the number and size of introduction events and the probability of success , as safety in numbers helps combat allee effects and stochastic extinction . in the event that propagule size is not large enough to overcome inverse density dependent effects , a population traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues ( pheromones and vocal / visual signals ) , dispersal / aggregation behavior , habitat preferences , mating synchronicity , and gamete morphology and performance ( see courchamp et al . direct evidence for the evolution of these traits as functional adaptations to allee effects is limited , but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection ( courchamp et al . 2008 ; gascoigne et al . an evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution . according to neutral quantitative genetic theory , a loss of genetic variation is expected from population bottlenecks and founder effects ( nei et al . however it is not neutral variation that matters , but rather evolvability depends on the variation relevant to selection . maintenance or even increases of this ( additive ) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations ( bryant et al . 1986 ; goodnight 1988 ; willis and orr 1993 ; cheverud and routman 1996 ; briggs and goldman 2006 ; turelli and barton 2006 ) . additionally , many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders ( lee 2002 ; allendorf and lundquist 2003 ; roman and darling 2007 and references therein ) , and that evolution can occur on contemporary timescales ( reznick and ghalambor 2001 ; carroll et al . 2007 ; kinnison and hairston 2007 ) . our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish , given any amount of genetic variation . in this paper , we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs . we model the growth and spread of this population according to a reaction - diffusion equation , and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise ( under the current , ecological paradigm ) the population would go extinct . the deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth ( i.e. , an allee effect ) and diffusive dispersal . this type of demographic model has been used as a compact and tractable representation of invasion ( e.g. , skellam 1951 ; lewis and kareiva 1993 ) . specifically , it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge ( drake et al . 2005 ; drury et al . 2007 ) . using this approach , drake et al . ( 2005 ) report acceptable volumes of discharge for various organisms ( with differing reproductive rates ) for a range of invasion risk tolerances . here , we consider populations that are introduced below the invasion risk threshold , but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system . the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space . this equation models the growth of the population ( the first term on the right hand side of eqn . 1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn . 1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig . growth dynamics of the model population ( a ) , and the solution of eqn . ( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 . trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 . there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn . the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al . 1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) . figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a ) there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth , there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) . the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 ) . this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) . this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity . in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) . this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch . this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth . thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand . we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model . the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) . the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success , this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival . specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time . this quantitative trait influences an organism 's ability to survive and reproduce in a small population . the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . therefore , it is important to investigate the possibility of evolution in the analysis of invasions . the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn . ( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) . thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) . this suggests that the genetic variance ( ) is small ( and constant in this model ) . this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection , quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn . the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities . the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal . we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time , the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient . the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system . the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space . this equation models the growth of the population ( the first term on the right hand side of eqn . 1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn . 1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig . growth dynamics of the model population ( a ) , and the solution of eqn . ( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 . trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 . there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn . the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al . 1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) . figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a ) there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth , there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) . the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 ) . this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) . this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity . in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) . this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch . this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth . thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand . we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model . the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) . the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success , we develop a quantitative genetic subsystem . this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival . specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time . this quantitative trait influences an organism 's ability to survive and reproduce in a small population . the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . therefore , it is important to investigate the possibility of evolution in the analysis of invasions . the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn . ( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) . thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) . this suggests that the genetic variance ( ) is small ( and constant in this model ) . this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection , quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn . the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities . the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal . we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time , the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient . the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system . the dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales ( kondrashov and khibnik 1996 ; webb 2003 ) . earlier , we assumed that the genetic variation ( ) was small ( to use mean fitness as a proxy for individual fitness ) , which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . when = 0 , the situation without evolution , the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem ( carrying capacity or extinction ) depending on its initial density ( greater than or less than the allee threshold respectively ; fig . when > 0 but small , the allee threshold evolves relatively slowly and influences the ecological system . whenever the population is below its carrying capacity ( u = 1 for each spatial coordinate x when space is explicit ) , eqn . ( 2 ) is negative , and decreases the mean allee threshold ( a ) , as the intensity of selection is density dependent . thus , fitness increases as allee effects are suppressed , and selection drives the allee threshold towards zero . if the population density is greater than the allee threshold , but still below the carrying capacity , it will progress towards carrying capacity more rapidly than it would without evolution as a decreases ; as the rate at which the population density changes ( eqn . 1 ) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction , but more slowly than it does without evolution . when u < a , eqn . ( 1 ) is negative , and the population density approaches extinction more rapidly with a constant ( as the difference between u and a increases ) , than it does with evolution as a decreases ( revealing a more pronounced time lag to extinction ) . during this time lag , as the population slowly declines , the opportunity for evolution to overcome inverse density dependent effects occurs . if the rate of evolution is fast enough , the allee threshold can fall below the population density , causing the rate of change of population density to become positive ( where u > a ) and the population grows and can successfully invade . the chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait ( i.e. , allee threshold ) and of population decline ( gomulkiewicz and holt 1995 ) ; hence the amount of genetic variance greatly impacts the ability to adapt and survive . a nonspatial example of this process , referred to as evolutionary rescue ( gomulkiewicz and holt 1995 ) , is shown in fig . 2 , where a population is introduced below the allee threshold . without evolution , the population declines to extinction ( fig . 2a , solid line ) as the allee threshold remains constant ( fig . 2b , solid line ) . when the population can evolve ( fig . 2 , dotted line ) , it declines at first until it can overcome the magnitude of inverse density dependence , and is then able to successfully establish . as it is difficult to measure the allee effect empirically ( tobin et al . 2007 ) , we use an extreme value that exaggerates density dependent effects in order to investigate the worst case scenario ( a = 0.3 , where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity ) . when evolution is included , we used a small value for the genetic variance , = 0.02 , in order to remain consistent with fast - slow dynamics , unless otherwise indicated . comparison of an invading population introduced at a density below the allee threshold , a = 0.3 ( u = 0.25 , r = 0.6 ) . the solid line represents the nonspatial system ( d = 0 ) described by eqns ( 1 ) and ( 2 ) without evolution ( = 0 ) which results in extinction ( a ) and a constant allee threshold ( b ) . the dotted line indicates population growth ( a ) when evolution ( = 0.02 ) acts to reduce the allee threshold ( b ) . in general , there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution ( instead of simple decline to extinction ) . as genetic variance increases , we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly . 3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . in this case , the rate of reproduction , r , also influences the potential for evolutionary rescue , as it impacts both population growth and rate of evolution ( eqns 1 and 2 , respectively ) . as we relax the assumption of fast - slow timescales , the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption . parameter combinations of reproductive rate : r ; genetic variance : , and initial population density : u , that result in extinction or evolutionary rescue . in this nonspatial scenario , initial population densities greater than the allee threshold ( a = 0.3 ) always succeed , thus the focus is on the parameter space that allows for evolutionary rescue ( i.e. , where the population growth changes from negative to positive ) . as the reproductive rate increases from 0.1 to 1 , there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects . nonetheless , the additional complexities result in qualitatively similar behavior to the nonspatial model . in this case , not only will evolution influence population growth , it affects the wave speed and the critical size threshold , rmin . as the population overcomes allee effects with a decreasing allee threshold , the wave speed accelerates and the critical patch size becomes smaller . thus , in addition to the initial density of the introduced population and the genetic variance , the initial radius or patch size of the initial invasion area , the ratio of diffusion to reproduction , and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system . ( 1 ) ( without the evolutionary subsystem ) in one - dimensional space , with an initial population density below the allee threshold , declines to extinction ( fig . this is contrasted by the results when the evolutionary subsystem is included . with the initial population density below the allee threshold , fig . the same type of rescue occurs for a population that starts near carrying capacity , but occupies an initial spatial size below that which is necessary for a population to successfully establish . figure 5a shows a rapidly declining population that goes extinct . under the same circumstance , but where evolution of the allee threshold occurs , fig . 5b shows the population density at first beginning to shrink and then growing and expanding . in addition to the time evolution of population density across space in figs 4 and 5 , the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution ( figs 4c and 5c ) . as the intensity of selection is density dependent ( and we assume constant genetic variance ) , locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection . the trait distribution over time , figs 4c and 5c , therefore reflect the population density distribution , but are also influenced by the trait values of the migrants . as individuals disperse out to new locations and push the boundaries of the species range , their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . this demographic allee threshold combines with their local population density to influence individual fitness and population growth ( where the distance between the density and mean trait value is the initial degree of maladaptation ) . diffusive dispersal of an introduced population at an initial density ( bold dashed line ) below the initial allee threshold , a = 0.3 ( u = 0.25 , r = 1 , d = 0.1 ) across a linear , one dimensional habitat . the population collapses over time to extinction ( a ) where there is no evolution ( = 0 ) , and succeeds ( b ) after an initial decline with evolution ( = 0.02 ) . ( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) . population density of a diffusion dispersed population across one dimensional space . the initial population density ( bold dashed line ) is near carrying capacity ( u = 0.95 , a = 0.3 , r = 1 , d = 0.5 ) , but introduced below the minimum radius of area determined to be critical for invasion success . ( a ) is collapsing to extinction without evolution ( = 0 ) , whereas ( b ) shows success of an invader with evolution ( = 0.02 ) after initial decline . ( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) . we explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . according to drake et al . ( 2005 ) , variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient , d. we therefore explored a range of values , and present those that best illustrate breadth of behavior . the parameter that controls the reproductive rate , r , was also varied substantially , but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction ( resulting in a measure of length ) ; we fixed r and varied d , unless otherwise noted . this was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios . the effects of the critical patch size , initial population density , ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig . the ratio of the diffusion coefficient ( d ) to the reproductive rate ( r ) determines whether the population will expand or collapse according to the initial radius of the introduced population . the areas under the curves denote combinations of genetic variance and initial population density that result in extinction . areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence . the parameter space between the vertical dashed lines refers to the different ways population survival is influenced . to the left of the initial allee threshold , the initial population density will either go extinct due to density dependent effects ( below the d / r curve ) , or given enough genetic variation , will be evolutionarily rescued ( above the d / r curve ) . the area to the right of the initial allee threshold [ and between the dashed lines in ( b ) and ( c ) ] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction , the population may go extinct without sufficient genetic variance ( below the d / r curve ) , otherwise it will evolve to overcome the critical patch size effect . for initial population densities thus , the area between the dashed lines in ( b ) and ( c ) truly delineates evolutionary rescue when d / r = 1 . the rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r . graphs ( a ) , ( b ) , and ( c ) represent different radii of the linear habitat that the introduced population initially occupies . when the size of the initial population is too small ( i.e. , a radius of 1 ) , a population at carrying capacity ( i.e. , u = 1 ) will go extinct without evolution due to the relative effect of diffusion to reproduction ( fig . if evolution occurs rapidly enough ( i.e. , > 0.02 ) , the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . when the initial radius of the population is increased ( fig . 6b , c ) , the chance of survival and establishment ( growth and expansion ) of populations above or below the allee threshold increases with initial density and genetic variance . therefore , the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance . this is demonstrated further in fig . 7a , where the rate of recovery ( i.e. , the inverse of the time lag before growth becomes positive and the population reaches carrying capacity ) for a population near carrying capacity depends on its initial size / radius and genetic variance . where size and variance are small , rescue never occurs . as these parameters increase , the rate of recovery gradually becomes faster until it essentially plateaus ( although with greater variance and initial radius , the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery ) . if the population occupies a large enough spatial extent , it will succeed without evolution ( where the genetic variance is zero ) , however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread ( e.g. , if spread is relatively fast compared to reproduction , d / r = 1 ) . the population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion , and the population can grow to carrying capacity . similar to the nonspatial case , a population ( greater than the allee threshold ) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without . rate of recovery in terms of the inverse of the time lag before population growth becomes positive , where one timestep equals 24 iterations of the model . in ( a ) , the initial population density is near carrying capacity ( u = 0.95 , a = 0.3 , d / r = 1 ) , and the initial radius and genetic variance , , varies . where the rate of recovery is zero , the population goes extinct as it initially occupies an area smaller than the critical patch size ( in this case , a radius of 1.4 ) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth ( in this case , for initial radii 3.8 and 0.036 ; for initial radii > 2.7 , the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery ) . when the initial population density varies ( indicating the initial degree of maladaptation where a = 0.3 ) , ( b ) shows the rate of recovery with the initial radius fixed ( as in fig . 5a where the radius = 1 and d / r = 1 ) . in this case , extinction will occur without evolution not only for an initial density below the initial allee threshold , but for any density as the initial radius is below the critical patch size . hence , a nonzero rate implies evolutionary rescue and a zero rate means extinction . as shown in fig . 7b , when evolutionary rescue is possible , the initial level of maladaptation ( a0u0 ) and the genetic variance ( ) also determine the rate at which evolutionary rescue proceeds . figure 7b uses parameters ( i.e. , radius and ratio of d to r ) for a population that would decline and go extinct without evolution regardless of the initial density . hence , it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation ( to both the critical density and spatial thresholds ) and/or genetic variance . as the rate at which this rescue occurs depends on the amount of genetic variance ( eqn . 2 ) , it may take an extremely long time ( as 0 , the rate of recovery 0 ) for the allee threshold to fall below the population density . in this circumstance , as the population density becomes very close to zero , the rate of change of the allee threshold is greater than that of the population density ( as u 0 , u/t 0 and a/t -2ra ) . thus , theoretically , rescue would always occur ( gomulkiewicz and holt 1995 ) . however , to maintain biological realism , when solving this system numerically , we always considered the population extinct when the maximum density ( across space , when diffusion is included ) becomes reasonably close to zero ( i.e. , u = 0.0001 ; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the pushed wave front behavior ) . overall , the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space . results for two - dimensional space are not shown as they are qualitatively similar to the simpler , one dimensional model . from these results , it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity . current management strategies ( e.g. , reducing population density or size ) are based on ecological theory ( e.g. drake et al . 2005 ) , but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . under the assumptions of an allee effect and diffusive dispersal , the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment ( lockwood et al . however , by incorporating evolution , we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . as the ratio of diffusion to reproduction decreases , the spatial constraint on population growth becomes weaker , and less genetic variance is needed to rescue populations with densities below the allee threshold . as the initial spatial radius of introduction increases , furthermore , the rate of this rescue depends on the initial genetic load or maladaptation ( i.e. , how far the population density is from the allee threshold ) , as well as the amount of genetic variance . because bottlenecks during founding events do not always result in highly reduced genetic variability , even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish , contrary to solely ecologically based predictions . additionally , we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context . as species are transported from their native environment into novel habitats or simply disperse on their own , it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . given enough genetic diversity , local populations can adapt to their local environment , but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . essentially , local population persistence depends on the race between the rate of evolution and the degree of maladaptation ( gomulkiewicz and holt 1995 ) . in this case , gene flow will play a major role in determining the outcome . as kirkpatrick and barton ( 1997 ) and garca - ramos and kirkpatrick ( 1997 ) demonstrate , individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . on the other hand , holt et al . ( 2003 , 2004 ) show that immigration can have a positive influence on adaptation to sink environments , in some circumstances . resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population 's ability to overcome it . in our model , dispersal impacts survival ecologically through the critical patch size , and genetically , as individuals may move from areas where they are well adapted ( i.e. , the population density is greater than the allee threshold or mean trait value ) to sink regions , where they are maladapted . as individuals disperse across space , they may be contributing positively in an ecological sense to the quality of their new local environment ( by increasing the local population density ) . however , dispersers are more likely to come from higher density areas where allee effects , and hence selection , are locally weak . these dispersers potentially introduce more maladaptation to their new location , because they increase the average phenotype ( allee threshold ) in the new location where density is likely to be lower . interestingly , the evolutionary impacts of migration in this model do not dramatically influence the dynamics . changes to the local mean phenotype through local selection and simple mixing ( i.e. , diffusion ) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection . as the selection intensity is density dependent and proportional to u 1 for each point in space , the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations . the gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities . however , this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term , as the negative effects of gene flow and the local rate of evolution ( which is relatively fast , based on the selection intensity ) essentially cancel each other out . in this context , similar to that of holt et al . ( 2003 , 2004 ) , the immigrants simply contribute to the local population density , which helps prevent extinction long enough for evolutionary rescue to occur locally ( i.e. , positive population growth ; note that whereas holt et al . ( 2003 ) attribute the main effect of immigration to the contribution of variation , this is not the case in our model , as we assume constant genetic variance ) . ( 2004 ) where immigration has a demographic effect on increasing fitness that can essentially outweigh the the primary determinant of invasion success depends on positive population growth at the center of the introduced range . this result comes from the allee effect [ and the solution to the pde ( 1 ) ] by forcing a pushed travelling wave front ( lewis and kareiva 1993 ) , where the wave speed causes population expansion , contraction , or propagation failure ( i.e. , pinning ; keitt et al . 2001 ) . intuitively , aggregation - like behavior emerges based on the strength of the allee effects . individuals that disperse too far from the whole are likely to die before they can pull others in their vicinity . in this regard , growth occurs from the inside out , where the population seemingly spills out and overflows to expand its range . consequently , in this study , and for biological invasions that exhibit similar dynamics , it is more important to focus on the center of the invader 's range and whether the initial beachhead can survive ( through evolutionary rescue ) , than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success . the overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur . even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population , additional invasion criteria need to be considered . when analyzing the model behavior in a spatially explicit context , there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . as previously mentioned , reproduction needs to compensate for the loss due to diffusion . including evolution and suppressing allee effects , actually contributes to the acceleration of the wave front ( i.e. , enhancing dispersal speed ) . a population then can more rapidly disperse as it evolves , and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth , the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area ( rmin ) . although there is no range contraction ( as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations ; filin et al . 2008 ) , as the critical patch size ( rmin ) becomes smaller with the decreasing allee threshold , rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model . this may seem like an oversimplification of the global dynamics ; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted , influencing the ways in which invasion biologists conduct their research ( see the other articles in this issue ) . specifically , adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size ( gascoigne et al . as it is difficult to conclusively support this claim empirically ( as the origin of the adaptation or the associated cost may be unknown ; courchamp et al . 2008 ; gascoigne et al . 2009 ) , mathematical models that incorporate evolution and compare the effects of various strategies ( e.g. , mitigating component allee effects ) can help decipher the mechanisms that both limit and facilitate population growth . two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone ( jonsson et al . another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation ( at low density ) bear a cost of decreased fitness at high density due to hybridization and competition ( levitan 2002 ) . in these cases , particular strategies are shown to influence population viability in addition to an associated tradeoff , whereas our investigation provides broad , albeit simplistic , results dealing with generalized demographic allee effects and evolution . in order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios , spatially explicit , individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge . in particular , tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion . however , this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species . this is due to the simplifying assumption of constant genetic variance based on mutation - selection balance ( lande 1976 ) . complex simulations could relax this assumption and permit genetic variation to change via mutation , selection , and drift , in tandem with the demographic processes in a heterogeneous environment , and explicitly investigate the costs associated with avoiding allee effects . hence , future models should incorporate how propagule pressure ( size and frequency of introduction events ) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species . although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations , this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions .

the mechanisms that facilitate success of an invasive species include both ecological and evolutionary processes . investigating the evolutionary dynamics of founder populations can enhance our understanding of patterns of invasiveness and provide insight into management strategies for controlling further establishment of introduced populations . our aim is to analyze the evolutionary consequences of ecological processes ( i.e. , propagule pressure and threshold density effects ) that impact successful colonization . we address our questions using a spatially - explicit modeling approach that incorporates dispersal , density dependent population growth , and selection . our results show that adaptive evolution may occur in small or sparse populations , providing a means of mitigating or avoiding inverse density dependent effects ( i.e. , allee effects ) . the rate at which this adaptation occurs is proportional to the amount of genetic variance and is a crucial component in assessing whether natural selection can rescue a population from extinction . we provide theoretical evidence for the importance of recognizing evolution in predicting and explaining successful biological invasions .

stroke is the second leading cause of death worldwide , and the leading cause of neurological disability in adults in developed countries.1 2 recognised risk factors include high blood pressure , heart disease , diabetes , obesity , smoking , alcohol and physical inactivity , but further environmental factors are likely to be relevant to stroke risk . exposure to psychosocial stress has been identified by several recently conducted studies as a possible stroke risk.35 psychosocial stress , particularly with chronic exposure , is a feasible risk for stroke through its influence on the hypothalamic pituitary adrenal ( hpa ) axis and sympathetic nervous system , which can result in inflammation and altered metabolic and cardiac autonomic control.6 moreover , stress may be related to lifestyle factors relevant to stroke risk , such as cigarette smoking , physical inactivity and alcohol intake.7 to date , the association between psychosocial stress and stroke has been investigated incompletely.8 recent case - control studies investigating this may have been limited by potential reporting bias , and exclusion of subjects who experienced fatal stroke , as the measure of exposure to stress was collected after onset of stroke.4 9 10 while some prospective studies have been performed,3 5 limited duration of follow - up has not allowed examination of chronic exposure , which may be of particular relevance . stress susceptibility or stress resilience is an important determinant of the consequences of exposure to stress.11 12 a poorly controlled stress response ( potentially signalled by low stress resilience ) results in a prolonged physiological response to stressful exposures , thus increasing the possible longer - term adverse consequences of psychosocial stress . stress resilience may be a useful and underused measure for investigating the consequences of chronic stress in relation to stroke risk . here , we examine the association of stress resilience in adolescence with subsequent stroke risk in middle age within a general population - based cohort of male swedish residents . stress resilience was measured during the assessment for military conscription over 10 years prior to stroke and , therefore , not subject to differential reporting bias . a secondary aim was to examine whether other risk factors might mediate associations of stress resilience with stroke risk . at the end of follow - up the subjects were still relatively young , under age 60 years , so the study is of stroke risk during working age rather than the later ages when stroke is more common . the study cohort consists of male swedish residents born between 1952 and 1956 , who were eligible for military conscription and included in the swedish military conscription register.13 at this time , conscription and the associated examinations were mandatory for all male citizens of the appropriate age ( 18 and 19 years , with a small number at later ages ) . during the years covered by this study , only men with significant disability or a severe chronic disease were exempted from conscription ( approximately 23% annually).14 the assessments included extensive and highly standardised physical and psychological examinations by physicians and psychologists . stroke risk was assessed from 1987 ( when the swedish national inpatient register15 attained full coverage ) to 2010 . from among a total of 284 198 men identified , we excluded 2564 subjects with unreliable data such as errors in the personal identification number or uncertain vital status . those with implausible values for height ( less than 144 cm ) , weight ( above 178 kg ) , body mass index , bmi , ( below 15 ) , systolic blood pressure ( below 50 or above 230 mm hg ) and diastolic blood pressure ( below 30 or above 135 mm hg ) , were also excluded from the analysis ; in total 225 men.16 after excluding individuals who emigrated , died , or had a diagnosis of stroke before study entry in 1987 , the sample comprised 271 767 men . men with missing data for stress resilience , potential confounding factors ( year of birth , geographic region , systolic and diastolic blood pressure , bmi , cognitive function , physical working capacity , parental socioeconomic index and household crowding ) were also excluded ( 37 196 men ; including 722 men with a stroke diagnosis at some time ) . the sample available for the main analysis comprised 237 879 men , 84% of the potential target population . the men underwent a psychological examination to assess their potential ability to cope with war - time stress,17 based on the ability to control and channel nervousness , tolerance of stress and disposition to anxiety.18 during this assessment , the potential conscripts met a psychologist for a semistructured interview which covered areas relevant to general everyday life ; including psychosocial dimensions , such as social maturity , leisure interests , psychological motivation , and emotional stability.18 this interview was used to produce a stress resilience score from 1 to 9 which we grouped into low ( 13 ) , medium ( 46 ) and high ( 79 ) . details of the test are available in swedish,19 and it has been used in published research.16 17 to ensure consistent evaluation , a central authority supervised the instruction and training of participating psychologists , supported by a written manual . height and weight were used to calculate bmi , which was categorised using the who criteria . systolic and diastolic blood pressure was measured after rest in recumbent men using a sphygmomanometer . physical working capacity was assessed using the cycle ergonometric test . after a normal resting electrocardiogram , 5 min of submaximal exercise was performed at different work rates depending on body mass , directly followed by a maximal test with gradually increasing load until volitional exhaustion , or an estimate was derived from a nomogram for men who did not complete the full duration . the cognitive test was a written assessment and comprised four domains : linguistic understanding , spatial recognition , general knowledge and ability to follow mechanical instructions : the results were transformed into a single score with a value ranging from 1 to 9 . from the medical assessment at conscription , we identified diagnoses , and we used the international classification of diseases eighth revision , icd-8 , icd-8 codes 393458 to indicate diagnosis of any cardiovascular disease at the time of conscription . using the swedish national inpatient register15 exlude and the cause of death register,20 we identified the dates of first fatal and non - fatal stroke diagnoses during the period 19692010 . the codes used for stroke were 430434 and 436 in icd-8 , 430 , 431 , 433 , 434 and 436 in icd-9 ; and i60 , i61 , i63 , i64 in icd 10 . for ischaemic stroke , they were 432434 in icd-8 , 433 , 434 in icd-9 , and i63 in icd-10 ; for intracerebral haemorrhagic stroke 431 in icd-8 , 431 and 432 in icd-9 , and i61 in icd-10 . the government organisation , statistics sweden , provided socioeconomic and demographic data including information on vital status and emigration from the total population register.21 childhood social and material circumstances were estimated using data from the 1960 census . the head of household 's occupation was classified as manual , agricultural , farm owners / mangers , office workers , business owners / mangers , and others . household crowding was divided into two categories to indicate a ratio of less than two persons per room , or more or equal to two persons per room . cox regression was used to examine the association of stress resilience in adolescence with subsequent stroke risk between ages 31 and 58 years . the follow - up period began on 1 january 1987 and ended at first stroke , death , emigration or 1 january 2010 , whichever occurred first . subjects with a diagnosis of stroke before 1987 were excluded ( n=218 ) . non - fatal and fatal stroke were examined together and separately . in the analyses of aetiological subtypes ( ischaemic and intracerebral haemorrhagic stroke ) , first event by each subtype was used . associations were examined using an unadjusted ( model 1 ) and three further adjusted models . in model 2 , adjustment was for demographic and socioeconomic factors for the family of origin : birth year , geographical region , parents socioeconomic index , and household crowding . model 3 was additionally adjusted for characteristics in adolescence : cognitive function ( continuous ) , systolic and diastolic blood pressure ( continuous ) , and cardiovascular disease at conscription . in model 4 lifestyle factors in adolescence ( here represented by physical fitness and body mass ) were also added to the model : bmi ( in 4 categories ) and physical working capacity score ( continuous ) . the proportional hazards assumption for stress resilience in relation to stroke was tested graphically , as well as using a test based on schoenfeld residuals , and no evidence was found that it was violated . as this was assessed using age as the underlying time scale , it indicates that the association between stress resilience and stroke was constant and did not diminish with increasing age . the study cohort consists of male swedish residents born between 1952 and 1956 , who were eligible for military conscription and included in the swedish military conscription register.13 at this time , conscription and the associated examinations were mandatory for all male citizens of the appropriate age ( 18 and 19 years , with a small number at later ages ) . during the years covered by this study , only men with significant disability or a severe chronic disease were exempted from conscription ( approximately 23% annually).14 the assessments included extensive and highly standardised physical and psychological examinations by physicians and psychologists . stroke risk was assessed from 1987 ( when the swedish national inpatient register15 attained full coverage ) to 2010 . from among a total of 284 198 men identified , we excluded 2564 subjects with unreliable data such as errors in the personal identification number or uncertain vital status . those with implausible values for height ( less than 144 cm ) , weight ( above 178 kg ) , body mass index , bmi , ( below 15 ) , systolic blood pressure ( below 50 or above 230 mm hg ) and diastolic blood pressure ( below 30 or above 135 mm hg ) , were also excluded from the analysis ; in total 225 men.16 after excluding individuals who emigrated , died , or had a diagnosis of stroke before study entry in 1987 , the sample comprised 271 767 men . men with missing data for stress resilience , potential confounding factors ( year of birth , geographic region , systolic and diastolic blood pressure , bmi , cognitive function , physical working capacity , parental socioeconomic index and household crowding ) were also excluded ( 37 196 men ; including 722 men with a stroke diagnosis at some time ) . the sample available for the main analysis comprised 237 879 men , 84% of the potential target population . the men underwent a psychological examination to assess their potential ability to cope with war - time stress,17 based on the ability to control and channel nervousness , tolerance of stress and disposition to anxiety.18 during this assessment , the potential conscripts met a psychologist for a semistructured interview which covered areas relevant to general everyday life ; including psychosocial dimensions , such as social maturity , leisure interests , psychological motivation , and emotional stability.18 this interview was used to produce a stress resilience score from 1 to 9 which we grouped into low ( 13 ) , medium ( 46 ) and high ( 79 ) . details of the test are available in swedish,19 and it has been used in published research.16 17 to ensure consistent evaluation , a central authority supervised the instruction and training of participating psychologists , supported by a written manual . height and weight were used to calculate bmi , which was categorised using the who criteria . systolic and diastolic blood pressure was measured after rest in recumbent men using a sphygmomanometer . physical working capacity was assessed using the cycle ergonometric test . after a normal resting electrocardiogram , 5 min of submaximal exercise was performed at different work rates depending on body mass , directly followed by a maximal test with gradually increasing load until volitional exhaustion , or an estimate was derived from a nomogram for men who did not complete the full duration . the cognitive test was a written assessment and comprised four domains : linguistic understanding , spatial recognition , general knowledge and ability to follow mechanical instructions : the results were transformed into a single score with a value ranging from 1 to 9 . from the medical assessment at conscription , we identified diagnoses , and we used the international classification of diseases eighth revision , icd-8 , icd-8 codes 393458 to indicate diagnosis of any cardiovascular disease at the time of conscription . using the swedish national inpatient register15 exlude and the cause of death register,20 we identified the dates of first fatal and non - fatal stroke diagnoses during the period 19692010 . the codes used for stroke were 430434 and 436 in icd-8 , 430 , 431 , 433 , 434 and 436 in icd-9 ; and i60 , i61 , i63 , i64 in icd 10 . for ischaemic stroke , they were 432434 in icd-8 , 433 , 434 in icd-9 , and i63 in icd-10 ; for intracerebral haemorrhagic stroke 431 in icd-8 , 431 and 432 in icd-9 , and i61 in icd-10 . the government organisation , statistics sweden , provided socioeconomic and demographic data including information on vital status and emigration from the total population register.21 childhood social and material circumstances were estimated using data from the 1960 census . the head of household 's occupation was classified as manual , agricultural , farm owners / mangers , office workers , business owners / mangers , and others . household crowding was divided into two categories to indicate a ratio of less than two persons per room , or more or equal to two persons per room . the men underwent a psychological examination to assess their potential ability to cope with war - time stress,17 based on the ability to control and channel nervousness , tolerance of stress and disposition to anxiety.18 during this assessment , the potential conscripts met a psychologist for a semistructured interview which covered areas relevant to general everyday life ; including psychosocial dimensions , such as social maturity , leisure interests , psychological motivation , and emotional stability.18 this interview was used to produce a stress resilience score from 1 to 9 which we grouped into low ( 13 ) , medium ( 46 ) and high ( 79 ) . details of the test are available in swedish,19 and it has been used in published research.16 17 to ensure consistent evaluation , a central authority supervised the instruction and training of participating psychologists , supported by a written manual . height and weight were used to calculate bmi , which was categorised using the who criteria . systolic and diastolic blood pressure was measured after rest in recumbent men using a sphygmomanometer . physical working capacity was assessed using the cycle ergonometric test . after a normal resting electrocardiogram , 5 min of submaximal exercise was performed at different work rates depending on body mass , directly followed by a maximal test with gradually increasing load until volitional exhaustion , or an estimate was derived from a nomogram for men who did not complete the full duration . the cognitive test was a written assessment and comprised four domains : linguistic understanding , spatial recognition , general knowledge and ability to follow mechanical instructions : the results were transformed into a single score with a value ranging from 1 to 9 . from the medical assessment at conscription , we identified diagnoses , and we used the international classification of diseases eighth revision , icd-8 , icd-8 codes 393458 to indicate diagnosis of any cardiovascular disease at the time of conscription . using the swedish national inpatient register15 exlude and the cause of death register,20 we identified the dates of first fatal and non - fatal stroke diagnoses during the period 19692010 . the codes used for stroke were 430434 and 436 in icd-8 , 430 , 431 , 433 , 434 and 436 in icd-9 ; and i60 , i61 , i63 , i64 in icd 10 . for ischaemic stroke , they were 432434 in icd-8 , 433 , 434 in icd-9 , and i63 in icd-10 ; for intracerebral haemorrhagic stroke 431 in icd-8 , 431 and 432 in icd-9 , and i61 in icd-10 . the government organisation , statistics sweden , provided socioeconomic and demographic data including information on vital status and emigration from the total population register.21 childhood social and material circumstances were estimated using data from the 1960 census . the head of household 's occupation was classified as manual , agricultural , farm owners / mangers , office workers , business owners / mangers , and others . household crowding was divided into two categories to indicate a ratio of less than two persons per room , or more or equal to two persons per room . cox regression was used to examine the association of stress resilience in adolescence with subsequent stroke risk between ages 31 and 58 years . the follow - up period began on 1 january 1987 and ended at first stroke , death , emigration or 1 january 2010 , whichever occurred first . subjects with a diagnosis of stroke before 1987 were excluded ( n=218 ) . non - fatal and fatal stroke were examined together and separately . in the analyses of aetiological subtypes ( ischaemic and intracerebral haemorrhagic stroke ) , first event by each subtype was used . associations were examined using an unadjusted ( model 1 ) and three further adjusted models . in model 2 , adjustment was for demographic and socioeconomic factors for the family of origin : birth year , geographical region , parents socioeconomic index , and household crowding . model 3 was additionally adjusted for characteristics in adolescence : cognitive function ( continuous ) , systolic and diastolic blood pressure ( continuous ) , and cardiovascular disease at conscription . in model 4 lifestyle factors in adolescence ( here represented by physical fitness and body mass ) were also added to the model : bmi ( in 4 categories ) and physical working capacity score ( continuous ) . the proportional hazards assumption for stress resilience in relation to stroke was tested graphically , as well as using a test based on schoenfeld residuals , and no evidence was found that it was violated . as this was assessed using age as the underlying time scale , it indicates that the association between stress resilience and stroke was constant and did not diminish with increasing age . all analyses are based on the 237 879 conscripts with complete information for the required variables , after exclusion of individuals who emigrated , died , or had a diagnosis of stroke before the beginning of follow - up in 1987 . during the follow - up period of 19872010 , 3411 men ( 1.4% ) received an inpatient diagnosis of stroke ( or stroke was recorded as the underlying cause of death ) . the stroke incidence rate was 64 per 100 000 person - years in our study population , compared with 101 per 100 000 person - years in the excluded subset with missing data on covariates . men with low resilience tended to have lower physical capacity , lower cognitive function scores , higher blood pressure , were underweight , overweight or obese , more often had a diagnosis of cardiovascular disease at conscription , and had parents with a lower socioeconomic index and experienced greater household crowding in childhood . population ( n=237 879 ) characteristics by stress resilience levels cvd , cardiovascular disease ; sei , socioeconomic index . the associations with stroke for stress resilience and the other measures are reported in table 2 . the lowest stress resilience group ( 21.8% with scores 13 ) compared with the highest ( 23.7% with score 79 ) was associated with increased risk of all stroke types . the intermediate category of stress resilience lay between the low and high groups , indicating a graded association . when adjusted for the demographic and socioeconomic factors in childhood , the association attenuated slightly but remained statistically significant . additional adjustment for health and development characteristics in adolescence further adjustment for physical fitness indicated by bmi and physical capacity had the most notable impact on the hrs , but despite the reduction in magnitude , statistical significance remained . blood pressure was modelled as continuous variables to ensure the most effective adjustment : the magnitude of the associations appears small as the hrs are for each unit change per mm hg , but statistical significance for associations with stroke remained after adjustment for all other measures . / material background factors ( birth year , region , parental sei and household crowding ) . model 3 . adjusted for 2 + characteristics in adolescence ( cognitive function , diastolic and systolic blood pressure and cvd diagnosis at conscription ) . model 4 . adjusted for 2 + 3 + physical fitness in adolescence ( physical working capacity and bmi ) . table 3 presents the outcomes for the stroke subgroups , fatal ( n=308 ) , ischaemic ( n=2060 ) and intracerebral haemorrhagic ( n=676 ) stroke . all showed a significant association with stress resilience , with higher magnitude associations for fatal than non - fatal stroke ; and for haemorrhagic than ischaemic stroke . in adjusted models , a similar pattern as for all stroke appeared , but the association of stress resilience and ischaemic stroke was more notably attenuated by adjustment for bmi and physical capacity . stress resilience and stroke subdivided by stroke characteristics model 2 . adjusted for socio / material background factors ( birth year , region , parents sei , household crowding ) . model 3 . adjusted for 2 + characteristics in adolescence ( cognition , systolic and diastolic blood pressure , cvd diagnosis at conscription ) . model 4 . adjusted for 2 + 3+physical fitness in adolescence ( physical working capacity and bmi ) . low stress resilience in adolescence was associated with a higher risk of stroke in men of working age between 31 years and 58 years , a relatively young age group to experience stroke . this association was independent of childhood socioeconomic circumstances , as well as health and developmental characteristics in adolescence . the association was attenuated more notably ( but not eliminated ) by adjustment for bmi and physical working capacity as indicators of physical fitness in adolescence , likely to signal future lifestyle characteristics . our results from a general population - based cohort of men are consistent with recent studies suggesting a role for psychosocial stress in influencing stroke risk.35 9 10 similarly , poorer adaptation to social adversity possibly signalling lower stress resilience has also been linked with an increased risk of stroke.22 stress resilience in adolescence does not measure exposure to stress but does indicate susceptibility , such that the adversities of daily life may have a greater impact and conspire to produce a more chronic pattern stress arousal over adult life . another study has found associations with stress independent of lifestyle factors.4 this difference with our findings may be due to methodological variations , as we have a measure of resilience rather than stress exposure , it was collected prospectively , many years before stroke , and we adjusted for some characteristics in adolescence that may themselves be consequences of low stress resilience , as discussed below . we hypothesise that low resilience to psychosocial stress could indicate exposure to psychosocial stress in earlier life , resulting in poorer control of the stress response that can continue across the life course . animal studies have demonstrated that early life stress can alter hpa axis function in a way that can persist over the life course , producing persistent low resilience to stress.23 24 stress resilience may also be a characteristic of personality type , and thus , a persistent trait . for example , low resilience may reflect something like type a behaviour which , although controversial , has previously been related to increased cardiovascular risk.25 it has been demonstrated that adverse socioeconomic conditions in childhood increase stroke risk26 ; so we adjusted for such factors , with little impact on the association of stress resilience with stroke . markers of early life exposure to stress have been linked with poorer cognitive development27 and higher blood pressure.28 thus , cognitive function and blood pressure may represent consequences of earlier stressful exposures and poorer stress resilience , and adjusting for these factors may have produced conservative estimates of association ; arguably an over - adjustment . markers of stressful exposures in early life are also linked with unhealthy weight gain,29 which may influence physical exercise or be a consequence of low exercise levels.30 thus , even in adolescence , bmi and fitness could , in part , be a consequence of lower stress resilience and earlier stressful exposures . we believe the results suggest that a significant proportion of the association of stress and stress resilience with stroke risk is due to lifestyle risk factors . this is consistent with recent findings from a british cohort study with older participants , where the association of psychological distress with cvd risk was largely explained by behavioural / lifestyle processes.31 our findings suggest such processes have a long natural history , beginning in childhood or adolescence . our results were consistent among the different stroke subtypes ( fatal , non - fatal , ischaemic and intracerebral haemorrhagic stroke ) although there may be pathological heterogeneity . previous studies investigating the association between psychosocial stress and stroke subtypes are few , but the results are mostly consistent with our findings suggesting a more notable association for fatal than for non - fatal stroke , and for haemorrhagic than ischaemic stroke . the stronger association with haemorrhagic than ischaemic stroke could also be a consequence of a greater aetiological heterogeneity in ischaemic stroke . physical working capacity and bmi in adolescence most notably attenuated that association of stress resilience with ischaemic stroke , suggesting more significant mediation through lifestyle factors . as this is a study of stress resilience measured in adolescence , it is possible that stressful exposures in adult life might represent a higher risk of stroke , particularly chronic exposures among the less stress resilient . overweight , obesity and poorer physical capacity in adolescence are likely to signal an accumulation of behavioural / lifestyle health risks that continue through adulthood.32 33 other lifestyle risks in the years between conscription and stroke , such as smoking , alcohol and diet , are also likely to be relevant mediating factors . further mediating factors might include type 2 diabetes , a stroke risk which tends to have its onset in adulthood some years after the time of conscription , which has also been linked with psychosocial stress.34 35 as behaviours and diagnoses occurring between conscription and stroke are intermediate in the causal pathway , we have not included them in the model . unfortunately , we have no data on smoking , diet or alcohol consumption , which could have added further to our understanding of lifestyle risks . thus , major mediation between poor stress resilience and stroke risk may be through lifestyle factors and fitness , suggesting interventions for psychosocial stress and behaviour might be most effective in lowering stoke risk among younger men . the use of prospectively recorded data from several linked registers , and the long - term follow - up subsequent to the measure of susceptibility , increases the validity and reliability of the study , as well as avoiding bias due to reverse causation or poor recall . the study population is largely representative of the male general population as only a small proportion of swedish men were exempted from enlistment examinations . extensive physical and psychological examinations were conducted in adolescence , so the information was objectively measured and were able to adjust for a variety of powerful stroke risk factors including , bmi and physical capacity which are objective physiological measures . the duration between conscription examination and start of follow - up means it is unlikely that prodromal characteristics of an impending stroke were driving the stress resilience measure , and it was possible to identify pre - existing cardiovascular diagnoses and adjust for blood pressure . the potential limitations of the study include the inclusion of only men , as women also suffer from stroke . this was necessary as the measure of stress susceptibility was collected during military conscription , which at the time was almost exclusively available to men . as stress resilience was only measured once , we can not be definitive about stability of this characteristic over time and how this may interact with stressful exposures . in these data , the association of stress resilience with stroke risk did not vary by age , indicating that this measure of stress resilience is a relatively stable characteristic over time , at least in terms of stroke risk . there is a possibility of misclassification of the stress resilience measure if potential conscripts attempted to obtain exemption from military service through false answers , if they exist , would most likely dilute the resilience measure and result in less precise estimates . the temporal relationship of stress resilience and physical fitness in adolescence can not be established , but we hypothesise that poor stress resilience has an adverse influence on physical fitness . as stressful exposures were not examined , it is possible that the magnitude of associations with stress resilience is conservative . a proportion of the cardiovascular diagnoses at conscription when the men in our cohort were 1819 years old may not be directly relevant to stroke risk . we included this information in the models to ensure that poorer stress resilience was not a function of pre - existing cardiovascular disease , where a proportion of these diseases may increase subsequent stroke risk . most stroke onset occurs at a higher age than in our study , so early stroke may be somewhat atypical and the aetiology may differ from stroke at older ages.36 however , stroke incidence in younger adults ( younger than 55 years of age ) is increasing.37 stroke at young ( working ) age is a particular concern , as a reduction of working capacity increases the personal , social as well as economic burden of the disease . this is of public health significance , as stroke at early ages has the potential for greater lifetime burden of disability , and because some risks may be modifiable . the proportion of stroke diagnoses in the analytic sample is lower than among those who were not included . this is for two reasons , and the first is that men who had a stroke at an early age , close in time to the conscription examination , were excluded deliberately to avoid associations due to reverse causation . some men in poor health , with very low levels of physical fitness , or low cognitive function at the time of the conscription examination , were identified as unsuitable for military service by the assessors and may not have completed all the examinations ( including for stress resilience ) . as these men excluded due to missing values on the assessments may have the greatest risk of stroke , we may have underestimated the proportion at risk and conceivably produced a conservative estimate of stroke risk . the validity of some diagnoses recorded in the swedish national inpatient register can be questionable , but the reliability of stroke diagnoses is satisfactory.38 39 the use of prospectively recorded data from several linked registers , and the long - term follow - up subsequent to the measure of susceptibility , increases the validity and reliability of the study , as well as avoiding bias due to reverse causation or poor recall . the study population is largely representative of the male general population as only a small proportion of swedish men were exempted from enlistment examinations . extensive physical and psychological examinations were conducted in adolescence , so the information was objectively measured and were able to adjust for a variety of powerful stroke risk factors including , bmi and physical capacity which are objective physiological measures . the duration between conscription examination and start of follow - up means it is unlikely that prodromal characteristics of an impending stroke were driving the stress resilience measure , and it was possible to identify pre - existing cardiovascular diagnoses and adjust for blood pressure . the potential limitations of the study include the inclusion of only men , as women also suffer from stroke . this was necessary as the measure of stress susceptibility was collected during military conscription , which at the time was almost exclusively available to men . as stress resilience was only measured once , we can not be definitive about stability of this characteristic over time and how this may interact with stressful exposures . in these data , the association of stress resilience with stroke risk did not vary by age , indicating that this measure of stress resilience is a relatively stable characteristic over time , at least in terms of stroke risk . there is a possibility of misclassification of the stress resilience measure if potential conscripts attempted to obtain exemption from military service through false answers , if they exist , would most likely dilute the resilience measure and result in less precise estimates . the temporal relationship of stress resilience and physical fitness in adolescence can not be established , but we hypothesise that poor stress resilience has an adverse influence on physical fitness . as stressful exposures were not examined , it is possible that the magnitude of associations with stress resilience is conservative . a proportion of the cardiovascular diagnoses at conscription when the men in our cohort were 1819 years old may not be directly relevant to stroke risk . we included this information in the models to ensure that poorer stress resilience was not a function of pre - existing cardiovascular disease , where a proportion of these diseases may increase subsequent stroke risk . most stroke onset occurs at a higher age than in our study , so early stroke may be somewhat atypical and the aetiology may differ from stroke at older ages.36 however , stroke incidence in younger adults ( younger than 55 years of age ) is increasing.37 stroke at young ( working ) age is a particular concern , as a reduction of working capacity increases the personal , social as well as economic burden of the disease . this is of public health significance , as stroke at early ages has the potential for greater lifetime burden of disability , and because some risks may be modifiable . the proportion of stroke diagnoses in the analytic sample is lower than among those who were not included . this is for two reasons , and the first is that men who had a stroke at an early age , close in time to the conscription examination , were excluded deliberately to avoid associations due to reverse causation . some men in poor health , with very low levels of physical fitness , or low cognitive function at the time of the conscription examination , were identified as unsuitable for military service by the assessors and may not have completed all the examinations ( including for stress resilience ) . as these men excluded due to missing values on the assessments may have the greatest risk of stroke , we may have underestimated the proportion at risk and conceivably produced a conservative estimate of stroke risk . the validity of some diagnoses recorded in the swedish national inpatient register can be questionable , but the reliability of stroke diagnoses is satisfactory.38 39 the association with stress resilience suggests that stress may be implicated in the aetiology of stroke , which is , in part , explained by poorer physical fitness among the less stress resilient . we hypothesise that the most effective interventions to prevent stroke would focus on behaviour and lifestyle factors , as well as psychosocial stress .

objectiveexposure to psychosocial stress has been identified as a possible stroke risk , but the role of stress resilience which may be relevant to chronic exposure is uncertain . we investigated the association of stress resilience in adolescence with subsequent stroke risk.methodsregister-based cohort study . some 237 879 males born between 1952 and 1956 were followed from 1987 to 2010 using information from swedish registers . cox regression estimated the association of stress resilience with stroke , after adjustment for established stroke risk factors.resultssome 3411 diagnoses of first stroke were identified . lowest stress resilience ( 21.8% ) compared with the highest ( 23.7% ) was associated with increased stroke risk , producing unadjusted hr ( with 95% cis ) of 1.54 ( 1.40 to 1.70 ) . the association attenuated slightly to 1.48 ( 1.34 to 1.63 ) after adjustment for markers of socioeconomic circumstances in childhood ; and after further adjustment for markers of development and disease in adolescence ( blood pressure , cognitive function and pre - existing cardiovascular disease ) to 1.30 ( 1.18 to 1.45 ) . the greatest reduction followed further adjustment for markers of physical fitness ( bmi and physical working capacity ) in adolescence to 1.16 ( 1.04 to 1.29 ) . the results were consistent when stroke was subdivided into fatal , ischaemic and haemorrhagic , with higher magnitude associations for fatal rather than non - fatal , and for haemorrhagic rather than ischaemic stroke.conclusionsstress susceptibility and , therefore , psychosocial stress may be implicated in the aetiology of stroke . this association may be explained , in part , by poorer physical fitness . effective prevention might focus on behaviour / lifestyle and psychosocial stress .

malignant salivary gland neoplasms account for < 0.5% of all malignancies and approximately 35% of all head and neck cancers . progress in understanding the cell biology of salivary gland carcinomas ( sgcs ) and detecting vulnerable molecular pathways may lead to the development of new targeted therapy options in these rare cancers with poor prognosis . anti - egfr agents include ( i ) monoclonal antibodies ( cetuximab or erbitux , panitumumab ) which block the binding of natural egfr ligands like egf or tgf- resulting in inhibition of downstream signal - transduction pathways and ( ii ) small molecule tyrosine kinase inhibitors ( tkis ) which act by binding the atp pocket within the kinase domain of the egfr and impairing its catalytic activity ( gefitinib , erlotinib , lapatinib ) . downstream signaling pathways triggered by egfr include the ras - raf - extracellular signal - regulated kinase / mitogen activated protein kinase ( mek / mapk ) pathway , which is mainly correlated to cell proliferation , and the p13k - pten - akt axis . recently , we were able to demonstrate that frequent egfr overexpression and the absence of drug - resistance egfr mutations in sgc plead in favor of further therapeutic trials with egfr - targeting monoclonal antibodies . one of the signaling effectors downstream of egfr , kras , was shown by us to be rarely mutated in sgc [ 2 , 3 ] . wildtype kras is one of the clinically proven prerequisites for a successful anti - egfr therapy and therefore anti - egfr monoclonal antibodies are approved only for metastatic colorectal cancer patients whose tumors display wildtype kras . in the absence of kras mutations , resistance to anti - egfr treatments could be caused by alterations of other members of the ras - raf - mapk pathway . braf ( v - raf murine sarcoma viral oncogene homolog b1 ) , a serine / threonine kinase , is the downstream effector of kras in the ras - raf - mapk signaling pathway . a somatic mutation ( v600e ) in exon 15 of braf has been identified in multiple human cancers with a mutation rate of 66% in malignant melanomas and at lower frequency in other human carcinomas . recently , it was demonstrated that wildtype braf is required for the response of patients with metastatic colorectal cancer to cetuximab and panitumumab . the aim of this study was to determine the braf v600e mutation frequency in a large cohort of sgcs of the main histopathological types and to design an allele - specific pcr as an effective screening method . surgically removed , formalin - fixed tumor samples were obtained from 65 patients ( 35 males and 30 females with a median age at diagnosis of 55 years ) treated with the histopathological diagnosis of an sgc according to the who classification . egfr - targeted therapy was not applied . the study cohort consisted of adenoid cystic carcinoma ( n = 25 ) mucoepidermoid carcinoma ( n = 10 ) , myoepithelial carcinoma ( n = 8) , acinic cell carcinoma ( n = 12 ) and adenocarcinoma ex pleomorphic adenoma ( n = 10 ) . genomic dna was extracted and pooled from oral mucosa samples of five healthy individuals . this pooled dna was used as a normal dna control for the development of the pcr assay . heterozygous mutant control dna was extracted from cells of the colorectal cancer cell line ht 29 which contains the heterozygous braf v600e mutation . the basis for discrimination using allele - specific pcr is that a pcr primer mismatched at its 3 end with the dna template will react less efficiently than one that is entirely complementary . our allele - specific multiplex pcr was designed with one common forward ( bf ) and two separate reverse primers ( br and bmu ) : bf : 5-ctcttcataatgcttgctctgatagg-3 , br : 5-agttgagaccttcaatgactttctagt-3 , bmu : 5-cccactccatcgagatttct-3. the forward primer bf and the reverse primer br amplify a 273 bp fragment of both mutant and wildtype alleles and thus serve as amplification control . the second reverse primer ( bmu ) is specific for the mutated allele at the 3 end . this primer together with bf generates an 143 bp product only in the presence of the v600e ( gtg > gag ) mutation ( figure 1(a ) ) . a series of annealing temperatures ( 52c60c ) , primer concentrations ( 0.10.4 mol / l ) , and mg concentrations ( 1.53.5 reactions consisted of : 80100 ng genomic dna ; 200 mol / l dntp ; 0.1 mol / l of primers ; 1.5 mmol / l mgcl2 ; 0.5 u taq polymerase ( roche diagnostics , penzberg , germany ) . final cycling conditions were as follows : 5 minutes of denaturing at 94c and 30 cycles of 94c for 30 seconds , annealing 55c for 45 seconds and 72c for 60 seconds . a volume of 10 l of the pcr products was electrophoresed on a standard 2.5% agarose gel stained with sybr - green i for visualization under uv light . the allele - specific pcr for the detection of the braf v600e mutation demonstrated high specificity ( i.e. , detection of only the normal or only the mutant allele ) , high sensitivity ( i.e. , no spurious pcr fragments ) , and acceptable yield . all 65 sgc in this cohort ( 100% ) presented the braf wildtype ( 95% exact confidence limit 00.07 ) . the 273 bp pcr fragment was always amplified confirming the integrity of the isolated dna from clinical tissue samples ( figure 1(a ) ) . to test the sensitivity of the mutation - specific pcr , we made a serial dilution of ht 29 dna ( which contains the heterozygous braf v600e mutation ) , in control dna with wildtype braf . no braf v600e mutation was detected in additionally screened dna samples from microdissected normal tissue adjacent to the tumor cells ( 5 cases ) . the activation of the egfr - ras - raf signaling cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules . egfr transmits signals to the nuclei instructing cancer cells to proliferate and metastasize , and kras and braf are those downstream signaling molecules . anti - egfr therapies interrupt the cancer - triggering signaling cascade , however , if the kras or the braf gene is mutated , their proteins are locked into an active conformation , regardless of whether the egfr is therapeutically blocked . cetuximab ( erbitux ) has already been tested in two phase ii studies in patients with recurrent and/or metastatic sgc including mainly acc , a cancer with generally poor outcome . in 50% of patients , clinical benefit ( i.e. , response or stable disease for 6 month ) gefitinib was associated with a 53% stable disease rate ( 10/19 ) in acc , but had no effects on patients with salivary duct tumors and mucoepidermoid cancer . lapatinib , inhibiting erbb1 and erbb2 tyrosine kinases , was studied in a phase ii trial and stabilized disease for greater than 6 months in 47% of acc patients . key molecules of the egfr - ras - raf signaling cascade and predictive markers of treatment outcome under anti - egfr therapies have not been comprehensively examined in sgc . investigations of the mutation status of proteins in the cascade downstream of egfr identified markers for egfr - targeted therapy in colorectal cancer . in recent studies , wildtype braf as well as wildtype kras and intact pten pik3ca were found to be required for the response of colorectal cancer patients treated with cetuximab or panitumumab [ 5 , 10 , 11 ] . we were able to demonstrate in this and in a former study that kras and braf mutations seem to be extremely rare in sgc . these findings imply that salivary gland carcinomas which rarely acquire mutations that result in constitutive activation of the signaling cascade downstream of egfr may be good candidates for anti - egfr therapies . molecular analyses of alternative members of the egfr signaling cascade , such as akt-1 and mek-1 , may further contribute to elucidating predictive markers of treatment outcome under anti - egfr therapies in sgc . because of its universal availability as a standard methodology in molecular medicine , we designed a braf mutation screening assay based on pcr . allele - specific pcr , also known as amplification refractory mutation system ( arms ) , is a well - established method for discriminating between different alleles at specific loci resulting from single base mutations [ 12 , 13 ] . we used the methodology to establish an assay with three pcr primers which allows the discrimination of allele - specific pcr fragments by agarose gel electrophoresis without the need of capillary electrophoresis devices . with our assay interpretation of the results can be made by simple visual inspection of the stained gel to determine whether or not a specific primer pair amplified a fragment with the template dna . because microdissected tumor areas were used for the allele - specific pcr , and a control amplification was incorporated into the pcr reaction to ensure dna integrity , we can exclude false negative results . so far , genomic screening for braf mutations has been based mainly on direct sequencing . our protocol provides an alternative rapid , sensitive , and cost - effective braf screening method .

braf is the main effector of kras in the ras - raf - mapk axis , a signaling pathway downstream of egfr . the activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules . recent studies in metastatic colorectal cancer found that an oncogenic activation of braf by a point mutation in exon 15 ( v600e ) could bypass the egfr - initiated signaling cascade with the effect that patients bearing the mutant braf allele are not likely to benefit from egfr - targeted therapies . we designed an allele - specific pcr and screened 65 salivary gland carcinoma ( sgc ) of the main histopathological types for the braf v600e mutation . all 65 sgc in this cohort ( 100% ) presented the braf wildtype . in a previous study , we found a kras wildtype in 98.5% of sgc . these findings imply that sgc rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of egfr and this pleads in favor of further therapeutic trials with egfr - targeting monoclonal antibodies .

the contour of the lower face , including the lips and soft - tissue chin , is thought to be closely related to the underlying dentoalveolar structure . both the upper and the lower lips are known to increase in length during growth and maintain a fairly constant vertical relationship with the edges of the corresponding central incisors after their full eruption.1 during lip closure , a subject with a normal maxillofacial skeletal pattern shows negligible contraction of the perioral musculature , and therefore , changes in the contour of the lip and soft - tissue chin are minimal.2,3 on the other hand , many orthodontic patients display visible tension in their soft - tissue chin during lip closure , which is often considered unesthetic . in particular , patients who have a long anterior vertical dimension or protruded incisors with a large interlabial gap show strain of their soft - tissue chin in an effort to close their lips , because the lower lip is elongated and the mentolabial sulcus moves upward and forward.2,4,5 one goal of orthodontic treatment is to achieve a desirable facial profile by obtaining not only satisfactory dental and skeletal relationships but also esthetic soft - tissue components including the lips and chin . any dental or skeletal factors found to be contributing to either tension in or deformation of the soft - tissue chin must be considered during diagnosis and treatment planning for better outcomes . the influence of diagnostic factors on pretreatment or posttreatment variables can be evaluated through various analytical methods such as logistic and multivariate regression analyses.6,7 however , these analyses often include complicated predictive equations , which occasionally present difficulties in result interpretation . a classification and regression tree ( cart ) is a useful analytical method that integrates the data of complex regression analyses and converts them into a simple tree model providing information on diagnostic factors in the order of their impact on the dependent variable with cut - off values.8,9 studies using cart as a mean of analytical method for the evaluation of the soft - tissue chin are scarce . the purpose of this study was to investigate the dentoskeletal factors which may predict soft - tissue chin strain during lip closure . two hundred and ninety - three women with angle 's class i or ii molar relationships and aged 18 - 30 years were recruited from three private orthodontic clinics . the exclusion criteria were as follows : ( 1 ) previous orthodontic treatment or orthognathic surgery ; ( 2 ) multiple missing teeth ; ( 3 ) presence of prostheses in edentulous areas ; ( 4 ) anb less than 0 ; ( 5 ) anterior edge - to - edge bite or crossbite ; and ( 6 ) congenital anomalies such as cleft lip and palate . the records included frontal and lateral facial photographs taken with the lips in the closed and relaxed positions and lateral cephalograms obtained with the teeth in centric occlusion . visually inspected the lips , mentolabial sulcus , and soft - tissue chin of the subjects in these records and classified the subjects into either the no - strain or the strain group according to the soft - tissue chin tension or deformation during lip closure . cochran 's q test was performed to compare the proportion of subjects classified into the strain group by the three examiners : the differences were found to be significant ( p = 0.02 ) . further , mcnemar 's test was used to compare the proportion of subjects classified into the strain group between the examiners . showed no significant difference ( p = 0.892 ) , with their concurrence being 81.5% . however , examiner j.p . showed significant discordance with both examiner y.k . and examiner y.y . ( p = 0.036 and 0.014 , respectively ) . the results of these tests suggested that the examiners showed subjectivity in classifying the women into the no - strain or strain group ; to enhance the credibility of this research , the data of only 209 subjects ( 114 and 95 women from the no - strain and strain groups , respectively ) who had been unanimously classified by all the three examiners were used . the lateral cephalogram of each subject was traced on standard 0.003 inch acetate tracing paper with a 0.3 mm mechanical pencil and measured on a scale with 0.5 mm and 0.5 intervals by one orthodontist . the description of cephalometric landmarks and references for the skeletal and dental measurements are shown in table 1 . the reliability of the measurements was assessed via intraclass correlation coefficients ( iccs ) and the wilcoxon signed - rank test . a t - test was performed to compare the differences in the mean values of the variables between the groups . three variables that did not pass the normality test were analyzed by the mann - whitney u - test . multivariate analysis of variance ( manova ) was conducted to determine the intergroup differences in the variables after age adjustment . the chi - square test was carried out to evaluate the significance of the group distribution according to angle 's classification of malocclusion . moreover , logistic regression analysis was used to determine the possible discriminants for each group . then , a cart model was constructed to obtain cut - off values for the variables selected as predictors . tracing and measurement errors were determined by retracing 20 cephalograms acquired through systematic random sampling . the results of the wilcoxon signed - rank test reflected no significant differences in the repeated measurements of all the variables ( p > 0.05 ; table 2 ) . the measurements were considered reliable and measurement errors were thought to be minimal because 26 of the 27 analyzed variables exhibited icc values of 0.95 or higher ( p < 0.001 ; table 2 ) . as shown in table 3 , the mean values of 22 of the 27 variables were significantly different between the groups . meanwhile , the age differences between the groups were not significant ( p = 0.362 ) . further , in the manova , the values of wilks ' lambda indicated that the intergroup differences in the mean values of the variables after age adjustment were comparable to those before age adjustment ( p < 0.05 ) . according to angle 's classification of malocclusion , 124 and 85 subjects had class i and ii malocclusions , respectively . the strain group included a lower number of subjects with class i malocclusion ( 48 [ 38.7% ] vs. 76 [ 61.3% ] ) and a slightly higher proportion of subjects with class ii malocclusion ( 47 [ 55.3% ] vs. 38 [ 44.7% ] ) than the no - strain group . the chi - square test showed that this trend was significant ( p = 0.018 ) . the variables analyzed in the forward stepwise ( likelihood ratio ) logistic regression analysis included the cephalometric measurements , age , and angle 's classification of malocclusion ( class i or ii malocclusion ) . four variables were chosen as the most prominently related factors for the group classification in the final model ( table 4 ) : overbite depth indicator ( odi ) , upper incisor to n - pog plane ( u1-npog , mm ) , overjet , and upper incisor to upper lip ( u1-upper lip , mm ) . a logit model using the four selected variables was derived as follows : where " a , " " b , " " c , " and " d " are the coefficient values of odi , u1-npog , overjet , and u1-upper lip , respectively ( table 4 ) . the logit model indicated that the lower the odi value and the higher the u1-npog , overjet , and u1-upper lip values , the greater was the probability of the subject being included in the strain group . by inverting the logit model with exponential function , the following equation was obtained : where " s " is the logit model and " pr(sg ) " is the probability of classification into the strain group . the prediction rate at which the group classification by using the four selected variables would concord with the actual classification was 81.8% ( table 5 ) . the cart model constructed by using the four selected variables provided the cut - off values of these variables ( figure 1 ) . the tree building began at the root ( or parent ) node ( node 0 ) , which included all the subjects in the dataset . the best splitter variable for each node was selected to maximize the average " purity " of the two child nodes . the best splitter variable for node 0 was found to be u1-npog , with a cut - off value of 14.2 mm . therefore , in node 1 , 80% of the subjects showing a u1-npog value less than or equal to 14.2 mm would be categorized into the no - strain group , and in node 2 , 79.8% of the subjects showing a u1-npog value greater than 14.2 mm would be categorized into the strain group . accordingly , u1-npog was considered the primary predictor , with the ability to classify approximately 80% of the total subjects into the strain or no - strain group at the value of 14.2 mm and without the necessity of the other variables . the next splitter variable for both node 1 and node 2 was considered to be overjet , although the number and condition of the subjects in these nodes were different ( i.e. , node 1 contained 120 subjects with a u1-npog value 14.2 mm and node 2 comprised 89 subjects with a u1-npog value > 14.2 mm ) . the tree building continued until one of the coupled nodes showed 100% purity in the categorization of the groups , as seen in nodes 12 , 14 , and 15 . the predictability of the group classification according to the cart model is shown in table 6 . during lip closure , the upper and lower lips cover two - thirds and one - third of the maxillary central incisors , respectively , and the main muscle activity is shown by the mentalis , followed by the lower and upper lips.1 the mentalis is essential in both esthetic and functional aspects because it is responsible for the central motion of the lower lip and determining the position of the chin point.4,10 patients with the dolichofacial type or maxillary protrusion tend to overwork the mentalis and lower lip to compensate for the lip incompetence . this condition leads to tension in the soft tissue at the insertion point of the mentalis and dimpling at the soft - tissue chin point , which is considered functionally and esthetically unfavorable.2 - 4 in this study , frontal and lateral extraoral photographs taken with the lips in their resting and closed positions and lateral cephalograms obtained with the teeth in centric occlusion were examined . to eliminate discrepancies related to gender or age , further , only the subjects who were unanimously categorized into the no - strain or strain group by all the three examiners were included to allow the maximal objectivity . to identify the cephalometric measurements highly related to strain of the soft - tissue chin , a cart is used in various dental and medical studies to evaluate diagnostic or predictive factors for a certain condition or disease.11 - 13 this diagnostic tree is a machine - learning method that allows the construction of a prediction model from the data.8,9 it exhibits independent variables ( e.g. , the cephalometric variables ) in the order of their influence on the dependent variables ( e.g. , no - strain or strain group ) in a stratified manner , with the earlier division or node ( higher in the tree ) demonstrating a greater effect . its main advantage is that it defines the cut - off values for dividing the parent node into the two child nodes that can simplify the decision for one way or the other . another advantage is the graphical presentation of the tree diagram , which effectively aids in the interpretation of complex statistical data . the child nodes can each be recursively divided into two nodes , forming additional child nodes with their own splitters and cut - off values . of the four discriminants selected in this study , the cart model showed that u1-npog had the highest differentiability at a cut - off value of 14.2 mm . in a previous study , subjects with class ii division 1 malocclusion and lip incompetence had higher perioral muscle activities than those with normal occlusion.14 further , in both maxillary and bimaxillary protrusion cases , a greater amount of muscle tension developed from the lower lip to the chin in the closed lip position , because the amount of lower lip movement increased in relation to the degree of maxillary incisor protrusion.3 these studies are highly comparable to the present study as they evaluated asian male and female patients equivalently or female patients only . accordingly , maxillary incisor protrusion has a notable effect on the lower lips and can lead to increased strain of the soft - tissue chin . moreover , the degree of the chin prominence relative to the lip and maxillary incisors should not be overlooked . the chin provides a fundamental base on which the lips rely for support and the balance of the lip position heavily depends on the strength of the chin.15 if the chin is too weak , the relative distance to the maxillary incisors would increase , which might cause the lower lip to protrude in order to reach the maxillary incisors during lip closure and lead to soft - tissue tension in the mentalis area . therefore , the optimal treatment plan to relieve strain of the soft - tissue chin should include retraction of the maxillary incisors or orthognathic surgery such as bimaxillary anterior subapical osteotomy where a more prominent chin profile is preferred.5,16 the next predictive variable in the cart model was overjet , with cut - off values of 4.8 and 4.2 mm . an increased overjet was associated with decreased activity of the upper lip and increased activity of the lower lip ; a positive correlation was noted between the overjet and the activity of the lower lip during lip closure , speech , and mastication.14 otuyemi17 reported that in a preadolescent nigerian population , an overjet greater than 6 mm was strongly related with a " trapped " lower lip and that greater than 7 mm led to inadequate coverage of the maxillary incisors by the lower lip . on the other hand , a direct correlation between the varying overjet and the different upper lip positions has not been shown , confirming a strong association between the overjet and the lower lip rather than with the upper lip.18 although these studies were performed with varying age groups and included male subjects , the results of the present study are in agreement with the previous findings concerning the influence of a large overjet on the movement of the lower lip . therefore , acquiring a normal overjet may minimize malfunction of the lower lip and distortion of the mentalis . u1-upper lip ( mm ) is a measurement reflecting the relative length of the lip . its average values in the strain and no - strain groups were 4.07 and 2.83 mm , respectively , indicating that maxillary incisor extrusion coincides with increased strain in the mentalis . this relationship was significantly different in the subjects with class ii division 1 and class i malocclusions , implying that the maxillary incisors tend to supra - erupt in class ii division 1 malocclusion.2 hayashida et al.19 reported that the most influential factor for the vertical position of the lower lip is the varying position of the tip of the maxillary incisor . an extruded maxillary incisor causes cushioning of the lower lip on the lingual surface and leads to eversion of the lower lip and distortion of the mentolabial sulcus and mentalis . therefore , the vertical position of the maxillary incisors should also be considered together with the smile arc in the esthetic aspect of orthodontic treatment.2,20,21 the odi was the only skeletal factor among the selected predictors and its influence was not as conspicuous as that of the other variables , which are related to the position of the maxillary incisors . although its influence is limited , the treatment plan should be oriented toward increasing the odi value to decrease tension in the mentalis . however , this may be difficult to achieve because the ab plane angle should not be increased in treating class i and ii malocclusions . therefore , prevention of extrusion or active intrusion of the maxillary molars is required to maintain or decrease the mandibular angle . the chi - square test showed an apparent discrepancy in the sample distribution of angle 's classification of malocclusion between the groups ( p = 0.018 ) . although the measurements were not compared between skeletal class i and skeletal class ii malocclusions , the differences in the cephalometric variables imply that the skeletal pattern between the groups was different , and the strain group showed a higher tendency for retrognathism , convex profile , and dolichofacial type . patients showing unfavorable strain at the soft - tissue chin may be managed by an orthodontic intervention - often involving retraction of anterior teeth - to establish satisfactory anterior dental relationship accordingly which induces changes in the lip posture , leading to lip closure without unnecessary tension in the lips and soft - tissue chin , and thus a more desirable profile is obtained.1 recognition of the variables predicting strain of the soft - tissue chin may enable the establishment of a guideline for the important dentoskeletal factors to be considered during diagnosis and treatment planning . to minimize strain of the soft - tissue chin , the treatment modality should be oriented toward decreasing the u1-npog , overjet , and u1-upper lip values while increasing the odi value . strain of the soft - tissue chin is influenced by not only the underlying dentoskeletal structures but also the thickness , length , and tone of the soft tissue.10 therefore , further studies should include soft - tissue variables to improve the predictability of the soft - tissue profile after orthodontic treatment . given that the present study was limited to only korean women , studies including men or various age or racial groups are warranted to compare the discrepancies arising from these predictive factors . odi , u1-npog , overjet , and u1-upper lip were found to be the discriminants for classifying the female adult subjects with class i or ii molar relationships into the no - strain or strain group , and the prediction rate of these variables for the group classification was 81.8%.in the cart model , u1-npog was the most predictive variable for categorizing the subjects into the no - strain or strain group , with a cut - off value of 14.2 mm.the subjects in the strain group showed a retruded mandible , vertical skeletal pattern , convex profile , and protruded dentoalveolar pattern compared with those in the no - strain group . odi , u1-npog , overjet , and u1-upper lip were found to be the discriminants for classifying the female adult subjects with class i or ii molar relationships into the no - strain or strain group , and the prediction rate of these variables for the group classification was 81.8% . in the cart model , u1-npog was the most predictive variable for categorizing the subjects into the no - strain or strain group , with a cut - off value of 14.2 mm . the subjects in the strain group showed a retruded mandible , vertical skeletal pattern , convex profile , and protruded dentoalveolar pattern compared with those in the no - strain group .

objectiveto investigate the dentoskeletal factors which may predict soft - tissue chin strain during lip closure.methodsthe pretreatment frontal and lateral facial photographs and lateral cephalograms of 209 women ( aged 18 - 30 years ) with angle 's class i or ii malocclusion were examined . the subjects were categorized by three examiners into the no - strain and strain groups according to the soft - tissue chin tension or deformation during lip closure . relationships of the cephalometric measurements with the group classification were analyzed by logistic regression analysis , and a classification and regression tree ( cart ) model was used to define the predictive variables for the group classification.resultsthe lower the value of the overbite depth indicator ( odi ) and the higher the values of upper incisor to nasion - pogonion ( u1-npog , mm ) , overjet , and upper incisor to upper lip ( u1-upper lip , mm ) , the more likely was the subject to be classified into the strain group . the cart showed that u1-npog was the most prominent predictor of soft - tissue chin strain ( cut - off value of 14.2 mm ) , followed by overjet.conclusionsto minimize strain of the soft - tissue chin , orthodontic treatment should be oriented toward increasing the odi value while decreasing the u1-npog , overjet , and u1 upper lip values .

subjects for this nested case - controlled study were selected from ongoing clinical investigations of skeletal muscle insulin resistance . only female subjects were included to provide a sex - controlled analysis . women aged 6075 years were recruited through advertisements in the pittsburgh , pa , area . a brief phone screening initially determined eligibility for study participation . potential subjects who were determined to be sedentary ( exercise 1 day / week ) , weight stable ( 3 kg in the previous 6 months ) , with a bmi > 30 kg / m , and nonsmokers were further evaluated at the clinical translational research center ( ctrc ) at the university of pittsburgh . exclusionary criteria included uncontrolled hypertension ( > 150/95 mmhg ) , anemia ( hematocrit < 34% ) , elevated liver enzymes ( 25% above normal ) , proteinuria , or hypothyroidism ( sensitive thyroid - stimulating hormone [ tsh ] 8 potential subjects were also excluded if they reported taking chronic medications known to affect glucose homeostasis . after the medical screening , potential subjects then completed a 2-h 75-g oral glucose tolerance test to determine glucose tolerance status . subjects with type 2 diabetes determined by fasting glucose > 126 mg / dl were excluded . all subjects gave written consent to the protocol , which was approved by the university of pittsburgh 's institutional review board . insulin sensitivity was determined by a 4-h hyperinsulinemic ( 40 mu m min ) euglycemic clamp and [ 6,6-h2]glucose infusion as previously described ( 28 ) . briefly , subjects reported to the ctrc the evening before the clamp and after a 48-h period , in which they refrained from any vigorous physical activity . a continuous intravenous infusion of insulin ( humulin ; eli lilly , indianapolis , in ) was begun at 7:00 a.m. , and euglycemia ( target 90 mg / dl ) was maintained using an adjustable infusion of 20% dextrose . to measure rate of insulin - stimulated glucose disposal and residual endogenous glucose production , a primed ( 0.22 mol / kg ) continuous ( 17.6 mol kg min ) infusion of [ 6,6-h2]glucose was administered ( 29 ) . [ 6,6-h2]glucose enrichment in plasma was determined by gas chromatography / mass spectrometry ( 6890 network/5973 series ; agilent , santa clara , ca ) as previously described ( 30 ) . plasma glucose was measured by the glucose oxidase method ( 2300 stat plus ; yellow springs instruments , yellow springs , oh ) . fasting plasma insulin concentration was determined by an enzyme - linked immunosorbent assay ( millipore , billerica , ma ) . rate of glucose disposal ( rd ) was calculated using the tracer : tracee method with a modified version of the steele et al . percutaneous muscle biopsy samples were obtained in the fasted state before the glucose clamp , as described previously ( 32 ) . briefly , muscle biopsies were obtained from the vastus lateralis , 15 cm above the patella . a portion of the specimen ( 30 mg ) was snap - frozen in liquid nitrogen and stored at 80c for sphingolipid and diacylglycerol analysis . a second portion ( 20 mg ) was snap - frozen and stored at 80c for gene expression analysis . a third portion of the specimen for histochemistry was mounted on a small piece of cork with mounting medium ( shandon cryochrome ; thermo electron , pittsburgh , pa ) , frozen in isopentane cooled with liquid nitrogen for 23 min ( 160c ) , and then placed into liquid nitrogen . briefly , liquid nitrogen frozen samples ( 30 mg ) were homogenized in ice - cold buffer ( 250 mmol / l sucrose , 25 mmol / l kcl , 50 mmol / l tris , and 0.5 mmol / l edta , ph 7.4 ) . total content as well as the various molecular species of diacylglycerol and sphingolipid were measured by high - performance liquid chromatography tandem mass spectrometry as previously described in detail ( 33 ) . plasma free fatty acid concentrations were determined by gas chromatography with flame ionization detection ( 6890n ; agilent , santa clara , ca ) using heptanoic acid as an internal standard . free fatty acids were derivatized with dimethylamine using deoxo - fluor reagent ( sigma - aldrich , st . histochemical analysis was performed on serial sections using a modified version of methods previously used in our laboratory ( 32 ) . briefly , mounted biopsy samples were sectioned ( 10 m ) on a cryostat ( cryotome e ; thermo shandon , pittsburgh , pa ) at 20c and placed on a glass slide . sections were then stained in a filtered solution of oil red o ( 300 mg / ml in 36% triethylphosphate ) for 30 min at room temperature . thereafter , sections were incubated with primary antibodies for anti - human myosin heavy chain ( myh)-7 ( type i myocytes ) and myh2 ( type iia myocytes ) overnight at room temperature and subsequently incubated with fluorescein ( fitc ) ( type iia myocytes ) and rhodamine ( type i myocytes ) conjugated secondary antibodies ( santa cruz biotechnology , santa cruz , ca ) . images were visualized using a leica microscope ( leica dm 4000b ; leica microsystems , bannockburn , il ) , digitally captured ( retiga 2000r camera ; q imaging , surrey , canada ) , and analyzed using specialized software ( northern eclipse , v6.0 ; empix imaging , cheektowaga , ny ) . oil red o staining intensity and cross - sectional area were determined in type i and type ii myocytes . gene expression analysis was carried out in a subset of subjects from each group , based on availability of vastus lateralis biopsy specimen ( six ir and six is ) . isolated rna was then further purified using a silicon - membrane spin column including an on - column dnase i treatment ( qiagen , valencia , ca ) . total rna was reverse - transcribed using moloney murine leukemia virus ( mmlv ) reverse transcriptase ( sabiosciences , frederick , md ) . gene expression was determined by quantitative real - time pcr ( qrt - pcr ) using sybr green - based custom rt profiler pcr arrays ( sabiosciences , frederick , md ) . qrt - pcr was performed on an abi prism 7900 ( applied biosystems , foster city , ca ) . baseline , threshold cycle ( ct ) and threshold were determined and set for all samples . the relative level of mrna expression for each gene in each sample was normalized to the average expression of 2-microglobulin , hypoxanthine phosphoribosyltransferase ( hprt1 ) , glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) , and -actin ( actb ) and expressed as 2 . total body fat and lean mass were assessed using dual - energy x - ray absorptiometry ( ge lunar prodigy and encore 2005 software v9.3 ) . subjects performed a vo2 peak test on an electronically braked cycle ergometer to determine aerobic fitness ( sensor medics , yorba linda , ca ) as previously described ( 34 ) . insulin sensitivity was determined by a 4-h hyperinsulinemic ( 40 mu m min ) euglycemic clamp and [ 6,6-h2]glucose infusion as previously described ( 28 ) . briefly , subjects reported to the ctrc the evening before the clamp and after a 48-h period , in which they refrained from any vigorous physical activity . a continuous intravenous infusion of insulin ( humulin ; eli lilly , indianapolis , in ) was begun at 7:00 a.m. , and euglycemia ( target 90 mg / dl ) was maintained using an adjustable infusion of 20% dextrose . to measure rate of insulin - stimulated glucose disposal and residual endogenous glucose production , a primed ( 0.22 mol / kg ) continuous ( 17.6 mol kg min ) infusion of [ 6,6-h2]glucose enrichment in plasma was determined by gas chromatography / mass spectrometry ( 6890 network/5973 series ; agilent , santa clara , ca ) as previously described ( 30 ) . plasma glucose was measured by the glucose oxidase method ( 2300 stat plus ; yellow springs instruments , yellow springs , oh ) . fasting plasma insulin concentration was determined by an enzyme - linked immunosorbent assay ( millipore , billerica , ma ) . rate of glucose disposal ( rd ) was calculated using the tracer : tracee method with a modified version of the steele et al . percutaneous muscle biopsy samples were obtained in the fasted state before the glucose clamp , as described previously ( 32 ) . briefly , muscle biopsies were obtained from the vastus lateralis , 15 cm above the patella . a portion of the specimen ( 30 mg ) was snap - frozen in liquid nitrogen and stored at 80c for sphingolipid and diacylglycerol analysis . a second portion ( 20 mg ) was snap - frozen and stored at 80c for gene expression analysis . a third portion of the specimen for histochemistry was mounted on a small piece of cork with mounting medium ( shandon cryochrome ; thermo electron , pittsburgh , pa ) , frozen in isopentane cooled with liquid nitrogen for 23 min ( 160c ) , and then placed into liquid nitrogen . briefly , liquid nitrogen frozen samples ( 30 mg ) were homogenized in ice - cold buffer ( 250 mmol / l sucrose , 25 mmol / l kcl , 50 mmol / l tris , and 0.5 mmol / l edta , ph 7.4 ) . total content as well as the various molecular species of diacylglycerol and sphingolipid were measured by high - performance liquid chromatography tandem mass spectrometry as previously described in detail ( 33 ) . plasma free fatty acid concentrations were determined by gas chromatography with flame ionization detection ( 6890n ; agilent , santa clara , ca ) using heptanoic acid as an internal standard . free fatty acids were derivatized with dimethylamine using deoxo - fluor reagent ( sigma - aldrich , st . histochemical analysis was performed on serial sections using a modified version of methods previously used in our laboratory ( 32 ) . briefly , mounted biopsy samples were sectioned ( 10 m ) on a cryostat ( cryotome e ; thermo shandon , pittsburgh , pa ) at 20c and placed on a glass slide . sections were then stained in a filtered solution of oil red o ( 300 mg / ml in 36% triethylphosphate ) for 30 min at room temperature . thereafter , sections were incubated with primary antibodies for anti - human myosin heavy chain ( myh)-7 ( type i myocytes ) and myh2 ( type iia myocytes ) overnight at room temperature and subsequently incubated with fluorescein ( fitc ) ( type iia myocytes ) and rhodamine ( type i myocytes ) conjugated secondary antibodies ( santa cruz biotechnology , santa cruz , ca ) . images were visualized using a leica microscope ( leica dm 4000b ; leica microsystems , bannockburn , il ) , digitally captured ( retiga 2000r camera ; q imaging , surrey , canada ) , and analyzed using specialized software ( northern eclipse , v6.0 ; empix imaging , cheektowaga , ny ) . oil red o staining intensity and cross - sectional area were determined in type i and type ii myocytes . gene expression analysis was carried out in a subset of subjects from each group , based on availability of vastus lateralis biopsy specimen ( six ir and six is ) . isolated rna was then further purified using a silicon - membrane spin column including an on - column dnase i treatment ( qiagen , valencia , ca ) . total rna was reverse - transcribed using moloney murine leukemia virus ( mmlv ) reverse transcriptase ( sabiosciences , frederick , md ) . gene expression was determined by quantitative real - time pcr ( qrt - pcr ) using sybr green - based custom rt profiler pcr arrays ( sabiosciences , frederick , md ) . qrt - pcr was performed on an abi prism 7900 ( applied biosystems , foster city , ca ) . baseline , threshold cycle ( ct ) and threshold were determined and set for all samples . the relative level of mrna expression for each gene in each sample was normalized to the average expression of 2-microglobulin , hypoxanthine phosphoribosyltransferase ( hprt1 ) , glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) , and -actin ( actb ) and expressed as 2 . total body fat and lean mass were assessed using dual - energy x - ray absorptiometry ( ge lunar prodigy and encore 2005 software v9.3 ) . subjects performed a vo2 peak test on an electronically braked cycle ergometer to determine aerobic fitness ( sensor medics , yorba linda , ca ) as previously described ( 34 ) . ten insulin - sensitive ( is ) and 12 insulin - resistant ( ir ) women were matched for age , body mass , bmi , and physical fitness ( vo2 peak ) ( table 1 ) . there was no significant group difference in fasting plasma glucose , insulin , or free fatty acid levels . however , by design , rd during the hyperinsulinemic - euglycemic clamp was greater ( p < 0.01 ) in the is group when compared with the ir group ( table 1 ) . the 2-h plasma glucose levels during the oral glucose tolerance test were also significantly higher in the ir group compared with the is group ( p = 0.016 ) . the ir ( n = 6 ) and is ( n = 6 ) subgroups for gene expression analysis had similar characteristics as their respective groups overall ( data not shown ) . type i and ii myocyte content and myocyte average cross - sectional area are presented in table 2 . the is group had a higher proportion of type i myocytes ( p = 0.002 ) and a lower proportion of type iia ( p = 0.014 and type iix ( p = 0.02 ) than the ir group . when data from both groups were combined , there was a higher proportion of type iia compared with type iix myocytes ( p = 0.008 ) . both groups combined , there was a significant positive correlation between the proportion of type i myocytes and insulin sensitivity and a negative correlation between the proportion of type ii myocytes ( type iia and iix combined ) and insulin sensitivity ( table 3 ) . muscle fiber type distribution and surface area in vastus lateralis muscle * significantly different from is group , p < 0.05 . data generated from analysis of subgroup ( ir , n = 10 ; is , n = 5 ) . correlation of rate of glucose disposal ( ml kg fat - free mass min ) with other study variables when data from both ir and is groups were combined * correlation is significant at the level p < 0.01 . correlation is significant at the level p < 0.05 . the data for muscle content of sphingolipid and diacylglycerol species is presented in fig . we observed higher total levels of ceramide ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , total unsaturated ceramide ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) , and total saturated ceramide ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) in the ir group compared with the is group . we also observed higher levels of specific ceramide species containing the following fatty acids : myristic ( c14:0 ) ( 4.0 0.8 vs. 1.9 0.4 pmol / mg protein , p = 0.044 ) , palmitic ( c16:0 ) ( 58 14 vs. 18 5 pmol / mg protein , p = 0.023 ) , and stearic ( c18:0 ) ( 72 10 vs. 40 7 pmol / mg protein , p = 0.02 ) in ir compared with is muscle . there also tended to be greater content of sphingolipids containing palmitoleic ( c18:1 ) ( 43 11 vs. 19 3 pmol / mg protein , p = 0.077 ) and lignoceric ( c24:0 ) ( 201 25 vs. 123 29 pmol / mg protein , p = 0.06 ) acids and sphingosine ( 3.4 0.6 vs. 2.2 0.3 pmol / mg protein , p = 0.08 ) in ir muscle . when data were combined from both groups , we observed a significant negative correlation between total unsaturated , total saturated , and total ceramide content and insulin sensitivity ( table 3 ) . total ceramide ( r = 0.517 , p < 0.05 ) and total saturated ceramide ( r = 0.523 , p < 0.05 ) were positively correlated with type ii myocyte percentage . however , no significant differences in total or individual diacylglycerol species were observed between is and ir subjects . sphingolipid ( a ) and diacylglycerol ( b ) content in vastus lateralis of ir and is subjects . ir data are expressed relative to is group , which was set to a value of 1 . * significantly different from is group , p < 0.05 total muscle imtg was greater in the ir group compared with the is group ( p = 0.009 ) . type i myocyte specific imtg content was also greater in the ir group compared with the is group ( p = 0.005 ) . specific imtg content between is and ir groups , nor was imtg content different in ir within subtypes of type iia and type iix myocytes . when both groups were combined , we observed a significant negative correlation between type i myocyte imtg content and insulin sensitivity , whereas there was no relationship between insulin sensitivity and type ii myocyte ( type iia and iix combined ) imtg content ( fig . e : imtg content in type i myocytes , type ii myocytes , and total imtg in vastus lateralis from ir and is groups . * significantly different from is , p < 0.05 . ( a high - quality color digital representation of this figure is available in the online issue . ) expression levels of key genes , including facilitated glut member 4 ( glut4 ) , insulin receptor substrate ( irs)-1 , and irs2 were lower in the ir group , indicating insulin resistance in this group at the tissue level in accord with their insulin resistance assessed in vivo . the expression of genes relating to oxidative phosphorylation transcription factors , fatty acid metabolism , ceramide metabolism , and lipid droplet associated proteins were examined . we found lower expression of the transcription factors peroxisome proliferator activated receptor ( ppar)- and ppar- in the ir group compared with the is group . also , expression of ppar- coactivator 1 ( pgc-1 ) and nuclear respiratory factor 1 ( nrf1 ) tended to be lower in ir muscle . furthermore , we observed a lower expression of carnitine palmitoyltransferase 1b ( cpt1b ) and malonyl - coa decarboxylase ( mlycd ) , genes that are related to fatty acid metabolism . gene expression of lipid droplet associated proteins hormone - sensitive lipase ( hsl ) , adipose differentiation related protein ( adrp ) , adipose triglyceride lipase ( atgl ) , and synaptosomal - associated protein 23 ( snap23 ) was lower in the ir group compared with the is group . interestingly , the expression of genes relating to ceramide metabolism was not different between groups . the expression of tumor necrosis factor ( tnf)- tended to be higher in the ir group compared with the is group ( p = 0.09 ) . gene expression in vastus lateralis of ir and is subgroups . ir gene expression is expressed relative to is group , which was set to a value of 1 . abhd5 , abhydrolase domain containing 5 ; acac , acetyl - coa carboxylase ; asah , n - acylsphingosine amidohydrolase ; cerk , ceramide kinase ; cpt , carnitine palmitoyltransferase ; mlycd , malonyl - coa decarboxylase ; nrf , nuclear respiratory factor ; s3 - 12 , plasma membrane associated protein kiaa1881 ; sptlc , serine palmitoyltransferase , long - chain base . * significantly different from is group , p < 0.05 . type i and ii myocyte content and myocyte average cross - sectional area are presented in table 2 . the is group had a higher proportion of type i myocytes ( p = 0.002 ) and a lower proportion of type iia ( p = 0.014 and type iix ( p = 0.02 ) than the ir group . when data from both groups were combined , there was a higher proportion of type iia compared with type iix myocytes ( p = 0.008 ) . both groups combined , there was a significant positive correlation between the proportion of type i myocytes and insulin sensitivity and a negative correlation between the proportion of type ii myocytes ( type iia and iix combined ) and insulin sensitivity ( table 3 ) . muscle fiber type distribution and surface area in vastus lateralis muscle * significantly different from is group , p < 0.05 . data generated from analysis of subgroup ( ir , n = 10 ; is , n = 5 ) . correlation of rate of glucose disposal ( ml kg fat - free mass min ) with other study variables when data from both ir and is groups were combined * correlation is significant at the level p < 0.01 . we observed higher total levels of ceramide ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , total unsaturated ceramide ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) , and total saturated ceramide ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) in the ir group compared with the is group . we also observed higher levels of specific ceramide species containing the following fatty acids : myristic ( c14:0 ) ( 4.0 0.8 vs. 1.9 0.4 pmol / mg protein , p = 0.044 ) , palmitic ( c16:0 ) ( 58 14 vs. 18 5 pmol / mg protein , p = 0.023 ) , and stearic ( c18:0 ) ( 72 10 vs. 40 7 pmol / mg protein , p = 0.02 ) in ir compared with is muscle . there also tended to be greater content of sphingolipids containing palmitoleic ( c18:1 ) ( 43 11 vs. 19 3 pmol / mg protein , p = 0.077 ) and lignoceric ( c24:0 ) ( 201 25 vs. 123 29 pmol / mg protein , p = 0.06 ) acids and sphingosine ( 3.4 0.6 vs. 2.2 0.3 pmol / mg protein , p = 0.08 ) in ir muscle . when data were combined from both groups , we observed a significant negative correlation between total unsaturated , total saturated , and total ceramide content and insulin sensitivity ( table 3 ) . total ceramide ( r = 0.517 , p < 0.05 ) and total saturated ceramide ( r = 0.523 , p < 0.05 ) were positively correlated with type ii myocyte percentage . however , no significant differences in total or individual diacylglycerol species were observed between is and ir subjects . sphingolipid ( a ) and diacylglycerol ( b ) content in vastus lateralis of ir and is subjects . ir data are expressed relative to is group , which was set to a value of 1 . * total muscle imtg was greater in the ir group compared with the is group ( p = 0.009 ) . type i myocyte specific imtg content was also greater in the ir group compared with the is group ( p = 0.005 ) . specific imtg content between is and ir groups , nor was imtg content different in ir within subtypes of type iia and type iix myocytes . when both groups were combined , we observed a significant negative correlation between type i myocyte imtg content and insulin sensitivity , whereas there was no relationship between insulin sensitivity and type ii myocyte ( type iia and iix combined ) imtg content ( fig . myocyte - specific imtg content in vastus lateralis of ir and is groups . a : representative image of oil red o stain ( 20 objective ) . e : imtg content in type i myocytes , type ii myocytes , and total imtg in vastus lateralis from ir and is groups . * significantly different from is , p < 0.05 . ( a high - quality color digital representation of this figure is available in the online issue . ) 3 . expression levels of key genes , including facilitated glut member 4 ( glut4 ) , insulin receptor substrate ( irs)-1 , and irs2 were lower in the ir group , indicating insulin resistance in this group at the tissue level in accord with their insulin resistance assessed in vivo . the expression of genes relating to oxidative phosphorylation transcription factors , fatty acid metabolism , ceramide metabolism , and lipid droplet associated proteins were examined . we found lower expression of the transcription factors peroxisome proliferator activated receptor ( ppar)- and ppar- in the ir group compared with the is group . also , expression of ppar- coactivator 1 ( pgc-1 ) and nuclear respiratory factor 1 ( nrf1 ) tended to be lower in ir muscle . furthermore , we observed a lower expression of carnitine palmitoyltransferase 1b ( cpt1b ) and malonyl - coa decarboxylase ( mlycd ) , genes that are related to fatty acid metabolism . gene expression of lipid droplet associated proteins hormone - sensitive lipase ( hsl ) , adipose differentiation related protein ( adrp ) , adipose triglyceride lipase ( atgl ) , and synaptosomal - associated protein 23 ( snap23 ) was lower in the ir group compared with the is group . interestingly , the expression of genes relating to ceramide metabolism was not different between groups . the expression of tumor necrosis factor ( tnf)- tended to be higher in the ir group compared with the is group ( p = 0.09 ) . gene expression in vastus lateralis of ir and is subgroups . ir gene expression is expressed relative to is group , which was set to a value of 1 . abhd5 , abhydrolase domain containing 5 ; acac , acetyl - coa carboxylase ; asah , n - acylsphingosine amidohydrolase ; cerk , ceramide kinase ; cpt , carnitine palmitoyltransferase ; mlycd , malonyl - coa decarboxylase ; nrf , nuclear respiratory factor ; s3 - 12 , plasma membrane associated protein kiaa1881 ; sptlc , serine palmitoyltransferase , long - chain base . despite a preponderance of evidence from cell culture and animal models suggesting a role for both ceramide and diacylglycerol in skeletal muscle insulin resistance ( 7,8 ) , there is actually no consensus as to whether this is relevant to human insulin resistance ( 12,13,15,17,19 ) . this study was conducted for three purposes : 1 ) to determine whether diacylglycerol or sphingolipid species are elevated in ir human skeletal muscle , 2 ) to investigate whether the associations between intramyocellular lipids are dependent on muscle fiber type , and 3 ) to explore whether the skeletal muscle expression of several key genes relating to lipid metabolism are altered in ir muscle corresponding to intramyocellular lipid content . a primary finding of our study was that total as well as saturated and unsaturated ceramide content was approximately twofold higher in ir skeletal muscle in human subjects independent of obesity , physical fitness , and plasma free fatty acid concentration . ( 13 ) , who observed elevated ceramide content in skeletal muscle of obese subjects . ( 12,14 ) also found that ceramide accumulation in skeletal muscle was inversely related to insulin sensitivity . they also suggested that higher ceramide content in lean offspring of patients with type 2 diabetes was due to decreased ceramide degradation , whereas ir of obesity was caused by an increase in de novo ceramide synthesis ( 12,14 ) . not all studies , however , have observed an association between muscle ceramide content and insulin resistance ( 18,19 ) . these discrepant results could be attributed to differences in the subject population studied , e.g. , age , sex , physical activity , and methodological differences , in assessing ceramide content and insulin sensitivity ( 11 ) . thus , our study provides important new information about the relationship between elevated ceramide content and muscle insulin resistance . this is the first to a comprehensive profile of the content of the various molecular species of ceramide in human ir muscle using tandem mass spectrometry while controlling for obesity , physical fitness , and age . this is the first study to demonstrate elevated levels of both saturated and unsaturated ceramides , as well as specific ceramides containing c14:0 , c16:0 , and c18:0 fatty acids in ir human muscle . none of the prior studies examining ceramide in human muscle have accounted for heterogeneity in muscle type that could account for differences in insulin sensitivity . our novel finding that ceramide content was associated with a higher percentage of type ii myocytes suggests that ceramide accumulation may be fiber - type dependent . in addition , the complementary observations that both the imtg content in type i myocytes and the lower proportion of type i myocytes are related to insulin resistance provide insight into muscle heterogeneity in lipid metabolism in insulin resistance . these data suggest a possibility that type i myofibers may have a greater capacity to buffer increases in free fatty acids by partitioning to lipid droplet imtg , whereas type ii fibers have a reduced capacity to incorporate excess fatty acids into imtg and are more inclined to partition to lipotoxic mediators such as ceramide species . an impairment of skeletal muscle oxidative capacity has been suggested to initiate or potentiate insulin resistance ( 35 ) . our data indicate that the proportion of type i and type ii myocytes is associated with higher ceramide content and insulin resistance and that the expression levels of several genes related to mitochondrial biogenesis and fatty acid metabolism are lower in insulin resistance . therefore , our complementary results support the concept that a low oxidative capacity of muscle may be associated with ceramide accumulation and insulin resistance . this is consistent with previous reports that the increase in whole muscle imtg content in obesity or type 2 diabetes is primarily due to increased imtg in type i myocytes ( 25 ) and that differences in fiber type proportion also play a role in insulin resistance of obesity and type 2 diabetes ( 36,37 ) . these observations are supported by a number of reports suggesting that type i myocytes are more insulin responsive than type ii myocytes ( 26,27,38 ) . yet , differences in fiber type proportion between obese subjects either with or without type 2 diabetes have been reported in some ( 36,37 ) , but not all previous studies ( 22,24 ) . despite evidence in ir animal models supporting a role for dag in insulin resistance ( 8,9 ) , we did not observe an increased level of dag in insulin - resistant muscle . ( 12 ) have also reported elevated levels of ceramide , but not dag , in obese impaired glucose tolerant versus obese normal glucose tolerant ( ngt ) subjects . a possible reason for lipid - induced insulin resistance by ceramide and not dag , and visa versa , may be that higher levels of palmitate result in greater ceramide accumulation , while unsaturated fatty acids partition toward dag ( 39 ) . thus , both ceramides and dag can potentially induce insulin resistance depending on the degree of saturation of the lipid overload . our finding that c16:0 ceramide was more than threefold higher in ir muscle is consistent with this . thus , it is plausible that basal levels of ceramide and dag in muscle are influenced by the degree of saturation and palmitate content in the diet of obese individuals . we also examined the differential expression of several other genes relating to lipid metabolism , which could underlie accumulation of imtg and other bioactive species , i.e. , dag and ceramide . we found that many genes involved in ceramide synthesis and degradation were not differentially expressed in ir muscle with increased ceramide content . a recent report suggests that over expression of acid ceramidase prevents the inhibitory effect of saturated fatty acids on insulin signaling ( 40 ) . also , inhibition of serine palmitoyltranseferase in zucker diabetic fatty ( zdf ) rats , by treatment with the amino acid antibiotic myriocin , prevented the onset of type 2 diabetes and inhibited an increase in ceramide content in muscle , when compared with nontreated zdf rats ( 41 ) . our results suggest that factors other than increased enzyme content , possibly increased enzyme activity or fatty acid flux , contribute to ceramide accumulation in ir muscle . in support of this , straczkowski et al . ( 12 ) demonstrated that the activity of neutral sphingomyelinase , the enzyme that converts sphingomyelin to ceramide , is increased in muscle of obese impaired glucose tolerant subjects compared with obese normal glucose tolerant subjects , concomitant with higher ceramide content . finally , the observation that gene expression of tnf- tended to be elevated in ir muscle , albeit statistically nonsignificant , concomitant with elevated ceramide content is consistent with data demonstrating that tnf- can stimulate de novo ceramide biosynthesis ( 42 ) . to further characterize intramyocellular lipid content and metabolism in ir skeletal muscle , we determined the expression of selected genes coding for lipid droplet proteins and proteins involved in lipid droplet hydrolysis . atgl and hsl expression / activity have been demonstrated to contribute to adipose tissue and skeletal muscle triacylglycerol lipolysis , elevated plasma fatty acid levels , and accumulation of ectopic lipid deposits associated with type 2 diabetes ( 43 ) . our observation that atgl and hsl mrna are lower in ir muscle is in line with reports of lower expression of these lipases in obesity and/or insulin resistance ( 44,45 ) . consistent with this , phillips et al . showed that skeletal muscle protein expression of adrp was upregulated in response to improvements in insulin action induced by weight loss and insulin sensitizer pharmacotherapy ( 46 ) . furthermore , atgl and adrp have been reported to be more highly expressed in type i muscle fibers ( 47,48 ) . the fiber type specific expression of atgl and adrp , and our observation of reduced type i fibers in obese ir subjects , potentially explains the lower level of atgl and adrp observed in whole muscle in the present study . it is tempting to speculate the same may be true for other lipases and that fiber type composition plays a greater role in insulin resistance than previously thought . furthermore , the trend toward higher tnf- gene expression in ir muscle is in line with reports that tnf- represses atgl expression ( 49 ) . we also observed for the first time in human muscle that the expression of snap23 , a member of the snare family of proteins whose function is to mediate intracellular vesicle fusion events including glut4 transport to the plasma cell membrane and lipid droplet fusion ( 50 ) , is lower in ir muscle . this novel finding potentially linking intramyocellular lipid droplets and glucose transport is supported by others describing an association between snap23 and fatty acid induced insulin resistance ( 50 ) . future studies should be conducted to examine whether or not interventions such as exercise or weight loss affect oxidative capacity , dag , ceramide , and insulin sensitivity to better understand these associations in humans . although we substantiated our insulin resistance phenotype with expression of key genes involved in insulin signaling and glucose transport , additional studies are needed to link intramyocellular lipids , fiber type , oxidative capacity , and activation of specific insulin - signaling proteins . taken together , however , these results suggest a role for muscle fiber type proportion and/or the oxidative capacity of muscle in insulin resistance , possibly through partitioning muscle lipids into either triglycerides or ceramides . in summary , ceramide but not diacylglycerol is elevated in ir human skeletal muscle independent of obesity , physical activity , or aerobic fitness . moreover , muscle fiber type as well as fiber type specific lipid content is associated with insulin resistance in muscle . importantly , key genes associated with fatty acid metabolism and lipid droplet regulation are decreased in ir muscle . together , these data suggest that mechanisms underlying the associations between intramyocellular lipids and insulin resistance likely involve ceramide , which in turn may depend on the muscle fiber type or its oxidative capacity .

objectivewe tested the primary hypotheses that sphingolipid and diacylglycerol ( dag ) content is higher within insulin - resistant muscle and that the association between intramyocellular triglycerides ( imtg ) and insulin resistance is muscle fiber type specific.research design and methodsa nested case - control analysis was conducted in 22 obese ( bmi > 30 kg / m2 ) women who were classified as insulin - resistant ( ir ; n = 12 ) or insulin - sensitive ( is ; n = 10 ) , determined by hyperinsulinemic - euglycemic clamp ( > 30% greater in is compared with ir , p < 0.01 ) . sphingolipid and dag content was determined by high - performance liquid chromatography tandem mass spectrometry . fiber type specific imtg content was histologically determined . gene expression was determined by quantitative pcr.resultstotal ( 555 53 vs. 293 54 pmol / mg protein , p = 0.004 ) , saturated ( 361 29 vs. 179 34 pmol / mg protein , p = 0.001 ) , and unsaturated ( 198 29 vs. 114 21 pmol / mg protein , p = 0.034 ) ceramides were higher in ir compared with is . dag concentrations , however , were similar . imtg content within type i myocytes , but not type ii myocytes , was higher in ir compared with is subjects ( p = 0.005 ) . insulin sensitivity was negatively correlated with imtg within type i myocytes ( r = 0.51 , p = 0.026 ) , but not with imtg within type ii myocytes . the proportion of type i myocytes was lower ( 41 vs. 59% , p < 0.01 ) in ir subjects . several genes involved in lipid droplet and fatty acid metabolism were differentially expressed in ir compared with is subjects.conclusionshuman skeletal muscle insulin resistance is related to greater imtg content in type i but not type ii myocytes , to greater ceramide content , and to alterations in gene expression associated with lipid metabolism .

do czstych powika mcs nale : incydenty zakrzepowe ( w tym zakrzepica urzdzenia ) , incydenty krwotoczne oraz hemoliza . ocena zmian w czasie wybranych parametrw ukadu krzepnicia u chorych ze schykow niewydolnoci serca leczonych za pomoc mcs i terapii przeciwzakrzepowej . przez 6 tygodni obserwowano 16 pacjentw ze schykow niewydolnoci serca ( sze pobra krwi dla kadego pacjenta ) . co tydzie oceniano rozszerzony panel bada ukadu krzepnicia , w tym czas czciowej tromboplastyny po aktywacji , czas protrombinowy , midzynarodowy wspczynnik znormalizowany , aktywno czynnika von willebranda ( vwf ) , aktywno czynnika viii , stenie fibrynogenu , d - dimeru , czynno pytek pod wpywem kwasu arachidonowego ( aspi test ) i dwufosforanu adenozyny ( adp test ) , aktywujcego receptora trombinowego peptydu 6 ( trap test ) i kolagenu ( col test ) . mediana czasu od momentu wszczepienia urzdzenia wspomagajcego prac lewej komory serca ( lvad ) wynosia 120 dni ( 100150 dni ) . podczas badania stenia d - dimeru oraz fibrynogenu byy podwyszone we wszystkich szeciu oznaczeniach , nie stwierdzono istotnych rnic midzy kolejnymi oznaczeniami . aktywno vwf i czynnika viii byy podwyszone w pierwszych dwch oznaczeniach , w kolejnych oznaczeniach obserwowano ich zmniejszenie . u pacjentw stosujcych mcs wykazano znaczne zmiany w parametrach hemostazy zarwno w ukadzie krzepnicia , jak i aktywnoci pytek krwi . heart transplantation remains the first - line therapy in the management of end - stage heart failure . however , the number of transplants is estimated at less than 3,500 yearly worldwide , which is obviously inadequate to meet the demand in this rapidly growing population of heart failure patients [ 1 , 2 ] . mechanical circulatory support ( mcs ) is an umbrella term describing various technologies used in both short- and long - term management of patients with either end - stage chronic heart failure ( hf ) or acute hf . bridge to transplant to support patients who are unable to wait any longer for a heart transplant , or , more recently , as destination therapy for older patients suffering from end - stage heart failure and who have contraindications to heart transplantation [ 3 , 4 ] . mechanical circulatory support includes a left ventricular assist device ( lvad ) or a bi - ventricular assist device ( bi - vad ) . however , mcs therapy is not without risk . recently , clinicians have identified common mcs therapy - associated complications : pump thrombosis , bleeding , and hemolysis . the incidence of lvad thrombosis is 213% of adult patients with a continuous - flow lvad ( axial flow 413% , centrifugal flow 2% ) . therapeutic options include surgical procedures ( device exchange , catheter - based thrombectomy ) and medical therapy . the latter may consist of : thrombolytic therapy with recombinant tissue plasminogen activator , intensified anticoagulation treatment with unfractionated heparin , bivalirudin , intensified antiplatelet treatment with intravenous gp iib / iiia inhibitors , or with thienopyridine - derivative p2y12 adp receptor inhibitor . however , there are no unified guidelines as to the antithrombotic therapy [ 7 , 8 ] . the incidence of lvad - associated bleeding , depending on its definition , varies widely between 10% and 50% , with no difference in the overall bleeding rates between axial- and pulsatile - flow devices . much of the bleeding risk is attributed to the antithrombotic regimen . however , there are reports that the observed increased risk of bleeding is higher than would be anticipated from antithrombotic therapy alone . given the coexistence of thrombo - embolic and hemorrhagic complications , monitoring of hemostasis using thromboelastometry/-graphy and platelet function analysis hemostasis assays should be used to reduce the risk of bleeding and thrombo - embolic complications during mcs therapy and antithrombotic management . therefore , we set out to determine time - dependant changes of selected hemostasis parameters in patients with end - stage heart failure treated with mcs and antithrombotic therapy . the study was approved by the local bioethics committee , and all patients gave written consent to participate in the study . sixteen patients with end - stage heart failure on lvad were followed for 6 weeks ( six blood samples for each patient ) . given the various effects of continuous - flow lvad and pulsatile - flow lvad on hemostasis , we included in the current study only continuous - flow lvad recipients . the pump is driven by a rotating magnetic levitated impeller and has a capacity of up to 15,000 rpm , resulting in a theoretical maximal blood flow of 810 l / min . it is a rotational pump with a magnetic levitating rotor ( similar to a propeller ) . the blood flow is not axial , because inflow and outflow axes are arranged in a 90 degrees angle . moreover , this pump runs with a lower rotation speed of 1,000 to 2,500 rpm and generates up to 10 l / min flow . every week an extended hemostasis panel was assessed , including activated partial thromboplastin time ( aptt ) , prothrombin time ( pt ) , international normalized ratio ( inr ) , von willebrand factor ( vwf ) activity , factor viii activity , fibrinogen level , d - dimer , platelet response to arachidonic acid ( aspi test ) and adenosine diphosphate ( adp test ) , thrombin receptor activating peptide-6 ( trap test ) and collagen ( col test ) . fasting venous blood samples were taken each week , and all tests were performed within one hour of blood samples collection . whole venous blood samples were collected from each patient and placed in 2 tubes of 4 ml with 109 mmol of sodium citrate ( 3.2% ) and in 2 tubes of 3 ml with > 15 g / ml of hirudin . plasma was separated by centrifugation at 2000 g for 15 min at ambient temperature ( 2025c ) . the vwf activity and d - dimers were evaluated by an immuno - turbidimetric assay , using a fully automated hemostasis analyzer ( bcs xp system , innovance siemens healthcare , usa ) . factor viii ( % activity of normal plasma ) and coagulant fibrinogen ( mg / dl ) were determined by chronometric techniques by means of fully automated hemostasis analyzers ( bcs xp system , siemens healthcare , usa ) . prothrombin time ( pt , % time of normal plasma ) , inr , and aptt ( s ) were also assessed by chronometric techniques . platelet aggregation was tested in physiological calcium conditions by the multiplate analyzer ( dynabyte , munich , germany ) , using agonists of thrombin receptor activating peptide-6 ( trap-6 ) , arachidonic acid ( aspi ) , adenosine diphosphate ( adp ) , and a collagen binding activity assay ( col ) . the antithrombotic protocol involved the use of unfractionated heparin according to the aptt ( 6080 s ) starting on postoperative day ( pod ) 1 . on pod 2 warfarin was started to achieve inr of 2.02.5 in continuous - flow pumps . in addition , aspirin and/or clopidogrel were used antiplatelet agents , starting from pod 2 . the type and dose of antithrombotic regimen were at the discretion of the attending physician . friedman analysis of variance ( anova ) test followed by bonferroni correction and the wilcoxon matched - pairs signed - ranks test were employed to test the difference between repeated laboratory tests during 6-week follow - up . friedman analysis of variance ( anova ) test followed by bonferroni correction and the wilcoxon matched - pairs signed - ranks test were employed to test the difference between repeated laboratory tests during 6-week follow - up . baseline clinical characteristic are presented in table i. the study population comprised 16 men with a median age of 41 ( interquartile range : 2449 ) . the d - dimer and fibrinogen concentrations were elevated but remained similar throughout all six measurements . meanwhile factor viii and vwf activities were elevated in the first two measurements and then subsequently declined . baseline clinical characteristics coagulation assay results in studied patients time - dependant changes in platelet function we set out to determine time - dependant patterns in hemostasis parameters in patients with end - stage heart failure treated with lvad . first , d - dimer and fibrinogen concentrations were elevated but remained similar throughout all six measurements . second , factor viii and vwf concentrations were elevated at the beginning of the study and then steadily declined . finally , despite the fluctuation in hemostasis parameters , there were no hemorrhagic or thrombo - embolic events during the study period . currently , long - term survival in carefully selected patients on mcs is much better than with medical therapy . however , mcs therapy is hampered by , sometimes life - threatening , complications including bleeding and device thrombosis . left ventricular assist device thrombosis etiology is multifactorial and thus presents complex and challenging problems in the diagnosis and management of such patients . the conditions associated with lvad thrombosis are divided into three large groups : ( a ) pump - related , ( b ) patient - related , and ( c ) management - related . left ventricular assist device thrombosis occurs in 213% of adult patients with a continuous - flow lvad ( axial - flow : 413% , centrifugal - flow : 2% ) . thrombus may be formed at various sites , i.e. left ventricle , inflow cannula , pump housing , outflow cannula , outflow graft , or the aortic root , thus leading to serious cardiovascular events including thromboembolic stroke , peripheral thromboembolism , lvad malfunction with reduced systemic flows or life - threatening hemodynamic impairment , cardiogenic shock , and even death [ 6 , 15 ] . the four clinical signs of lvad thrombosis recognized are as follows : ( a ) isolated power elevations , ( b ) isolated ldh rise , ( c ) evidence of hemolysis , and ( d ) new heart failure symptoms . the multidisciplinary and multi - institutional ishlt group proposed an algorithm for the diagnosis and management of lvad thrombosis . left ventricular assist device patients experience significant baseline activation of endothelial and coagulation systems , further accentuated in the early postoperative period . more importantly , prolonged activation of the endothelial and coagulation systems was also reported , which may indicate activation of the extrinsic ( tissue factor ) pathway of thrombosis mediated by sustained endothelial dysfunction in these patients . elevated d - dimer concentrations , as found in our study , may reflect chronic , ongoing activation of the coagulation system . elevated concentrations of fibrinogen , factor viii , and vwf might result in a hypercoagulable state resulting in lvad thrombosis . more importantly , we have to keep in mind that factor viii is an acute - phase protein ( malignancy , chronic diseases , infections ) . thus , genetic or acquired conditions determining high factor levels might predispose patients to lvad thrombosis . the vwf has a pivotal role in thrombogenesis , and high plasma levels of vwf have been associated with increased risk of thrombosis . a varied response to the antiplatelet regimen , as demonstrated during the study period , one study found that asa doses at or below 81 mg / day were an independent predictor of device thrombosis . reported that pump thrombosis was preceded by an almost surprising increase in platelet aggregation induced by aspi and adp . thus the authors argue that an increase of platelet reactivity or non - response ( or low response ) to antiplatelet therapy may , in part , play a role in pump thrombosis . indeed , elevated expression of platelet membrane receptors , namely cd62p and cd63 , have been reported in patients on lvad . left ventricular assist device thrombosis has been reported in non - responders to aspirin and clopidogrel . in addition to thrombosis , bleeding has recently been identified as one of the most common adverse events of lvad therapy and is the major cause of morbidity . having said that , some authors report that the increased risk of bleeding is higher than would be attributed to antithrombotic therapy . the most commonly reported sources of bleeding are epistaxis , gi bleeding , bleeding of the mediastinum and thorax , and intracranial hemorrhage . the incidence of bleeding , depending on its definition , varies widely between 10% and 50% , with no difference in the overall bleeding rates between axial- and pulsatile - flow devices . the mechanisms underlying bleeding in lvad patients are complex and not yet fully understood , and include colonic dysplasia , concomitant use of anticoagulant and antiplatelet agents , and the presence of acquired bleeding diathesis ( acquired von willebrand syndrome type 2a resulting from the deficiency of high - molecular - weight vwf multimers ) . as such , high - molecular - weight vwf multimers declined in patients with lvad [ 27 , 28 ] , but returned to normal in 6 patients after heart transplantation . however , in the present study we measured only vwf antigen , which is generally within the normal range or elevated and can not be used in the confirmation or exclusion of acquired von willebrand syndrome type 2a . the sensitivity of various laboratory tests for vws2a in patients with bleeding gi dysplasia is as follows : gel electrophoresis ( quantification of hmwm ) > pfa-100 closure time > vwf : rco > bleeding time > vwf : ag [ 15 , 30 ] . it would seem that lvad recipients achieve a new equilibrium between prothrombotic and prohemorrhagic states . more importantly , there is a fine and complex balance in the management of such patients of overcoagulation and undercoagulation . management protocols for lvad are usually institution - dependent , and unfortunately , there is a large variability in clinician - related factors . we have to keep in mind that the one size fits all approach is impractical and ineffective . therefore , individually tailored antithrombotic therapy protocols must exist in centers managing lvad recipients . we did not assess the dysfunction of vwf ( in particular the deficit of high molecular weight multimers of vwf assessed in gel electrophoresis ) . moreover , we did not use thromboelastographic ( teg ) monitoring , which could have provided additional information , as teg is uniquely capable of showing the combined interaction of coagulation factors , platelet content , and platelet function in the process of clot production in whole blood . patients with end - stage heart failure on lvad therapy demonstrate significant time - dependant changes in hemostasis parameters that could be attributable to the risk of developing both thrombotic and bleeding events . however , further studies are needed to determine whether these changes could serve as biomarkers of such events . the creation of the paper was supported by a grant funded by the medical university of silesia ( no .

introductionmechanical circulatory support ( mcs ) therapy is associated with the improvement of long - term prognosis in patients with end - stage heart failure . for years it has been used as a bridge to transplant . however , more recently it is even being used as a destination therapy . recently , clinicians have identified common mcs therapy - associated complications : pump thrombosis , bleeding , and hemolysis . these complications are very challenging with regard to both diagnosis and management.aimto determine time - dependant changes of selected hemostasis / coagulation parameters in patients with end - stage heart failure treated with mcs and antithrombotic therapy.material and methodssixteen patients with end - stage heart failure on left ventricular assist device ( lvad ) were followed for 6 weeks ( six blood samples for each patient ) . every week an extended hemostasis panel was assessed , including activated partial thromboplastin time , prothrombin time , international normalized ratio , von willebrand factor ( vwf ) activity , factor viii activity , fibrinogen level , d - dimer , platelet response to arachidonic acid ( aspi test ) and adenosine diphosphate ( adp test ) , thrombin receptor activating peptide-6 ( trap test ) and collagen ( col test).resultsthe study population comprised 16 men . the median time from lvad implantation was 120 days ( 100150 days ) . during the study period the d - dimer and fibrinogen concentrations were elevated but remained similar throughout all six measurements . meanwhile factor viii and vwf activities were elevated in the first two measurements and then subsequently declined . inhibition of platelet aggregation was greater early after lvad implantation . during subsequent weeks the inhibition of platelet aggregation was less pronounced . no patient developed any bleeding or thrombo - embolic event during the study period.conclusionspatients on mcs therapy demonstrate significant time - dependant changes in hemostasis parameters ( both in the coagulation system and platelet aggregation ) .

the antimetabolite 5-fluorouracil ( fu ) is widely used in the treatment of solid tumors , including gastrointestinal , breast , head , and neck cancers . cardiotoxicity , a rare adverse effect of 5-fu , has a reported incidence of 1.2% to 18% . the patient presented with typical angina and electrocardiographic changes suggestive of an ischemic coronary event during the continuous infusion of 5-fu . the ischemia recurred when the infusion was stopped , and was relieved by administration of nitroglycerin followed by a sublingual calcium channel blocker . an 83-year - old man who had been diagnosed with stage iiic ( t4n2m0 ) adenocarcinoma of the ascending colon and who had undergone a right hemicolectomy was admitted to inje university seoul paik hospital to begin adjuvant chemotherapy , using the folfox4 regimen ( 400 mg / m 5-fu bolus infusion followed by the continuous infusion of 600 mg / m 5-fu for 22 hours on days 1 and 2 ; 200 mg / m leucovorin as a continuous infusion for 2 hours before 5-fu infusion on days 1 and 2 ; and infusion of 85 mg / m oxaliplatin on day 1 ) . baseline echocardiography performed 1 month before admission showed normal left ventricular systolic function and no regional wall motion abnormality . he was treated with 1,500 mg high - dose 5-fu ( 1,000 mg / m ) per day . on the morning of the 3rd day , he developed a severe , substernal , crushing chest pain during the continuous intravenous infusion of 5-fu ( cumulative dose 1,679 mg / m ) , which was partially relieved by administering sublingual nitroglycerin . the electrocardiogram ( ecg ) showed st segment elevation with a tall t wave in leads i , avl , and v4 - 6 , and reciprocal st segment depression in leads v1 - 2 ( fig . the troponin - i and ck - mb levels were 0.010 ng / ml ( reference range , 0.1 ) and 3.73 ng / ml ( reference range , 4.94 ) , respectively . severe hypokinesia of the lateral wall of the left ventricle was noted on a portable bedside echocardiogram . the 5-fu infusion was stopped , and the chest pain and electrocardiographic changes resolved after intravenous infusion of nitroglycerin at 30 g / min . emergency coronary angiography was then performed , which revealed significant stenosis in the proximal left circumf lex coronary artery ( lcx ) . intracoronary nitroglycerin ( 200 g ) was injected to exclude coronary vasospasm , but no change occurred ( fig . 2 ) . intravenous ultrasound ( ivus ) showed severe luminal narrowing with a heavy concentric plaque in the proximal lcx . percutaneous coronary intervention of the proximal lcx lesion was performed successfully with the implantation of a drug - eluting stent ( 3.5 16 mm ; taxus , national medical center , seoul , korea ) ( fig . the patient was transferred to the coronary - care unit , where 8 hours later , he reported a recurrence of the anterior chest pain . the ecg also showed st segment elevation and reciprocal st changes similar to those seen in the previous ischemic events ( fig . the chest pain and ecg changes persisted despite a 100 g / min nitroglycerin infusion . to rule out acute stent thrombosis , the chest pain and ecg changes were relieved after sublingual administration of 10 mg nifedipine . repeated coronary angiography showed a widely patent stent ( fig . 4 ) . the postprocedural troponin - i and ck - mb levels were 0.010 and 4.62 ng / ml , respectively . echocardiography performed the next day also showed the absence of the regional wall motion abnormality and normal left ventricular systolic function . we present here a case of severe cardiotoxicity mimicking acute anterolateral myocardial infarction occurring in a patient receiving 5-fu chemotherapy for adenocarcinoma of the colon . the cessation of 5-fu administration and the subsequent initiation of treatment with a sublingual calcium channel blocker and nitrate resulted in a successful outcome . although the mechanism by which 5-fu exerts its cardiotoxic effects is unknown , the resolution of the patient 's chest pain and the normalization of his ecg changes with a vasodilator strongly support the vasospastic hypothesis of 5-fu cardiotoxicity . one study postulated that 5-fu - associated cardiotoxicity is due to the uncoupling of the electromechanical mechanisms that underlie normal myocardial function , which might be mediated at the level of the cell membrane . recently , kuzel et al . suggested that 5-fu promotes a hypercoagulable state ( e.g. , coronary artery thrombosis ) and observed a significant increase in fibrinopeptide a and a decrease in protein c activity during 5-fu administration . the incidence of clinically apparent 5-fu cardiotoxicity is less than 10% in patients receiving the drug . patients with a history of coronary artery disease ( cad ) have a significantly increased risk of 5-fu - induced cardiotoxicity . although our patient did not have a history of cad , a large atheromatous plaque was found on coronary angiography and ivus . therefore , during 5-fu infusion , close , careful monitoring of patients , especially those with pre - existing cad or cad risk factors , is mandatory . prophylactic calcium channel blockers or nitrates should be administered to patients with cad during 5-fu administration , to prevent vasospasm . one study proposed that impaired renal function is also a risk factor for 5-fu cardiotoxicity . although it is not clear whether the cardiotoxic metabolites undergo renal excretion , the pathophysiological effect of 5-fu on the myocardium is likely to increase with decreased renal function . thus it is necessary to clarify which patients may benefit from optimum anti - angina prophylaxis and careful , close monitoring . the incidence of 5-fu - related cardiotoxicity appears to be dependent on the dosage and delivery system . infusion of 5-fu , which is now being used more frequently and at higher doses instead of bolus therapy , may be a significant factor in the development of 5-fu cardiotoxicity . in one study , nine patients treated with a higher - dose ( > 800 mg / m ) continuous infusion of 5-fu died suddenly . interestingly , despite stopping the 5-fu , the chest pain and ecg changes recurred in our case . one series reported that 19% of the patients developed reversible angina pectoris during treatment , which lasted for up to 12 hours after cessation of the infusion . the possible mechanisms of delayed angina are the late release of potent vasoactive 5-fu metabolites , which accumulate over time due to degradation of 5-fu . therefore , a calcium channel blocker or nitrates should be administered after stopping the 5-fu when 5-fu - induced cardiotoxicity occurs . the long - term outcome of patients with 5-fu - related cardiotoxicity has not been investigated extensively . as with our case , patel et al . recently reported interval improvements in the left ventricular wall motion abnormalities in echocardiography performed 8 to 15 days following the initial study . when cardiotoxicity occurs , 5-fu treatment is usually discontinued due to its very high recurrence rate ( 90% ) . the re - administration of 5-fu is not recommended , and a different chemotherapy regimen should be considered . interestingly , meydan et al . continued 5-fu chemotherapy in one group who experienced 5-fu cardiotoxicity due to the absence of an alternative drug and found that subsequent serious , hemodynamic consequences were easily controlled with nitrate treatment . for the remaining patients , either 5-fu was removed from the combination regimen or an alternative drug was started and no cardiotoxicity developed subsequently . in summary , although 5-fu - associated cardiac toxicity is rare , it may cause angina , myocardial infarction , and even sudden death . physicians should be aware of this potentially lethal side effect and should start the proper treatment when 5-fu cardiotoxicity develops . this case supports the vasospastic hypothesis of 5-fu cardiac toxicity , indicating that a calcium channel blocker may be effective for prevention or treatment of 5-fu cardiotoxicity .

cardiotoxicity associated with 5-fluorouracil ( fu ) is an uncommon , but potentially lethal , condition . the case of an 83-year - old man with colon cancer who developed chest pain during 5-fu infusion is presented . the electrocardiogram ( ecg ) showed pronounced st elevation in the lateral leads , and the chest pain was resolved after infusion of nitroglycerin . a coronary angiogram ( cag ) revealed that the patient had significant atherosclerosis in the proximal left circumflex artery . coronary artery spasm with fixed stenosis was considered , and a drug - eluting stent was implanted . after 8 hours , the patient complained of recurring chest pain , paralleled by st elevation on the ecg . the chest pain subsided after administration of intravenous nitroglycerin followed by sublingual nifedipine . repeated cag showed patency of the previous stent . this case supports the vasospastic hypothesis of 5-fu cardiac toxicity , indicating that a calcium channel blocker may be effective in the prevention or treatment of 5-fu cardiotoxicity .

genetic disorders caused by mutations in the -globin gene are widely known as the human -hemoglobinopathies , in which -thalassemia and sickle cell disease ( scd ) are the most prevalent ones , particularly in the mediterranean , africa , and southeast asia , leading to great mortality and morbidity [ 14 ] . the high occurrence of the -thalassemia and scd mutations is due to the reason that both cause mild severity of malarial infection in the heterozygous state [ 57 ] . however , in the homozygous state , these mutations shorten the lives of affected ones . -thalassemia is caused by the inherited mutations in the -globin gene complex , resulting a total absence or severe decrease in the production of -globin chains [ 8 , 9 ] . the lack of -globin chain production leads to the accumulations and precipitations of free intracellular -globin chains , which may consequently result in premature hemolysis of red blood cells and apoptosis of erythroid precursor [ 8 , 10 , 11 ] . therefore , the combining effects of ineffective erythropoiesis , hemolysis , and hypersplenism are the main culprit of severe anemia in -thalassemia patients . it is characterized by the abnormal appearance of the red blood cells which are rigid and sickled . scd is attributed to a point mutation at the coding sequence of the -globin gene which causes the substitution of glutamate by valine in the glutamic acid at the sixth position of -globin protein , and thus forming a sickle hemoglobin ( hbs , 22 ) when incorporating into a hemoglobin tetramer [ 13 , 14 ] . hbs will polymerize inside the red blood cells under hypoxic condition , resulting in the alternation of the shape of red blood cells as well as their function . however , long - term transfusion therapy may cause iron overload in patients from the gradual breakdown of transfused blood which may eventually result in cardiac failure and/or even death . though the advance in iron chelation can help to remove excess iron in patients , the survival rate is greatly dependent on the iron chelation regimens . allogeneic hematopoietic stem cell transplantation ( hsct ) is one of the gene transfer therapies aimed at the underlying molecular causes of scd and -hemoglobinopathies . several hundred scd and thalassemia patients have successfully experienced hsct with promising results [ 16 , 17 ] . nevertheless , there is a great likelihood that hsct will be limited to a small proportion of hemoglobinopathy patients as evidence has shown merely younger patients and those who have not developed significant disease complications have gained the best results in hsct . also , most successful transplantations have to utilize stem cells from matched sibling donors making hsct a challenging therapy for some patients . therefore , hsct may not be applicable to many current patients . transferring of - or -hematopoietic stem cells of patients can be another therapy option for -thalassemia patients . it has taken a long period of time to have the clinical gene transfer protocol being approved since the transduction of human hematopoietic stem cells and gene expression must reach certain efficiency and high level . despite the fact that this approach has successfully passed its initial human trial , previous studies reported the issues regarding low autoploidy recombination , insertional mutagenesis , and effect of inserted vectors on the expression of nearby genes could possibly limit the application [ 21 , 22 ] . apart from gene therapy , fetal hemoglobin reactivation by chemical agents appears promising enough to develop into effective interventions to cure human -hemoglobinopathies . previous studies have revealed that homozygous -thalassemia patients will not suffer severe anemia until fetal -globin genes are silenced and that patients carrying hereditary persistence of fetal hemoglobin ( hpfh ) , meaning fetal hemoglobin ( hbf ) is abnormally persisted at high level in adults , will only suffer mild anemia [ 8 , 13 , 2327 ] . more evidences also supported that hpfh can improve the severity of both -thalassemia and scd . therefore , it have been suggested that increasing the synthesis of fetal hemoglobin ( hbf ) by reactivating fetal -globin gene can be a potential therapy in patients suffering -thalassemia or scd . it is expected that the pharmacological induction of hbf can correct the globin chain imbalance in -thalassemia patients , while inhibit hbs polymerization in scd patients [ 2832 ] . in recent years , much effort has been made to identify the naturally occurring inducers and drug treatments which can increase the synthesis of hbf and promote the expression of fetal -globin gene . some chemotherapeutic agents , for example , 5-azacytidine and hydroxyurea , ( hu ) have been reported due to their ability to enhance hbf production [ 31 , 33 , 34 ] . yet , most of these currently identified hbf - inducing agents exhibit low efficacy and specificity , myelotoxicity , and carcinogenesis as well as modest responses to treatment which greatly limit their usefulness in the clinical practice [ 5 , 35 , 36 ] . owing to this , ( i ) discovering novel screening platform for identifying potential inducers with high efficiency and accuracy and ( ii ) identifying new hbf - inducing agents from the natural world with the combination of efficacy , safety , and ease of use will be high on the agenda . with the aim of determining the therapeutic potency of the novel inducing compounds and studying the underlying regulatory mechanism of the embryonic and fetal human globin genes expression , various in vitro and in vivo screening platforms have been widely utilized . for in vitro models , there are six human cell lines carrying an embryonic - hbf phenotype ; they are k562 human chronic myelogenous leukemia cells , m - tat , nsmeg , ocim1 , ocmi2 , and as - e2 , while k562 cell line is one of the most well - known and widely used screening platforms for hbf inducers [ 13 , 30 ] . another seven human cell lines which are capable of synthesizing both - and -globin chains are jk-1 , kmoe-2 , ku812 , lama-84 , tf-1 , tn922 , and ap217 ; yet , ku812 cell line is comparatively unique and useful from the others as it can undergo a spontaneous differentiation which can be observed [ 30 , 3739 ] . moreover , human bone marrow cd34 + hematopoietic progenitors drawn from -thalassemia patients and primary erythroid progenitors stem cells ( epscs ) obtained from peripheral blood are also great in vitro models to study the effect of different hbf - inducing agents [ 1 , 13 ] . back to the 1980s , baboons , a nonhuman primate model , have already been used for the study of fetal and adult hemoglobin synthesis during fetal development [ 40 , 41 ] . there was an influential research conducted by de simone and colleagues that the adult baboon has been shown to respond to erythropoietic stress with the reverse hemoglobin switch during which an increase in the number of hbf - containing erythrocytes ( f cells ) and an increase in hbf synthesis can be observed in adult baboon [ 40 , 42 , 43 ] . however , this animal model is expensive to purchase , feed , and maintain in conditions appropriate to modern animal husbandry . in order to understand the influence of hbf synthesis and its induction mechanism , till the 90s , researchers have successfully discovered that transgenic mice carrying human a -globin gene , which can act as a new in vivo model for screening novel pharmaceutical compounds for hbf induction in the adult [ 44 , 45 ] . these inducers can be categorized into several classes based on their mechanisms of action [ 13 , 46 ] as listed in table 1 . some of them are classified as chemotherapeutic agents , for example , hydroxyurea ( hu ) , 5-azacytidine ( 5-aza ) , decitabine , and citarabine . hu is also known as a ribonucleotide reductase inhibitor due to its ability of inhibiting dna analysis , while 5-aza , decitabine and citarabine are dna methyltransferase inhibitors who responsible for the hypomethylation of dna [ 8 , 13 ] . several short - chain fatty acids ( scfas ) specifically stimulate transcription in the -globin gene promoter through histone deacetylase hdac inhibition , resulting in global histone hyperacetylation [ 5 , 47 ] . in contrast , some studies argue that globin histone hyperacetylation induced is not the primary mechanism of scfa ; yet , hdac inhibitors are often potent -globin inducers [ 47 , 48 ] . rapamycin preferentially induce -globin mrna accumulation , while being only minor for -globin and none for -globin mrnas . as its hbf - inducing effect is not related to cytotoxicity and cell growth inhibition , scientists are very interested in further studying if the enhancement of -globin mrna medicated by rapamycin is associated with xmni polymorphism . there are studies showing that k562 cells treated with dna - binding agents , such as mithramycin , have led to erythroid differentiation and sharp enhancement of -globin mrna level . through pcr arrested experiments , it is found that these dna - binding drugs were capable of interacting with -globin promoter of human genomic dna . in recent years , researches have been done on immunomodulatory drugs , such as thalidomide , revlimid , and pomalidomide . their exhibited hbf - inducing activity has been revealed in k562 or primary human erythroid cultures . further study has demonstrated their activity is associated with the increase in histone acetylation at -globin gene promoter . erythropoietin ( epo ) is a cytokine that have been shown to induce hbf production during in vitro differentiation of primary human cells in several trials [ 47 , 5255 ] . it also stimulates red blood cells production , prolongs the survival of erythroid cells , and decreases the incident of programmed cell death . in recent years , scientists have conducted numerous studies in order to identify the natural remedies that could be possibly applied in treating -hemoglobinopathies , including scd and -thalassemia , summarized in table 2 . the extract of aegle marmelos containing bergatene was found to be responsible for the activation of erythroid differentiation and hbf induction in human leukemic k562 cells [ 31 , 69 ] . they are powerful inducers of erythroid differentiation , -globin gene expression and hbf synthesis in human erythroid cells . thus , it is known as a potential therapeutic approach for both -thalassemia and sickle cell anemia . in addition , nicosan ( formerly known as niprisan ) , an ethanol / water extract from nigeria indigenous plants , has successfully demonstrated a significant anti - sickling effects in vitro as well as in vivo [ 70 , 71 ] . there is evidence demonstrating that angelicin is a powerful inducer of erythroid differentiation , enhancement of the hbf synthesis in erythroid progenitors and -globin mrna accumulation of human leukemia k562 cells [ 8 , 73 ] . red wine , especially the skin of grapes , contains resveratrol which mimics the hbf - inducing activity of hu . since -thalassemia cells exhibit a high level of oxidative stress , which eventually shorten the survival of erythroid cells in -thalassemia patients , resveratrol which exhibits both antioxidant activity and hbf inducing property can become a very promising hbf inducer from the natural world . rapamycin is isolated from streptomyces hygroscopicus , a bacterial species being found in the soil of easter island . it has the ability to increase hbf production in cultures of erythroid precursors from -thalassemia patients without cytotoxicity or growth - inhibitory effect [ 8 , 13 ] . it is a dna - binding drug which has the potential to induce -globin mrna accumulation and hbf production in erythroid cells from healthy human subjects as well as -thalassemia patients . recently , it is reported that an indian almond , called terminalia catappa , has long been used as a traditional herbal treatment for sca in nigeria . has then demonstrated terminalia catappa distilled water active fraction ( tcdwf ) from terminalia catappa leaves exhibit a stimulatory effect on the hbf production in primary erythroid prohenitor stem cells ( epscs ) . terminalia catappa consists of flavonoids , alkaloids , and anthraquinones . also , through gas chromatography - mass spectrometry ( gcms ) , it is shown that a group of highly related long - chain fatty acids , for example , hexadecanoic acid , 10-octadecenoic acid , and octadecenoic acid , are present in the tcdwf . yet , further investigation is required in order to confirm the biological activities of these active compounds present in the tcdwf . for the chinese herbal medicine , yisui shengxue granule ( yssxg ) , a complex prescription made up of 11 different kinds of chinese herbal medicines , has shown to be effective in enhancing the hbf expression and inhibiting ineffective hematopoiesis [ 76 , 79 ] . current research has further confirmed this complex prescription has the ability to increase -globin gene expression and alter the expression of genes that involved in the survival , proliferation , and terminal differentiation of erythroid cells . recently , some researchers found that cucurbitacin d ( cud ) in a chinese medicinal herb , called fructus trichosanthis ( ft ) , exhibits a higher potency in hbf induction compared with hydroxyurea since there is evidence showed cud results in a higher fetal cell percentage and greater hbf content in k562 cells with much lower cytotoxicity . scientists have started to put effort on appreciating their underlying molecular mechanisms as well as verifying their target molecules . the first piece of evidence regarding the cell signaling of the hbf induction has suggested that , in k562 cells , butyrate induces erythroid differentiation and hemoglobin production through p38 mapk pathway [ 1 , 80 ] . several years later , another group of scientists has interestingly revealed that , without drug treatment , the -globin mrna level is increased sufficiently solely by overexpressing mapkk3 and mapkk 6 which are the direct upstream activators of p38 in k562 cells . according to mabaera et al . , p38 mapk signaling pathway is critical for the upregulation of the production of hbf . different environmental stresses could activate the p38 mapk signal pathway which will subsequently cause apoptosis , cell growth and erythroid differentiation . various studies have also revealed the effect of numerous hbf - inducing agents , such as butyrate , apicidin , and trichostatin a , are associated with p38 mapk signaling pathway . therefore , they both indicated p38 mapk pathway plays a vital role in promoting -globin gene expression . during the past few years , researchers come up with different mechanistic models of hbf induction while most of the models are generally based on what are thought to be the primary actions of hbf - inducing agents , for instance , global dna hypomethylation induced by dna methyltransferase inhibitors ( dnmt inhibitors ) or global histone hyperacetylation induced by histone deacetylase inhibitors ( hdac inhibitors ) , including scfa derivatives . however , when we come across with the recent key experimental findings , including the fact that 5-aza can promote hbf production without dna hypomethylation , that -globin promoter hypomethylation is inadequate to stimulate gene expression , and that the ability of hdac inhibitors to induce hbf is regardless of the potency of hdac inhibitions , some of these proposed mechanistic models are no longer capable to explain those results . therefore , it is of essence to propose a new model of hbf induction which is valid to most hbf - inducing agents and can adequately account for the recent experimental results . , through integrating recent results of cell - signaling experiments with the stress erythropoiesis model , have proposed a new model called the cell stress signaling model . they suggested that the key effect of most hbf - inducing agents is to activate the cell stress signaling pathways during adult erythropoiesis which subsequently lead to -globin gene expression and hbf production . it is found that certain hbf - inducing agents , such as hu , butyrate ( scfa ) , thalidomide ( imid ) , trichostatin a ( hdac inhibitor ) , and anisomycin , can activate the corresponding cell stress responses , including nitric oxide , oxidative stress ( ros ) , osmotic shock , and protein synthesis inhibition . these cell stress responses will eventually activate the downstream p38 mapk signaling pathway , including downstream kinases and transcription factors , and thus result in -globin gene expression and hbf production . besides p38 mapk signaling pathway , the potential of camp signaling pathway in hbf production has also been mentioned in the cell stress signaling model . there are findings suggested that in primary erythroid cell cultures , camp response element binding protein ( creb ) is activated by camp - activated protein kinase a instead of p38 mapk pathway . the phosphorylated creb will then activate the downstream transcription factors and eventually lead to -globin gene expression . therefore , not only is the cell stress signaling model applicable to most of the hbf - inducing agents but also is able to explain the key findings of some of the previous experiments . in the previous studies , researchers have found out that stimulating erk signaling pathway leads to megakaryocytic differentiation ; contrastingly , suppressing erk signaling pathway results in the enhancement of the erythroid phenotype as well as the -globin mrna expression . moreover , the evidence that erk inhibitor u0126 has the ability to stimulate -globin gene expression and hbf production in human erythroid progenitor cells suggested the inhibition of erk can possibly lead to the promotion of hbf production . the involvement of jnk in erythroid differentiation still remained debatable [ 76 , 8587 ] . it is reported that pretreatment with hu in k562 cells has led to a significant inhibition of jnk . moreover , short chain fatty acid derivatives ( scfad ) , such as butyrate and valproate , did not result in jnk phosphorylation ; thus , there was not any significant changes on jnk pathway in k562 cells [ 76 , 87 ] . nevertheless , in mouse erythroleukemia cells , experimental findings have suggested that activation of jnk is crucial for erythropoietin - induced erythroid differentiation [ 86 , 88 ] . as a result , whether jnk plays a significant role in hbf induction still remains to be investigated in the future . the potential of reactivating fetal hemoglobin via manipulating the gene transcription of - or -globin has been gained much attention recently . there is escalated number of publications regarding the genetic regulatory mechanism of developmental stage - specific expression of -globin genes . the mechanisms accounting for switching globin - genes expression during the development are highly regulated by cis - acting elements and trans - acting factors which include the locus control region ( lcr ) [ 85 , 86 ] . previous studies have also revealed that lcr has an important of enhancing globin - gene switching potently . understanding the fetal - to - adult hemoglobin switching is believed to have a clinical relevance of developing novel approaches of reversal of fetal hemoglobin production from adult hemoglobin in patients . several researchers have applied the knowledge of the regulatory mechanisms of globin - genes expression to design an artificial zinc finger dna - binding domain ( zf - dbd ) with the aim of manipulating the expression patterns of globin - genes [ 85 , 87 ] . therefore , techniques involving small molecule inhibitors or genetic knockdown can be potential applications to reactivate the fetal hemoglobin in the future . interestingly , besides the discovery of the upstream signaling pathway of activating the -globin gene expression , there are findings supporting the fact that posttranscriptional regulation also involves in regulating the -globin gene expression in response to different stimuli [ 89 , 90 ] . weinberg et al . revealed that instead of acting on the transcription rate of -globin gene in patients , butyrate has the ability to enhance the efficiency of translation of -globin mrna . moreover , there is evidence proving that the key effect of gtp and doxorubicin derivatives is the elevation of the amount of -globin in patients , at least partially , by enhancing the stability of -globin mrna [ 91 , 92 ] . liu et al . also suggested that , similar to gtp and doxorubicin derivatives , cud can therapeutically induce the production of hbf due to its ability of increasing -globin mrna stability . they have showed even though the change in the half - life of -globin mrna is small , it leads to remarkable changes in the total amount of stable -globin mrna and consequently the amount of functional -globin present in the cell . therefore , the control of -globin mrna stability can be known as a significant regulatory mechanism of -globin gene expression . in -thalassemia , due to the precipitation of excess -globin chains , rapid cellular apoptosis of early erythroblasts and red blood cell membrane damage are well characterized [ 9496 ] . 5-azacytidine , hydroxyurea , myleran , and butyrate had long been applied in clinics for -thalassemia treatment with the aim of stimulating hbf production [ 97100 ] . despite the ability of inducing the production of hbf in -thalassemia , a large portion of identified hbf - inducing agents , such as 5-azacytidine and hydroxyurea , are chemotherapeutic agents which inhibit cell proliferation and cause cell growth arrest . further , due to their cytotoxic nature , the dose limiting myelotoxicity and potential carcinogenicity have always led to concerns . therefore , in order to correct the pathophysiology of -thalassemia , it is of essence to improve the underlying erythroid cell survival and proliferation , with the intention that hbf - inducing agents can exert their effect on stimulating the fetal globin expression prior to the activation of irreversible programmed cell death pathway . owing to this issue , perrine et al . ( 2002 ) have conducted a pilot study revealing the combination of butyrate and rhu - erythropoietin ( epo ) , the hematopoietic growth factor that stimulates erythroid proliferation , decreases apoptosis , and prolongs erythroid cell survival and differentiation , has addictive effects in inducing hematologic responses in any -thalassemia patients . this result further suggested that definitive treatment to correct -thalassemia will likely require more than one type of therapeutic regimen ; in other words , hematopoietic growth factor , such as exogenous epo will be required for -thalassemia patients in order to respond optimally to any hbf - inducing agent . hydroxyurea ( hu ) was first approved by the food and drug administration ( fda ) for the treatment of sickle cell disease ( scd ) in 1996 ; yet , hu only increases hbf production in approximately half of scd patients and is even less effective in enhancing the hbf level for -thalassemia patients [ 104106 ] . additionally , there is a recent clinical trial conducted to assess the therapeutic potential of hqk-1001 , an oral butyrate derivative 2,2-dimethylbutyrate sodium salt to treat -thalassemia patients . although results revealed hqk-1001 exhibits a stimulatory effect on hbf production and -globin gene expression , further study is needed to find out if the surge of hbf production is sufficient to alleviate the complications of -thalassemia including anaemia , chronic haemolysis , and so forth . nevertheless , up to date , there is no such pharmacological agent(s ) has been officially approved by fda for treating patients with -thalassemia . the underlying reason of this is that for pharmacological hbf induction therapy in patients with thalassemia major , it requires the production of -globin chains ( plus -globin chains in thalassemia major ) reaches 50% of the production of -globin chains in order to result in optimal therapeutic correction of the anemia in patients with -thalassemia ; while for that in patients with scd , it only requires the production of hbf reaches 2030% of total circulating hemoglobin for sufficient prevention of sickling effect in scd . therefore , it is necessary to identify novel pharmacological hbf inducer or alternate therapeutic approaches which can effectively enhance the hbf production to the optimal level and sufficiently reduce the chain imbalance in homozygous -thalassemia . in recent years , ( 2008 ) have proposed p38 mapk cell stress signaling pathway and other stress - related pathways may be the keys to understanding hbf induction , owing to the fact that most hbf - inducing agents are , as mentioned before , cytotoxic and many activate the p38 mapk cell stress signaling pathway . this stress signaling model predicts a variety of diverse hbf - inducing compounds and stimuli will activate cell stress signaling pathway which will then activate similar response genes , such as the -globin genes . it is supported by several observation and evidence , for example , the stimulation of -globin gene expression and hbf production in human primary erythroid cells by 5-azacytidine is closely related to p38 mapk phosphorylation and this stimulation is inhibited when treated with p38 mapk inhibitor sb203580 . on the other hand , based on the stress signaling model , mabaera and colleagues ( 2008 ) have also made some predictions which require further investigation , for example , the members of stress signaling pathways , ranging from the sensors of cellular stress to the activated transcription factors that bind to the -globin gene promoters , are needed for activating the -globin gene expression as well as hbf production . nevertheless , concerns have been raised about the possibility of triggering rapid cellular apoptosis in erythroid cells and consequently leading to low blood count when patients , especially those with thalassemia , are receiving chemotherapeutic agents including 5-azacytidine , decitabine , hu and butyrate . comparing with scd patients , -thalassemia patients are more susceptible to chemotherapeutic agents because treating with chemotherapeutic agents may further encourage rapid cellular apoptosis which has already been well - characterized in -thalassemia patients even they are not exposed to the agents . also , as mentioned before , accelerated cellular apoptosis of erythroid progenitors in -thalassemia is a significant barrier to definitive therapy because there is a high possibility that the programmed cell death will be established earlier before hbf - inducing agents act its beneficial effect on the globin chain balance in cells . consequently , chemotherapeutic agents , such as hu , butyrate and 5-azacytidine , may not be the best hbf - inducer for -thalassemia due to the fact that the hbf - inducing property of those agents are largely dependent on the activation of cell stress signaling pathway . in contrast , those chemotherapeutic agents may have beneficial effect on activating -globin gene expression and hbf production in scd ; yet , the dose of inducing agents must be strictly monitored in order to ensure it is high enough to activate stress signaling pathway but not too high to trigger cell - cycle arrest or apoptosis in hematopoietic precursor cells that will result in dangerously low blood counts . in light of the lack of promising hbf - inducing agents for -thalassemia treatment , up to now , different research groups have paid lots of effort on identifying the existing or novel natural chinese herbal medicines which have the possibility to effectively induce hbf synthesis without any apparent growth - inhibitory effects . yisui shenxu granule ( yssxg ) , a complex prescription consisting of 11 chinese herbal medicines , had been used for treating -thalassemia for more than over 20 years [ 76 , 79 ] . recently , effort has been put on investigating the efficacy and safety of yssxg by a randomized single - blinded trial . result has demonstrated it has obvious clinical efficacy , while hepatauxe and splenomegaly were relieved and no adverse reaction was observed . the underlying mechanism for the effect of yssxg is possibly by activating the expression of -globin gene and increase hbf production in order to compensate for the functional deficiency of -globin gene . zhang and wu have found out that stimulating the gene expression of -globin , epor , spi , and fklf , while hindering the gene expression of ckit , gata1 , and gata2 , could promote the -globin gene expression and alter the expression of gene which is responsible for the regulation of -globin gene expression and the expression of other genes that are participated in the survival , proliferation , and terminal differentiation of erythroid cells . ( 2011 ) have presented the first piece of evidence on the hbf - inducing property of ethanol extract of fructus trichosanthis ( ft ) , one of the most frequently used chinese herbal medicine . their study has demonstrated ft has the ability to enhance the -globin gene expression as well as hbf synthesis through activating p38 mapk and inhibiting erk signaling pathway . despite the promising result shown , acute and chronic toxicity test in vivo is strongly required in the future in order to ensure the ethanol extract of ft is safe for clinical use on human . also , the efficacy of the extract should be examined in clinical evaluation before actual clinical practice . during the same year , another group of researchers have identified cucurbitacin d ( cud ) , a chemical inducing agent that can be found in fructus trichosanthis , is a novel therapeutic agent for treating -thalassemia . there are evidences suggesting that cud could act as a good hbf - inducing agent for -thalassemia patient comparing with hu since cud exhibits a higher amplitude and rapidity in enhancing the hbf production than hu does in k562 cells , and , unlike hu , cud does not show any growth - inhibitory effect even when it is at its optimal activity . taken all together , natural herbal medicines , which exhibit a higher potency in hbf induction compared with chemotherapeutic agents and have much lower cytotoxicity , will definitely be the novel therapeutic candidates for -thalassemia by targeting the activation of -globin gene expression ; yet , some of the candidates are still required further investigation on their safety and efficacy . chemotherapeutic agents , such as 5-azacytidine , hydroxyurea , myleran , and butyrate , had long been used for -thalassemia treatment by stimulating hbf synthesis ; yet , cytotoxicity , growth - inhibitory effect , fear of long - term carcinogenesis , and only modest hbf - inducing activity have limited the clinical usage of these agents in -thalassemia and scd treatment . also , through understanding the pathology of -thalassemia , it is revealed that most of the identified hbf - inducing agents have limitation on treating -thalassemia . it is because the rapid cellular apoptosis of erythroid progenitors in -thalassemia causes a significant obstacle that overstimulating the cell stress signaling pathway by the hbf inducer may possibly lead to irreversible cellular apoptosis before -globin gene expression and hbf synthesis can be stimulated . with the advancement of biotechnologies , increasing number of studies will be done to explore and optimize new interventions and nature remedies to reactivate hbf synthesis for -thalassemia patients . in the future , it is expected that increasing number of hbf inducing agents could be found from natural remedies and folk medicines all over the world . in this context , further studies are required with the aim of exploring more natural herbal medicines as well as studying the efficacy and safety of from the laboratory to clinical use for the individuals with -hemoglobinopathies .

for the treatment of -thalassemia and sickle cell disease ( scd ) , pharmacological induction of fetal hemoglobin ( hbf ) production may be a promising approach . to date , numerous studies have been done on identifying the novel hbf - inducing agents and understanding the underlying mechanism for stimulating the hbf production . in this review , we have summarized the identified hbf - inducing agents by far . by examining the action mechanisms of the hbf - inducing agents , various studies have suggested that despite the ability of stimulating hbf production , the chemotherapeutic agents could not be practically applied for treating -hemoglobinopathies , especially -thalassemia , due to the their cytotoxicity and growth - inhibitory effect . owing to this therapeutic obstacle , much effort has been put on identifying new hbf - inducing agents from the natural world with the combination of efficacy , safety , and ease of use . therefore , this review aims to ( i ) reveal the novel screening platforms for identifying potential inducers with high efficiency and accuracy and to ( ii ) summarize the new identified natural remedies for stimulating hbf production . hopefully , this review can provide a new insight into the current status and future perspectives in fetal hemoglobin reactivation for treating -thalassaemia and scd .

according to the statistical analysis of the international society of heart and lung transplantation , survival after lung transplantation systematically improves . 88% of recipients survive three months after operation , 79% survive for one year , and about 50 - 60% of recipients survive five years after transplantation . the most common indication for lung transplantation is chronic obstructive pulmonary disease ( copd ) ( 34% ) . the major early causes of death are graft failure and non - cmv infections , bronchitis obliterans syndrome ( bos ) , chronic lung rejection , and graft failure after one year . a 58-year - old female with hyperthyroidism , osteoporosis , and copd in the fourth stage was classified for lung transplantation in 2011 . additional tests demonstrated pao2 ( partial pressure of oxygen ) 61.2 mmhg , paco2 ( partial pressure of carbon dioxide ) 44 mmhg ( on oxygen therapy 2 l / min ) , fev1 ( forced expiratory volume in 1 second ) 0.44 l 19.7%sd , fvc ( forced vital capacity ) 1,38,8% sd , 6-mwt ( six - minute walk test ) 174 metres , rvsp ( right ventricular systolic pressure ) 36 mmhg , and pra ( panel reactive antibody ) 3% . after four months of expectation a 57-year - old female donor , who died because of cerebral haemorrhage and was ventilated for 48 hours with pao2/fio2 360 mmhg , was identified . both women were characterised by comparable growth and identical blood group . the day after donor identification the transplantation was performed . for this purpose the right pulmonary vein cuff proved to be very short and tight , so it was decided to implant the left donor lung in the recipient 's right emphysemal lung locus . elements of the recipient 's right lung hilum were dissected , cut distally with clamping of the pulmonary artery and pulmonary veins cuff . after preparation the left donor 's lung was implanted in the contralateral position . in conjunction with different bronchus diameters the donor bronchus was telescopically placed in the recipient 's bronchus and stitched with single absorbable sutures 3 - 0 . two drains were inserted in the pleural cavity , and closure of the thorax was carried out . metal suturing of the sternum and the suturing of the periosteum of the fifth rib and layered lid suture were performed ( fig . the patient was transferred to the intensive care unit ( icu ) , where she stayed for eight days . because of retrospectively positive cross - match it was necessary to implement five automatic plasmapheresis and to apply human immunoglobulin and rituximab to diminish concentration of lymphocytotoxic antibodies against hla antigens . on the third day after transplantation the primary graft dysfunction and rising emphysema of the recipient 's own lung shifted the mediastinum towards the graft side . as well as standard treatment , c1-esterase was provided to deplete the extracapillary exudation , and the operative ( lung volume reduction surgery lvrs ) reduction of emphysemal changed lung tissue was pursued with complication of prolonged air leakage ( fig . the patient needed infusion of 16 units of fresh frozen plasma ( ffp ) , 2 units of packed red blood cells ( prbcs ) , and 100 ml of 20% albumin . the right lung drainage was removed after seven days and the left one after 35 days . on the 44 day after transplantation the patient was discharged from hospital with the following study results : po2 68.9 mmhg , pco2 44.0 mmhg ( without oxygen therapy ) , fev1 1.12 - 50.5% , fvc 1.38 - 52.2% , 6-mwt 246 metres , and with recommendation of taking immunosuppressing medications such as : 50 mg of cyclosporine twice a day , 1000 mg of mycophenolate mofetil twice a day , and 10 mg of prednisone once a day . 3 ) . it was histopathologically confirmed and antibodies against donor antigens were detected ( anti - hla class ii 158.57 - 701.89 mfi ) . on the third day the therapy with methylprednisolone was pursued . in chronically treatment instead of cyclosporine the tacrolimus of 0.5 mg a day was implemented . additionally , the hospitalisation was complicated by moraxella catarrhalis infection , so amoxicillin with clavulanic acid was applied . diagnostic tests performed meanwhile showed transient renal failure , ion disorders , hypoproteinaemia , and leukopaenia . at the turn of fifth and sixth month after transplantation the next complications occurred : bronchitis caused by corynebacterium pseudodiphtericum , and escherichia coli urinary tract infection , both treated with amoxicillin and clavulanic acid , and a second episode of acute graft rejection . during the next two months e. coli sepsis was seen and was treated with ceftazidime . then the cytomegalovirus disease treated with ganciclovir , pulmonary embolism , and anxiety disorder treated with opipramol and venlafaxine occurred . on account of leukopenia 2900/l after the next month the third episode of acute graft rejection appeared , following bacterial infection caused by pseudomonas aeruginosa cured with ciprofloxacin and ceftazidime and bilateral otitis with sinus and temporal bone polyposis development , hence clindamycin and budesonide inhalation were applied . because of hypogammaglobulinaemia it was decided to assign human immunoglobulins every three weeks . during the 11 and 12 month after the transplantation ( fig . 4 ) the next bacterial infections caused by acinetobacter baumanii , proteus mirabilis , candida albicans , c. glabrata and aspargillus species ensued . the therapy contained cefuroxime , imipenem , ampicillin with sulbactam , amikacin , amphotericin b , itraconazole , and colistin . meanwhile drug - induced diabetes with rapid decompensation occurred , which initially needed glimepiride and then insulin therapy . later , renal failure and a fourth episode of acute lung rejection appeared . between the 14 and 16 month after lung transplantation the fifth episode of rejection appeared , renal failure deepened , and destabilisation of diabetes occurred . the cytomegalovirus disease activated and valganciclovir was used in therapy , but the leukopaenia deteriorated and it was necessary to reduce the dose of the drug . in addition , acinetobacter baumani , klebsiella pneumoniae , and candida albicans infections appeared . in therapy the ceftazidime , ciprofloxacin , ampicillin with sulbactam , colistin , metronidazole , tazobactam , itraconazole , and nystatin were used . at this time unfortunately , the psychological state of the patient gradually worsened , and she manifested symptoms of depression . collateral complication appeared as posttraumatic haematoma of the shin , which required vac - therapy ( vacuum assisted closure vac ) . 498 days after transplantation the patient died due to symptoms of sepsis after unsuccessful reanimation . two drains inserted in right pleural cavity and catheter in right carotid internal vein are visible chest x - ray performed after lung volume reduction surgery ( lvrs ) chest x - ray performed during first episode of acute rejection chest x - ray performed one year after transplantation lung transplantation in medically incurable patients is the only method that gives a chance to lengthen their lives . currently it is recommended that the cross - match be pursued before performing the transplantation to avoid such a situation , although there are some scientific reports discussing the primary existence of these antibodies in the recipient 's organism or their appearance as the consequence of transplanted organ dysfunction . there are some case reports that adduce false positive and false negative pre - transplant virtual cross - matches . however , virtual cross - match should be considered as a routine technique used before transplantation procedure in poland . virtual cross - match is the admitted method of prediction if the recipient produces hla - antibodies . an appropriate computer program determines every hla antigen immunogenicity that is incompatible with full recipient hla haplotype as an inconsistent immunogenic loci ( miss - match amino acids eplets mmep ) . safe immunogenic incompatibility that does not result in humoral response can also be predicted with this method . it is a huge advantage in patients who need multiple transplantations . extending the scope of immunological selection to hla - cw , dq , dp increases the effectiveness of virtual cross - match ( 91% ) in comparison to serological selection on hla - a , b , dr used in poland . difficulties in proper donor selection are related with imperfection of chemical reagents and antibody detection techniques . in the first case the chance for transplantation is wasted , in the second the patient is exposed to serious postoperative complications . furthermore , the role of antibodies against the host 's own ( personal ) antigens in pathogenesis of chronic dysfunction of transplanted organs is discussed . however , no matter which hypothesis is accepted , only quickly implemented therapy can improve the patient 's condition . there are some ambiguous reports about monoclonal antibodies although there are some indications that such treatment can be effective [ 6 , 7 ] . we proceeded with mandatory cross - match before lung transplantation in every recipient with pra > 0% . also , the age of qualified donors is limited to 55 years old , whereas the criterion of pao2/fio2 was raised to > 400 mmhg . the recommended operation technique is bilateral lung transplantation because of the reservoir of microorganisms in the recipient 's own lung , so nowadays in such a clinical situation probably reconstruction of the donor 's right pulmonary vein cuff would be performed . additionally , lvrs , according to present knowledge , would not be pursued , because the shading of the graft after transplantation was caused by primary graft dysfunction and not by the recipient 's own lung . finally , meticulous training of the transplantation team in surgical techniques should be continued , which is achieved in our case by practicing microsurgical techniques on rats and by using a cadaver workshops .

lung transplantation is a method useful in such non - malignant end - stage parenchymal and vascular diseases as : chronic obstructive pulmonary disease ( copd ) , idiopathic interstitial pulmonary fibrosis , cystic fibrosis , or primary pulmonary hypertension . the main aim of this procedure is to extend the patient 's lifespan and quality of life . however , the availability of the operation is limited by organ access . in this paper we present the case of a 58-year - old female in the fourth stage of copd , who was classified to have a single lung transplantation . because of some technical problems it was decided to transplant a left donor lung in the right recipient lung locus . positive cross match was demonstrated retrospectively , and we applied five courses of plasmapheresis . human immunoglobulin and rituximab treatment were performed to decrease the impact of lymphocytotoxic antibodies . the patient survived 498 days after transplantation , 271 in the hospital .

a 53-year - old asian woman with a history of lupus erythematosus was initially treated with hydroxychloroquine ( plaquenil , unknown dose ) for 5 years . approximately 3 years ago , the patient 's medication was changed to chloroquine ( 400 mg daily ) . within a few years of taking chloroquine , she noticed a circular shadow in her central vision , which she described as an arch surrounding her central vision . at first , she only noticed a superiorly oriented half - moon shadow that lasted for a few seconds during reading . the shadow was seen while both eyes were open and disappeared after a few seconds . because of these symptoms and the presence of bull 's eye maculopathy on fundus examination , chloroquine was discontinued and the patient 's medication was changed to dapsone ( unknown dose ) . the best - corrected visual acuity ( corrected with current spectacles ) of the highly myopic patient during her most recent examination was 20 / 25 in both eyes , with no evidence of an afferent pupillary defect . slit - lamp examination indicated that the anterior segment was unremarkable and goldmann applanation intraocular pressure was normal ( 16 mmhg ) in both eyes . however , a significant central defect was noted on humphrey visual field ( hvf ) examination ( humphrey visual field analyzer 10 - 2 ; humphrey instruments , san leandro , ca , usa ) in both eyes . dilated fundoscopy revealed tilting of the optic nerves in both eyes , consistent with myopia , and a ring - shaped area of rpe depigmentation in the parafoveal area of the left eye ( fig . fundus autofluorescence ( faf ) imaging revealed a hypofluorescent lesion in the foveal and perifoveal areas correspond with the bull 's eye retinopathy . in the left eye , a prominent hypofluorescent lesion was also noticed on faf imaging , reflecting marked atrophy of the rpe layer ( fig . cross - sectional retinal scans , obtained with sd - oct ( spectralis hra+oct ; heidelberg engineering , heidelberg , germany ) showed obvious thinning of the macula and disruptions in the outer retina . more specifically , loss of the photoreceptor inner / outer segment ( is / os ) junction corresponded to the location of the maculopathy as identified on fundus photography , faf , and hvf 10 - 2 . the outer retinal layer had a moth - eaten appearance at the is / os junction , but the thickness and appearance of the inner retina were within normal limits . additionally , a downward displacement of the overlying retina was seen in perifoveal areas and corresponded to the hypofluorescent lesion in faf images . unlike the anatomically disrupted areas , which corresponded to the bull 's eye lesion , the central fovea had normal thickness and structure on sd - oct in both eyes ( fig . the ao system used in the current study utilized a dual liquid crystal on silicon spatial light modulators ( lcos - slms ; x10468 - 02 , hamamatsu photonics , morimoto , japan ) first introduced by hirose et al . . although deformable mirrors have been widely used , their performance is limited with respect to compensating large amounts of aberration of human eyes . meanwhile , although lcos - slm is able to compensate large amounts of aberration by the phase wrapping method utilizing a continuity of the wavefront of a laser beam , it is only capable of compensating a particular polarization component . this system is very efficient at following changes in aberration of a subject according to change in imaging location , but is not suitable for obtaining high contrast images because the power of the lights used for simultaneous imaging and measurement / compensation of time is limited for safety . in this study , we overcame these problems by adopting dual lcos - slm to compensate the two orthogonal polarization components and sequential processing of slo imaging and measurement / compensation of aberrations . ao - slo images were obtained and cone density was measured 0.5 , 1.0 , and 1.5 mm from the center of the fovea in each of the 4 major directions ( superior , inferior , nasal , and temporal ) . a montage was then created and verified by checking the correspondence between the ao - slo and wide - field images . cone density was calculated using photoreceptor analysis software ( canon , tokyo , japan ) . to obtain an accurate cone density , the axial length , anterior chamber depth , and keratometry values were all considered when counting cone cells , which were represented in images as bright spots 2 to 5 m in size . we manually selected areas of interest , and excluded retinal vessels and artifacts that obscured the underlying cone mosaic . ao - slo images showed patch cone mosaic lesions , indicating abnormal regions of missing or lost cones . in addition , the observed cones were asymmetrical in shape and size and varied in brightness ( fig . cone densities in the right eye were 14,927 , 10,377 , and 11,853 cones / mm at 0.5 , 1.0 , and 1.5 mm from the center of the fovea , respectively . the corresponding cone densities in the left eye were 16,692 , 12,564 , and 13,100 cones / mm , respectively . the cone density at 1.5 mm from the center of the fovea was closer to normal than the other distances ; however , the photoreceptors located 1.5 mm from the foveal center exhibited asymmetrical shapes and sizes , and varied in brightness in ao - slo images . additionally , sd - oct images showed an abnormal is / os cytoarchitecture 1.5 mm from the foveal center . when we matched the disrupted areas on the ao - slo images with the visual field test , the areas with abnormal cone mosaic patterns and low cone density corresponded with the location of visual field defects on hvf ( fig . ocular side effects associated with chloroquine can be primarily divided into keratopathy , ciliary body changes , and retinopathy . bull 's eye maculopathy , a type of retinopathy , is associated with the use of antimalarial agents and is relatively rare . however , bull 's eye maculopathy is the most serious of the ocular adverse effects and is of serious pathologic concern , as the associated visual changes can be severe and there is little chance of visual recovery . in addition , visual loss associated with bull 's eye maculopathy can progress even after the drug treatment is terminated . patients with retinopathy usually present with central visual loss , visual field defect , color vision deficiency , photoaversion , night blindness , and entopic phenomenon . easterbrook reported that patients with minimal paracentral scotomas do not complain of symptoms and have normal color vision . in these patients , however , in nearly 70% of patients with ocular symptoms , abnormal color vision , and fundus changes , retinopathy progresses even after termination of drug use . together , these studies illustrate the importance of early detection , ideally before the onset of visual symptoms . fluorescein angiography ( fa ) can reveal the bull 's eye pattern of granular hyperfluorescence , but is not useful for the diagnosis of early retinopathy . in full - field electroretinography ( erg ) , there may be a deepening of the a - wave or an amplitude reduction in the scotopic b - wave . however , in most cases , erg findings are normal with minimal macular damage and decrease only after diffuse retinal damage has occurred . it was also reported that sd - oct images are able to show the perifoveal interruption of the photoreceptor is / os junction in eyes with chloroquine retinopathy . however , despite this new information , there is currently no established criteria for diagnosing chloroquine retinal toxicity prior to the development of irreversible visual loss . studies performed in mice have shown that photoreceptors are the primary targets affected by chloroquine maculopathy , and that further degeneration occurs despite cessation of medication . additionally , rosenthal et al . showed that use of chloroquine in rhesus monkeys causes significant changes in both the inner and outer retinal layers . exposure to ultraviolet radiation causes chloroquine to fluoresce , and based on this property , clinical studies have shown that a widespread , severe reduction of rod and cone processes occur in the outer nuclear and plexiform layers . still , because of aberrations of ocular optics , none of the currently available imaging methods are capable of observing cells with disrupted photoreceptors in living human eyes . recently , ao technology has been developed that allows for individual cones to be clearly visualized in vivo by compensating for aberrations in ocular optics [ 6 - 8 ] . using ao - slo technology , several studies have shown that successful visualization of photoreceptor microstructure is possible in both normal eyes and eyes with various retinal diseases . consistent with previous ao studies , our ao - slo images from normal eyes showed a regular cone mosaic pattern , in which the cone density decreased with increasing distance from the center of the fovea . in the patient with bull 's eye maculopathy , we observed a disrupted cone mosaic pattern with individual cones having irregular shapes and sizes . additionally , compared with normal patients , patients with bull 's eye maculopathy can exhibit irregular cone brightness as well as dark and patchy lesions where cones are either missing or lost . in the present study , along with the disrupted cone mosaic pattern , measured cone densities were diminished in all areas . to better interpret the findings in our study , comparative data was obtained from both previous studies and historical controls . in normal eyes the average cone densities 0.5 and 1.0 mm away from the foveal center are approximately 30,000 and 15,000 cones / mm , respectively . these values were in agreement with histological studies reporting cone densities of 37,000 , 16,000 , and 14,000 cones / mm at 0.5 , 1.0 , and 1.5 mm away from the foveal center , respectively . stepien et al . previously reported an ao image of bull 's eye maculopathy associated with antimalarial agent use . although similar abnormal images of cones have been obtained using their ao system , they were not as high quality as the images obtained in the present study . specifically , we were able to achieve improved image quality and contrast because our ao - slo utilized a dual liquid crystal on lcos - slms to correct for wavefront aberrations , while the system used by stepien et al . moreover , creating a montage of ao - slo images made it possible to compare each ao - slo image to visual field test results . using this technique , we were able to demonstrate that areas with visual defects could be matched with cone pattern and density abnormalities . because ao - slo can image the retina with such high resolution , it is more sensitive to photoreceptor abnormalities than sd - oct . in our study , sd - oct images revealed equal disruption of the outer retinal layer at 1.0 and 1.5 mm away from the fovea . however , ao - slo images revealed a higher level of cone pattern disruption and a lower cone density at 1.0 mm away from the fovea , compared with 1.5 mm away from the fovea . there were several limitations of the present study that should be mentioned . because the central foveal structure was conserved , our patient had a visual acuity of 20 / 25 . however , we could not obtain images of the central fovea for reasons described in previous studies [ 6 - 8 ] . unfortunately , the inability to obtain these images made it impossible to compare cellular images of the fovea with those obtained from the bull 's eye lesion . another limitation of our study was that the ao - slo system could not acquire images from the central fovea or the peripheral retina . therefore , we only investigated cone properties at 0.5 , 1.0 , and 1.5 mm away from the center of the fovea . because of this limitation , we were unable to compare preserved areas adjacent to the bull 's eye lesion , which may possibly have contained cones in the preclinical stage of the disease . as described above , the visual acuity of the patient was 20 / 25 , and we hypothesized that the reason for the relatively mild visual decrease was because the structure of the central fovea remained relatively preserved . however , we could not confirm this possibility because the center of the fovea could not be precisely imaged by ao - slo . similarly , because the ao - slo can not obtain images from the peripheral retina , it was not possible to compare the disrupted macula to the relatively preserved peripheral retina . depending on the specific disease , patients may be treated with either a chronic maintenance or acute high - level dose of chloroquine . a daily dose of chloroquine > 3 mg / kg and a treatment duration of > 5 years therefore , patients who are taking chloroquine or hydroxychloroquine should have regular ophthalmic examinations to detect retinal changes as early as possible in order to minimize retinal toxicity . in the present study , we showed that ao - slo can image cellular structures of the retina in living eyes in order to detect preclinical stages of chloroquine associated retinal damage that may not be apparent on standard clinical tests ( e.g. , fundus photography , fa , hvf , and electrophysiological testing ) . importantly , the ability to identify photoreceptor disruptions in the preclinical stages of toxicity should allow for chloroquine withdrawal during the reversible stages of maculopathy . despite the limitations of our ao system , our results suggest that ao - slo should provide a non - invasive , quantitative , high - resolution modality for imaging chloroquine retinopathy patients . likewise , the abilities of ao - slo have the potential to provide a new approach for the diagnosis and monitoring of chloroquine toxicity .

a 53-year - old asian woman was treated with hydroxychloroquine and chloroquine for lupus erythematosus . within a few years , she noticed circle - shaped shadows in her central vision . upon examination , the patient 's visual acuity was 20 / 25 in both eyes . humphrey visual field ( hvf ) testing revealed a central visual defect , and fundoscopy showed a ring - shaped area of parafoveal retinal pigment epithelium depigmentation . fundus autofluorescence imaging showed a hypofluorescent lesion consistent with bull 's eye retinopathy . adaptive optics scanning laser ophthalmoscope ( ao - slo ) revealed patch cone mosaic lesions , in which cones were missing or lost . in addition , the remaining cones consisted of asymmetrical shapes and sizes that varied in brightness . unlike previous studies employing deformable mirrors for wavefront aberration correction , our ao - slo approach utilized dual liquid crystal on silicon spatial light modulators . thus , by using ao - slo , we were able to create a photographic montage consisting of high quality images . disrupted cone ao - slo images were matched with visual field test results and functional deficits were associated with a precise location on the montage , which allowed correlation of histological findings with functional changes determined by hvf . we also investigated whether adaptive optics imaging was more sensitive to anatomical changes compared with spectral - domain optical coherence tomography .

cardiovascular diseases ( cvd ) are becoming the major cause of mortality in developing countries.1 the 2002 world health report indicates that the most important risk factors for noncommunicable diseases include high blood pressure , high blood cholesterol , inadequate fruit and vegetable intake , overweight and obesity , physical inactivity and tobacco use.2 a recent community - based national study , involving adult saudis between the ages of 30 and 70 years , showed that the overall prevalence of coronary heart disease ( chd ) in the kingdom of saudi arabia is 5.5%.3 however , the expected increase in mortality as a result of ischemic heart disease in the middle east region in 2020 compared to 1990 was estimated as the highest of all the regions of the world ( 146% increase in women and 174% increase in men).4 this is mostly attributed to the high prevalence of major chd risk factors . the latest report involving saudis between the ages of 30 and 70 years showed the following prevalence : 23.7% for diabetes ; 26% for hypertension ; 12.8% for smoking ; 53.9% for hypercholesterolemia , and 35.6 for obesity.3 this high prevalence of chd risk factors in the saudi population confirms many earlier findings on chd risk factors involving different segments of the saudi society.58 prevention of cvd depends on controlling the modifiable risk factors , such as , physical inactivity and obesity . in fact , physical inactivity represents an independent risk factor for a number of chronic diseases , including chd , diabetes mellitus , obesity , osteoporosis.911 however , physical activity has been shown to both prevent and treat many established atherosclerotic risk factors.101214 the world health organization ( who ) , which has long recognized the heavy burden of noncommunicable diseases on the health services , recently launched its global strategy on diet , physical activity and health.15 the overall goal of the strategy is to improve public health through healthy eating and physical activity . this concerted effort on the part of who to improve global public health through physical activity is a continuation of previous actions taken during the last decade by numerous health and medical organizations , including the american heart association,916 the american college of sports medicine,131718 and centers for disease control and prevention.1119 the present paper , therefore , provides an overview of the importance of physical activity in health promotion and disease prevention , and discusses the public health burden of physical inactivity . it is also the aim of this paper to briefly review the status of physical inactivity in saudi arabia and to calculate the attributable risk of physical inactivity in the saudi population . in addition , some preliminary findings from the longitudinal study on physical activity and cardiovascular health of the saudi youth will be presented throughout this paper . physical activity is defined as any bodily movement produced by the skeletal muscles that results in energy expenditure above the basal level.20 physical activity is considered a complex set of behaviors . our ability to relate physical activity to health indicators depends on accurate , precise and dependable measures . physical activity is commonly measured by either self - report or direct monitoring through mechanical / electronic or physiological measurements . table 1 presents an important distinction between the definitions of physical activity and physical fitness . it also shows the meaning of metabolic equivalent ( met ) , which is increasingly becoming a very common term for quantifying the intensity of physical activity or energy expenditure , especially for the purpose of exercise prescription or physical activity assessment . definitions of basic terms today , it is estimated that 60 - 85% of adults around the world are simply not active enough to achieve the health benefits of physical activity.21 to deal with this epidemic of physical inactivity , who has recently established the physical activity unit as part of the department of noncommunicable disease prevention and health promotion.22 the goal of this unit is to promote higher levels of physical activity within the world population of all ages and conditions , men and women . the unit 's strategic products include : ( 1 ) developing a global strategy and database on physical activity ; ( 2 ) promoting the annual global move for health initiative ; ( 3 ) supporting a country 's actions and facilitating the development of multisectorial national policies and programmes on physical activity ; and ( 4 ) developing partnership and networks for increasing population participation in physical activity with a special focus on young people . despite the fact that most medical institutions currently do not provide their graduates with the proper training and necessary skills in physical activity assessments and exercise prescription,23 many medical organizations are now urging health care professionals to provide counseling on physical activity for their patients . the american heart association ( aha ) for example , stated in its recent guidelines for the primary prevention of cvd and stroke , that the assessment of the risk factors in adults should commence at 20 years of age . physical activity is a primary chd risk factor , and should be assessed at every routine evaluation.24 the aha goes further in its recommendations and asks that : ( a ) schools should be encouraged to teach skills required for physically active lifestyle ; ( b ) health care professionals should be educated about exercise as a therapeutic modality , and the importance of lifelong physical activity for patients ; ( c ) they should routinely prescribe exercise for their patients ; and ( d ) exercise testing should be performed before vigorous exercise in selected patients with cvd or those at high risk.24 furthermore , the importance of physical activity in health promotion and disease prevention strategy was evident in the healthy people 2010 report , published by the centers for disease control and prevention ( cdc ) of the united states department of health and human services.25 in that report , 10 leading health indicators ( lhi ) were identified . the lhi reflect the major public health concerns in the united states , and highlight the importance of health promotion and disease prevention on the societal level . physical activity , not surprisingly , is the first on the list of lhi , followed by obesity in the healthy people 2010 report . among the target goals for the healthy people 2010 was an increase in the proportion of adults and adolescents who engage in moderate physical activity ( 3 - 6 mets ) for at least 30 minutes on five or more days per week . during the past three decades , the kingdom of saudi arabia has undergone tremendous changes in lifestyle , including physical activity and eating habits . these dramatic lifestyle changes have definitely had a considerable negative impact on the health of the society . indeed , this lifestyle transformation is thought to be responsible for the epidemic of noncommunicable diseases and their complications in the country.32627 unfortunately , there exists no physical activity surveillance system in the country . however , mere observation indicates that there is a reduction in daily physical activity and energy expenditure of the saudi people relative to earlier times . moreover , findings from a limited number of studies on the prevalence of physical inactivity in saudi population confirm that a sedentary life style is on the rise.262829 across all segments of the saudi population , physical inactivity ranged from 43% to 99% , depending on gender , age , location , and target population.30 figure 1 presents some data for physical inactivity among saudi children , youth and adults , conducted on samples from riyadh.293132 in the studies conducted on children2631 and youth,32 continuous 12-hour heart rate telemetry was used to measure physical activity . in the adult study,29 the overall prevalence rates of physical inactivity among saudi children , youth and adults were roughly 60% , 70% , and 80% , respectively . the prevalence rates for inactivity among saudis , shown in figure 1 , agree with those estimates reported worldwide by who.21 prevalence of physical inactivity in saudi children , youth and adults . it is quite clear from the data presented in figure 1 that physical inactivity is prevalent among saudi children and youth . findings from a recent study on 12 - 20-year - old schoolboys living in riyadh have also shown inactivity prevalence of about 50%.33 promoting physical activity among children and adolescents is very important in order to offset any decline in their activity level as they grow - up . data from several countries indicated a decline in physical activity among the youth starting at age 12 and continuing up to 20 years.34,35 preliminary findings from our own longitudinal study on saudi youth showed a 30% increase in physical inactivity from childhood ( 7 - 12 years ) to early adulthood ( 18 - 23 years ) . this was coupled with a three - fold increase in television viewing during the same period of time.36 data from our laboratory3236 also demonstrated that inactivity prevalence in youth is far more pronounced than any of the other chd risk factors , as shown in figure 2 . therefore , a reduction in the prevalence of physical inactivity among saudi youth would have a far greater impact on risk reduction than a reduction in any of the other traditional chd risk factors . the council on cardiovascular disease in the youth of the aha has issued a statement for health professionals asking them to counsel their young patients on physical activity , including physical activity assessment and exercise prescription.37 therefore , routine counseling by local physicians on physical activity and the health of their young patients should be initiated . prevalence of coronary heart disease ( chd ) risk factors among a sample of saudi youth the burden of mortality , morbidity and disability attributable to noncommunicable diseases , including sedentary life , is considerably high and continuing to grow.15 according to preliminary data from a who study on risk factors , inactivity is one of the 10 leading global causes of death and disability.21 worldwide , physical inactivity was estimated to cause 1.9 million deaths and 19 million disability - adjusted life years.2 physical inactivity was also estimated to globally cause about 22% of ischemic heart disease and about 10 - 16% of cases each of diabetes mellitus , breast , colon and rectal cancer.2 research on the epidemiology of physical activity revealed that it appears to be a far more important risk factor than previously estimated.3840 quantitative estimates from the united states indicated that sedentary life is responsible for 35% of chd deaths , 32% of deaths from colon cancer and 35% of deaths from diabetes.41 furthermore , inactivity - related disease in the united states causes over 14 times more deaths annually than acquired immune deficiency syndrome ( aids).42 in saudi arabia , the prevalence of physical inactivity is extremely high , especially in women , and may be considered among the highest in the world.30 recent local data also showed a high prevalence of other chd risk factors among saudi population.3 in addition , type 2 diabetes mellitus is becoming increasingly more prevalent among saudis.343 obesity has also reached epidemic proportions , especially among saudi females.344 it is our own belief that strong associations do exist between the high prevalence of physical inactivity in the saudi population and the epidemic of modern chronic diseases and risk factors in saudi arabia . therefore , reducing the proportion of inactive saudis would have a tremendous impact on lowering these lifestyle - related diseases and risk factors , and thus reduce future health care costs in the kingdom . population attributable risk ( par ) is one of the more useful methods of estimating the proportion of a public health burden resulting from a particular risk factor.41 par is the risk in total population minus the risk in the unexposed group . thus , par provides an estimate of how much of a particular disease could be prevented if exposure to the risk factor was eliminated.45 it can be calculated from the estimate of relative risk ( rr ) and the population prevalence of the risk factor . rr is used to assess the magnitude of risk of exposed individuals to a particular disease relative to unexposed individuals . however , the societal impact of exposure depends not only on the magnitude of the relative risk but also on the prevalence of the risk factor in the population.45 the concept behind par is to help provide a balanced view between a relatively strong risk factor that affects fewer people and a relatively weaker risk factor that is more prevalent in a population.39 figure 3 shows par for major chd risk factors in the united states ( usa)40 and the united kingdom ( uk).46 par for physical inactivity in the us and the uk were 35% and 37% , respectively . this means that approximately one third of chd mortality could be attributed to physical inactivity in the us and the uk . in both countries , physical inactivity as a contributing risk factor to chd deaths was next in magnitude to hypercholesterolemia . estimated population attributable risk ( par ) for major chd risk factors in the usa and uk par of sedentary living for mortality from chd and diabetes to the data from saudi population were applied . physical inactivity prevalence data were taken from a recent study on saudi adult males living in riyadh , where there were three levels of exposure and prevalence estimates.29 for relative risk , estimates that were previously reported elsewhere were used.4041 of course , the relative risk means the number of deaths among active population divided by the number of deaths among the inactive population . as indicated in table 2 , par of sedentary living for mortality from chd and diabetes in saudi arabia was considerably high and much greater than what is reported in figure 3 for the uk and the usa since physical inactivity is much higher in saudi arabia than in the usa or in the uk . estimated population attributable risk ( par ) of sedentary living for mortality from coronary heart disease ( chd ) and diabetes mellitus in saudi arabia table 3 presents the relative risk , prevalence and par of some major chd risk factors applied to the saudi population . the relative risk data were taken from powell , et al.47 prevalence data for physical inactivity were taken from an earlier study on adult males in riyadh.29 the prevalence data of other chd risk factors were from a newly published paper on saudi adults between the ages of 30 and 70 years.3 as clearly shown in table 3 , physical inactivity represents a far more important risk factor than was previously thought . therefore , reducing the proportion of inactive saudi adults to 40% from the current figure of 80% would definitely reduce the burden of physical inactivity on public health . in the usa , healthy people 2010 calls for reducing to no more than 20% , the proportion of people 18 years and older who are inactive.25 relative risk ( rr ) , prevalence ( % ) and population attributable risk ( par ) of major coronary heart disease risk factors applied to saudi population it was clear from the available evidence that physical inactivity is becoming more prevalent in the saudi population of different ages and both sexes . this high prevalence of inactivity in saudi arabia represents a major public health burden , as evidenced by the high par of physical inactivity , compared with those of the usa and uk . moreover , due to the high prevalence of other chd risk factors among saudis , the rate of lifestyle - related diseases ( chd , diabetes , obesity , etc ) in the society may keep escalating to epidemic proportions in the near future . unless concrete steps are taken to reduce physical inactivity in the saudi population , the future public health cost will be heavily burdened . physical activity is associated with numerous health benefits and plays a major role in modifying many other chd risk factors . the following recommendations for reducing physical inactivity and promoting active living are made : ( 1)national policy and legislative initiatives are urgently needed to encourage active lifestyle and discourage sedentary living habits . this recommendation was clearly stated as an important objective in the global strategy on diet and physical activity , launched recently by who.(2)there is a need to establish a surveillance system to monitor physical activity in the saudi population . monitoring physical activity of the saudi population at regular intervals would definitely provide important database . such a database would represent a cornerstone for any programs that would aim at combating physical inactivity and promoting active living.(3)medical communities and health care providers must play a leading role in promoting physical activity , by providing routine assessments and counseling on physical activity and exercise prescription for their patients . this is consistent with many recent appeals from leading medical and public health organizations , such as the american heart association , the american academy of pediatrics , the american college of sports medicine , the centers for diseases control and prevention , and the who.(4)implementation of daily physical education for students from kindergarten to grade 12 is necessary to promote life - long physical activity among saudis . emphasis should be on quality curricula and instructions that help students develop the knowledge , attitudes , motor skills and confidence needed to adopt and maintain physically active lifestyles.(5)opportunities for physical activity should be available for a wide range of people , including the elderly , children and women . given that walking is acceptable across sociodemographic subgroups of the saudi population , efforts must be made to increase outdoor as well as indoor walking trails . promoting brisk walks as a means of physical activity could markedly increase the proportion of physically active saudis . national policy and legislative initiatives are urgently needed to encourage active lifestyle and discourage sedentary living habits . this recommendation was clearly stated as an important objective in the global strategy on diet and physical activity , launched recently by who . there is a need to establish a surveillance system to monitor physical activity in the saudi population . monitoring physical activity of the saudi population at regular intervals would definitely provide important database . such a database would represent a cornerstone for any programs that would aim at combating physical inactivity and promoting active living . medical communities and health care providers must play a leading role in promoting physical activity , by providing routine assessments and counseling on physical activity and exercise prescription for their patients . this is consistent with many recent appeals from leading medical and public health organizations , such as the american heart association , the american academy of pediatrics , the american college of sports medicine , the centers for diseases control and prevention , and the who . implementation of daily physical education for students from kindergarten to grade 12 is necessary to promote life - long physical activity among saudis . emphasis should be on quality curricula and instructions that help students develop the knowledge , attitudes , motor skills and confidence needed to adopt and maintain physically active lifestyles . opportunities for physical activity should be available for a wide range of people , including the elderly , children and women . given that walking is acceptable across sociodemographic subgroups of the saudi population promoting brisk walks as a means of physical activity could markedly increase the proportion of physically active saudis .

because of the enormous changes in the lifestyle of saudis in the last three decades , the risk factors of coronary heart disease ( chd ) , including physical inactivity , are increasingly becoming prevalent in the society . this paper provides an overview of the importance of physical activity in health promotion and disease prevention , and discusses the public health burden of physical inactivity in saudi arabia . available evidence clearly indicates that physical inactivity is extremely prevalent in the different ages and sex of the saudi population . this high prevalence of inactivity in saudi society presents a major public health burden , as evidenced by the high risk in the saudi population as a risk of physical inactivity compared with the populations of united states and the united kingdom . unless concrete steps are taken to reduce physical inactivity in the saudi population , the future public health cost would be enormous . it is well known that physical activity is associated with numerous health benefits and plays a major role in modifying many other chd risk factors . finally , several recommendations for reducing physical inactivity and promoting active life in the saudi population have been discussed .

cancer , which is commonly a harbinger of imminent patient death , is a group of diseases characterized by uncontrolled growth and spread of abnormal cells and aberrant cell behavior , which leads to expansive masses that destroy surrounding normal tissue and is able to attack vital organs resulting in disseminated disease . it is an inevitable matter of concern in the medicinal chemistry era and an increasing burden to the population . according to estimates from the international agency for research on cancer ( iarc ) , the global burden is expected to grow to 21.4 million new cancer cases and 13.2 million cancer deaths by 2030 . there were 12.7 million new cancer cases in 2008 worldwide , of which 5.6 million were in the economically developed countries and 7.1 million were in the economically developing countries . the corresponding estimates for total cancer deaths in 2008 were 7.6 million ( about 21,000 cancer deaths a day ) , 2.8 million in the economically developed countries and 4.8 million in the economically developing countries [ 1 , 2 ] . pathophysiology of all cancers involves the malfunction of genes that control cell growth , division , and death . cancers evolve through multiple changes resulting from a combination of hereditary and environmental factors , which mutate genes encoding critical cell - regulatory proteins . the new generations of anticancer drugs affect the signals that promote or regulate the cell cycle , growth factors and their receptors , signal transduction pathways , and pathways affecting dna repair and apoptosis rather than targeting the direct synthesis of dna . chalcone derivatives of diverse chemical architectures are quite significant in anticancer drug discovery and hence are in the center of attention of drug hunters . anticancer activity of chalcone might be due to molecular alteration such as induction of apoptosis , dna and mitochondrial damage , inhibition of angiogenesis , tubulin inhibition , kinases inhibition , and also drug efflux protein activities . chalcone is chemically 1,3-diaryl-2-propen-1-one ( figure 1 ) in which the two aromatic rings are joined by a three - carbon ,-unsaturated carbonyl system , representing a class of flavonoids that occur naturally in fruits and vegetables . chalcones are also metabolic precursors of some flavonoids and isoflavonoids . the plants containing chalcone are traditionally employed for therapeutical purposes [ 37 ] . chalcones were exploited well for their wide application in pharmacological area . it is reported that chalcones have several advantages such as poor interaction with dna and low risk of mutagenicity . hence , some clinically useful anticancer drugs have reported genotoxicity due to their interaction with amino groups in nucleic acids ; chalcones may be devoid of these side effects due to their structural flexibility . literature reveals that natural and synthetic chalcones are desirable to elicit cytotoxic and apoptotic activity ( figure 2 ) . they have been reviewed not only in the articles mentioned in the text but also in more recent articles , which have eluded the authors [ 911 ] . this review ( surveyed database , 20132015 ) is complementary to the researchers for optimizing the lead chalcone as potential anticancer scaffold with increased potency . cell death in tumors is a passive degradative reaction known as necrosis , most likely due to inadequate angiogenesis within the tumor cell . on the other hand , apoptotic cell death includes a number of gene families , and altering proapoptotic pathways specifically in tumors is something of a holy grail for oncology and medicinal chemistry . when cell production rate exceeds the cell loss rate through mitosis , it results in net tumor . apoptosis is often referred to as an altruistic cell suicide process , where damaged dna in the cell produces signals , resulting in both repair and apoptotic pathways , and if repair can not be done then the cell undergoes apoptosis in a manner like better dead than wrong . the cells which harbour mutant p53 will have a survival advantage over normal cells . in response to dna damage , normal cells upregulate p53 which acts as a transcription factor for cell cycle arrest and apoptosis ; thereafter , p53-mutant cells become unable to carry out this protective arrest or apoptosis and might survive with what otherwise would turn to lethal genetic damage , perhaps explaining why p53 mutations are so common in human cancers . the decision of apoptosis is largely played out on the mitochondrial surface between three major families : the so - called three horsemen of apoptosis . the final executioner proteases called caspases play pivotal role by cleaving critical substrates such as dna repair enzymes and cytoskeletal proteins , but they are stored as zymogens bound to an apoptotic adenosine triphosphate , apoptosis - activating factor-1 ( apaf-1 ) , which is the mammalian homologue of the nematode caenorhabditis elegans cell death protein , ced-4 . once cytochrome c is released , it binds to the cytosolic protein apaf-1 to facilitate the formation of apoptosome , which in turn activates apoptotic caspases . however , proapoptotic bcl-2 family proteins such as bax which is upregulated by p53 can activate apoptosis by releasing cytochrome c ( cyt - c ) from mitochondria , where apaf-1 activation takes place [ 2 , 6 , 12 , 13 ] . literature on anticancer chalcones highlights the employment of three pronged strategies , namely , structural manipulation of both aryl rings , replacement of aryl rings with heteroaryl scaffolds , and molecular hybridization through conjugation with other pharmacologically interesting scaffolds for enhancement of anticancer properties . various substitutions on both aryl rings ( a and b ) of the chalcones , depending upon their positions in the aryl rings , appear to influence anticancer activity by interfering with various biological targets . similarly , heterocyclic rings , either as ring a or as ring b in chalcones , also influence the anticancer activity shown by this class of compounds . hybrid chalcones formulated by chemically linking chalcones to other prominent anticancer scaffolds such as benzodiazepines , benzothiazoles , and imidazolones have demonstrated synergistic or additive pharmacological activities too . anticancer activity of three naphthyl chalcones , 1 , 2 , and 3 , shown in figure 3 was investigated by winter et al . , these chalcones were found to possess concentration- and time - dependent cytotoxicity on murine lymphoblastic leukemia cell line ( l1210 ) . chalcones 1 , 2 , and 3 induced apoptosis via an activated caspases - dependent pathway . the activities of caspase-8 , caspase-9 , and caspase-12 were found to increase after the treatment of l1210 cells with the cc50 of 30 m of chalcone 1 and 40 m of chalcones 2 and 3 . it was found that chalcones 1 and 2 induced an increase of proapoptotic proteins bax , bid , and bak ( only chalcone 2 ) , as well as a decrease in antiapoptotic bcl-2 expression . these chalcones also induced an increase in death receptor fas and a decrease in p21 and p53 expression . chalcones 1 and 2 exert a statistically significant increase in cytochrome c release from mitochondria to cytoplasm . cytochrome c release mediates caspase-9 activation , while the apoptosis inhibitors from bcl-2 family prevent the efflux of cytochrome c from mitochondria . chalcone 3 seems to act by a different mechanism to promote cell death , as it did not change the mitochondrion - related proteins nor did it induce the cytochrome c release . chalcones 1 , 2 , and 3 induced an increase in cell calcium concentration and an increase in chop ( c / ebp homologous protein ) expression which is the transcription factor involved in endoplasmic reticulum stress , together with an increase in caspase-12 activity , suggesting that chalcones could induce an endoplasmic reticulum ( er ) stress . they did not result in the arrest of g0/g1 , s , or g2/m phases of the cell cycle in contrast to paclitaxel , which was used as a positive control in this study and induced a statistically significant enrichment of g2/m fraction . they presented a selectivity index ( si ) higher than one , indicating that chalcones show higher selectivity to leukemic cells when compared to nontumoral cells ( vero = monkey kidney cells and nih/3t3 = murine fibroblast ) . the only exception was chalcone 3 which presented the same toxicity in l1210 and vero cells . the selectivity index ( si ) corresponds to cc50 ( concentration of the compounds , which results in 50% of cell viability ) of chalcones on nontumoral cell lines ( vero and nih/3t3 ) divided by cc50 determined for cancer cells ( l1210 ) . 2,4,5-trimethoxychalcones 4 , 5 ( figure 3 ) were found to reduce the viability of human k562 acute myeloid leukemia cell and human jurkat acute lymphoid leukemia cell significantly and caused a cytotoxic effect in both cell lines in a concentration- and time - dependent manner . they were found to have low ic50 values ( 4 to 8 m ) against both cell lines and did not have a cytotoxic effect on normal human lymphocytes . the mechanism of action involves the reduction of cell proliferation which has been shown by a reduction in the expression of cell proliferation marker ki67 . cell death induced by 2,4,5-trimethoxychalcones was due to induction of apoptosis through the reduction of the mitochondrial membrane potential , reduction in bcl-2 expression , increase in bax expression , increase in active caspase-3 , and activating the intrinsic pathway and execution phase of apoptosis . histone deacetylases ( hdacs ) are enzymes which catalyze removal of acetyl groups from lysine residues . hdacs play a critical role in epigenetic gene regulation and hence control multiple cellular events . hence , hdacs expressions and/or activity are deregulated in various cancer subtypes , leading to a target for anticancer therapy . coumarin - containing compounds 6a , 6b , and 6c ( figure 3 ) were found to inhibit total hdac activity by 2050% at 100 m in chronic myeloid leukemia k562 and histiocytic lymphoma u937 cell lines . the activity of the compounds was compared to peripheral blood mononuclear cells ( pbmcs ) of healthy donors , which showed no effect on pbmc viability . in comparison to 6a , compound 6b , with a methoxy group at r3 , presented increased levels of inhibition . compound 6b was tested against seven hdac isoenzymes ( 1 , 2 , 3 , 6 , 8 , 10 , and 11 ) and acted as a pan - hdaci with ic50s between 12 and 85 m . 6b inhibited hdac3 with an ic50 at 12 m and may serve as a lead for targeting this nuclear isoenzyme . chalcone 7 ( figure 3 ) with monomethoxy group at the ortho position showed a significant effect on downregulation of cancer cell proliferation and viability in three different leukemia cell lines ( k562 , jurkat , and u937 ) . the executioner caspase cleavage analyses indicated that the cytotoxic effect mediated by chalcone 6 is due to induction of apoptotic cell death by caspase-3 and caspase-7 activation . the cytotoxic effect was cell type - specific and targeted preferentially cancer cells as peripheral blood mononuclear cells ( pbmcs ) from healthy donors were less affected by the treatment compared to k562 , jurkat , and u937 leukemia cells . it was observed that compound 6 obeyed lipinski 's rule of five which indicates drug - likeness property of that molecule . 2-hydroxy-2,4,6-trimethoxy-5,6-naphthochalcone , 8 ( figure 4 ) , had most effectively inhibited the clonogenicity of sw620 colon cancer cells . the ic50 value for compound 8 was 14.5 1.1 m against sw620 cells . mechanistically , compound 8 triggered cell cycle arrest at g2/m phase , followed by an increase in apoptotic cell death , which involves the disruption of microtubular networks . the mitotic spindle network is essential for appropriate segregation of chromosomes between the two daughter cells at cell division , where disruption leads to apoptosis . compound 8 was found to destabilize microtubule assembly during mitosis , associated with failure of mitotic spindle formation , in turn blocking mitosis at the prometaphase or metaphase - anaphase transition . however , the study did not distinguish between the dna damage checkpoint and mitotic checkpoint pathways of apoptosis . trail ( tumor necrosis factor- ( tnf- ) related apoptosis - inducing ligand ) is a member of the tnf superfamily of cytokines . tnf family members contain highly conserved carboxyl - terminal domains and induce receptor trimerization for the transduction of intracellular signal . trail binds to four different receptors , two of which are death receptors 4 and 5 ( dr4 and dr5 , resp . ) , induces apoptosis , and decoys receptors , dcr1 and dcr2 , which lack the cytoplasmic death domain for transducing apoptotic death signals and protect cells from trail induced cell death by interfering with signaling through dr4 and dr5 . it is generally believed that transformed tumor cells are more susceptible to trail - mediated cell death owing to the selective loss of decoy receptors . chalcone 9 ( figure 4 ) ( ic50 = 4.39 m ) containing an amino ( -nh2 ) group was the most potent and selective against trail - resistant human colon cancer ( ht-29 ) and nontoxic against normal human embryonic kidney ( hek-293 ) cells . it was observed that a large portion of ht-29 cells had undergone late apoptosis and/or necrosis after treatment with 5-fluorouracil ( 5 m ) . these findings were consistent to show that chalcone 33 induces apoptosis rather than necrosis in ht-29 cells . it increases the death receptors expression ( trail - r1 and trail - r2 ) and proapoptotic markers ( p21 , bad , bim , bid , bax , smac , caspase-3 , and caspase-8 ) as well as reducing the antiapoptotic markers ( livin , xiap , and hsp27 ) . anthraquinone based chalcone compounds were synthesized and evaluated for their anticancer potential , where compounds 10a , 10b , and 10c ( figure 5 ) showed promising activity in inhibition of cervix adenocarcinoma ( hela ) cells with ic50 values ranging from 2.36 to 2.73 m and low cytotoxicity against healthy human fetal lung fibroblast cell lines ( mrc-5 ) . all the compounds cause the accumulation of cells in s and g2/m phases in a dose - dependent manner and induce caspase-3-dependent apoptosis and noncovalent interaction with the minor groove of the double - helical ct - dna and the damage of dna double helix structure . significant increase of p18 bax in treated hela cell line yielded consistency with the higher cytotoxic efficacy of compounds which implies the involvement of mitochondria in apoptotic process . synthesis and antiproliferative activity of a series of novel coumarin - chalcone hybrids were done by singh et al . . the most potent molecule of the series was compound 11 ( figure 5 ) . regression of tumors induced by hela cell xenografts in nod scid mice was found after oral administration of this molecule . it inhibited proliferation of cervical cancer cells ( hela and c33a ) by inducing apoptosis and arresting cell cycle at g2/m phase . apoptosis was due to induction of caspase - dependent intrinsic pathway and alterations in the cellular levels of bcl-2 family proteins . the mitochondrial transmembrane potential also got highly depleted in compound 11 treated cells due to an increase in bax / bcl-2 ratio and intracellular ros . compound 11 induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspase-3 and caspase-7 . tumor suppressor protein p53 and its transcriptional target puma ( p53 upregulated modulator of apoptosis ) were upregulated , suggesting their role in mediating the cell death . based on dose response curves , the calculated ic50 ( after 48 h treatment ) was 4.7 1.0 m for c33a cells and 7.6 2.0 m for hela cells . similar results were found in colony formation assay , where significant decrease in cellular colonies was observed in compound treated groups . chalcone 11 was apparently nontoxic to the animals as they did not show any loss of weight during experiment . cell population with fragmented nuclei increased in a time - dependent manner with the addition of chalcone 11 . significant increase in apoptotic cells with fragmented nuclei , which is consistent with the changes in nuclear morphology and flow cytometry analysis , indicated induction of apoptosis by chalcone 11 . novel isoxazolyl chalcones were synthesized and evaluated for their activities in vitro against human non - small lung cancer cells ( h1792 , h157 , a549 , and calu-1 ) . compounds 12a and 12b ( figure 6 ) were identified as the most potent anticancer agents with ic50 values ranging from 1.35 to 2.07 m and from 7.27 to 11.07 m against h175 , a549 , and calu-1 cell lines , respectively . the structure - activity relationship study showed that compounds bearing electron withdrawing groups ( ewg ) at the 2-position of the phenyl ring in aryl group were more effective . compounds 12a and 12b increased the percentage of cell in sub - g0 phase and decreased the percentage of cell in g0/g1 phase . these results suggested that 12a and 12b induced apoptosis in a549 cells in a dose - dependent manner . they have upregulated the dr5 in a dose - dependent manner which suggests the extrinsic pathways of apoptosis . cleaved bands of caspase-8 , caspase-9 , caspase-3 , and poly ( adp - ribose ) polymerase ( parp ) also indicated the induction of apoptosis . imine derivatives of hybrid chalcone analogues 13a , 13b , 13c , and 13d ( figure 6 ) containing anthraquinone scaffold were synthesized and evaluated for their in vitro cytotoxic activity against hela , ls174 , and a549 cancer cells . compound 13c with furan ring linked to imino group showed potent activity against all target cells with ic50 values ranging from 1.76 to 6.11 m , which are comparable to cisplatin . generally , introduction of electron withdrawing substituents such as -cl and -cf3 in metaposition of the phenyl ring was found to exert good cytotoxicity . compounds did not have tendency to significantly accumulate the cells in sub - g1 phase , except compound 13a , which led to an increase in sub - g1 ( observed only in cells undergoing apoptosis ) phase in a549 cells as well as changes in g2/m arrest of a549 and ls174 cells . compounds induced the activities of caspase-3 and caspase-8 in treated hela cells and exhibited strong antiangiogenic activity by showing potent inhibitory potential against matrix metalloproteinases ( mmp-2 ) secretion . strong implication of mmp-2 is found in angiogenesis and promotes tumor metastatic potential playing crucial role not only in invasion but also in determination of cancer cell transformation , growth , apoptosis , and signal transduction pathways . 3-phenylquinolinylchalcone derivatives , 14 and 15 ( figure 6 ) , were synthesized and evaluated for their antiproliferative activities . compound 14 was active against the growth of non - small cell lung cancer cells h1299 and skbr-3 breast cancer cells with ic50 values of 1.41 and 0.70 m , respectively , which was more active than the positive topotecan ( ic50 values of 6.02 and 8.91 m , resp . ) . compound 15 exhibited an ic50 value of less than 0.10 m against the growth of mda - mb231 in a dose- and time - dependent manner and was noncytotoxic to the normal mammary epithelial cell ( h184b5f5/m10 ) . the proportion of cells was decreased in g1 and accumulated in g2/m phase after 24 h treatment of compound 15 , while the hypodiploid ( sub - g0/g1 phase ) cells increased . compound 15 caused activation of caspase-3 , cleavage of parp , and consequently the cell death . caspase-3 is an executioner caspase whose activation leads to the cleavage of key cellular proteins including dna repair enzyme poly ( adp - ribose ) polymerase which is involved in dna repair predominantly in response to environmental stress and is important for the maintenance of cell viability . -cyano bis(indolyl)chalcone 16 ( figure 6 ) was found to be the most potent and selective against a549 lung cancer cell line ( ic50 = 0.8 m ) in vitro which is time - dependent . the activity is due to the presence of methoxy and fluoro substituents on indole rings . it was found to enhance tubulin polymerization suggesting the activity as microtubule stabilizing agents , though it was a preliminary study . nf-b pathway has been shown to be critical to survival of lung cancer cells , and many natural products obtained from plants were found to inhibit the activation of this pathway . two cardamonin analogs , 4,4-dihydroxylchalcone , 17 , and 4,4-dihydroxy-2-methoxychalcone , 18 ( figure 6 ) , were found to suppress the activation of nf-b pathway in lung cancer cells . compound 17 inhibited the survival of primary lung cancer cells with ic50 values of 0.44 0.05 and 0.16 0.03 m , respectively , against two lung cancer cells tested . compound 18 inhibited their survival with similar ic50 values ( 0.56 0.12 m and 0.65 1.36 m ) . immunoblot analysis displayed that caspase-3 and parp were both cleaved in the two lung cancer cell lines treated with compounds 17 and 18 , indicating the activation of apoptotic pathway . subsequent estimation of the phosphorylation level of ikk , nf-b pathway stimulating kinase , by immunoblotting assay demonstrated that both decreased ikk phosphorylation in lung cancer cell lines including a549 , nci - h460 , and nci - h1668 and lead to the accumulation of ib and ib and the export of nuclear p65 transcription factor . from the immunofluorescent staining , it was shown that transcription factor p65 , which is a downstream effect of nf-b pathway , was sequestered in the cytoplasm of a549 cells treated by compound 17 or compound 18 . incorporation of luciferase reporter system also results in transcription activity of nf-b in lung cancer cells treated with compound 17 or compound 18 and demonstrated that relative luciferase expression was significantly reduced in compound 17 or compound 18 treated lung cancer cells . in addition , the transcription of target genes of nf-b pathway , bcl-2 , and survivin was found to be suppressed in lung cancer cells treated with the two compounds . . higher concentration of the two compounds greatly inhibited the growth of tumors by 71.0 and 79.1% , respectively , although inhibitory effect was not significant at lower concentration . compared with compound 17 , compound 18 appeared to be slightly more potent to retard the growth of established tumors . -carboline based chalcones were synthesized and evaluated for their cytotoxic activity against a panel of human cancer cell lines . compounds 19a and 19b ( figure 6 ) showed very good antiproliferative activity against breast cancer cell line ( mcf-7 ) , where ic50 values were recorded at 2.25 m and 3.29 m , respectively . furthermore , compound 19a markedly induced dna fragmentation and apoptosis in breast cancer cells , which is an essential step in the cells undergoing apoptosis . the molecular mechanism to induce apoptosis in human hepatoma smmc-7721 cells was investigated by using 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone , 20 ( figure 7 ) , isolated from the buds of cleistocalyx operculatus . compound 20 induced apoptosis via a mitochondria dependent pathway involving inhibition of bcl-2 expression leading to disintegration of the outer mitochondrial membrane . further downstream of the apoptosis cascade , compound 20 , increases caspase-3 and caspase-9 activities . angiogenesis and invasion are two highly interconnected events in cancerous cells where involvement of multiple molecules and pathways takes place . hypoxia inducible factor-1 ( hif-1 ) has been identified as an important protein involved in those events . activated hif-1 controls the expression of genes involved in tumor cell immortalization , stem cell maintenance , metabolic reprogramming , autocrine growth , and treatment failure , thereby making it an important target for the development of new antitumor therapy . a novel series of chalcone derivatives were synthesized and their biological activities against hif-1 were evaluated by wang et al . it has been found that compound 21 ( figure 7 ) exhibited inhibitory effects on hif-1 at 10 m concentration by downregulating the expression of hif-1 and hif-1 under hypoxic conditions . the hif inhibition rate has been found to be 83.0 16.9% , while topotecan , hif-1 inhibitor , was used as control and for comparison it has the inhibition rate of 82.8 2.9% . in addition , compound 21 displayed inhibitive effect against the migration and the invasion of hep3b ( human liver cancer cell line ) and human umbilical vein endothelial cells ( huvec ) and tube formation of huvecs and significant antiangiogenic and anti - invasive activities . from the in vivo study it has been found that 21 could retard tumor growth of hep3b xenograft models and reduced cd31 and mmp-2 expression in tumor tissues . cd31 ( cluster of differentiation 31 ) is platelet endothelial cell adhesion molecule ( pecam-1 ) , which is a protein found to be expressed in certain tumors . compound 27 was well tolerated and was found to be nontoxic up to 200 mg / kg in swiss mice in acute intravenous toxicity . the molecular mechanisms of growth inhibition in human bladder cancer cell lines t24 and ht-1376 by chalcone 22 ( figure 8) are due to the induction of apoptosis , cell cycle progression inhibition by regulating cell cycle related factor like significant reduction in the expression of cyclin a and cyclin b1 , decreases in the expression of cdc2 , and also increases in the expression of p21 and p27 . so , chalcone may cause cell cycle arrest by reducing the complex of cdc2/cyclin b due to downregulation of multiple g2/m regulating proteins . it triggers the mitochondrial apoptotic pathway by releasing cytochrome c from the cytoplasm with an increase in caspase-9 and activating caspase-3 . it is also increases the expression of proapoptotic proteins bax and bak , which are responsible for the initiation of mitochondrial apoptotic pathway , and decreases the expression of antiapoptotic proteins bcl-2 and bcl - xl , while overexpression of bcl-2 and bcl - xl is associated with enhanced oncogenic potential . chalcone inhibits the nf-b activation by increasing the expression of ib in cytoplasm , leading to apoptosis . the copper chelate complex 23 ( figure 8) showed promising activity with ic50 value of 5.95 m against pc3 cancer cell line in vitro . the estimated ic50 value for doxorubicin was 8.7 m , which was used as positive control . it was found that cancer cells have high affinity to copper ions ; thus , upon chelation of chalcones with copper they are thought to be more liable to target cancer cells in microdendron architecture . it was assumed that copper ions might act as penetration enhancers of chalcones into cancer cells . even if copper ions are noncytotoxic to cancer cells , they might act as inhibitors of drug efflux proteins characteristic to cancer cells . drug efflux proteins , such as p - glycoproteins ( pgp ) , breast cancer resistance protein ( bcrp ) , and multidrug resistance - associated protein 1 ( mrp1 ) , are responsible for drug resistance of cancer cells . the high cytotoxic activity is attributed to the presence of the p - methylphenyl moiety of ring b and the copper ion . hybrids of n-4-piperazinyl - ciprofloxacin - chalcone were prepared by abdel - aziz et al . national cancer institute ( nci ) selected 5 compounds , namely , 24a , 24b , 24c , 24d , and 24e ( figure 9 ) , to carry out in vitro one - dose anticancer assay against full nci 60 cell lines derived from leukemia , lungs , colon , renal , melanoma , cns , prostate , ovarian , and breast cancer cell lines . of the selected compounds , 24a and 24d exhibited the highest ability to inhibit the proliferation of different cancer cell lines while undergoing one - dose anticancer test . among the nine tumor subpanels tested for , compound 24a was found to have broad - spectrum antitumor activity with selectivity ratios ranging between 0.42 and 1.87 at the gi50 level without producing selectivity , while compound 24d showed high selectivity towards the leukemia subpanel with selectivity ratio of 6.71 at gi50 level . moreover compound 24d showed remarkable anticancer activity against most of the tested cell lines with gi50 ranging from 0.21 to 57.6 m . furthermore , compounds 24c and 24e have shown remarkable topo - ii inhibitory activity compared to etoposide at 100 m and 20 m concentrations . compounds 24c and 24e were found to be potent topo - i inhibitors at 20 m concentration when compared to camptothecin . the results that were obtained indicated that introduction of n-4-piperazinyl moiety of ciprofloxacin into the chalcones derivatives dramatically increases the anticancer activity of the tested compounds severalfold higher than that of the activity shown by weak anticancer ciprofloxacin . increase in antiproliferative activity of the tested compounds might be the result of either synergistic effect of the chalcone derivatives and/or alteration of the physicochemical properties of ciprofloxacin . to determine the dna unwinding properties of 24c and 24e , amsacrine ( m - amsa ) , a well - known dna intercalator , was chosen as a standard where a supercoiled phot1 dna was used as a substrate since unwinding of the double strand of dna helix is a practical characteristic of intercalating drugs . amsacrine blocked unwinding of phot1 dna in the presence of excess topo - i in dose - dependent manner , while compounds 24c and 24e did not block dna unwinding at high concentration up to 1000 m treatment . the assay indicated that 24c and 24e do not interact with dna but interact with topoisomerases for their inhibitory activity . compound 25 ( figure 9 ) with a propionyloxy group at the 4-position of the left phenyl ring showed the most potent inhibition activity which inhibited the growth of hepg2 , a549 , a375 , smmc-7221 , and k562 cancer cell lines with ic50 values of 0.15 , 0.36 , 0.62 , 0.61 , and 0.52 m , respectively . compound 25 was also shown to arrest cells in the g2/m phase of the cell cycle and inhibit the polymerization of tubulin . molecular docking study suggested that compound 25 binds into the colchicine binding site of tubulin . in summary , these findings suggest that compound 23 is a promising new antimitotic compound for the potential treatment of cancer . a new series of pyrano chalcone derivatives containing indole moiety synthesized by wang et al . ( 2014 ) were evaluated for antiproliferative activities . among those , compound 26 ( figure 9 ) , with a propionyloxy group at the 4-position of the phenyl ring a and n - methyl-5-indolyl on the b ring displayed the most potent cytotoxic activity against all tested cancer cell lines including multidrug resistant phenotype , which inhibits cancer cell growth with ic50 values ranging from 0.22 to 1.80 m . the tested cell lines were smmc-7221 , hepg2 ( liver ) , pc-3 ( prostate ) , a549 ( lung ) , k562 ( leukemia ) , hct116 ( colon ) , skov3 ( ovarian ) , mcf-7 ( breast ) , vincristine resistant hct-8 ( hct-8/v ) , and taxol resistant hct-8 ( hct-8/t ) . compound 26 was the most potent towards hepg2 cells with ic50 value of 0.22 m . colchicine ( 2.5 m ) and paclitaxel ( 2.5 m ) were used as a reference . compound 26 produced cytotoxic activity in normal human liver cell line ( lo2 ) above the concentration of 10 m . it significantly induced cell cycle arrest in g2/m phase and inhibited the polymerization of tubulin . it was also shown from molecular docking analysis that compound 26 binds at the colchicine binding site of tubulin . chalcone 26 also exerted potent anticancer activity in hepg2 xenografts in balb / c nude mice , where the growth of tumors in mice treated with chalcone 26 ( 30 mg / kg ) was reduced by 56.58% compared with that in control mice treated with vehicle only at day 35 . positive control reduced the growth of tumors by 25.93% . cell division cycle 25 ( cdc25 ) , which includes cdc25a , b , and c homologues , a subfamily of dual - specificity protein tyrosine phosphatases , plays a role in the regulation of the cell cycle . the cdc25a and cdc25b isoforms are overexpressed in primary tissue samples from various human cancers , which is strongly associated with tumor aggressiveness and poor prognosis . cdc25b controls cell cycle progression by catalyzing removal of covalently attached contiguous phosphates and the subsequent activation of cyclin - dependent kinase 1 ( cdk1 ) . cdc25b overexpression has been documented in a variety of human cancers , including head and neck and colon , and non - small cell lung cancer validates its oncogenic potential . a series of 2-hydroxy-4-isoprenyloxychalcone derivatives were evaluated for the inhibition of cdc25b activity by zhang et al . ( 2014 ) . among those , two compounds , 27 and 28 ( figure 9 ) , have been found to have the potent inhibition activity and significantly inhibited cdc25b with inhibition rates of 99.95% and 99.75% , respectively , at a dose of 20 g / ml , which is similar to the known tyrosine phosphatase inhibitor sodium orthovanadate . in vitro cytotoxic activity assays showed that compounds 27 and 28 were potent against hct116 , hela , and a549 cells . they were particularly cytotoxic against colon hct116 cancer cell line with ic50 values of 0.89 and 1.76 irinotecan ( ic50 = 2.14 mol / l ) , a clinically relevant camptothecin , was chosen as a reference drug . in colo205 xenograft balb / c nude male mice , compound 27 produced a tumor volume inhibition of about 50% . a new series of pyrazole chalcones were screened for the in vitro anticancer activities against mcf-7 ( human breast adenocarcinoma ) and hela ( human cervical tumor cells ) cell lines . compound 29 ( figure 9 ) showed the highest inhibition in human mcf-7 and hela cell lines with an ic50 value of 0.018 g / ml and 0.047 g / ml , respectively . the ic50 for standard doxorubicin in hela and mcf-7 cells was 1.15 g / ml and 1.84 g / ml , respectively . new prenylated chalcone , 30 ( figure 9 ) , named as renifolin c , was isolated from whole desmodium renifolium plants and cytotoxicity was evaluated against five human tumor cell lines ( nb4 , a549 , shsy5y , pc3 , and mcf7 ) using the mtt method , using paclitaxel as a positive control . the ic50 values were 6.4 m and 8.5 m against nb4 and pc3 cell lines and above 10 m for the other cell line tested . various benzylidene pregnenolones were synthesized and in vitro cytotoxicity studies were performed by banday et al . compounds 31a and 31b ( figure 9 ) were found to be active against the hct-15 ( colon ) and mcf-7 ( breast ) cell lines . chalcone 31a has ic50 value of 1.02 m and 0.79 m , respectively , against hct-15 and mcf-7 , while 31b has 0.81 m and 1 m , respectively . chalcone - coumarin hybrid 33 ( figure 10 ) ( ic50 = 0.53 m ) displayed cytotoxicity against human lymphoblastic leukemia cell line ( molt-3 ) , where etoposide ( ic50 = 0.05 m ) was taken as a standard . compounds 32 and 33 shown in figure 10 displayed higher cytotoxic potency against hepg2 cells than the control drug , etoposide . the hybrid 33 ( ic50 = 4.26 m ) was potent but was found to be toxic toward noncancerous african green monkey kidney cell line ( vero ) . though analog 32 displayed cytotoxic activity against hepg2 with ic50 value of 8.18 m , it was found to be nontoxic against vero cells . it has been shown from molecular docking study that compounds could snugly occupy the colchicine binding site of -tubulin . ( 2013 ) . among them , potent chalcone 34 ( figure 10 ) induced apoptotic cell death of a wide variety of tumor cell lines including multidrug resistant human tumor cell lines in the nanomolar range by attacking microtubules . flow cytometric study showed that chalcone 34 significantly induced cell cycle arrest in g2/m phase at 0.2 m concentration . in vitro immunofluorescence staining and tubulin polymerization assay displayed that chalcone 34 promoted tubulin polymerization into microtubules and caused microtubule stabilization similar to paclitaxel , which is suggesting that chalcone 34 is a microtubule stabilizer via enhancing the rate of tubulin polymerization . then , in in vivo study , 34 exhibited potent inhibitory activities in human hepg2 xenograft tumor models . chalcone derivatives , named parasiticins c , 35 and 36 ( figure 10 ) , were isolated from the leaves of cyclosorus parasiticus . the in vitro cytotoxic activities were evaluated against lung cancer a549 , hepatocellular carcinoma hepg2 , breast cancer mcf-7 and mda - mb-231 , leukemia all - sil , and pancreatic cancer sw1990 . compounds 35 and 36 exhibited substantial cytotoxicity against all six cell lines , especially toward hepg2 with the ic50 values of 1.60 and 2.82 m , respectively , which is comparable to the doxorubicin positive control ( 1.19 m ) . they induced apoptosis in the hepg2 cell line . in the clonogenicity assay on hepg2 cell line , capacity of single cancer cells to generate colonies compound 35 showed more potent cytotoxicity than 36 , suggesting that the presence of an additional lactone ring fused enhances the cytotoxic activity . a fission yeast , schizosaccharomyces pombe , was utilized in bioassay guided screening on several plant extracts to search for anticancer agents by snchez - pic et al . it was found that an extract from leaves of corema album belonging to family ericaceae had antiproliferative activity . 2,4-dihydroxychalcone , 37 ( figure 11 ) , the promising cytotoxic compound , was found in this extract and cytotoxic activity is likely due to its ability to induce dna damage which blocks cell cycle progression at the g2/m transition in a concentration of 8 m to 20 m . compound 37 could induce dna damage or interfere with dna replication , which would eventually result in the activation of the cell cycle checkpoints , thus blocking cell cycle in g2 . in subsequent study , deletions of rad3 , chk1 , and cds1 , three key kinases required for dna checkpoints operating at g2/m , were done . rad3 is required in order to signal downstream of either dna replication problems or dna damage , whereas chk1 transmits dna damage - induced signals and cds1 signals in response to defective dna replication . it was found that both rad3 and chk1 deletions suppressed the cell cycle block produced by compound 37 , while cds1 deletion showed partial suppression , suggesting that the g2/m block observed in compound 37 is mostly caused by the activation of dna damage checkpoint response . methyl methanesulfonate ( mms ) , an alkylating compound that induces dna damage , was used as control . thus , compound 37 ( 820 m ) directly or indirectly causes damage to the dna which results in a rad3-dependent and chk1-dependent cell cycle block at the g2/m transition . cathepsins have emerged as a potential target for chemoprevention and anticancer therapy . increased levels of cathepsin b and cathepsin h in a variety of tumors have shown their contribution towards invasion and metastasis . molecule 38 ( figure 12 ) has been found to be potent among the synthesized inhibitors , which inhibits cathepsin b and h. the ki ( inhibition constant ) value against cathepsin b was 6.18 10 m and 2.8 10 m for cathepsin h. the inhibition was of competitive type . 2-hydroxychalcones have been evaluated as potential inhibitors to these cysteine proteases probably due to presence of ,-unsaturated carbonyl moiety in the molecule . -ch2sh moiety present at the active site of enzymes can interact with the electron deficient center present in the chalcones inhibiting the enzymes activity . nitrosubstitution is useful for an effective interaction with cathepsins b and h. its inhibition to cathepsin b as compared with cathepsin h indicates that cathepsin b 's active site is more susceptible to these compounds as compared to cathepsin h. the results were also found to be consistent when compared with in silico docking studies . cotreatment with a chemosensitizer such as compounds 39a and 39b ( figure 13 ) and trail was found to be more efficient to treat gbm ( glioblastoma multiforme ) than treatment with trail alone . gbm is one of the most aggressive forms of human malignant brain tumors , characterized by rapid growth , extensive invasiveness , and robust neoangiogenesis . against human crt - mg astroglioma cells by using the lactate dehydrogenase ( ldh ) release assay , it was found that compounds 39a and 39b with trail ( 25 ng / ml ) showed good cytotoxicity . 2-ethylamino and substituted triazole groups are promising templates in the backbone of chalcone to develop trail sensitizers as anticancer agents . mdr ( multidrug resistance ) can be defined as the ability of cancer cells to survive exposure to different chemotherapeutic agents and is a major cause of treatment failure in human cancers . 4-hydroxy-2,6-dimethoxychalcone , 40 ( figure 14 ) , was isolated from polygonum limbatum with ic50 values of 2.54 m against multidrug resistant cem / adr5000 leukemia cells . p - glycoprotein - expressing and multidrug resistant cem / adr5000 cells were much more sensitive toward compound 40 than doxorubicin ( ic50 = 195.12 m ) , which is a reference drug . compound 40 ( figure 14 ) has been found to arrest the cell cycle between g0/g1 phases , which is via the strong induction of apoptosis by disrupting mitochondrial membrane potential ( mmp ) and an increased production of reactive oxygen species ( ros ) in the studied leukemia cell line . it was found that caspases were not involved in the apoptotic pathway induced by compound 40 . the presence of hydroxyl ( -oh ) and methoxy ( -och3 ) substituents influences the activity of compound 40 . the membrane transporter p - glycoprotein ( p - gp ) , encoded by mdr1 gene , is the main mechanism responsible for resulting in decreased intracellular drug accumulation in human mdr cancers . mitogen activated protein ( map ) kinase pathway is involved in p - gp - mediated mdr . dimer of chalcone named as tomoroside b , 41 ( figure 14 ) , was isolated from the aerial parts of helichrysum zivojinii . the combined effects of chalcone dimer 41 and tipifarnib ( the inhibitor of map kinase signaling cascade , a farnesyltransferase inhibitor ) in mdr cancer cell line , nci - h460/r , were found . these results point to the ability of compound 41 to enhance tipifarnib efficacy in mdr cancer cells as it reduced the ic50 for tipifarnib from 9.0 m ( applied alone ) to 3.8 m . activity was compared to tipifarnib employing human sensitive non - small cell carcinoma cell line nci - h460 ( ic50 = 198 m for compound 41 alone ) , its multidrug resistant ( mdr ) counterpart nci - h460/r ( ic50 = 172 m for compound 41 alone ) , and spontaneously immortalized normal human keratinocyte cell line hacat . the concentration around 100 m of compound 41 significantly inhibited the growth of normal hacat cells , considering that hacat are normal , but transformed cells with immortalized phenotype ; these results point to the potential of compound 41 to exert the cancer preventive effects . compound 41 increased the expression of hif-1 , probably by exerting antioxidant and redox status modulator effect . compound 41 increased the expression of hif-1 and acted synergistically with tipifarnib probably by inhibiting the map kinase prosurvival signaling . to identify potent anticancer agents against the cisplatin - resistant human ovarian cancer cell line the two most active compounds were 42 ( gi50 = 3.87 m ) and 43 ( gi50 = 3.95 m ) ( figure 14 ) . it was found that compounds 42 and 43 trigger cell cycle arrest at the g2/m phase and apoptotic cell death in a2780/cis cancer cells . aurora a kinase is known to be involved in cell cycle arrest at the g2/m phase . in vitro aurora a kinase assay of compound 42 showed the half - maximal inhibitory concentration ( ic50 ) of 66.4 m . because aurora a kinase may not be a real molecular target of compound 42 , gi50 value of compound 42 is higher than the ic50 value of 42 . chalcone architecture linked with various substitutions , such as methoxy , amino , and hydroxyl , and alteration of both rings has been found to be effective in making it a potential candidate in the anticancer armamentarium . the related studies showed excellent cytotoxic activities of chalcones in the different cell death pathways , not just acting in blocking the process of cell division . inhibitory concentrations in nanomolar range were impressive to show its ability to arrest cell division . furthermore , besides preclinical study , clinical study of chalcone may yield the development of research in this field . it is expected that this review may give the medicinal chemists a basis to get in touch with the recent updates and will be helpful for enrichment in this field .

cancer is an inevitable matter of concern in the medicinal chemistry era . chalcone is the well exploited scaffold in the anticancer domain . the molecular mechanism of chalcone at cellular level was explored in past decades . this mini review provides the most recent updates on anticancer potential of chalcones .

premature beats arising from foci other than pulmonary veins have been related to its pathogenesis . a 64-year - old female underwent superior vena cava ( svc ) isolation after triggers were identified originating from the svc following pulmonary vein isolation ; immediately after svc isolation , she developed junctional rhythm with symptomatic hypotension requiring emergent management . apical motion abnormalities were noticed in the echocardiography suggesting stress - induced cardiomyopathy which resolved 48 hours later . although received a dual chamber pacemaker , intact sinus node function returned 2 weeks later . superior vena cava isolation in those with trigger mediated atrial fibrillation following pulmonary vein isolation ( pvi ) is performed to enhance long - term outcomes . we present the first case of time course of recovery of sinus node function , injured during svc isolation . atrial fibrillation ( af ) is the most common sustained arrhythmia ; its incidence increases with aging . this condition could increase morbidity and mortality due to its high potential to cause heart failure , thromboembolic events and the related conditions associated to its treatment such as bleeding . the cornerstones for the management of atrial fibrillation are anticoagulation to prevent embolic events , control symptoms and avoid deterioration of heart function . af arising from the pulmonary veins ( pv 's ) was recognized by haissaguerre et al . , in a study that showed 94% of the time pvs are implicated . however , there is some evidence showing that non - pulmonary veins ( npv 's ) premature beats could trigger af in up to 28% of cases . the most common npv foci include the left atrial posterior wall ( 38% ) and superior vena cava svc (37% ) . other areas acting as npv include crista terminalis , coronary sinus , ligament of marshall and interatrial septum . catheter techniques looking for the electrical isolation of pv antrum and other atrial tissue originating premature beats has emerged as therapeutic option for symptomatic patients with atrial fibrillation , providing symptomatic benefit . we present a clinical case of a patient who developed a transient sinus node block as a complication during pvi and superior vena cava isolation ( svci ) . , although patient did receive a dual chamber pacemaker , two weeks later , sinus node function returned back to baseline , identifying the time course of recovery of sinus node function . a 64 year - old female with drug refractory af and atrial flutter underwent pvi and cavotricuspid isthmus(cti ) ablation . basal measurements and conduction study were considered normal . during the isoproterenol testing the patient developed recurrent episodes of recurrent atrial tachycardia ( at ) , left bundle branch block ( lbb ) and rapid ventricular response af ( cycle length up to 410 msec ) . pvi was carried out with irrigated radiofrequency ( rf ) ablation and lesions were tagged on geometry created using carto 3d sound system ( biosense webster , diamond bar , ca , usa ) ; we did a wide area circumferential ablation on all four pv 's including carinal ablations , with evidence of entrance block . however , triggered af was inducible with isoproterenol , and triggers in vein of marshall was successfully ablated . further testing revealed svc origination of npv triggers and svci was performed ( see fig . 1 , fig . 2 ) . during immediate observation , junctional rhythm at 36 bpm ( fig . 3 ) with hypotension requiring emergency administration of epinephrine and temporary transvenous pacemaker insertion was needed . an echocardiogram showed apical motion abnormalities , and coronary angiogram was negative for coronary artery disease . patient was implanted with a permanent pacemaker after failure of recovery of sinus node function at 48 hours . on weekly observations in clinic , recovery of sinus node function yamaguchi et al . showed that in those patients with npv foci pvi alone is not enough to reach long term responses suggesting that they should always be addressed during the index ablation procedure . controversies still exist about the npv foci management especially with svc triggers and conflicting evidence exists based on randomized control trials ( rct ) . . showed in 2003 that there were no difference between patients randomly assigned to pvi versus pvi + svci regarding af recurrence after ablation . however in a more recent clinical trial evaluating 2 randomly assigned groups to pvi compared to pvi + svci , the authors found that those patients who underwent both procedures were significantly less prone to paroxysmal af after 12 months of follow - up . the most common are svc stenosis , phrenic nerve damage with diaphragmatic paralysis and sinus node injury ( sni ) . described 6 patients who developed sni after svci , in this clinical study 5 patients had transient junctional rhythm that resolved in the first 48 hours postprocedure and just one patient required permanent pacemaker implantation . probably the sni is due to the damage to the atrial muscle sleeve that extends into the svc and contains sinus node pacemaker activity . to the best of our knowledge ours is the first report of sni associated with pvi + svci where time course of recovery of sinus node function was noted . identification of npv triggers and subsequent successful ablation is performed in order to get higher responses in the long term follow up after the index procedure . premature beats arising from svc triggering af is infrequent but when present has to be approached cautiously due to proximity of phrenic nerve and inadvertent sinus node injury .

introductionatrial fibrillation is the most common sustained heart arrhythmia . premature beats arising from foci other than pulmonary veins have been related to its pathogenesis.methods and resultsa 64-year - old female underwent superior vena cava ( svc ) isolation after triggers were identified originating from the svc following pulmonary vein isolation ; immediately after svc isolation , she developed junctional rhythm with symptomatic hypotension requiring emergent management . apical motion abnormalities were noticed in the echocardiography suggesting stress - induced cardiomyopathy which resolved 48 hours later . although received a dual chamber pacemaker , intact sinus node function returned 2 weeks later.conclusionsuperior vena cava isolation in those with trigger mediated atrial fibrillation following pulmonary vein isolation ( pvi ) is performed to enhance long - term outcomes . sinus node injury has been related previously to this procedure . we present the first case of time course of recovery of sinus node function , injured during svc isolation .

bilateral ectopic pregnancy is a rare condition and is divided in two subgroups , primary and secondary , based on history of assisted reproductive technology . a 30 year old primigravid woman with history of infertility and ovulation induction presented to a hospital in kashan in year 2013 . she had vaginal bleeding , abdominal pain and ultrasound findings suggested early pregnancy . due to high titer of -hcg , gestational trophoblastic disease was proposed and d8c was done in referral and admission to gyneco - oncology ward in tehran . repeat sonography suggested ectopic pregnancy in left side and repeat -hcg level showed an increase of 19435 miu / ml . bilateral ectopic gestation should be considered as a rare differential diagnosis for ectopic pregnancy . in this study , the implantation and development of fertilized ovum outside the uterine cavity is observed in approximately 2% of all pregnancies . over 95% of ectopic pregnancies because of the increase in incidence of sexually transmitted diseases , tubal surgery and more frequent use of ovulation induction and assisted reproductive technologies ( art ) , the incidence of ectopic pregnancies has grown in the last 30 years according to many reports from developed countries . ectopic pregnancy is still the leading cause of pregnancy - related deaths in developed countries ( 1 ) . however , bilateral tubal pregnancy is a rare clinical condition which occurs in only 1 per 200,000 pregnancies . this condition is divided in two subgroups , primary and secondary , and because of different physiopathological mechanisms , these two entities should be studied separately . however , as studies have shown , the cause of bilateral ectopic pregnancy after art is clearly different from spontaneous cases . clinical and paraclinical findings of this condition are the same as unilateral ectopic pregnancy so the distinct diagnosis is difficult ( 2 ) . in the present article , a case of bilateral tubal pregnancy a 30 year old primigravid woman has been hospitalized in kashan in year 2013 due to minimal vaginal bleeding and low abdominal pain . she had been under different treatments including sequential treatment with clomiphene , fsh and hmg in previous cycle . her last menstrual period was 8 weeks ago . in admission , vital signs were normal . the first transvaginal ultrasound revealed a suspicious gestational sac without yolk sac in uterus and normal adnexal area was reported . due to high level of -hcg ( 16000 miu / ml ) , without normal viable intrauterine pregnancy , dilatation and curettage had been done to clarify existence of intrauterine pregnancy or its complications or ep . due to high level of -hcg , gestational trophoblastic neoplasm was considered in differential diagnosis besides ectopic pregnancy and it resulted in gyneco - oncology consultation . the rise of -hcg level to 21770 miu / ml , 9 days after operation resulted in referral and admission of patients to gynecology - oncology ward of our center in tehran . positive findings in this admission to gynecooncology ward in tehran were low abdominal pain , minimal vaginal bleeding with stable vital signs similar to the ones observed in previous admission . through re - checking the -hcg level , titer of 19435 miu / ml was observed . the next ultrasound reported a normal uterus with endometrial thickening up to 11 mm , a heterogeneous mass measuring 49 mm in left adnex , suspicious to ectopic pregnancy and two simple cysts , 69 mm in left ovary and 50 mm in right ovary . there was no free fluid in the pelvic cavity . because of persistently high level of -hcg , surgical intervention was done . atypical findings and diagnosis of gtn besides fp led to choice of laparatomy instead of laparascopy . surgical findings were two bluish , 5 cm in left side and 2 cm in right side , swelling in both ampullary parts of fallopian tubes with minimal bleeding from fimbria . two simple cysts , about 5 cm in right ovary and 4 cm in left ovary were seen . bilateral salpingostomy was performed and some trophoblastic - like tissue was extracted and sent for pathologic evaluation separately . the patient received 50 mg / m intramuscular methotrexate due to bipateral fp and non - radical approach . secondary bilateral ectopic pregnancy is a condition with localization of trophoblastic tissue in both tubes , following a kind of manipulation in physiology such as through the use of art drugs . the classic triad of pain , vaginal bleeding and missed period was present , similar to unilateral ectopic pregnancy . in a study , 19 secondary bilateral tubal pregnancy cases were reviewed in an attempt to examine the symptoms and signs . diagnosis in this group was done due to discrepancy between sonography and -hcg level . in 6 out of 19 ( 31.6% ) cases , due to involvement of both fallopian tubes , probability of rupture is higher in comparison to unilateral ectopic gestation ( 2 ) . in the case of the present study , as a secondary bilateral ectopic gestation , high level of -hcg without intrauterine pregnancy suggested ectopic gestation and atypical high titer of hcg resulted in referral to a tertiary gyneco - oncology ward . in review of studies , mean gestational age at the time of diagnosis was about 6.7 weeks ( 5 - 9 weeks ) after the last menstruation ( 2 ) . sonographic findings and -hcg level , usually , do n't lead to a correct diagnosis of bilateral ectopic pregnancy . due to ample amount of -hcg during normal gestation , its level is not diagnostic ( 13 ) . in a review of hcg level in 16 cases of secondary bilateral tubal pregnancy , mean level of -hcg was 20878 miu / ml with a wide range of 27 - 226768 miu / ml . three cases out of 16 revealed -hcg titers of 13296 , 62520 and 226768 miu / ml ( 2 , 47 ) . in unilateral ectopic pregnancy , there is lower level of -hcg . in contrast to normal pregnancy with predictable range of -hcg in each week of gestation , in ectopic pregnancy it is more variable ( 2 , 3 ) . in our case , empty uterus coexisting with high level of -hcg suggested gestational trophoblastic disease ( gtn ) and ectopic pregnancy was less probable due to high atypical titer 21770 miu / ml hcg . high level of -hcg titer in bilateral ectopic gestation , such as above 10,000 in 3 case reports and our case and even 226768 miu / ml in a case report , proposed gtn as a reasonable differential diagnosis . however , pre - operative diagnosis of secondary bilateral ectopic pregnancy was made just in 10% of patients ( 2 ) . in our case , bilateral rupture of tubes was rare . in 5 out of 19 cases in a review of secondary bilateral ectopic pregnancy , unilateral tubal rupture in operative field bilateral unruptured tubal pregnancy in ampulary portion of tubes was seen . in our case , left tube was more distended , about 5 cm and right tube was 2 cm . in the review of bilateral tubal gestation in literature including primary and secondary cases , left tubal pregnancy was larger and more prone to rupture in comparison to right one . an attempt was made to review cases of medical literature which the side and size of rupture in bilateral tubal gestation was defined ( table 1 ) . medical treatment of these patients is not fully studied when pre - operative diagnosis is made . there are three surgical approaches for ectopic pregnancy : salpingotomy , salpingostomy and radical salpingectomy . regarding the future plan of fertility procedures , conservation or resection of fallopian tubes might be planned in the operation field ( 1 , 2 , 810 ) . in this case , size of right and left tube distention according to surgical findings in cases with bilateral ectopic pregnancy bilateral ectopic gestation , although rare , should be regarded as the differential diagnosis for ectopic pregnancy , especially in assisted reproductive technology cases and high titers of -hcg ( more than 100000 ) . left tubal side of bilateral tubal pregnancy was more probable to rupture and it revealed bigger size in review of cases .

backgroundbilateral ectopic pregnancy is a rare condition and is divided in two subgroups , primary and secondary , based on history of assisted reproductive technology.case presentationa 30 year old primigravid woman with history of infertility and ovulation induction presented to a hospital in kashan in year 2013 . she had vaginal bleeding , abdominal pain and ultrasound findings suggested early pregnancy . due to high titer of -hcg , gestational trophoblastic disease was proposed and d8c was done in referral and admission to gyneco - oncology ward in tehran . repeat sonography suggested ectopic pregnancy in left side and repeat -hcg level showed an increase of 19435 miu / ml . laparotomy findings revealed bilateral ampullary ectopic pregnancy . bilateral salpingostomy followed by one course of methotrexate was prescribed.conclusionbilateral ectopic gestation should be considered as a rare differential diagnosis for ectopic pregnancy . in this study , bigger size and rupture in left side was observed .

sevoflurane has a low blood / gas partition coefficient and does not irritate the airways . inhalation induction should provide rapid loss of consciousness to ensure patient 's comfort and satisfaction and to avoid adverse effects such as excitations , breath holding , coughing and/or laryngospasm . several adjuvants and techniques have been proposed to ensure a rapid induction of anesthesia with sevoflurane , such as intravenous ( iv ) midazolam , clonidine , fentanyl , breathing different gas flows , or ventilation with larger tidal volumes . the effect of nitrous oxide ( n2o ) when added to the inhalation of sevoflurane in oxygen has been controversial . n2o added to oxygen was reported to make shorter the induction time with sevoflurane , an effect similar to that produced by clonidine . these results are not consistent with the results obtained from other studies , which conclude that the addition of n2o to the sevoflurane / oxygen mixture is not advantageous regarding the speed of induction of anesthesia . in contrast , results from previous studies point out that the addition of n2o to the sevoflurane / oxygen mixture may be associated with more , though minor , excitatory phenomena . our hypothesis was that the speed of inhalation induction with sevoflurane may benefit from the pretreatment with n2o , and subsequent volume contraction before sevoflurane administration . the aim of the present study was to investigate the effect of pretreatment with 50% n2o in oxygen on the speed of inhalation induction of anesthesia with sevoflurane in women undergoing minor gynecological procedures . the study was approved by the institutional review board ( n-79/04 - 04 - 06 ) . eighty patients american society of anesthesiology physical status i or ii scheduled for hysteroscopy under general anesthesia , who accepted an inhalation induction with no premedication , gave their written informed consent to participate in the study protocol . patients with body weight exceeding the ideal body weight by 25% , with central nervous system disease , respiratory disease , hyper - or hypothyroidism , intake of sedatives , hypnotics , and antidepressants were excluded from the study before randomization . patients were randomly allocated to the air ( fio2 = 0.21 ) or n2o ( n2o / o2:50/50 , fio2 = 0.5 ) group . randomization was done by means of odd and even numbers retrieved from a computer - generated table ( http://www.randomizer.org ) corresponding to the n2o and to the air group respectively . the pretreatment gas was administered via n2o - oxygen flow meters or an air flow meter connected to the wall pipelines , all covered by drapes so the investigators were blinded to the gas administered . group allocation to intervention was done by an independent anesthesiologist not participating in the study who arranged the gases before induction ( air or n2o / o2 ) , covered the flow meters with drapes and recorded the group each patient was assigned . in the operating room monitoring consisted of pulse oximetry , electrocardiogram , heart rate , noninvasive arterial pressure , end - tidal carbon dioxide ( etco2 ) , inspired and end - tidal sevoflurane concentrations , tidal volume and respiratory rate ( s/5 anesthesia monnitor , datex - ohmeda division , datex - ohmeda , helsinki , finland ) and bispectral index ( bis xp a-2000 monitor , system rev 3.21 , aspect medical systems , leiden , the netherlands ) . according to the group allocation , in the preinduction period patients were fully awake , breathing spontaneously either 50% n2o in oxygen ( fio2 = 0.5 ) or air ( fio2 = 0.21 ) for 10 min via a mapleson c breathing system and a tightly fitting face mask . the anesthetic circuit was primed with 8% sevoflurane : an oxygen flow 6 l / min was allowed to pass through the circle anesthetic system with the sevoflurane vaporizer dial being set at 8% for 5 min . an endotracheal tube was attached to the end of the anesthetic system with the distal end tightly surrounded by a plastic bag . after the completion of the 10 min administration of the n2o / oxygen or air , the face mask was connected to the sevoflurane primed anesthetic circuit and all patients were allowed to breathe spontaneously sevoflurane in oxygen with the sevoflurane vaporizer set at 8% and an oxygen flow rate 6 l / min . after a 5 min period of sevoflurane inhalation , the study protocol and data collection were completed . a short acting opioid was given iv and a supraglottic airway device was inserted for the ventilation of patients during surgery . during the 10 min preinduction period , bis , respiratory rate , tidal volume , etco2 , heart rate and oxygen saturation ( spo2 ) were recorded every minute . the same parameters were recorded every 30 s during the first 5 min induction period with the patient breathing sevoflurane in oxygen . during the induction period inspired and end - tidal sevoflurane concentrations were also recorded every 30 s. arterial blood pressure was recorded only before starting the pretreatment with the gas and after completion of measurements to avoid stimulus during cuff inflation affecting bis measurements . we hypothesized that the speed of inhalation induction with sevoflurane will be enhanced by n2o pretreatment , and possible subsequent volume contraction occurring before sevoflurane administration . bis was the primary outcome measure , the rest variables being secondary outcome measures . at each time point , three consecutive bis values were read and the median one was selected . initial sample size estimation showed that approximately 40 patients are needed in each group to detect a clinically relevant reduction of bis after induction of anesthesia by 30% with a power of 0.80 and level of significance of 5% . to assess differences in patients characteristics between the two groups , two independent samples t - tests for normally distributed responses and mann - whitney tests for nonnormally distributed responses were carried out . overall group differences in all variables were assessed by using repeated measures anova . a paired samples t - test or a mann - whitney test was used to assess differences at each time point wherever appropriate . all analyses were performed using the statistical package for social sciences ( spss ) software , version 11.0 ( spss inc . , chicago , il , usa ) . in the operating room a peripheral vein was catheterized and a ringer 's solution infusion was started . monitoring consisted of pulse oximetry , electrocardiogram , heart rate , noninvasive arterial pressure , end - tidal carbon dioxide ( etco2 ) , inspired and end - tidal sevoflurane concentrations , tidal volume and respiratory rate ( s/5 anesthesia monnitor , datex - ohmeda division , datex - ohmeda , helsinki , finland ) and bispectral index ( bis xp a-2000 monitor , system rev 3.21 , aspect medical systems , leiden , the netherlands ) . according to the group allocation , in the preinduction period patients were fully awake , breathing spontaneously either 50% n2o in oxygen ( fio2 = 0.5 ) or air ( fio2 = 0.21 ) for 10 min via a mapleson c breathing system and a tightly fitting face mask . the anesthetic circuit was primed with 8% sevoflurane : an oxygen flow 6 l / min was allowed to pass through the circle anesthetic system with the sevoflurane vaporizer dial being set at 8% for 5 min . an endotracheal tube was attached to the end of the anesthetic system with the distal end tightly surrounded by a plastic bag . after the completion of the 10 min administration of the n2o / oxygen or air , the face mask was connected to the sevoflurane primed anesthetic circuit and all patients were allowed to breathe spontaneously sevoflurane in oxygen with the sevoflurane vaporizer set at 8% and an oxygen flow rate 6 l / min . after a 5 min period of sevoflurane inhalation , the study protocol and data collection were completed . a short acting opioid was given iv and a supraglottic airway device was inserted for the ventilation of patients during surgery . during the 10 min preinduction period , bis , respiratory rate , tidal volume , etco2 , heart rate and oxygen saturation ( spo2 ) were recorded every minute . the same parameters were recorded every 30 s during the first 5 min induction period with the patient breathing sevoflurane in oxygen . during the induction period inspired and end - tidal sevoflurane concentrations were also recorded every 30 s. arterial blood pressure was recorded only before starting the pretreatment with the gas and after completion of measurements to avoid stimulus during cuff inflation affecting bis measurements . we hypothesized that the speed of inhalation induction with sevoflurane will be enhanced by n2o pretreatment , and possible subsequent volume contraction occurring before sevoflurane administration . bis was the primary outcome measure , the rest variables being secondary outcome measures . at each time point , three consecutive bis values were read and the median one was selected . initial sample size estimation showed that approximately 40 patients are needed in each group to detect a clinically relevant reduction of bis after induction of anesthesia by 30% with a power of 0.80 and level of significance of 5% . to assess differences in patients characteristics between the two groups , two independent samples t - tests for normally distributed responses and mann - whitney tests for nonnormally distributed responses overall group differences in all variables were assessed by using repeated measures anova . a paired samples t - test or a mann - whitney test was used to assess differences at each time point wherever appropriate . all analyses were performed using the statistical package for social sciences ( spss ) software , version 11.0 ( spss inc . , chicago , il , usa ) . the n2o and the air groups did not differ in age , body weight , and height . similarly , they did not differ in systolic and diastolic arterial pressure before or after the end of measurements [ table 1 ] . patient characteristics , sap and dap blood pressures before and after the measurements of the 80 patients recruited for the study , 43 patients were assigned to the n2o and 37 to the air group . four patients in the n2o group and one patient in the air group did not accept the facemask from the very beginning and no data were collected from these patients . one patient in the n2o group presented excitatory movements and removed the mask after n2o inhalation for 7 min and no further data were collected . in another patient in the air group bis values were not at any time point obtained due to technical reasons , but all the other variables were recorded and analyzed [ figure 1 ] . the flow diagram of the study during sevoflurane inhalation in the n2o group the study was interrupted in one patient at 120 s due to persisting cough and salivation , in one patient at 180 s due to tachypnea , tachycardia and upper airway obstruction , in one patient at 210 s due to tachypnea and tachycardia , in one patient at 240 s due to upper airway obstruction , and in one patient at 270 s due to prolonged apnea . the incidence of complications during pretreatment with n2o or air followed by sevoflurane induction in the n2o and air groups during the 10 min period of breathing 50% n2o in oxygen or air , the bis , etco2 , tidal volume , respiratory rate and heart rate did not differ between the group given n2o and that given air , nor was there a trend to a difference . spo2 was slightly but significantly greater in patients given n2o , consistent with the higher inspired oxygen concentration of oxygen breathed . during sevoflurane induction bis values were lower in the n2o group [ table 3 and figure 2 ] . at the time points 60 , 90 , 120 and 150 s , the bis values in the n2o versus air group were : 77 19.0 versus 92 8.6 ( p < 0.001 ) , 44 19.8 versus 73 21.4 ( p < 0.001 ) , 36 12.4 versus 55 24.7 ( p = 0.002 ) and 32 11.0 versus 45 20.8 ( p = 0.014 ) , respectively . etco2 and tidal volume values were lower and respiratory rate higher in the n2o group when compared to the air group . spo2 , heart rate , inspired and end - tidal sevoflurane concentration did not differ significantly between the two groups during the first 300 s of sevoflurane inhalation [ table 3 ] . bis , etco2 , vt , rr , spo2 , hr and fi and fe sevoflurane concentrations in the n2o- and air - breathing groups during induction of anesthesia with sevoflurane bispectral index values ( mean standard deviation ) during inhalation with sevoflurane in the nitrous oxide and air groups the results of the present study suggest that pretreatment with n2o in 50% oxygen for 10 min before inhalation induction with sevoflurane enhances induction as assessed by decreased bis values . these results are not consistent with other studies when n2o was administered concomitantly with sevoflurane for inhalation induction . nevertheless , these studies were based on clinical criteria , which are subjective and therefore more difficult to be quantified . found that the addition of 50% n2o to the sevoflurane / oxygen mixture had no effect on the speed of induction and was associated with more excitatory effects . the investigators defined induction time as the time needed for the raised arm to fall to the horizontal level . also , according to siau and liu , 66% n2o added to oxygen did not decrease the induction time with 8% sevoflurane when compared to the induction time of 8% sevoflurane in oxygen alone . these investigators administered higher concentrations of n2o than those administered in the present study but did not prime the circle breathing circuit with sevoflurane . in a previous study , we have shown that pretreatment with 100% oxygen enhances the inhalation induction with 7% sevoflurane as assessed by bis monitoring . replacing nitrogen in the lungs with a more soluble gas like oxygen , results in lung collapse , decreased functional residual capacity and increased alveolar concentration of the inhaled anesthetic , that is , sevoflurane . in contrast to oxygen , in the present study the n2o was discontinued during induction . it is worthy to note that during induction , the end - tidal ( fe ) sevoflurane concentration ( though no statistically significant ) was higher during the first 120 s. the higher fe is consistent with increased ventilation in the n2o group , as indicated by the higher respiratory rates and the lower co2 values . , oxygen is also expected to enhance the sevoflurane uptake and to diminish the difference between the n2o and air groups as it is more soluble than nitrogen and part of the lungs will collapse to some extent ( absorption atelectasis ) . similarly , n2o - being 32 times more soluble than nitrogen - is rapidly up taken by the blood , resulting in lung contraction and increased alveolar concentration of sevoflurane . however , eventually alveolar collapsing will be associated with pulmonary shunt which will delay sevoflurane uptake . the time points at which bis values differ significantly in the present study are between 60 and 150 s. the inspired and expired sevoflurane concentrations did not differ between the n2o and the air groups during the 300 s period of induction , though the end - tidal / inspired concentration ( f e / fi ) ratio for sevoflurane was consistently higher by 2 - 7% in the n2o group . sevoflurane due to its low blood gas partition coefficient might not benefit from the second gas effect phenomenon . however taheri and eger have nicely demonstrated the second gas effect for desflurane , an agent even less blood soluble . demonstrated a higher ratio of alveolar / inspired ( f a / fi ) sevoflurane concentration when n2o was added to the inspired mixture . peyton et al . reported a two to three times more powerful second gas effect of n2o on arterial versus the end - expired sevoflurane pressures and attributed this effect to ventilation perfusion inhomogeneity by general anesthesia . in both studies , our data were collected with the patients breathing spontaneously and receiving solely sevoflurane without other anesthetics or adjuvants . pretreatment with n2o may not ensure an overall faster inhalation induction , but highlights the failure of previous studies to demonstrate enhancement of sevoflurane induction by concomitant n2o administration . previous studies failed to show a more rapid induction of anesthesia with sevoflurane for various reasons . as sevoflurane is an agent with low solubility , it may not benefit the very first minutes of induction by the concomitant administration of n2o . so we remain with our hypothesis that this difference between the n2o and air groups may be attributed to the pretreatment rather than the co - administration of n2o during sevoflurane induction of anesthesia . a very early increased concentration effect due to volume reduction and before the shunt effect , as n2o is taken up by the pulmonary capillaries or increased ventilation , as shown by the lower etco2 , in the n2o group may have influenced the speed of the sevoflurane uptake . in clinical practice , fast inhalational induction may be beneficial for both patients and hospitals ; it may shorten the second stage of anesthesia , thus reducing the risk of agitation / adverse respiratory events , and increase patient 's satisfaction , while - depending on the time saved - it may also decrease the overall operating room time and facilitate fast turnover . our measurements are limited by the sensitivity of the device we used to measure sevoflurane and n2o , thus the infrared analyzer , as the gold standard to measure concentrations of inhaled anesthetics is the gas chromatography or the mass spectrometry . other limitations of the study are the number of dropouts , which may have an impact on the study power and the relatively long period of n2o pretreatment which weakens the clinical implementation of our results and shorter pretreatment periods like 2 or 3 min may be clinically applicable . under the present study design , breathing of 50% n2o in oxygen before induction with sevoflurane enhances the speed of induction in the very early period as assessed by the bis monitor .

background and aims : inhalation anesthesia with sevoflurane may be enhanced by several drugs or techniques . the aim of the present study was to investigate the effect of nitrous oxide ( n2o ) pretreatment on the speed of anesthesia induction with sevoflurane.material and methods : eighty patients scheduled for hysteroscopy under general anesthesia were randomly assigned to inhale for 10 min before induction 50% n2o in oxygen or air via a facemask . anesthesia was induced with 7 - 8% sevoflurane in oxygen via a facemask . bispectral index ( bis ) , end - tidal carbon dioxide ( etco2 ) tidal volume , respiratory rate , oxygen saturation ( spo2 ) , and heart rate were recorded every minute during the 10 min pretreatment periods and every 30 s during the first 300 s of induction with sevoflurane . during induction of anesthesia inspired and end - tidal sevoflurane concentrations were also recorded.results:during the 10 min of inspired 50% n2o or air bis , etco2 , tidal volume , respiratory rate and heart rate values did not differ between the two groups except for the spo2 , which was higher in the n2o group ( p < 0.001 ) . during induction of anesthesia the n2o group exhibited lower bis values ( p = 0.001 ) , being significant at 60 - 150 s ( p < 0.001 , p < 0.001 , p = 0.002 , p = 0.014 ) as well as at 270 s ( p = 0.004 ) . etco2 and tidal volume were consistently lower in the n2o group ( p = 0.001 , p = 0.041 respectively ) and respiratory rate was higher ( p = 0.007).conclusion : our results show that pretreatment of the patients with 50% n2o for 10 min enhances the speed of induction with sevoflurane as assessed by the bis monitoring .

three types of calibrated microspheres have recently been approved in japan as embolic agents for hypervascular tumors and arteriovenous malformations . to date , two types of embolic microspheres capable of being loaded with drug have been introduced : dc bead ( biocompatibles , farnham , uk ) and hepasphere ( biosphere medical , roissy cdg cedex , france ) . in a phase ii randomized controlled study of transarterial chemoembolization ( tace ) using a drug - eluting bead ( deb - tace ) for hepatocellular carcinoma ( hcc ) , the precision - v study , the response rate after 6 months was 52% in the deb - tace group and 44% in the tace group using a lipiodol ( laboratoire guerbet , aulnay - sous - bois , france)/anticancer drug mixture solution ( lip - tace ) , showing no significant difference or superiority of deb - tace . however , complication by postembolization syndrome , such as abdominal pain , fever , nausea , vomiting and liver enzyme elevation , developed in 2080% after lip - tace [ 3 , 4 ] , whereas symptoms developed in only 1837% after deb - tace [ 5 , 6 ] , showing that the complications were milder than those after lip - tace . on the other hand , deb - tace induced liver abscesses at a relatively high frequency , which could be fatal [ 5 , 7 , 8 , 9 ] . adverse events after deb - tace have been reported occasionally [ 5 , 7 , 8 , 9 , 10 , 11 ] , but as it has just been introduced in japan , many points regarding adverse events are unclear with respect to the developmental mechanism and risk factors . we encountered a hcc patient who underwent deb - tace and experienced delayed intratumoral hemorrhage . an 81-year - old male regularly visited his physician for treatment of hepatitis c virus - related cirrhosis . on routine abdominal ultrasonography ( us ) , on esophagogastroduodenoscopy , a type 0-iia early gastric carcinoma with a diameter of 25 mm was detected in the lesser curvature of the middle body . the patient was referred to our hospital for further examination and treatment . during the first examination , the liver function was classified as child - pugh class a , and the tumor markers were normal ( afp level : 3.4 ng / ml , afp - l3 fraction level < 0.5% ) , but the pivka ii had a level of 118 mau / ml , showing a mild elevation . it was capsulated and enhanced in the arterial phase on dynamic computed tomography ( ct ) ( fig . 1 ) , and showed a mosaic pattern accompanied by a marginal hypoechoic zone on gray - scale us . the mass was enhanced in the vascular phase ( 040 s ) on contrast - enhanced us ( ceus ) using sonazoid ( daiichi sankyo , tokyo , japan ) , and showed a hypoechoic area in the postvascular phase ( 10 min after an intravenous injection of sonazoid ) . based on the tumor marker and imaging findings , the patient was diagnosed with hcc . deb - tace was performed because the patient declined to undergo surgical resection of hcc . access for tace was performed under sterile conditions and under local anesthesia , via the right femoral artery using a 3-fr sheath ( medikit , tokyo , japan ) and in a retrograde fashion . when a microcatheter was advanced to the anterior segmental artery of the right hepatic artery and contrast imaging was applied , an intensely stained tumor was observed , with a5 as the feeding artery . a solution impregnated with 100300 m dc bead and 50 mg epirubicin hydrochloride , with a dc bead volume of 0.35 ml , was administered via the feeding artery to perform deb - tace . disappearance of the intensely stained tumor image was confirmed by right hepatic arterial angiography , and the treatment was completed . the course after deb - tace was favorable , and no adverse events above grade 3 according to the common terminology criteria for adverse events ( ctcae ) version 4.0 were observed . endoscopic submucosal dissection ( esd ) of the early gastric carcinoma was performed about 1 month after deb - tace . right hypochondriac pain suddenly developed 3 days after esd , but no adverse events were assumed to be caused by esd ( i.e. , free air , noted on dynamic ct ) . however , the tumor diameter had increased from that before deb - tace , and the tumor showed a high - intensity area on unenhanced ct ( fig . 2a ) , which was not enhanced in the arterial phase on dynamic ct , suggesting intratumoral hemorrhage . the hemodynamics of the tumor were followed using ceus over the time before and after deb - tace . on ceus carried out on the day after deb - tace , the whole intratumoral enhancement decreased , and nonenhanced patchy regions , assumed to be necrosis , were noted in the tumor in the vascular phase . on ceus performed 4 weeks after deb - tace , i.e. , immediately before esd , the tumor size was 33 31 mm , and the enhancement area was increased in the vascular phase ( fig . however , the tumor had enlarged to 41 36 mm on ceus when the right hypochondriac pain developed ( 5 weeks after deb - tace ) , and changes in the echogenicity on gray - scale us and nonenhancement of almost the entire tumor in the vascular phase on ceus were noted ( fig . based on the above findings , the cause of right hypochondriac pain may have been deb - tace - associated intratumoral hemorrhage . since the hemorrhage was limited to inside the tumor , it stopped spontaneously with rest . the advantages of deb - tace are as follows : the infusion dose of the chemotherapeutic agent can be modified by adjusting the chemotherapeutic agent carrier , while altering the depth of embolization with beads , the duration of tumor exposure to chemotherapeutic agent can be fine - tuned through sustained release of the chemotherapeutic agent , and systemic adverse reactions of chemotherapeutic agent can be attenuated by continuous release of high - dose chemotherapeutic agents into the tumors . deb has proven to be extremely potent and safe by allowing large amounts of the chemotherapeutic agent to concentrate within the tumor over time , thereby maximizing the cytotoxic effect , which also makes it attractive as an effective treatment for large lesions . favorable therapeutic effects have been reported occasionally [ 8 , 9 ] and have increased the expectations of hcc treatment with deb - tace ; however , the beads exhibit characteristic physical properties and behavior that are different from those of gelatin particles . it is important to fully investigate these characteristics to avoid inadequacy of the therapeutic effect and to prevent the onset of adverse events . previous studies have reported that the incidence of mild adverse events , such as postembolization syndrome , is lower in the deb - tace group compared with the lip - tace group [ 3 , 4 , 5 , 6 ] . meanwhile , liver abscesses were induced at a relatively high frequency ( about 1.67.4% ) [ 5 , 7 , 8 , 9 ] . malagari et al . reported that the 30-day mortality was 1.26% among 237 hcc patients treated with deb - tace with dc bead loaded with doxorubicin , and that the cause was liver abscess in all cases . regarding other adverse events , acute pancreatitis and cholecystitis developed with incidences of 15 and 16.3% , respectively . previous studies have shown that rare adverse events included liver failure and infection , bile duct injury , upper gastrointestinal bleeding , pulmonary embolism , splenic infarction , or spinal embolization [ 1 , 7 , 10 , 11 ] . unlike lip - tace , many adverse events occurred due to ischemic changes induced by the inflow of beads into organs other than the hcc lesion , which is characteristic of deb - tace . tumor hemorrhage has also been reported after deb - tace [ 1 , 7 , 10 , 11 , 13 ] with large tumors , with a diameter of 516 cm , and the tumor locations were subcapsular in all previous reports . to our knowledge , there has been no case of a tumor with a diameter < 5 cm distinct from the subcapsular , as was observed in our patient . there has been no report of a mechanism underlying the deb - tace - specific tumor hemorrhage . in general , the mechanism of tumor rupture after tace might be related to increased intratumoral pressure as a result of rapid edematic expansion due to tumor necrosis or vascular injury , secondary to embolization . the risk is particularly high in subcapsular lesions , and we must be careful because the spread of hemorrhage in the abdominal cavity can not only lead to hemorrhagic shock , but also peritoneal metastasis . unlike the reported cases of tumor hemorrhage after deb - tace , in the present case , the tumor diameter was not large and the tumor was distinct from the subcapsular region . however , intratumoral hemorrhage occurred about 1 month after deb - tace . local pooling of the contrast agent within the target tumor could occur during deb - tace , and seki et al . reported that it occurred in 25.2% of 135 hcc patients treated with deb - tace ( epirubicin - loaded hepasphere ) . while the cause of this local pooling remains unclear , it is similar to extravasation or blood sinus , suggesting that it was a local hemorrhage due to intratumor vascular collapse as a consequence of the treatment . when local pooling of the contrast agent was noted , seki et al . added a small amount of gelatin sponge particles , with a diameter ranging from 1 to 2 mm , to the feeding artery until the pooling disappeared . based on the above findings , the observation of local pooling of the contrast agent within the target tumor during treatment suggests that intratumoral pressure has already increased , which was unlikely in our patient . treatment - induced intratumoral hemorrhage continued immediately after deb - tace because no pooling was noted during treatment or on confirmation angiography following treatment . however , it is possible that the amount of dc bead was insufficient and embolization was incomplete . it was assumed that incomplete embolization subsequently caused intratumoral vascular collapse , i.e. , a phenomenon similar to pooling , and that the hemorrhage expanded to the necrotic intratumoral area by sustained release of the chemotherapeutic agent and embolization , leading to complication of intratumoral hemorrhage despite the low risk of tumor hemorrhage . many points remain unclear , i.e. , the appropriate bead sizes for each case and the level of embolization , but it may be important to apply sufficient embolization because insufficient embolization is likely to lead to tumor hemorrhage . deb - tace is considered to rarely cause adverse events , but fatal complications have been reported [ 5 , 7 , 8 , 9 ] , and reports of adverse events may increase with augmentation of the numbers of deb - tace - treated patients . if reports of tumor hemorrhage after deb - tace increase , the mechanism of intratumoral hemorrhage in cases similar to our patient may be elucidated . intratumoral hemorrhage was suspected due to the presence of a high - intensity area in the tumor on unenhanced ct and a rapid increase in the tumor diameter and accompanying enlargement of the unenhanced area on ceus performed before and after deb - tace . based on these findings it was difficult to assess the therapeutic effect of deb - tace by ct , based on lipiodol accumulation , unlike that after conventional lip - tace , and evaluation of the intratumoral hemodynamics could be important . ceus is superior in evaluating intratumoral hemodynamics in real time , and the utility of ceus to evaluate the therapeutic effect after deb - tace was suggested . we encountered a patient who experienced intratumoral hemorrhage about 1 month after deb - tace . deb - tace for hcc is a new treatment method in japan , and reports of adverse events may increase in the future . to obtain the therapeutic effect of deb - tace while preventing the adverse events , it may be important to understand the characteristics of the beads themselves and to apply the appropriate embolization to each individual case . it was also suggested that ceus is useful to evaluate the therapeutic effect after deb - tace .

transarterial chemoembolization ( tace ) using a drug - eluting bead ( deb - tace ) for hepatocellular carcinoma ( hcc ) is a new treatment method . we report on a case of delayed intratumoral hemorrhage after deb - tace . an 81-year - old male with hepatitis c virus - related cirrhosis was diagnosed with a hcc of 35 mm in diameter in s5 detected by dynamic computed tomography ( ct ) and contrast - enhanced ultrasonography ( ceus ) . deb - tace with dc bead and epirubicin hydrochloride was performed because the patient declined to undergo surgical resection . the treatment was completed , and the course after deb - tace was favorable . however , right hypochondriac pain suddenly developed about 1 month after deb - tace . unenhanced ct showed an increase of the tumor diameter and intratumoral high - intensity area , which was not enhanced in the arterial phase . ceus performed at the time of right hypochondriac pain ( 5 weeks after deb - tace ) showed nonenhancement of almost the entire tumor in the vascular phase . the cause of the symptom may have been deb - tace - associated intratumoral hemorrhage . tumor hemorrhage has been reported after deb - tace with tumors > 5 cm in diameter , and the tumor locations were subcapsular in all previous reports . there has been no case of a tumor with a diameter < 5 cm distinct from the subcapsular , as was observed in our patient . incomplete embolization might be the cause of the intratumoral hemorrhage experienced by this case presenting a few risks . to obtain the therapeutic effect of deb - tace while preventing the adverse events , it may be important to understand the characteristics of the beads and to apply the appropriate embolization to each individual case .

men , aged > 30 years , with type 2 diabetes were recruited from barnsley hospital nhs trust , barnsley , u.k . most of the subjects were recruited from the retinal screening program , which is attended by virtually all patients with diabetes in the barnsley area . subjects were given written and verbal information regarding the study , and 355 men gave their informed consent to take part . the study population comprised patients with diabetes usually managed in primary care facilities as well as secondary care patients . other measurements included blood pressure , a1c , nonfasting lipid levels , height , weight , and waist circumference . the main data from the study were published previously in diabetes care ( 2 ) . patients were seen in the morning between 8:00 and 10:00 a.m. symptoms of hypogonadism were assessed by completion of the androgen deficiency in the aging male ( adam ) questionnaire , which was validated to assess hypogonadism in aging men ( 19 ) . serum total testosterone , total estradiol , and shbg were measured by an enzyme - linked immunosorbent assay using commercially available kits ( drg diagnostics , marburg , germany ) . bioavailable testosterone was determined by a modification of the ammonium sulfate precipitation method described by tremblay and dube ( 20 ) . free testosterone was calculated from total testosterone and shbg by the formula of vermeulen et al . . these methods of assessing bioavailable and free testosterone have been used in previous studies from our research team and have determined to be reliable for the assessment of men with diabetes and vascular disease ( 17 ) . a1c was assessed using a menarini analyser ha8160 ( menarini diagnostics , florence , italy ) . serum lipid parameters were assessed by olympus analyzers ( olympus diagnostics , hamburg , germany ) . testosterone and shbg were log - normally distributed and were converted to a normal distribution to allow use of student 's t test for comparison of group means . with smaller groups , the normal distribution was assessed using single - sample kolmogorov - smirnov testing . the two - sample kolmogorov - smirnov test was used to compare groups when data did not fulfill the normal distribution . results were initially considered statistically significant at p < 0.05 . because of multiple statistical testing patients were seen in the morning between 8:00 and 10:00 a.m. symptoms of hypogonadism were assessed by completion of the androgen deficiency in the aging male ( adam ) questionnaire , which was validated to assess hypogonadism in aging men ( 19 ) . serum total testosterone , total estradiol , and shbg were measured by an enzyme - linked immunosorbent assay using commercially available kits ( drg diagnostics , marburg , germany ) . bioavailable testosterone was determined by a modification of the ammonium sulfate precipitation method described by tremblay and dube ( 20 ) . free testosterone was calculated from total testosterone and shbg by the formula of vermeulen et al . . these methods of assessing bioavailable and free testosterone have been used in previous studies from our research team and have determined to be reliable for the assessment of men with diabetes and vascular disease ( 17 ) . a1c was assessed using a menarini analyser ha8160 ( menarini diagnostics , florence , italy ) . serum lipid parameters were assessed by olympus analyzers ( olympus diagnostics , hamburg , germany ) . testosterone and shbg were log - normally distributed and were converted to a normal distribution to allow use of student 's t test for comparison of group means . with smaller groups , the normal distribution was assessed using single - sample kolmogorov - smirnov testing . the two - sample kolmogorov - smirnov test was used to compare groups when data did not fulfill the normal distribution . results were initially considered statistically significant at p < 0.05 . because of multiple statistical testing , we performed a bonferroni correction to minimize the chance of type 2 errors . in our cross - section of 355 men with type 2 diabetes , 186 were not treated with statins . of 169 men treated with statins , 81 were treated with atorvastatin , 66 with simvastatin , 15 with pravastatin , and 7 with other statins ( 3 with fluvastatin , 1 with cerivastatin , and 3 with unknown statin ) . patients treated with statins did not significantly differ from untreated individuals in age , waist circumference , bmi , a1c , or blood pressure ( table 1 ) . total cholesterol and ldl cholesterol levels were lower in men taking statins , but there were no significant differences in hdl cholesterol or triglyceride levels ( data not shown ) . mean sex hormone levels , measures of obesity , and cholesterol levels in men treated with any statin , atorvastatin , and simvastatin compared with untreated men p values given for student 's t test comparing treated patients with the untreated group . total testosterone was significantly lower in the statin and atorvastatin groups but not in the simvastatin group . bioavailable and free testosterone levels were not significantly lower in any group . both statins were associated with lower cholesterol levels , but none of the groups were significantly different in terms of obesity . total testosterone levels were significantly lower in men treated with statins , and there was a trend toward lower shbg levels , but this did not reach statistical significance ( table 1 ) . bioavailable testosterone , calculated free testosterone , and serum estrogen levels were not significantly different between the groups . the symptom score from the adam questionnaire was not significantly altered in those men receiving statin therapy . there were sufficient numbers of men treated with simvastatin and atorvastatin to consider these groups more closely ( tables 1 and 2 ) . these groups were not significantly different from untreated men in terms of bmi , waist circumference , blood pressure , a1c , or age . total cholesterol and ldl cholesterol levels were lower in both groups compared with those in untreated men but were not significantly different between simvastatin- and atorvastatin - treated groups . men treated with simvastatin did not have significantly different total , bioavailable , or free testosterone , estradiol , or shbg levels than untreated men ( fig . 1 and table 1 ) . in contrast , men treated with atorvastatin had an average total testosterone level 1.96 nmol / l ( 56 ng / dl ) less than that in untreated men . there was a trend toward lower shbg levels in this group ( 35.3 vs. 27.6 nmol / l , p = 0.022 ) . further analysis revealed an apparent dose - response relationship , with the lowest testosterone levels seen in men taking higher doses of atorvastatin ( table 3 ) . nmol / l ( 108 ng / dl ) less than that of untreated men ( table 3 ) , but this value did not reach statistical significance ( p = 0.017 ) . shbg was also reduced without reaching significance ( p = 0.043 ) , but free and bioavailable testosterone levels were unaltered . no significant differences were seen in sex hormone levels when the simvastatin - treated men were split into similar groups ( data not shown ) . there were no significant differences in age , bmi , waist circumference , blood pressure , or a1c in any of the atorvastatin- or simvastatin - treated subgroups . characteristics of patients untreated with statins and those treated with atorvastatin and simvastatin p values given for student 's t test . there were no significant differences in age , anthropomorphic data , glycemic control , or blood pressure . total and ldl cholesterol are lower in the statin - treated group , reflecting the primary action of the drugs . mean testosterone levels in untreated men and those treated with atorvastatin or simvastatin ; 95% cis are shown . international guidelines suggest that testosterone replacement is invariably warranted when the total testosterone level is < 8 nmol / l ( 231 ng / dl ) or the free testosterone level is < 0.18 nmol / l ( 346 ng / dl ) or a free testosterone between 0.18 and 0.25 nmol / l . * p < 0.01 ; + p < 0.05 . average testosterone , shbg , and measures of obesity in men treated with atorvastatin split into two groups : those treated with 10 mg and those treated with 20 mg or more the 10-mg atorvastatin group was compared with the no statin group with student 's t test ; significant results are shown in boldface . the 20-mg atorvastatin group was compared with the no statin group using a two - sample kolmogorov - smirnov test . there is a trend toward lower total testosterone and shbg levels in men taking the higher doses of atorvastatin , but this does not reach statistical significance . in our cross - section of 355 men with type 2 diabetes , 186 were not treated with statins . of 169 men treated with statins , 81 were treated with atorvastatin , 66 with simvastatin , 15 with pravastatin , and 7 with other statins ( 3 with fluvastatin , 1 with cerivastatin , and 3 with unknown statin ) . patients treated with statins did not significantly differ from untreated individuals in age , waist circumference , bmi , a1c , or blood pressure ( table 1 ) . total cholesterol and ldl cholesterol levels were lower in men taking statins , but there were no significant differences in hdl cholesterol or triglyceride levels ( data not shown ) . mean sex hormone levels , measures of obesity , and cholesterol levels in men treated with any statin , atorvastatin , and simvastatin compared with untreated men p values given for student 's t test comparing treated patients with the untreated group . total testosterone was significantly lower in the statin and atorvastatin groups but not in the simvastatin group . bioavailable and free testosterone levels were not significantly lower in any group . both statins were associated with lower cholesterol levels , but none of the groups were significantly different in terms of obesity . total testosterone levels were significantly lower in men treated with statins , and there was a trend toward lower shbg levels , but this did not reach statistical significance ( table 1 ) . bioavailable testosterone , calculated free testosterone , and serum estrogen levels were not significantly different between the groups . the symptom score from the adam questionnaire was not significantly altered in those men receiving statin therapy . there were sufficient numbers of men treated with simvastatin and atorvastatin to consider these groups more closely ( tables 1 and 2 ) . these groups were not significantly different from untreated men in terms of bmi , waist circumference , blood pressure , a1c , or age . total cholesterol and ldl cholesterol levels were lower in both groups compared with those in untreated men but were not significantly different between simvastatin- and atorvastatin - treated groups . men treated with simvastatin did not have significantly different total , bioavailable , or free testosterone , estradiol , or shbg levels than untreated men ( fig . 1 and table 1 ) . in contrast , men treated with atorvastatin had an average total testosterone level 1.96 nmol / l ( 56 ng / dl ) less than that in untreated men . there was a trend toward lower shbg levels in this group ( 35.3 vs. 27.6 nmol / l , p = 0.022 ) . further analysis revealed an apparent dose - response relationship , with the lowest testosterone levels seen in men taking higher doses of atorvastatin ( table 3 ) . nmol / l ( 108 ng / dl ) less than that of untreated men ( table 3 ) , but this value did not reach statistical significance ( p = 0.017 ) . shbg was also reduced without reaching significance ( p = 0.043 ) , but free and bioavailable testosterone levels were unaltered . no significant differences were seen in sex hormone levels when the simvastatin - treated men were split into similar groups ( data not shown ) . there were no significant differences in age , bmi , waist circumference , blood pressure , or a1c in any of the atorvastatin- or simvastatin - treated subgroups . characteristics of patients untreated with statins and those treated with atorvastatin and simvastatin p values given for student 's t test . there were no significant differences in age , anthropomorphic data , glycemic control , or blood pressure . total and ldl cholesterol are lower in the statin - treated group , reflecting the primary action of the drugs . mean testosterone levels in untreated men and those treated with atorvastatin or simvastatin ; 95% cis are shown . international guidelines suggest that testosterone replacement is invariably warranted when the total testosterone level is < 8 nmol / l ( 231 ng / dl ) or the free testosterone level is < 0.18 nmol / l ( 346 ng / dl ) or a free testosterone between 0.18 and 0.25 nmol / l . * p < 0.01 ; + p < 0.05 . average testosterone , shbg , and measures of obesity in men treated with atorvastatin split into two groups : those treated with 10 mg and those treated with 20 mg or more the 10-mg atorvastatin group was compared with the no statin group with student 's t test ; significant results are shown in boldface . the 20-mg atorvastatin group was compared with the no statin group using a two - sample kolmogorov - smirnov test . there is a trend toward lower total testosterone and shbg levels in men taking the higher doses of atorvastatin , but this does not reach statistical significance . this is the first study to analyze fully testosterone levels and hypogonadal symptoms in comparison with statin use in a population receiving routine medical management . those treated with atorvastatin had lower levels of total testosterone with a trend toward lower shbg compared with untreated men , whereas those treated with simvastatin were not significantly affected . statin treatment did not significantly affect the biologically active fractions of testosterone , symptoms of hypogonadism , or estradiol levels . this study demonstrates that treatment with statins in this group can be a confounding factor in the assessment of hypogonadism . total testosterone is the primary test used in the diagnosis of hypogonadism , and the effect of atorvastatin could potentially lead to misdiagnosis of the condition in men with normal free and bioavailable testosterone levels . alternatively , it is possible that reductions in total testosterone without changes in bioactive testosterone fractions have clinical effects in view of findings suggesting that shbg - bound testosterone may be biologically active ( 13 ) . the adam questionnaire failed to detect an increase in hypogonadal symptoms in groups with lower total testosterone levels , but the questionnaire does not correlate closely with testosterone levels , and men with diabetes have a high level of false - positive hypogonadal symptoms ( 2 ) such as erectile dysfunction owing to vascular and neuropathic factors , medications , and depression . atorvastatin is a more potent statin than simvastatin , thus causing a greater reduction in the total cholesterol and hence total testosterone levels . no patients in our study were treated with other potent statins such as rosuvastatin so we are unable to confirm that atorvastatin reduces testosterone and shbg levels secondary to more potent effects on cholesterol levels . indeed , cholesterol and other lipid fraction levels were not significantly different between simvastatin- and atorvastatin - treated groups . in a normal physiological state , luteinizing hormone ( lh ) promotes uptake of cholesterol by the testis and stimulates testosterone synthesis . a reduction in testosterone is sensed by the hypothalamic - pituitary axis and leads to greater lh release , which completes a negative feedback loop and maintains testosterone levels . the apparent failure of the hypothalamic - pituitary - testicular axis to respond and maintain testosterone levels in the statin - treated patients in our study may be explained by the hypogonadal - obesity - adipocytokine hypothesis ( 1 ) . the majority of the patients in our study were overweight or obese . in this population , greater production of adipocytokines such as tumor necrosis factor- , interleukin-6 , and leptin and increased estradiol from metabolism of testosterone by aromatase in adipose tissue inhibit lh release from the pituitary gland , which leads to lower circulating testosterone levels . in our study , gonadotrophin levels were only tested in men with total testosterone levels < 12 mmol / l ( 2 ) , leaving us unable to test this hypothesis . the most likely reason that bioavailable and free testosterone levels are unaltered despite lower total testosterone is a homeostatic mechanism via reduced shbg production . an alternative hypothesis is that atorvastatin causes a primary reduction in shbg with consequent reductions in total testosterone . there was a consistent trend toward low shbg levels in all groups with reduced levels of total testosterone , although these low levels did not reach statistical significance . shbg is produced in the liver , the primary site of action of the statins , but the mechanism by which statins could alter shbg is unknown . shbg levels are known to be modulated by a number of factors including downregulation by insulin resistance and upregulation by estrogens ( 21 ) . we are aware of one clinical trial assessing the effects of atorvastatin on testosterone levels in men . the results showed a nonsignificant fall in total testosterone and no change in shbg ( 4 ) . these data are insufficient to confirm or refute our findings relating to atorvastatin , and it may be that the study allowed insufficient time for changes to develop . some trials have shown that simvastatin reduces serum testosterone levels ( 911 ) , whereas others have shown no effect ( 68 ) . our data suggest that any effect of simvastatin in reducing serum testosterone levels is not clinically significant in men with type 2 diabetes . we are not aware of any previous evidence for an effect of any statin on shbg levels . nmol / l ( 231 ng / dl ) are hypogonadal , that men with total testosterone levels > 12 nmol / l ( 346 ng / dl ) do not have hypogonadism , and that men with levels between 8 and 12 nmol / l need consideration for treatment depending on their clinical picture ( 18 ) . an endocrine society clinical practice guideline suggested a diagnostic cutoff value of testosterone of < 10.4 our findings are important in this context because statin treatment was associated with lower total testosterone levels of 11.9 nmol / l ( 340 ng / dl ) ( versus 13.4 nmol / l [ 384 ng / dl ] ) . men treated with 20 mg / day atorvastatin had an average total testosterone level of only 9.6 nmol / l ( 275 ng / dl ) , which is 3.8 nmol / l ( 109 ng / dl ) lower than that of men not treated with statins . thus , statin treatment may lead to reductions in total testosterone levels to < 12 or 10.4 nmol / l in many men but will not significantly alter bioavailable or free testosterone levels or hypogonadal symptoms . we recommend a low threshold for measuring or calculating bioavailable or free testosterone in men receiving statin therapy . it has now been confirmed that reductions in total testosterone are accompanied by similar changes in free and bioavailable testosterone and a high prevalence of hypogonadal symptoms ( 2 ) . furthermore , short - term studies in small numbers of hypogonadal diabetic men have shown improvements in glycemic control , central obesity , and serum leptin during testosterone replacement therapy ( 23,24 ) . other short - term studies have shown beneficial effects on further cardiovascular risk factors including total cholesterol levels ( 25 ) . in this context , the assessment of hypogonadism in men with type 2 diabetes is likely to increase in frequency . assessment of the clinical syndrome of hypogonadism can be challenging in this group because of the confounding effects of vascular disease , psychological factors , and medications on symptoms such as erectile dysfunction . this assessment is complicated by low shbg levels , which have long been associated with insulin resistance , leading to suggestions that low testosterone levels in type 2 diabetes are due to low shbg rather than to reductions in bioactive testosterone fractions . this suggestion has been refuted by studies showing low free and bioavailable testosterone levels in men with type 2 diabetes ( 2 ) and meta - analysis data suggesting relatively small changes in shbg in men with diabetes ( 22 ) . the realization that statin treatment may reduce total testosterone and shbg levels in this patient group serves to refocus attention to a subgroup who are particularly likely to have low shbg levels with the potential for a misdiagnosis of hypogonadism . in summary , this large data set is the first to suggest a significant effect of statins in lowering total testosterone and shbg levels in a population of men with type 2 diabetes . the findings have important implications for the diagnosis of hypogonadism in men receiving statin treatment . the opportunity to conduct similar studies of men with type 2 diabetes , comparing men treated with statins to untreated men , has now probably passed owing to the almost ubiquitous use of statins in this group . limitations of this study are its observational nature and the resultant inability to prove causality . statin - treated men did not differ from other men in terms of age , blood pressure , obesity , or glycemic control , but unidentified confounders can not be excluded . therefore further longitudinal or interventional studies are also needed to assess the effects of statins on androgen status in various patient groups and could include assessment of gonadotrophins and prolactin , which were not measured here . researchers should also investigate mechanisms that lead to changes in shbg and total testosterone in this context .

objectivethere is a high prevalence of hypogonadism in men with type 2 diabetes . this will lead to an increase in assessments of hypogonadism . statins could potentially decrease testosterone levels by reducing the availability of cholesterol for androgen synthesis . we compared testosterone levels and hypogonadal symptoms with statin use in a cross - sectional study of 355 men with type 2 diabetes.research design and methodstotal testosterone , sex hormone binding globulin ( shbg ) , and estradiol were measured by an enzyme - linked immunosorbent assay . bioavailable testosterone was measured by the modified ammonium sulfate precipitation method . free testosterone was calculated using vermeulen 's formula . symptoms of hypogonadism were assessed using the androgen deficiency in the aging male questionnaire.resultsstatins were associated with lower total testosterone ( 11.9 vs. 13.4 nmol / l , p = 0.006 ) and a trend toward lower shbg ( 29.4 vs. 35.3 nmol / l , p = 0.034 ) compared with no treatment . bioavailable testosterone , free testosterone , estradiol , and hypogonadal symptoms were not affected . subanalysis showed that atorvastatin was associated with reduced total testosterone ( 11.4 vs. 13.4 nmol / l , p = 0.006 ) and a trend toward reduced shbg ( 27.6 vs. 35.3 nmol / l , p = 0.022 ) compared with no treatment , and there was an apparent dose - response effect with the lowest levels of total testosterone seen in men treated with 20 mg atorvastatin ( 9.6 nmol / l , p = 0.017 ) . simvastatin use was not associated with significant reductions in testosterone or shbg levels.conclusionsassessing androgen status using total testosterone in men with type 2 diabetes treated with statins , particularly atorvastatin , may potentially lead to diagnostic error . levels of bioavailable testosterone or free testosterone are recommended for the assessment of hypogonadism in this group if total testosterone levels are borderline .

heart transplantation continues to provide patients with end - stage heart disease with extended survival with a half - life of 9.3 years between 2000 and june 2008 . . however , despite substantial advancements in immunosuppression , patients continue to be at significant risk for allograft rejection early after cardiac transplantation . the two recognized forms of allograft rejection are acute cellular rejection and antibody - mediated rejection ( amr ) . while acute cellular rejection has historically been the most common cause of allograft dysfunction , amr has only recently become widely accepted . during the 2004 international society of heart and lung transplantation ( ishlt ) , cellular rejection grades were revised and amr was formally defined . in april 2010 , a publication from the ishlt consensus conference assessed the status of amr in heart transplantation and a pathologic grading scale was devised . despite the advent of new technology , such as gene expression profiling and echocardiograms , endomyocardial biopsy remains the standard for detecting rejection . to minimize the risk of a false negative , multiple specimens ( usually 35 ) are obtained from 3 different sites . though rare , false negatives do exist either through sampling error , artifact , quilty lesions , or pathology misread . prior to the 2010 consensus conference , hemodynamic compromise , in the absence of acute cellular rejection , was termed biopsy - negative rejection . although the prevalence of the so - called biopsy - negative rejection has declined with the clinical diagnosis of amr , there are incidences of patients who present with cardiac dysfunction ( left ventricular ejection fraction , lvef 45% ) but have no biopsy findings of cellular or antibody - mediated rejection . since the outcome of patients with bnr ( which includes cardiac dysfunction ) has not been well established , we sought to review the characteristics and treatment response of patients who have developed bnr after heart transplantation . we retrospectively reviewed our cohort of patients who underwent heart transplantation between 2002 and 2012 and found 12 cases of bnr in 11 heart transplant patients , as defined by patients presenting with cardiac dysfunction , characterized by lvef 45% , and who had no biopsy findings of cellular rejection or amr . as severe infections are also known to cause left ventricular dysfunction , patients with a clear clinical picture of sepsis with fever , increase in white blood count , or positive cultures were excluded . continuous variable was presented as mean standard deviation while categorical variable is presented as percentages . we identified 11 patients who underwent heart transplantation between 2002 and 2012 and were treated for bnr . none of the bnr patients had previous blood transfusion or previous transplant . 5 ( 45% ) patients had a previous vad placement and 1 ( 9% ) patient was african american . the baseline immunosuppression medications are as follows : 5 patients ( 45% ) underwent induction therapy with antithymocyte globulin ( atg ) , 9 patients ( 82% ) initiated with tacrolimus and mycophenolate mofetil , 1 patient ( 9% ) initiated with cyclosporine and mycophenolate mofetil , and 1 patient ( 9% ) initiated with cyclosporine and azathioprine . 2 patients switched to cyclosporine from tacrolimus because of reduced seizure threshold ; 1 patient switched to a renal sparing protocol of sirolimus with mycophenolate mofetil because of renal function concerns . the 12 cases of bnr presented with an average lvef of 34% 10% and 11 ( 92% ) treated cases occurred during the first - year after transplant ( table 3 ) . the mean time to the first incidence of bnr is 7.8 7.5 months and no patient had treated rejection before onset of bnr except for the patient at the second onset . among the 106 biopsies that have been done before bnr , 24 ( 23% ) demonstrated low grade acute cellular rejection ( 1r , 1a , or 1b ) and 1 ( 0.9% ) showed suspicious antibody - mediated rejection ( amr 1 ) . de novo circulating antibodies developed in 3 cases ( in 3 patients ) and 1 of them had donor - specific antibodies . 4 out of the 12 cases presented with lvef 35% and presented with heart failure symptoms . of these 4 heart failure cases , 2 required inotropic support both received intravenous ( iv ) methylprednisolone ( 500 mg1000 mg per day for 3 days ) , atg ( 125150 mg per day for 35 days ) , and iv immunoglobulin ( ivig , 70 g per day for 3 days ) , followed by oral corticosteroids ( prednisone 80100 mg per day bolus and taper ) . for the remaining 2 patients who presented with heart failure symptoms but did not necessitate inotropes , 1 was treated with atg and iv methylprednisolone followed by prednisone bolus and taper and the other was given iv methylprednisolone followed by prednisone only . for the 8 out of 12 cases without heart failure symptoms , 7 were treated with only high dose oral corticosteroids . overall , 7 cases ( 58% ) ( in 7 patients ) favorably resulted with return to normal left ventricular function ( lvef of 57% 6% ) within a mean of 14 10 days from the initial negative biopsy . of these 7 patients , one expired approximately 6 months after the date of the normalized lvef and another one expired approximately 4 months after the date of normalized lvef . the remaining 5 cases ( in 5 patients , including 1 patient 's second case of bnr ) maintained persistent left ventricular dysfunction beyond 90 days . a flow chart of treatment and effect could be found in figure 1 . in summary , the lvef status at 90 days is as follows : 5 cases ( in 5 patients , including 1 case as second bnr on a patient ) had persistent lv dysfunction , and 7 cases ( in 7 patients ) experienced normalized lvef . as our understanding of the biological mechanisms underlying cardiac allograft rejection increases , the number of undiagnosed rejection decreases . the concept of bnr has evolved from its initial definition as hemodynamic compromise in the absence of acute cellular rejection to its current definition of patients presenting with cardiac dysfunction in the absence of biopsy findings of cellular rejection or amr . amr has changed from being suggested as bnr to its own ishlt biopsy grading scale . however , even though the majority of rejection episodes of unclear etiology have now been resolved to be cases of amr , there are still cases where the biopsy findings are inconsistent with the clinical prognosis , that is , patients with a decrease in lvef with no biopsy findings of rejection , cellular , or humoral . due to the inconsistencies of endomyocardial biopsies , the existence of bnr is questioned . this argument is furthered by the fact that prior to the 2010 amr consensus conference , 1020% of cardiac allograft recipients were diagnosed with bnr when in actuality the majority of patients most likely experienced amr . this brings up the question of whether bnr should exist as a separate category of rejection or is merely a by - product of the problems inherent with endomyocardial biopsies . in one case study , sampling error or nonuniformity of histopathologic changes resulted in a false - negative biopsy . in this case study , a heart transplant recipient with repeatedly unremarkable endomyocardial biopsies and a negative evaluation for humoral rejection was found to have severe subepicardial myocyte necrosis with classic cellular rejection in the subsequent autopsy . this is contrary to other studies which have found that rejection is evenly distributed throughout the right ventricular endomyocardium . although endomyocardial biopsy is the primary resource to diagnose acute rejections in all the cases discussed in this study , there are a few noninvasive diagnostics that have been demonstrated to be helpful in diagnosing acute allograft rejection after heart transplantation . in one multicenter clinical trial , pham et al . reported that using gene - expression profiling to monitor rejection for patients 6 months after heart transplantation was not associated with increased risk of serious adverse outcomes and could reduce the need for biopsy . in two pilot studies , wu et al . applied cardiac magnetic resonance ( cmr ) in vivo to detect immune - cell infiltration at sites of rejection by monitoring macrophages . the investigators subsequently developed a functional index from local strain analysis and proved it to be correlated with rejection grades [ 8 , 9 ] . although clinical applications have not been implemented , cmr was demonstrated to be capable of providing the rejection status of whole - heart perspective , and thus might be a potential tool of optimizing diagnosis of bnr . another potential tool is speckle - tracking 2-dimensional strain echocardiography ( 2dse ) . by using rat cardiac transplantation models , pieper et al . demonstrated that 2dse was able to differentiate myocardial function between rejection in allografts and nonrejection in isografts . perhaps bnr is another form of amr but due to the newness of amr and the lack of a complete understanding of amr , no definite conclusions can be made . despite the ishlt revised biopsy rejection scale , this revised definition of bnr has been noted to result in a decrease in 3-year subsequent survival , lower subsequent freedom from cardiac allograft vasculopathy ( cav ) , and a decrease in freedom from nonfatal major adverse cardiac events ( nf - mace ) . previous studies characterized bnr as occurring on average 43 38 months following transplantation while the data collected in our study indicates that the time to the first incidence of bnr is 7.8 7.5 months . the mechanism associated with bnr unfortunately not too many reports in the field looked at the treatment protocols for bnr . in our single center experience , who patients presented with heart failure symptoms were given more aggressive treatment , that is , atg , iv methylprednisolone , and ivig while patients without heart failure symptoms were mostly given prednisone bolus and taper only . as bnr is an infrequent phenomenon , of which there is considerable debate regarding its validity , further work needs to be instigated on whether there is a correlation between different factors and bnr episodes . overall , from this set of data , it is inconclusive as to whether or not there are any characteristics of bnr that differs from other types of rejections . a point of interest in this study is the patient who experienced two episodes of bnr . due to the small sample size , it is uncertain whether having one episode of bnr increases the risk of recurrent bnr episodes . during the first bnr episode , in contrast , it took the patient 6 months to experience a return to normalized lvef ( 51% ) during the second bnr episode . for the first episode of bnr , there was a favorable return to normal cardiac function following treatment with oral corticosteroids bolus and taper . in the case of the second episode of bnr , ivig was given instead which led to an increase in lvef , although it took a longer time ( 6 months ) for the patient to experience a return to the range of normalized lvef . prior to experiencing normalized lvef the patient 's lvef remained in the 40% range , fluctuating between 43% and 48% . the repeat bnr patient was the only african americans in this study and did not experience normalized lvef at 90-day after second onset . african americans have been reported to have a higher risk of rejection after transplant compared to any other race due to their unique metabolism in regard to immunosuppression [ 14 , 15 ] . a possible immunosuppression regimen with respect to bnr could require more aggressive treatment , employing the use of atg and ivig in addition to steroids . potential cause for this second onset of bnr could be the combined factor of this patient not receiving an adequate treatment for the second bnr and the ethnicity of the patient . as cases of bnr tend to respond favorably to appropriate rejection therapy 2 ( 17% ) of 12 bnr cases required inotropes and 7 ( 58% ) bnr cases without heart failure symptoms were treated with oral steroid bolus and taper . seven of 12 ( 58% ) treated bnr cases resulted in a return to a normal lvef of 57% 6% within a mean of 14 10 days from the initial negative biopsy . due to the small sample size , it is uncertain whether bnr should be considered as a new category in the biopsy grading scale or if it is merely difficult to diagnose based on endomyocardial biopsies alone . if it is the former , then mechanism needs to be elucidated . if it is the latter , then perhaps other methods for detecting rejection need to be considered . bnr is a rare phenomenon ( 12 cases of bnr in a 10-year period ) and can be a severe form of rejection in that there is cardiac dysfunction . however , this type of rejection is not apparent on biopsies due to either lack of knowledge regarding further types of rejection or other factors . characteristics of these cases of rejection are described above with most cases responding favorablly to rejection therapy .

purpose . the most recent international society for heart and lung transplantation ( ishlt ) biopsy scale classifies cellular and antibody - mediated rejections . however , there are cases with acute decline in left ventricular ejection fraction ( lvef 45% ) but no evidence of rejection on biopsy . characteristics and treatment response of this biopsy negative rejection ( bnr ) have yet to be elucidated . methods . between 2002 and 2012 , we found 12 cases of bnr in 11 heart transplant patients as previously defined . one of the 11 patients was treated a second time for bnr . characteristics and response to treatment were noted . results . 12 cases ( of 11 patients ) were reviewed and 11 occurred during the first year after transplant . 8 cases without heart failure symptoms were treated with an oral corticosteroids bolus and taper or intravenous immunoglobulin . four cases with heart failure symptoms were treated with thymoglobulin , intravenous immunoglobulin , and intravenous methylprednisolone followed by an oral corticosteroids bolus and taper . overall , 7 cases resulted in return to normal left ventricular function within a mean of 14 10 days from the initial biopsy . conclusion . bnr includes cardiac dysfunction and can be a severe form of rejection . characteristics of these cases of rejection are described with most cases responding to appropriate therapy .

this investigation includes a series of blood investigations and imaging ; when these investigations found no evidence for a demyelinating disorder ( through mri features principally ) , systemic autoimmune condition ( e.g. , antinuclear antibodies , dna , aquaporin-4 , and myelin oligodendrocyte glycoprotein antibodies ) , or infectious or metabolic causes , a granulomatous cause was considered , and further investigations were undertaken . those in whom systemic features of sarcoidosis were identified during investigation and those already known to have active systemic sarcoidosis , in whom the diagnosis of a granulomatous optic neuropathy was considered by the authors to be highly probable , were recorded prospectively and included in the study . the records were kept according to data protection regulations in the neuroophthalmology departments and collated by 2 of us ( b.j.b . and d.p.k . ) . criteria for the diagnosis of sarcoidosis involved histologic proof ( in 40 cases ) of a granulomatous disorder currently active or requiring continuing treatment or ( in 12 cases ) adequate proof on clinical grounds that no alternative explanation existed for the systemic disorder in the minds of the treating physicians : for example , clear radiologic evidence for lung and mediastinal involvement together with clear evidence for another characteristic feature of the disease , such as hypercalcemia , raised serum angiotensin - converting enzyme ( ace ) or other tissue involvement , associated with a typical pattern of isotope uptake using gallium scintigraphy or pet . ethical approval for the study was obtained , and patients gave informed consent to be included . ethical approval for the study was obtained , and patients gave informed consent to be included . twenty - four were caucasian , 24 african or caribbean , and 4 were from south asia . the diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . the remaining 18 had already been diagnosed , between 6 months and 20 years before the onset of the optic neuropathy . a histologic diagnosis was made from respiratory tissue ( transbronchial or mediastinal lymph gland biopsy ) in 28 cases , lymph gland biopsy in 3 , liver biopsy in 1 , orbital biopsy in 3 , and neurologic tissue in 5 . all fulfilled the criteria for the highly probable diagnosis of the wasog ( world association of sarcoidosis and other granulomatous diseases ) sarcoidosis organ assessment instrument . in 12 cases , the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians , with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above ; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . the predominate systemic clinical syndrome was respiratory in 38 cases , the orbit and lacrimal glands in 6 , cervical lymph glands in 4 , the skin in 3 , the nose and sinuses in 3 , and the liver and joints in 1 each . the serum ace was elevated in 24 ( 57% ) of available data at the time of onset of the optic neuropathy ; the median serum ace was 111 ( 18205 ) u / l ( normal range 850 gallium scans were undertaken in 10 instances , in which 1 was normal , 6 showed a panda pattern of uptake in the lacrimal and parotid glands , 1 a lambda pattern of mediastinal uptake , and 2 a combination of both patterns . all patients presented with symptoms and signs of an optic neuropathy , defined as a reduction in central visual acuity and color vision , reduction in the visual field , and when seen , an abnormality of the pupillary response . patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course , in which the symptoms evolved over weeks . the former were considered to show an optic neuritis syndrome , the latter a progressive syndrome . forty - three cases showed an optic neuritis syndrome , of which 27 were unilateral . twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . the median ( range ) acuity at presentation was 0.05 ( 01.0 ) in unilateral cases and 0.13 ( 01.0 ) in bilateral involvement . mri showing ( a ) swelling and high signal within the nerve , and ( b ) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . with oral corticosteroid treatment ( c ) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . ( d , e ) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . a wide range of visual field defects was noted ; most had central or centrocecal scotomas , but hemianopic and altitudinal defects were also seen . clinical characteristics and ocular findings at presentation in the 52 patients studied intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases ( 36% ) : a granulomatous anterior uveitis was seen in 10 cases , an isolated vitritis in 1 , a panuveitis in 1 , isolated choroidal involvement in 1 , features of a retinal vasculitis in 2 , cotton wool spots in 2 , macular exudates in 1 , and episcleritis in 1 . in no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . one patient underwent ct ; the remainder had mri studies of the orbits and brain , of which 13 were adjudged to be normal . twenty patients had abnormalities restricted to the optic nerve or sheath , of whom 14 showed enhancement of the optic nerve and sheath ( figure 1 ) with intrinsic lesions of one or both optic nerves , while the remainder showed high signal within the nerve without enhancement . in addition , 3 cases ( and the one who had ct ) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . the chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . both patients with a perineuritis showed features of this , i.e. , the predominate feature was of enhancement and thickening of the sheath , not the whole complex ( figure 2 ) . ( b , c ) optic perineuritis : t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . this was the presenting feature of the disease , which was diagnosed subsequently on histologic analysis of a cervical lymph node . a minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease : 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves , enhancement of the complex , which also involved the orbital apex ( 5 cases ) ( figure 3a ) , the floor of the middle cranial fossa ( 4 cases ) ( figure 3b ) , and the frontal lobes ( 1 case ) . ( a , b ) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs , mediastinum , and heart . ( c , d ) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . despite intensive treatment with corticosteroids and immunosuppression , in general , the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen ( figure 4 ) . histogram showing the relationships between mri features at onset / diagnosis ( abscissa ) and initial ( nadir ) visual acuity then best acuity after treatment ( ordinate ) . those whose clinical syndrome was of a progressive optic neuropathy ( 6 cases ) all showed meningeal enhancing lesions that had formed masses , in each case at the intracranial portion of the orbital apex ( figure 3 ) . csf results were available in 33 cases , including 2 with mass lesions and one with perineuritis . seven ( 21% ) showed csf protein levels greater than 0.6 g / dl ( median 0.95 g / dl , range 0.791.92 ) . five ( 15% ) showed a leukocyte pleocytosis ( > 5 cells / cm ) ( median 19 , range 670 ) . twenty - eight ( 85% ) had no oligoclonal bands , and 5 showed matched bands in both csf and serum . patients were treated in an individual way in 3 different hospitals so there was a wide range of medications , dosage regimens , and treatment durations . forty - nine were treated with corticosteroids , of whom 14 commenced with iv methylprednisolone followed by oral prednisolone , while the remainder received treatment with oral steroids from the outset . the daily dose of prednisolone varied between 30 and 80 mg per day . the duration of treatment varied greatly ( 4320 weeks ) . the median ( range ) length of follow - up was 8 ( 020 ) years . the median ( range ) change in visual acuity ( defined as the best acuity following treatment , even after relapse ) was + 0.46 ( 0.01 to + 1.22 ) in bilateral cases and + 0.175 ( 0.05 to + 1.25 ) in unilateral cases . the median final acuity in these 2 groups was 0.7 ( 01.25 ) and 0.5 ( 01.25 ) . those with a subacute syndrome showed a median improvement of + 0.225 ( range 0.01 to + 1.22 ) . those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . while there was a suggestion that mri abnormalities at onset ( figure 4 ) correlated with nadir acuity and an improvement following treatment , this was not significant ( p > 0.3 ) . considering treatment in those without a progressive disease course or perineuritis , 12 were treated with iv then oral steroids ; median ( range ) acuity was 0.02 ( 01 ) and increased to 0.75 ( 0.021 ) , median change + 0.3 , and those treated with oral steroids at onset 0.1 ( 01 ) increasing to 0.75 ( 01.25 ) , and median change + 0.5 ( p = 0.77 ) . for the most part , this occurred early on ; for example , patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg , and the signs settled with an escalation of treatment . immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . the patient treated with methotrexate had had repeated relapses , which stopped on the immunosuppressive drug . one patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened ; she was treated with cyclophosphamide then infliximab . in those who relapsed , the improvement in acuity following treatment was + 0.25 ( 0.15 to + 1.2 ) , and relapse did not appear to influence the outcome in any way . we have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace ( median 100 [ range 80178 ] u / l ) . the clinical syndrome was not different from the others described above ; half were caribbean , half caucasian , and half were bilateral . the median ( range ) acuity in each eye was 0.1 ( 01.0 ) . in each case , the affected discs were seen to be swollen . imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . csf in 4 cases showed normal protein and negative oligoclonal bands in 3 ; in the other case , matched serum and csf bands were found . in this case one was considered to have a disc granuloma and was watched without treatment ; he improved over 2 years . these cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . they appear to have neither sarcoidosis nor multiple sclerosis . twenty - four were caucasian , 24 african or caribbean , and 4 were from south asia . the diagnosis of systemic sarcoidosis was made concurrently in 33 patients and in one subsequently . the remaining 18 had already been diagnosed , between 6 months and 20 years before the onset of the optic neuropathy . a histologic diagnosis was made from respiratory tissue ( transbronchial or mediastinal lymph gland biopsy ) in 28 cases , lymph gland biopsy in 3 , liver biopsy in 1 , orbital biopsy in 3 , and neurologic tissue in 5 . all fulfilled the criteria for the highly probable diagnosis of the wasog ( world association of sarcoidosis and other granulomatous diseases ) sarcoidosis organ assessment instrument . in 12 cases , the diagnosis had been deemed secure on clinical grounds by the treating respiratory physicians , with clear evidence on clinical grounds for a multisystem disorder typical of sarcoidosis as noted in the methods section above ; these were all patients presenting with an optic neuropathy already diagnosed with systemic sarcoidosis . the predominate systemic clinical syndrome was respiratory in 38 cases , the orbit and lacrimal glands in 6 , cervical lymph glands in 4 , the skin in 3 , the nose and sinuses in 3 , and the liver and joints in 1 each . the serum ace was elevated in 24 ( 57% ) of available data at the time of onset of the optic neuropathy ; the median serum ace was 111 ( 18205 ) u / l ( normal range 850 gallium scans were undertaken in 10 instances , in which 1 was normal , 6 showed a panda pattern of uptake in the lacrimal and parotid glands , 1 a lambda pattern of mediastinal uptake , and 2 a combination of both patterns . all patients presented with symptoms and signs of an optic neuropathy , defined as a reduction in central visual acuity and color vision , reduction in the visual field , and when seen , an abnormality of the pupillary response . patients with a subacute optic neuropathy that evolved over days were considered separately from those with a more progressive clinical course , in which the symptoms evolved over weeks . the former were considered to show an optic neuritis syndrome , the latter a progressive syndrome . forty - three cases showed an optic neuritis syndrome , of which 27 were unilateral . twelve of the bilateral cases showed a sequential optic neuropathy with a range of latency between the 2 episodes of 1 to 60 months . the median ( range ) acuity at presentation was 0.05 ( 01.0 ) in unilateral cases and 0.13 ( 01.0 ) in bilateral involvement . mri showing ( a ) swelling and high signal within the nerve , and ( b ) enhancement in a patient who presented with a painless subacute optic neuropathy to no perception of light over 5 days . with oral corticosteroid treatment ( c ) t1-weighted axial mri showing swelling and enhancement of the anterior half of the left optic nerve . ( d , e ) t1-weighted coronal mri showing 2 other cases in which the nerve is seen to be swollen and enhancing following administration of contrast . a wide range of visual field defects was noted ; most had central or centrocecal scotomas , but hemianopic and altitudinal defects were also seen . intraocular inflammation was seen at the same time as the optic neuropathy in 19 cases ( 36% ) : a granulomatous anterior uveitis was seen in 10 cases , an isolated vitritis in 1 , a panuveitis in 1 , isolated choroidal involvement in 1 , features of a retinal vasculitis in 2 , cotton wool spots in 2 , macular exudates in 1 , and episcleritis in 1 . in no case was the intraocular disease longstanding and associated with chronic glaucoma or retinal ischaemia . one patient underwent ct ; the remainder had mri studies of the orbits and brain , of which 13 were adjudged to be normal . twenty patients had abnormalities restricted to the optic nerve or sheath , of whom 14 showed enhancement of the optic nerve and sheath ( figure 1 ) with intrinsic lesions of one or both optic nerves , while the remainder showed high signal within the nerve without enhancement . in addition , 3 cases ( and the one who had ct ) showed intrinsic lesions within the nerve and thickening of the adjacent optic nerve sheath complex . the chiasmal lesion was associated with enhancement of the chiasm extending forward into one of the nerves . both patients with a perineuritis showed features of this , i.e. , the predominate feature was of enhancement and thickening of the sheath , not the whole complex ( figure 2 ) . the lesion was biopsied and granulomatous inflammation identified . the optic neuropathy improved with steroids . ( b , c ) optic perineuritis : t1-weighted axial and coronal mri showing enlargement of and enhancement of the optic nerve and its sheath throughout its entire length . this was the presenting feature of the disease , which was diagnosed subsequently on histologic analysis of a cervical lymph node . a minority of patients whose clinical syndrome was one of a subacute optic neuropathy resembling an optic neuritis showed additional imaging features of more widespread involvement of the nervous system by the disease : 8 patients with unilateral optic neuropathies and 2 with bilateral involvement showed high signal within the nerves , enhancement of the complex , which also involved the orbital apex ( 5 cases ) ( figure 3a ) , the floor of the middle cranial fossa ( 4 cases ) ( figure 3b ) , and the frontal lobes ( 1 case ) . ( a , b ) orbital apex mass with a progressive painful optic neuropathy and sarcoidosis of the lungs , mediastinum , and heart . ( c , d ) two cases of progressive optic neuropathy with striking meningeal enhancement and swelling to form mass lesions . despite intensive treatment with corticosteroids and immunosuppression , neither case showed a meaningful improvement in visual acuity . in general , the initial acuity was better in those with normal imaging and no enhancement than in those in whom enhancement was seen ( figure 4 ) . histogram showing the relationships between mri features at onset / diagnosis ( abscissa ) and initial ( nadir ) visual acuity then best acuity after treatment ( ordinate ) . those whose clinical syndrome was of a progressive optic neuropathy ( 6 cases ) all showed meningeal enhancing lesions that had formed masses , in each case at the intracranial portion of the orbital apex ( figure 3 ) . csf results were available in 33 cases , including 2 with mass lesions and one with perineuritis . seven ( 21% ) showed csf protein levels greater than 0.6 g / dl ( median 0.95 g / dl , range 0.791.92 ) . five ( 15% ) showed a leukocyte pleocytosis ( > 5 cells / cm ) ( median 19 , range 670 ) . twenty - eight ( 85% ) had no oligoclonal bands , and 5 showed matched bands in both csf and serum . patients were treated in an individual way in 3 different hospitals so there was a wide range of medications , dosage regimens , and treatment durations . forty - nine were treated with corticosteroids , of whom 14 commenced with iv methylprednisolone followed by oral prednisolone , while the remainder received treatment with oral steroids from the outset . the daily dose of prednisolone varied between 30 and 80 mg per day . the duration of treatment varied greatly ( 4320 weeks ) . the median ( range ) length of follow - up was 8 ( 020 ) years . the median ( range ) change in visual acuity ( defined as the best acuity following treatment , even after relapse ) was + 0.46 ( 0.01 to + 1.22 ) in bilateral cases and + 0.175 ( 0.05 to + 1.25 ) in unilateral cases . the median final acuity in these 2 groups was 0.7 ( 01.25 ) and 0.5 ( 01.25 ) . those with a subacute syndrome showed a median improvement of + 0.225 ( range 0.01 to + 1.22 ) . those in the progressive subgroup showed no improvement in visual acuity with treatment although in each case there was resolution of meningeal enhancement over time . while there was a suggestion that mri abnormalities at onset ( figure 4 ) correlated with nadir acuity and an improvement following treatment , this was not significant ( p > 0.3 ) . considering treatment in those without a progressive disease course or perineuritis , 12 were treated with iv then oral steroids ; median ( range ) acuity was 0.02 ( 01 ) and increased to 0.75 ( 0.021 ) , median change + 0.3 , and those treated with oral steroids at onset 0.1 ( 01 ) increasing to 0.75 ( 01.25 ) , and median change + 0.5 ( p = 0.77 ) . for the most part , this occurred early on ; for example , patients treated with 60 mg prednisolone developed recurrence of symptoms and signs at 40 to 20 mg , and the signs settled with an escalation of treatment . immunosuppressive agents were added in 7 cases azathioprine in 4 and mycophenolate and methotrexate in one each . the patient treated with methotrexate had had repeated relapses , which stopped on the immunosuppressive drug . one patient had an intracranial relapse of neurosarcoidosis and her optic neuropathy worsened ; she was treated with cyclophosphamide then infliximab . in those who relapsed , the improvement in acuity following treatment was + 0.25 ( 0.15 to + 1.2 ) , and relapse did not appear to influence the outcome in any way . we have seen 6 additional patients who presented with an optic neuritis and were found during investigation to have a raised serum ace ( median 100 [ range 80178 ] the clinical syndrome was not different from the others described above ; half were caribbean , half caucasian , and half were bilateral . the median ( range ) acuity in each eye was 0.1 ( 01.0 ) . in each case , the affected discs were seen to be swollen . imaging was normal in 3 and showed high signal within the affected nerve in the other 3 . csf in 4 cases showed normal protein and negative oligoclonal bands in 3 ; in the other case , matched serum and csf bands were found . in this case one was considered to have a disc granuloma and was watched without treatment ; he improved over 2 years . these cases have been followed and none has developed further or recurrent neurologic or systemic symptoms . this work forms the largest series of patients with optic neuropathy in sarcoidosis identified and each has been followed prospectively . the age range was wide but the condition was more prevalent in the first half of life . the majority of patients were women and there were equal numbers in caucasian and african or caribbean ethnic groups . according to the epidemiologic findings in access , the ethnic subgroups in london are 60% caucasian , 14% african and caribbean , and 18% south asian , and our results therefore suggest a greater prevalence of the disease in african and caribbean people who live in london . in general , the clinical syndrome resembled an optic neuritis , although in some , the disease course was more slowly progressive . pain was a less prevalent feature than is seen in demyelinating optic neuritis , and the median visual acuity at nadir was equivalent , although more were no perception of light ( table 2 ) . sixty - four percent had a unilateral involvement , 28% presented at a later time with involvement of the second eye , while synchronous bilateral involvement was less common ( 9% ) . it is noteworthy that there was evidence for concurrent intraocular inflammation in only a minority of cases . predominately this was mild ( although more severe cases had been excluded because of the uncertainty of the pathogenesis of the optic neuropathy ) . indeed , in 64% of patients , there was no history of ocular involvement at any stage of the disease to date . imaging showed clear abnormalities , which accounted for the clinical features in 75% of cases . comparison of clinical features with results of the onsg spontaneous improvement occurred ( as has been noted previously ) , although the majority of patients were treated with steroids . following this , an often substantial improvement in vision took place , even after several weeks without treatment . relapse occurred in 25% of cases , necessitating recommencement or an escalation of treatment , and in the majority , this occurred early , as steroids were withdrawn . six cases ( 11% ) had a more progressive disease course and were shown to have a more widespread neurologic involvement , each showing evidence for adjacent meningeal inflammation . visual outcome in this subgroup was poor , even though the disease process required , in general , a higher and more prolonged dose of steroids , concurrent immunosuppression , and in 2 cases , a requirement for biological agents with refractory disease . evidence for neurosarcoidosis defined as the presence of intracranial meningeal enhancement arose in 16 cases overall all of those with a progressive course and 10 with a subacute course . involvement of the optic nerve in sarcoidosis may arise through a variety of mechanisms : inflammation of the nerve itself , compression or infiltration by an inflammatory mass adjacent to the anterior visual pathway ( e.g. , the orbital apex or pituitary gland ) , secondary involvement through ischemic complications of retinal and choroidal inflammation and glaucoma , granulomas within the disc itself , meningeal inflammation within the optic nerve sheath leading to an optic perineuritis , and finally as a consequence of hydrocephalus . it was seen in 5% of one series of 202 patients with ocular disease in sarcoidosis , but only one in a series of 649 patients with systemic sarcoidosis and none in another of 285 . in the literature before 2000 , 57 cases had been published , of which 35 involved the optic nerve itself , 9 compression due to an intracerebral lesion , 6 due to hydrocephalus , and 6 due to the complications of uveitis . many noted that bilateral involvement occurred and that imaging showed enlargement of the optic nerve others noted that the acuity was lower than that seen in a demyelinating optic neuritis . one study described 24 cases with optic neuropathy : 71% had not been known previously to have systemic sarcoidosis and 42% showed previous or current intraocular inflammation . another showed that 14 of 67 patients known to have neurologic sarcoidosis had some form of optic neuropathy . we have identified a small subgroup of patients in whom the serum ace is elevated at presentation and then subsequently normalizes without relapse . on further investigation , these patients do not have sarcoidosis and on follow - up show no sign of transformation into a more widespread disease . the serum ace appears to increase in a nonspecific way and appears not to indicate a systemic granulomatous disease . it is important to identify this subgroup , which requires no additional treatment , merely close follow - up and observation . this large series of patients with optic neuropathy in conjunction with sarcoidosis has shown that more than half are not previously diagnosed with sarcoidosis , only a third have concurrent intraocular inflammation , and the clinical characteristics and in most cases the imaging features are not substantially different from that seen in a regular demyelinating optic neuritis . however , patients should be identified early because the response to treatment may lessen with time , and the identification of more widespread disease , particularly within the nervous system , will require prolonged , often high - dose treatment with steroids , immunosuppressive drugs , and modern biological therapies . modern imaging investigations including pet may make identification of these cases easier , but a high index of clinical suspicion is still important , particularly in non - caucasian patients presenting with a painless optic neuritis . once identified , we recommend that patients be treated with high - dose ( oral or iv ) corticosteroids , followed by a slow taper watching carefully for relapse as the dose is reduced . repeated relapses , particularly preventing steroid dose reduction , should be treated with an additional immunosuppressive agent . the presence of neurosarcoidosis requires more prolonged higher - dose treatment with steroids and immunosuppression from the outset , with early recourse to biological agents should the clinical and radiologic features of the disorder fail to improve quickly . further study is required to identify precisely which treatments are best and over what period of time . dr . kidd was responsible for the study design , data acquisition , analysis and interpretation , and writing the manuscript . burton served on the scientific advisory board for bayer , novartis , received travel funding from novartis , bayer , is subeditor for eastern european journal of neurology , received research support from novartis alcon .

objective : to identify and follow a series of 52 patients with optic neuropathy related to sarcoidosis.methods:prospective observational cohort study.results:the disorder was more common in women and affected a wide age range . it was proportionately more common in african and caribbean ethnic groups . two clinical subtypes were identified : the more common was a subacute optic neuropathy resembling optic neuritis ; a more slowly progressive optic neuropathy arose in the remaining 17% . sixteen ( 31% ) were bilateral . concurrent intraocular inflammation was seen in 36% . pain arose in only 27% of cases . an optic perineuritis was seen in 2 cases , and predominate involvement of the chiasm in one . mri findings showed optic nerve involvement in 75% of cases , with adjacent and more widespread inflammation in 31% . treatment with corticosteroids was helpful in those with an inflammatory optic neuropathy , but not those with mass lesions . relapse of visual signs arose in 25% of cases , necessitating an increase or escalation of treatment , but relapse was not a poor prognostic factor.conclusions:this is a large prospective study of the clinical characteristics and outcome of treatment in optic neuropathy associated with sarcoidosis . patients who experience an inflammatory optic neuropathy respond to treatment but may relapse . those with infiltrative or progressive optic neuropathies improve less well even though the inflammatory disorder responds to therapy .

epidemiological studies during the past decade have shown a positive correlation between the general consumption of cruciferous vegetables and decreased incidence of some cancers , including lung , stomach , colon , breast,[23 ] bladder,[45 ] prostate,[67 ] and non - hodgkin lymphoma . lung cancer risk is reduced especially by dietary intake of isothiocyanates or cruciferous vegetables in persons with genotypes of glutathione s - transferase mu 1-null and glutathione s - transferase theta 1-null , highlighting an important interplay between genetic susceptibility factors and chemopreventive agents.[1910 ] the cancer chemopreventive effect of cruciferous vegetables is due to glucosinolates . sulforaphane ( sfn ) is formed by enzymatic transformation of a glucosinolate , glucoraphanin , present in cruciferous vegetables , during chewing or through conversion through the gut microflora . sfn acts as a chemopreventive agent by inducing phase - ii enzymes by activating the nuclear factor erythroid-2 related factor 2 ( nrf2 ) pathway.[1213 ] evidence is mounting that sfn acts through other chemopreventive mechanisms such as blocking cancer initiation via inhibition of cytochrome p-450-dependent monoxygenases that convert procarcinogens into active carcinogens and modulating signaling pathways that regulate cell growth and apoptosis to suppress cancer progression.[1415 ] several groups have reported that sfn is effective in preventing chemical carcinogen - induced breast , stomach , pancreas , and colon cancer , as well as decreased polyp formation in apc mice . in the lungs , sfn and its n - acetylcysteine conjugate inhibit malignant progression of adenomas induced by tobacco carcinogens . in addition to its anticarcinogenic effect , sfn treatment inhibits the growth of established prostate,[2123 ] breast , pancreas,[2527 ] and lung cancer xenografts in mice . preclinical study results have also shown that sfn - mediated activation of nrf2 improves bacterial clearance and corticosteroid sensitivity in mice with chronic cigarette smoke exposure.[2930 ] several clinical trials are underway for evaluating sfn for treatment of various diseases ( www.clinicaltrials.gov ) . somatic loss of function mutations in kelch - like ech - associated protein 1 ( keap1 ) and oncogenic gain of function mutations in nrf2 have been reported in lung , prostate , skin , esophagus , gallbladder , and ovarian cancer . more recently , oncogenic activation of k - ras , myc , and b - raf have been shown to activate nrf2 signaling , and this cross talk has been shown to be important for oncogene - induced senescence evasion and tumorigenesis.[3637 ] for future development of sfn as a chemopreventive or therapeutic agent , we have evaluated the risk of long - term sfn administration on lung tumorigenesis by using oncogenic k - ras mouse model and human lung cancer xenograft mouse models . d , l - sulforaphane was purchased from toronto research chemicals , ontario ( cat#s699115 ) and stored at -20c . the sfn was freshly diluted in phosphate - buffered saline ( pbs ) before treatment . a549 and h1975 cells were purchased from american type culture collection ( manassas , virginia , united states ) and cultured under recommended conditions . cells were cultured in dulbecco modified eagle medium ( invitrogen , carlsbad , california , united states ) containing 10% fetal bovine serum and 1% penicillin streptomycin . ad - cmv - cre ( 1 10 pfu / ml ) was obtained from vector biolabs . kras ( strain number 01xj6 ) mice were obtained from a mouse repository ( nci , frederick ) and were bred in our facility . athymic ncr - nu / nu ( strain number 01b74 ) nude mice ( 6 - 8 weeks ) were obtained from nci . mice were fed the 2018sx diet ( harlan teklad ) and had access to water ad libitum ; they were housed under controlled conditions ( 232c ; 14-h light/ 10-h dark cycles ) . all experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . eight - week - old kras mice were anesthetized with ketamine ( 100 mg / kg of body weight ) and xylazine ( 10 mg / kg of body weight ) . five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration ( 50 l , twice at an interval of 5 min ) according to the standard protocol . five days after adenovirus cre treatment , mice were randomly placed into one of the two groups . sfn ( 0.5 mg ) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer ( aeroneb lab ) in the sfn group ( n = 9 ) . the lesions were assessed subjectively for the percentage of lesion area to total lung section area . the lesions were categorized as mild hyperplasia , severe hyperplasia , adenoma , or adenocarcinoma . either a549 cells ( 5 10 ) or h1975 cells ( 1 10 ) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . when the volume of the tumor reached 50 20 mm , the mice were separated randomly into different treatment groups , and sfn treatment was started . the mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . the tumor volumes were calculated by using the following formula : length ( mm ) width ( mm ) width ( mm ) 0.52 . the subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . the control mice in all the experiments received the same volume of pbs as did their respective cohorts . the mice were euthanized by means of cervical dislocation , and the tumors were removed without skin and weighed . design for xenograft experiments total rna was extracted from tissues and cells by using the rneasy kit ( qiagen ) and was quantified by means of ultraviolet absorption spectrophotometry . the reverse transcription reaction was performed by using a high - capacity cdna synthesis kit ( applied biosystems , foster city , california , united states ) and the multiscribe first strand synthesis system ( applied biosystems , foster city , california , united states ) in a final volume of 20 l containing 1 g of total rna , 100 ng of random hexamers , 1 reverse transcription buffer , 1 mm deoxyribonucleotide triphosphate , multiscribe reverse transcriptase , and nuclease - free water . a quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase , quinone 1 ( nqo1 ) , heme oxygenase 1(ho-1 ) , and glutamate - cysteine ligase , catalytic subunit ( gclc ) was performed by using assay - on - demand primers and probe sets from applied biosystems . tumor volume , tumor weight , and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . d , l - sulforaphane was purchased from toronto research chemicals , ontario ( cat#s699115 ) and stored at -20c . the sfn was freshly diluted in phosphate - buffered saline ( pbs ) before treatment . a549 and h1975 cells were purchased from american type culture collection ( manassas , virginia , united states ) and cultured under recommended conditions . cells were cultured in dulbecco modified eagle medium ( invitrogen , carlsbad , california , united states ) containing 10% fetal bovine serum and 1% penicillin streptomycin . ad - cmv - cre ( 1 10 pfu / ml ) was obtained from vector biolabs . kras ( strain number 01xj6 ) mice were obtained from a mouse repository ( nci , frederick ) and were bred in our facility . athymic ncr - nu / nu ( strain number 01b74 ) nude mice ( 6 - 8 weeks ) were obtained from nci . mice were fed the 2018sx diet ( harlan teklad ) and had access to water ad libitum ; they were housed under controlled conditions ( 232c ; 14-h light/ 10-h dark cycles ) . all experimental protocols conducted on the mice were performed in accordance with national institutes of health guidelines and were approved by the johns hopkins university animal care and use committee . eight - week - old kras mice were anesthetized with ketamine ( 100 mg / kg of body weight ) and xylazine ( 10 mg / kg of body weight ) . five million adenovirus cre - recombinase particles diluted in 100 l of minimal essential medium were administered by oropharyngeal aspiration ( 50 l , twice at an interval of 5 min ) according to the standard protocol . five days after adenovirus cre treatment , mice were randomly placed into one of the two groups . sfn ( 0.5 mg ) suspended in 50 l of pbs was administered 5 d / wk for 14 weeks by using a nebulizer ( aeroneb lab ) in the sfn group ( n = 9 ) . the lesions were assessed subjectively for the percentage of lesion area to total lung section area . the lesions were categorized as mild hyperplasia , severe hyperplasia , adenoma , or adenocarcinoma . either a549 cells ( 5 10 ) or h1975 cells ( 1 10 ) were injected subcutaneously into the right flank of the anesthetized athymic nude mice . when the volume of the tumor reached 50 20 mm , the mice were separated randomly into different treatment groups , and sfn treatment was started . the mice were weighed and the tumor dimensions were measured with calipers once a week until 1 week before harvesting of the tumor . the tumor volumes were calculated by using the following formula : length ( mm ) width ( mm ) width ( mm ) 0.52 . the subcutaneous sfn injections were administered 0.51 cm adjacent to the left side of the tumor . the control mice in all the experiments received the same volume of pbs as did their respective cohorts . the mice were euthanized by means of cervical dislocation , and the tumors were removed without skin and weighed . total rna was extracted from tissues and cells by using the rneasy kit ( qiagen ) and was quantified by means of ultraviolet absorption spectrophotometry . the reverse transcription reaction was performed by using a high - capacity cdna synthesis kit ( applied biosystems , foster city , california , united states ) and the multiscribe first strand synthesis system ( applied biosystems , foster city , california , united states ) in a final volume of 20 l containing 1 g of total rna , 100 ng of random hexamers , 1 reverse transcription buffer , 1 mm deoxyribonucleotide triphosphate , multiscribe reverse transcriptase , and nuclease - free water . a quantitative real - time polymerase chain reaction analysis of nad(p)h dehydrogenase , quinone 1 ( nqo1 ) , heme oxygenase 1(ho-1 ) , and glutamate - cysteine ligase , catalytic subunit ( gclc ) was performed by using assay - on - demand primers and probe sets from applied biosystems . tumor volume , tumor weight , and body weight were measured and presented as mean plus or minus standard error and compared by means of a t - test or anova . we determined whether chronic sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . the k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . in this study , we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . one week after administration of a high titer adenoviral cre to induce mutant k - ras expression , 1 cohort of mice was treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer [ figure 1a ] . we have reported that sfn ( 0.5 mg / mice ) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation , mice were sacrificed , and lung sections were screened for neoplastic foci . histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . although tumor number , histological subtypes , and grades were not significantly different between the two groups , we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [ figure 1b ] . extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1 , gclc , and ho-1 in the lungs [ figure 1c ] . thus , our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . ( a ) schematic of the experimental design . ( b ) bar graph showing relative number of neoplastic lesions ( hyperplasia , adenoma , adenocarcinoma ) and percentage of the area of the lung covered by the tumor cells . ( c ) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . p - values are calculated using t test to evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts , we selected two cell lines , a549 and h1975 . a549 cells with a mutant k - ras and keap1 allele , and h1975 with a mutant epidermal growth factor receptor ( egfr ) allele , are the two commonly used lung cancer xenograft models . a549 and h1975 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volume reached 50 20 mm , mice were randomly allocated into two groups , and sfn treatment was initiated [ figures 2a and 3a ] . mice were treated with vehicle ( pbs ) or sfn by means of intraperitoneal injection ( 5 d / wk ) , and tumor volume was measured weekly . sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight , has been reported to be well tolerated . we did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn , and thus , we increased the sfn dose and repeated the experiment with 3 and 6 mol . again , sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 2b and c and 3b and c ) . for h1975 cells expressing mutant egrf allele , we increased the sfn dose further ( 9 mol ) since these tumors proliferate rapidly in vivo . similar to our results with a549 tumors , sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 3b and 3c ] . to ascertain whether systemic sfn administration causes weight loss , we recorded body weights of the control and treated mice . average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [ figures 2d and 3d ] . systemic delivery of sfn does not promote growth of keap1 and k - ras mutant , a549 , lung cancer xenografts . tumor volume is not significantly different between the three cohorts of mice ( anova ) . ( e ) real - time rt - pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) . systemic delivery of sfn does not promote growth of egfr mutant ( h1975 ) lung cancer xenografts . tumor volume is not significantly different between the two cohorts of mice ( t test ) . ( c ) tumor weights ( day 30 ) are not significantly different between the two groups ( t test ) . ( e ) real - time pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice , we measured nqo1 expression in the liver [ figures 2e and 3e ] . in addition to the liver , we also measured the induction of nrf2 signaling in a549 and h1975 tumors . however , sfn treatment did not upregulate nrf2 signaling in tumors [ figures 2e and 3e ] . these data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . to determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors , we selected a549 cells as the model system . a549 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volumes reached 50 20 mm , mice were randomly allocated into 2 groups , and treatment groups were administered sfn ( 1 mol , 5d / wk ) by means of subcutaneous injection adjacent to the tumor [ figure 4a ] . the average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors ( p < 0.05 ) [ figure 4b ] . tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors ( p = 0.05 ) [ figure 4c ] , with no apparent difference in total body weight [ figure 4d ] between the two groups . graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p < 0.05 ( t test ) . ( c ) tumor weights were significantly lower in the sfn - treated group . * p < 0.05 ( t test ) . ( e g ) relative tumor growth , average tumor weight , and body weight in the cohort of mice treated with 500 nmol of sfn . ( h j ) relative expression of nrf2-dependent genes in the liver and tumor tissues . * significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose ( 0.5 mol ) as well . treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [ figures 4e and 4f ] . there was no adverse effect of localized sfn treatment on animal health or body weight , although we noticed slight thickening of skin in the area surrounding the injection spot [ figure 4 g ] . to demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression , we measured nqo1 , ho-1 , and gclc expression in the skin and liver . in addition to these two tissues , we measured the induction of nrf2 signaling in a549 tumors . sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [ figures 4h j ] . since sfn was administered locally , we did not notice significant change in nrf2 dependent gene expression in the liver . these data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . we determined whether chronic sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . the k - ras mutant mouse develops progressively less differentiated lung tumors after the administration of adenoviral cre - recombinase . in this study , we evaluated the effect of sfn on k - ras mediated lung tumorigenesis post oncogenic k - ras activation . one week after administration of a high titer adenoviral cre to induce mutant k - ras expression , 1 cohort of mice was treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer [ figure 1a ] . we have reported that sfn ( 0.5 mg / mice ) administered using nebulizer significantly induces nrf2-dependent gene expression in lungs . fourteen weeks after adenovirus - cre - recombinase administration and mutant k - ras activation , mice were sacrificed , and lung sections were screened for neoplastic foci . histological analyses of the lung sections from the k - ras mutant mice showed the presence of atypical adenomatous hyperplasia and adenomas with infiltration of inflammatory cells . although tumor number , histological subtypes , and grades were not significantly different between the two groups , we noticed a trend toward reduced tumor burden in the sfn - treated cohort of mice [ figure 1b ] . extended exposure to sfn transcriptionally induced the expression of the nrf2 target genes nqo1 , gclc , and ho-1 in the lungs [ figure 1c ] . thus , our data demonstrate that prolonged exposure to sfn via aerosol inhalation does not promote or increase tumorigenesis in the mutant k- ras - driven mouse model of lung tumorigenesis . ( a ) schematic of the experimental design . ( b ) bar graph showing relative number of neoplastic lesions ( hyperplasia , adenoma , adenocarcinoma ) and percentage of the area of the lung covered by the tumor cells . ( c ) real - time rt - pcr data showing induction of nrf2-dependent gene expression in the lungs of the sfn - treated cohort of mice . to evaluate the effect of prolonged sfn treatment on the growth of established lung cancer xenografts , we selected two cell lines , a549 and h1975 . a549 cells with a mutant k - ras and keap1 allele , and h1975 with a mutant epidermal growth factor receptor ( egfr ) allele , are the two commonly used lung cancer xenograft models . a549 and h1975 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volume reached 50 20 mm , mice were randomly allocated into two groups , and sfn treatment was initiated [ figures 2a and 3a ] . mice were treated with vehicle ( pbs ) or sfn by means of intraperitoneal injection ( 5 d / wk ) , and tumor volume was measured weekly . sfn dose was selected based on previously published results where sfn administration at a dose 250400 mol / kg body weight , has been reported to be well tolerated . we did not notice a significant reduction in the tumor growth rate / weight of a549 xenografts treated with 3 mol dose of sfn , and thus , we increased the sfn dose and repeated the experiment with 3 and 6 mol . again , sfn treatment at a dose of 3 or 6 mol did not inhibit the growth of a549 tumors significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 2b and c and 3b and c ) . for h1975 cells expressing mutant egrf allele , we increased the sfn dose further ( 9 mol ) since these tumors proliferate rapidly in vivo . similar to our results with a549 tumors , sfn at a dose 9 mol did not attenuate the growth of h1975 xenografts significantly , although we noticed a trend toward lower tumor volume and weight in the sfn - treated group [ figures 3b and 3c ] . to ascertain whether systemic sfn administration causes weight loss , we recorded body weights of the control and treated mice . average body weights of the control and sfn - treated mice did not differ significantly throughout the treatment protocol [ figures 2d and 3d ] . systemic delivery of sfn does not promote growth of keap1 and k - ras mutant , a549 , lung cancer xenografts . tumor volume is not significantly different between the three cohorts of mice ( anova ) . ( e ) real - time rt - pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) . systemic delivery of sfn does not promote growth of egfr mutant ( h1975 ) lung cancer xenografts . tumor volume is not significantly different between the two cohorts of mice ( t test ) . ( c ) tumor weights ( day 30 ) are not significantly different between the two groups ( t test ) . ( e ) real - time pcr showing expression of the nrf2-dependent gene , nqo1 , in the liver and tumor tissues.*p < 0.05 ( t test ) to demonstrate that systemic delivery of sfn induced nrf2-dependent target gene expression in mice , we measured nqo1 expression in the liver [ figures 2e and 3e ] . in addition to the liver , we also measured the induction of nrf2 signaling in a549 and h1975 tumors . however , sfn treatment did not upregulate nrf2 signaling in tumors [ figures 2e and 3e ] . these data indicate that systemic sfn treatment induces nrf2 signaling but does not promote growth of lung cancer xenografts . to determine whether localized delivery of sfn that results in higher accumulation of sfn in the tumor and neighboring areas can retard the growth of subcutaneous tumors , we selected a549 cells as the model system . a549 cells were injected into the flanks of nude mice , and change in tumor volume was recorded . once the tumor volumes reached 50 20 mm , mice were randomly allocated into 2 groups , and treatment groups were administered sfn ( 1 mol , 5d / wk ) by means of subcutaneous injection adjacent to the tumor [ figure 4a ] . the average change in tumor volume was significantly different between the vehicle- and sfn - treated tumors ( p < 0.05 ) [ figure 4b ] . tumor weights were significantly higher in the vehicle - treated tumors than in the sfn - treated tumors ( p = 0.05 ) [ figure 4c ] , with no apparent difference in total body weight [ figure 4d ] between the two groups . ( b ) graph showing relative growth of a549 tumors treated with 1000 nmol of sfn . * p < 0.05 ( t test ) . ( c ) tumor weights were significantly lower in the sfn - treated group . * p < 0.05 ( t test ) . ( e g ) relative tumor growth , average tumor weight , and body weight in the cohort of mice treated with 500 nmol of sfn . ( h j ) relative expression of nrf2-dependent genes in the liver and tumor tissues . * p < 0.05 . significant inhibition of tumor growth by sfn at a dose of 1 mol prompted us to determine whether sfn exerts a similar effect at a lower dose ( 0.5 mol ) as well . treatment of a549 xenograft tumors with a lower dose of sfn significantly retarded the growth of a549 tumors in nude mice . average tumor volume and weight were significantly lower in the sfn - treated cohort of mice [ figures 4e and 4f ] . there was no adverse effect of localized sfn treatment on animal health or body weight , although we noticed slight thickening of skin in the area surrounding the injection spot [ figure 4 g ] . to demonstrate that localized delivery of sfn induced nrf2-dependent target gene expression , we measured nqo1 , ho-1 , and gclc expression in the skin and liver . in addition to these two tissues , we measured the induction of nrf2 signaling in a549 tumors . sfn treatment significantly induced the expression of ho-1 in the skin with no apparent effect on nrf2-dependent gene expression in the tumors [ figures 4h j ] . since sfn was administered locally , we did not notice significant change in nrf2 dependent gene expression in the liver . these data indicate that localized sfn treatment probably results in greater accumulation of sfn in subcutaneous tumors leading to significant growth inhibition . since the early 1980s , naturally occurring antioxidants and vitamins have been investigated and promoted as chemopreventive agents . antioxidants have been popular as chemopreventive agents because there is strong scientific evidence supporting reduced incidence of epithelial cancers associated with high dietary intake of fruits and vegetables . because of this evidence , major clinical trials were launched to test -carotene , alone or in combination with vitamin e or vitamin a. the largest trials for chemoprevention of lung cancers in high - risk smoker populations were atbc , the alpha tocopherol ( vitamin e ) , beta - carotene trial in finland , and caret , the beta - carotene and retinol efficacy trial smokers in the united states . the results were devastatingly similar , with excess lung cancer incidence and mortality rates in the active treatment arm , compared with those in the placebo arm of each trial . the unexpected and unwanted outcomes from the caret and atbc trials prompted us to reconsider issues related to both the effectiveness and the safety of micronutrient supplementation and careful evaluation of chemopreventive agents in preclinical cancer models . sfn derived from broccoli sprouts is currently under clinical evaluation in diseases in which oxidative stress and inflammation play important roles in disease pathogenesis , namely , pulmonary diseases , such as chronic obstructive pulmonary disease , asthma , and cystic fibrosis , and cardiovascular disease , and protection against radiation dermatitis . in addition to these , sfn is being evaluated as a potential chemopreventive agent in melanoma and breast cancer and as an anticancer agent in patients with recurrent prostate cancer . at present , there are 17 clinical trials listed at www.clinicaltrials.gov that are using sfn or an enriched broccoli sprout preparation for treatment of a variety of diseases although sfn is known to modulate several signaling pathways , it is a potent inducer of nrf2-keap1 signaling , and the anticarcinogenic and anti - inflammatory activities of sfn are mediated primarily by nrf2-dependent induction of phase - ii detoxification and antioxidant enzymes.[13142930 ] results from recent studies suggest that sfn offers protection against tumor development and progression during the post - initiation phase by modulating diverse cellular processes that inhibit the growth of transformed cells . the ability of sfn to induce reactive oxygen species production , apoptosis , and cell cycle arrest is associated with regulation of many molecules , including the bcl-2 family of proteins , caspases , p21 , cyclins , cyclin - dependent kinases , and histone deacetylases and nuclear factor - kappa - b pathways . sfn also suppresses angiogenesis and metastasis by downregulating hypoxia - inducible factor 1 alpha , matrix metallopeptidase 2 , matrix metallopeptidase 9 , and vascular endothelial growth factor in various preclinical models of cancer however , in the past few years , loss of function mutations in keap1 and activating mutations in nrf2 leading to gain of nrf2 have been reported in lung cancer . in addition to lung cancer , gain of nrf2 function has been reported in other cancers such as prostate , gallbladder , esophagus , breast , skin , and ovarian cancer . in addition to mutations , results from recent studies have demonstrated that the oncogenes k - ras , b - raf , and myc upregulate nrf2 signaling to evade senescence and apoptosis and promote tumorigenesis . a comprehensive profiling of sfn bioavailability and tissue distribution kinetics revealed a dose - dependent increase in tissue concentrations after oral administration of sfn in mice . sfn accumulation peaked at 2 h in lung , liver , kidney , brain , and plasma , but in small intestine , colon , and prostate , the highest concentrations were recorded at 6 h. maximum sfn accumulation was detected in small intestine . furthermore , tissue concentrations of sfn metabolites varied as much as 100-fold between different tissues suggesting that peak plasma concentrations do not always align precisely with target tissues . thus , route of administration may have a critical impact on sfn tissue distribution and accumulation . in this study , we compared three routes of sfn administration ; ( a ) direct delivery to the target organ ( lung ) using nebulizer , ( b ) localized delivery to tumor bearing area ( subcutaneous injection ) , and ( c ) systemic delivery with minimal risk of potential side effects due to systemic distribution ( intraperitoneal injection ) . to evaluate the effect of prolonged sfn treatment on growth of lung cancer xenografts , we selected two models : ( 1 ) a549 cells harboring mutant k - ras oncogene and mutant keap1 gene and ( 2 ) h1975 cells harboring mutant egfr oncogene but wild - type keap1 and nrf2 . prolonged systemic sfn treatment did not promote the growth of these tumors in vivo . on the contrary , localized delivery of sfn significantly abrogated the growth of keap1 mutant a549 tumors in vivo suggesting that the tumor suppression effects of sfn are mediated by non - nrf2-dependent signaling mechanisms . importantly , significant tumor growth inhibition and a trend toward reduction with localized and systemic route of sfn administration , respectively , suggests that localized delivery of sfn probably results in better target tissue distribution and accumulation as compared to the systemic route . next , we investigated whether prolonged sfn administration affects oncogenic k - ras ( k - ras)-driven lung tumorigenesis . a study by denicola et al . showed that oncogenic k - ras signaling upregulates nrf2 expression and its transcriptional network . interestingly , although sfn administration via aerosol inhalation only modestly affected the nrf2 signaling , we still noticed a trend toward reduced tumor burden in sfn treated cohort of mice . this raises a possibility that alternative route of administration resulting in localized accumulation and robust induction of nrf2 signaling may significantly inhibit k - ras mediated lung tumor growth . in summary , sfn treatment however , one limitation of our study is that we only evaluated the effect of sfn on k - ras - mediated lung tumorigenesis post oncogenic k - ras activation and tumor foci formation . it remains to be investigated if sfn administration prior to cre - recombinase mediated oncogenic k - ras activation may impact the overall tumor burden . pharmacological activators of nrf2 ( dithiolethiones , isothiocyanates , and triterpenoids ) including sfn have been evaluated as modifiers of multistage carcinogenesis in animal models and no protumorigenic effects have been reported to date . our data from three preclinical mouse models of lung cancer suggest that prolonged sfn treatment does not promote tumor growth in mice . these findings , along with those from previously published studies on the chemopreventive properties of sfn against chemical carcinogens , support its future clinical development as a drug for chronic obstructive pulmonary disease and other pulmonary diseases . dr . anju singh , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . shyam biswal , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland department of oncology , sidney kimmel comprehensive cancer center , school of medicine , johns hopkins university , baltimore , maryland mr . shengbin yang , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . edward gabrielson , department of oncology , sidney kimmel comprehensive cancer center , school of medicine , johns hopkins university , baltimore , maryland dr . jinfang ma , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . rajesh k thimmulappa , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland dr . ponvijay kombairaju , department of environmental health sciences , bloomberg school of public health , johns hopkins university , baltimore , maryland

background : sulforaphane ( sfn ) , an activator of nuclear factor erythroid-2 related factor 2 ( nrf2 ) , is a promising chemopreventive agent which is undergoing clinical trial for several diseases . studies have indicated that there is gain of nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor kelch - like ech - associated protein 1 ( keap1 ) . more recently , several oncogenes have been shown to activate nrf2 signaling as the main prosurvival pathway mediating ros detoxification , senescence evasion , and neoplastic transformation . thus , it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of sfn using mouse models of cancer.materials and methods : we evaluated the effect of prolonged sfn treatment on oncogenic k - ras ( k - raslsl - g12d)-driven lung tumorigenesis . one week post mutant - k - ras expression , mice were treated with sfn ( 0.5 mg , 5 d / wk ) for 3 months by means of a nebulizer . fourteen weeks after mutant k - ras expression ( k - raslsl - g12d ) , mice were sacrificed , and lung sections were screened for neoplastic foci . expression of nrf2-dependent genes was measured using real time rt - pcr . we also determined the effect of prolonged sfn treatment on the growth of preclinical xenograft models using human a549 ( with mutant k - ras and keap1 allele ) and h1975 [ with mutant epidermal growth factor receptor ( egfr ) allele ] nonsmall cell lung cancer cells.results:systemic sfn administration did not promote the growth of k - raslsl - g12d - induced lung tumors and had no significant effect on the growth of a549 and h1975 established tumor xenografts in nude mice . interestingly , localized delivery of sfn significantly attenuated the growth of a549 tumors in nude mice , suggesting an nrf2-independent antitumorigenic activity of sfn.conclusions:our results demonstrate that prolonged sfn treatment does not promote lung tumorigenesis in various mouse models of lung cancer .

the ultimate goal of periodontal therapy is to arrest the progress of periodontal disease and regenerate the lost periodontal support . hyaluronic acid ( ha ) is a high molecular weight , nonsulphated glycosaminoglycan component which is produced during various phases of cell 's life cycle . it contributes in tissue hydrodynamics , cell migration , and proliferation , and improves healing properties of the tissue . its physiological , structural , and biochemical properties prove that it provides elasticity and stability to tissues and is critically beneficial in tissue regeneration . pharmacologically , the content of ha in the tissues is often broken down by the blood stream or by lymphatic drainage and its turnover rate is found to be 2030% . after being broken down , ha was discovered in 1934 from cow 's eye by karl meyer and john plamer and was first used in 1942 by endre balazs as a substitute for egg white in bakery products . the chemical structure of ha shows alternate units of n - acetyl glucosamine and d - glucorounic acid , both of the components linked through glycosidic bonds . ha acts as an antioxidant by scavenging reactive oxygen species , which helps in the regulation of immune response implying its anti - inflammatory properties . it has also been reported that ha shows osteoinductive properties , which is useful for treatment of periodontal disease . other beneficial effects have also been seen for the treatment of recurrent apthous ulcer , for treating gingival lesions , and promote healing in extraction socket . ha has also reported as a diagnostic biomarker of inflammation in gingival crevicular fluid and repair of tissues . recent investigations have indicated that ha induces mineralization of dental pulp cells through cd44 cell surface glycoprotein and is considered to be a principle ligand for receptor cd44 . hyaluronic acid was found to have extensive actions in various periodontal therapies such as topically applied in subgingival regions reduces microbial activity , bone regeneration in deep periodontal bony defects , guided bone regeneration , nonsurgical treatment of peri - implantitis pockets , peri - implant maintenance of immediately placed implants , and gingival augmentation in mucogingival surgery . ha may act as a scaffold for other molecules such as bone morphogenic protien-2 and platelet derived growth factor - bb , used in guided bone regeneration techniques and tissue engineering research . ha when applied to patients with chronic periodontitis showed reduction in bleeding on probing ( bop ) , probing pocket depth ( ppd ) , and clinical attachment level , and hence , can be used as an adjunct to scaling and root planning . ha gel , injections , or oral ( by mouth ) should not be used in patients with allergies . a 24-year - old female reported to the department of periodontics with her esthetic concern and complaint regarding the loss of interdental papilla , i.e. black triangle , in the anterior maxillary region [ figure 1 ] . patient was concerned for esthetics ; class ii type of interproximal tissue loss without any bone loss and no extra oral deformities were detected . a written informed consent was taken from the patient before the procedure , and ethical clearance certificate was obtained from the institution . preoperative : black triangle between maxillary right central incisor and lateral incisor nordland and tarnow proposed a classification using three reference points , i.e. , contact point , facial and apical extent of cementoenamel junction ( cej ) , and interproximal extent of cej ( icej ) , and was classified into the following four classes : normal : interdental papilla occupies embrasure space to the apical part of the interdental contact point . class i : tip of interdental papilla occupies space between the interdental contact point and the most coronal part of cej . class ii : tip of interdental papilla lies at / or the apical to the icej but coronal to the apical most part of the cej on facial aspect . class iii : tip of interdental papilla lies at level with or apical to the facial cej . the loss of interdental papillae can be due to many reasons such as gingivitis , oral hygiene procedures with trauma , tooth shape with abnormal anatomy , or improper contours of the restoration . inclusion criterion for the study were : age range : 2075 yearsmaxillary anterior teethplaque index less than 20%teeth should not have caries and no fixed prosthesis or orthodontic appliancenonsmoker individualsno systemic disease that may affect the periodontal statusavoid consumption of drugs causing gingival overgrowth . age range : 2075 years maxillary anterior teeth plaque index less than 20% teeth should not have caries and no fixed prosthesis or orthodontic appliance nonsmoker individuals no systemic disease that may affect the periodontal status avoid consumption of drugs causing gingival overgrowth . patient was completely informed about the study . after introducing a local anesthetic agent , less than 0.2 ml of the ha gel was injected at the sites 23 mm apical to the coronal tip of the papilla [ figure 2 ] . the patient was instructed not to brush on the day of treatment but maintain oral hygiene , and after 24 hours , using a soft bristle toothbrush at the anterior teeth to start their routine oral hygiene measures and avoid using dental floss at the sites of treatment . the patient was on follow - up for 3 weeks and was recalled for a total of 4 times . after first follow - up , 3 weeks later , the treatment patient did not show any improvement , therefore ; another shot of 0.2% ha injection was given . measurements of the black triangle were done using photography . injecting 2% hyaluronic acid into interdental papilla after 3 months , photographs were taken and comparison was done using these images . this technique resulted in significant gain in papillary volume and esthetic improvements were notable [ figure 3 ] . because using 0.2% ha for the reconstruction of lost interproximal tissue gave significant changes , it can be used as a noninvasive method , which will reduce the use of surgical procedures for regenerating soft tissues in upcoming years . this study can be limited by the amount of the interproximal tissue loss , depending upon the size of the black triangle , which might require surgical intervention to gain complete coverage . the main advantage of this study is that it is nontoxic to the patient and there is reduced discomfort after the procedure as compared to surgical procedure done . furthermore , this study can be elaborated by more number of patients depending upon the size and type of the black triangle . becker et al . concluded that ha gel is a synthetic material and can be used with no drug interference and is a safe material , which significantly decreases the interdental black triangle in the esthetic zone . vedamurthy reported ha to be a dermal filler and applied it for soft tissue augmentation , observing significant improvements . monheit et al . discussed the inherent properties of ha that make them ideal for cosmetic surgeries . studied gingival augmentation with an autologous cell ha and reported significant results with the complete coverage . found that antioxidant capacity of ha is inversely proportional to the severity of inflammation and can be used as a biomarker in periodontitis . it is acceptable that injecting ha to periodontal wound sites had shown significant effects in periodontal tissue regeneration . engstrm et al . reported bone regenerative effects of ha in nonsurgical and surgical groups and showed no statistical difference when evaluated on radiographs in the nonsurgical group ; however , there was remarkable decrease in the height of alveolar bone after oral prophylaxis in both the nonsurgical and surgical group . ha when involved with soft and hard tissues showed negligible effect on the immune system of the patient . stated enhanced accelerating capacity of new bone formation in the infrabone defects when combined with autologous bone graft . this study indicates possible improvements in regenerating lost interdental papilla and removal of black triangle by injecting ha into the lost papilla using a nonsurgical approach . because of its property of modulating periodontal wounds , this nonsurgical approach limits the use of surgical procedures for regenerating lost papilla , and hence , it is a noninvasive method and also reduces patient discomfort . therefore , this study demonstrates ha to be a nonsurgical approach for regenerating lost papilla and gave significant and satisfactory results . to overcome the limitations of using of ha for regenerating lost papilla , this study need to be elaborated with more number of patients depending upon the size of the black triangle .

interdental papilla construction , especially in the esthetic area , is one of the most challenging tasks . interdental papilla loss might occur due to several reasons as a consequence of periodontal surgery or trauma . the purpose of this study is to report the reconstruction of lost interdental papilla using hyaluronic acid gel . hyaluronic acid is a glycosaminoglycan molecule with anti - inflammatory , anti - edematous properties on periodontal tissues invaded by submicrobial flora . it enhances wound healing and accelerates periodontal repair and regeneration . in addition to the field of dentistry , it has been used in other fields such as orthopedics , ophthalmology , and dermatology . it shows growth factor interaction , regulates osmotic pressure , and enhances tissue lubrication , which helps in maintaining the structural and homeostatic integrity of tissues , hence resulting in beneficial effect on lost interdental papilla . this study was aimed to reconstruct the lost interdental papilla by injecting 0.2% hyaluronic acid via nonsurgical approach . it is a noninvasive approach which reduces patient 's postoperative discomfort with marked variations in the volume of interdental papilla before and after the procedure . as sufficient information is not available regarding the effectiveness of hyaluronic acid in interdental papilla construction , this study was conducted .

the global labor force is about 2,600 million and 75% of this force is working in developing countries . the total labor force in india is estimated to be 317 million , in which the organized sector employs only 26.8 million ( 8.5% ) while the unorganized sector employs as many as 290.2 million ( 91.5% ) . the indian industry remains labor - intensive and often employs relatively inexpensive and hazardous technology due to financial constraints . occupational safety and health ( osh ) , also commonly referred to as occupational health and safety ( ohs ) or workplace health and safety ( whs ) , is an area concerned with the safety , health , and welfare of people engaged in work or employment . joint international labour organization ( ilo)/world health organization ( who ) committee on occupational health focuses on three different objectives : ( i ) the maintenance and promotion of workers health and working capacity , ( ii ) the improvement of the working environment and work to become conducive to safety and health , and ( iii ) development of work organizations and working cultures in a direction , which supports the health and safety at work and in doing so also promotes a positive social climate and smooth operation and may enhance productivity of the undertakings . the concept of a working culture is intended in this context to mean a reflection of the essential value systems adopted by the undertaking concerned . such a culture is reflected in practice in the managerial systems , personnel policy , principles for participation , training policies , and quality management of the undertaking . the occupational health safety network ( ohsn ) is a secure electronic surveillance system developed by the national institute for occupational safety and health ( niosh ) to address health and safety risks among health care personnel . ohsn uses existing data to characterize the risk of injury and illness among health care workers . hospitals and other health care facilities can upload the occupational injury data they already collect to the secure database for analysis and benchmarking with other de - identified facilities . it is a study to evaluate the effectiveness of video - assisted teaching program on safety measures followed by the employees working in the silica - based industry in puducherry , india . to assess the level of knowledge , attitude , and practice of the subjects on safety measures against occupational hazard among employees working in the silica - based industryto evaluate the effectiveness of video - assisted teaching program on safety measures against occupational hazard among employees working in the silica - based industry . to assess the level of knowledge , attitude , and practice of the subjects on safety measures against occupational hazard among employees working in the silica - based industry to evaluate the effectiveness of video - assisted teaching program on safety measures against occupational hazard among employees working in the silica - based industry . to associate the pretest knowledge , attitude , and practice score with selected demographic variables of the employees working in the silica - based industry to assess the level of knowledge , attitude , and practice of the subjects on safety measures against occupational hazard among employees working in the silica - based industryto evaluate the effectiveness of video - assisted teaching program on safety measures against occupational hazard among employees working in the silica - based industry . to assess the level of knowledge , attitude , and practice of the subjects on safety measures against occupational hazard among employees working in the silica - based industry to evaluate the effectiveness of video - assisted teaching program on safety measures against occupational hazard among employees working in the silica - based industry . to associate the pretest knowledge , attitude , and practice score with selected demographic variables of the employees working in the silica - based industry h1there will be a significant difference in the knowledge , attitude , and practice among the employees on safety measures followed in the silica - based industry before and after video - assisted teaching program . h2there will be a significant association among the knowledge , attitude , and practice , and selected demographic variables . the study was conducted in m / s ace glass containers ltd . , which is situated in puducherry , india . based on a pilot study report , 105 samples were selected from the workmen category . out of which , 45 samples were permanent workers and 60 were casual laborers . reliability of the tool was established using split - half technique and it was found to be reliable with the reliability values for knowledge the value alpha = 0.87 , for attitude = 0.87 , and for practices = 0.70 . video - assisted teaching program was provided to the employees in the workmen category who were selected for the study . the video - assisted teaching program included a brief knowledge about occupational health , occupational hazards , occupational diseases , general safety measures , personal protective equipment , medical measures , engineering measures , and legislation . informed consent was taken from each subject and the data were collected from the workers . the knowledge of the subjects was assessed by using a self - administered questionnaire , their attitude was assessed by the likert scale , and their practice of using protective devices was assessed by a checklist . it took 10 - 15 min for each worker to complete the pretest . each day , around 15 - 20 workers were gathered in the factory dispensary following which preassessment video - assisted teaching programs were given to the subjects on occupational health , occupational hazards , and safety measures . booklet was also given to each subject to enrich and reinforce his / her knowledge on the use of safety measures to promote the health of the workers . after video - assisted teaching program , the investigator informed the date and time of posttest to all the subjects and posttest was conducted by using the same questionnaire after 1 week as per the schedule . the demographic data revealed that out of 105 subjects , 53 ( 50.5% ) were in the age group of 30 - 39 years ; 54 ( 51.4% ) of them were from rural areas , 81 ( 77.1% ) of them were married , 40 ( 38.1% ) of them were technically educated , 60 ( 57.1% ) subjects earned less than 3,000 per month , and 19 ( 18.1% ) were working for 4 - 5 years in the same company . most , i.e. , 97 ( 92.4% ) the subjects were on shift duty and the remaining 8 ( 7.6% ) subjects were on regular duty from 8 pm to 4 pm . table 1 shows the comparison of overall pretest and posttest knowledge score of the subjects where 57 ( 54.3% ) subjects had inadequate knowledge and only 18 ( 17.1% ) had adequate knowledge in the pretest . but after video - assisted teaching program , 98 ( 93.3% ) subjects had adequate knowledge and 7 ( 6.7% ) had moderately adequate knowledge . comparison of overall pretest and posttest knowledge score of the subjects on safety measures table 2 shows that in the pretest , about 96 ( 91.4% ) subjects had an uncertain attitude and only 9 ( 8.6% ) subjects had a positive attitude . but in the posttest , almost all of them , i.e. 105 ( 100% ) had a positive attitude toward the use of safety measures . comparison of attitude score of the subjects between pretest and posttest table 3 represents that 84 ( 80% ) subjects had adequate practice of using safety measures and 21 ( 20% ) only had inadequate practice in pretest . but in posttest , 105 ( 100% ) subjects were found to be adequately utilizing the safety measures . it was evident that the video - assisted teaching program was effective in improving their level of practice in the utilization of personal protective equipment from occupational hazards . comparison of the practice score of the subjects between pretest and posttest the mean knowledge score of the subjects in pretest was found to be 21.06 with standard deviation but after video - assisted teaching program , the mean knowledge score on safety measures was increased to 34.38 ( out of 40 ) with sd of 2.33 , which was statistically significant at p < 0.001 level . the mean attitude score on safety measures in pretest but after video - assisted teaching program , the mean attitude score on safety measures was increased to 54.48 with sd of 3 , which was also found to be statistically significant at p < 0.001 level . the mean practice score on safety measures before video - assisted teaching program was 25.06 with sd of 0.92 . but in the posttest , the mean practice score was found to be increased to 27.43 with the sd of 0.5 , which was statistically significant at p < 0.001 level from which it was evident that the video - assisted teaching program was effective in improving the level of knowledge , attitude , and practice of the subjects regarding safety measures . comparison of the mean pretest and posttest scores on knowledge , attitude , and practice of the subjects auni fatin nadia chiek desa , et al . , ( 2013 ) stressed that the safety culture , employee involvement , employee attitude , leadership style , safety and health training , and effective communication may have an impact on osh administration performances . ( 2011 ) recommended raising awareness of the concerns of the recycling workers safety and health by advertising through the media and conducting comprehensive and intensive research so that the public would have a better understanding of the situation and start to be aware of occupational hazards . the study result revealed that there was a statistically significant association between the pretest knowledge and attitude score of the subjects with selected demographic variables such as their age , education , income , section of work and their experience . as the subjects ages were above 50 - 60 years , they had a high mean knowledge score of 24.2 6.38 and those who had technical educational qualification had a mean knowledge score of 24.6 5.38 , which was highly significant while comparing with the others . the subjects experienced for more than 5 years had a higher mean knowledge score of 23.52 5.41 , which was highly significant ( p = 0.000 * * * ) . further , those who were working in the foundry section had mean knowledge score of 50.33 , 3.53 , which was highly significant when compared to the others working in various other sections . similarly as the subjects age increases above 50 - 60 years , their mean attitude score was high 24.2 6.38 and those who had technical education qualification had a favorable mean attitude score of 24.6 5.38 , which was highly significant while comparing with the others . the subjects who were experienced for more than 5 years had a favorable mean attitude score of 23.52 5.41 , which was highly significant ( p = 0.000 * * * ) while comparing with the others experiences . those who were working in the foundry section had a favorable mean attitude score of 50.33 3.53 , which was highly significant comparing with others working in the other sections . ( 2015 ) highlighted the current issues in occupation - related injuries ( oris ) and occupational safety and health training ( osht ) . some of the elements of ori identified in the literature were differences in age , young workforce , aging workforce , multigenerations , working experience , cognitive abilities , and occupations . the elements of osht have indicated that the elements of learning effectiveness in safety training are training instructions , training method , adult learner , and training design . the study has revealed that video - assisted teaching program on ohs measures was effective in improving the knowledge , attitude , and practice of the subjects , which was statistically significant at p < 0.001 level . there was a statistically significant association between the pretest knowledge and attitude score of the subjects with selected demographic variables as their age , education , income , section of work , and their experience .

introduction : employees constitute a large and important sector of the world 's population . the global labor force is about 2,600 million and 75% of this force is working in developing countries . occupational health and safety ( ohs ) must be managed in every aspect of their work . occupational safety and health ( osh ) , also commonly referred to as ohs or workplace health and safety ( whs ) is an area concerned with the safety , health , and welfare of people engaged in any employment . the goal of osh is to foster a safe and healthy work environment . the aim of this study is to evaluate the effectiveness of video - assisted teaching program on safety measures.materials and methods : a total of 105 employees were selected from m / s ace glass containers ltd . at puducherry , india using the convenience sampling technique . pretest was conducted using a self - administered questionnaire . subsequent video - assisted teaching was conducted by the investigator after which posttest was conducted.results and conclusion : video - assisted teaching program was found to be effective in improving the knowledge , attitude , and practice of the subjects . periodical reorientation on safety measures are needed for all the employees as it is essential for promoting the well - being of employees working in any industry .

glaucoma is a leading cause of blindness worldwide.1 despite the growing understanding of its pathophysiology and advances in its treatment , challenges remain regarding how to predict and stop its progression . the treatment plan for glaucoma depends on its progression rate . in practice , most ophthalmologists determine the glaucoma progression rate based on the results of examinations of the patient s visual field ( vf ) . it is difficult to predict the rate of glaucoma progression.2 many studies on the progression of glaucoma have been reported . candidate factors associated with glaucoma progression include increased baseline intraocular pressure ( iop ) , increased mean iop , increased peak iop , and greater iop fluctuation.38 de moraes et al reported that the peak iop is a better predictor of vf deterioration compared to the mean iop.9 the study is supported by an investigation of an animal model of glaucoma which demonstrated a more predictive role of the maximum iop in structural change , compared to the mean iop.10 it is difficult to identify the peak iop because it might occur while the patient is asleep . if the peak iop is to be determined using goldmann applanation tonometry ( gat ) , the iop must be measured many times . the iop is known to fluctuate , and iop fluctuation might be a better predictor of the progression of glaucoma than the mean iop.6,8,11 the existing studies on iop fluctuation can be divided into short - term ( throughout the day ) and long - term ( several years ) studies . the relationship between short - term and long - term iop fluctuations has not been clear . since an assessment of short - term iop fluctuation requires that the iop be measured at night , it burdens both the patient and his or her examiner(s ) . short - term ( 24-hour ) iop fluctuation can be measured using a contact lens sensor ( cls ) . the cls was developed to continuously monitor habitual iop fluctuations over a 24-hour period by measuring changes in the eye s circumference in the area of the corneoscleral junction.12,13 once a cls is placed on an eye , the iop fluctuation can be measured automatically without interference with the subject s sleep . the measurement obtained with a cls has been shown to be accurate and reproducible.14 in the present study , we investigated the relationship between short - term and long - term iop fluctuations . this was a retrospective study . fifty adult glaucoma patients ( 25 men , 25 women ) all 50 patients underwent a comprehensive ophthalmic examination , including test of refraction and visual acuity , goldmann gonioscopy , gat , a fundus examination , and automated perimetry ( humphrey field analyzer ; carl zeiss meditec ag , jena , germany ) . the study and the research protocol were approved by the institutional review board of the university of toyama ( approval no 23 - 100 ) , and the procedures used conformed to the tenets of the declaration of helsinki . after the nature and possible consequences of the study were explained to the subjects , written informed consent was obtained from each subject . the diagnosis of glaucoma was made if both of the following criteria were satisfied : 1 ) the presence of glaucomatous optic disk neuropathy ( a cup / disk ratio of 0.7 or the presence of notching ) accompanied by corresponding vf defects , and 2 ) a threshold sita 30 - 2 examination showing a glaucoma hemifield test result outside normal limits , and a cluster of three contiguous points on the pattern deviation plot depressed at the p<0.05 level ( occurring in age - matched normal subjects ) not crossing the horizontal meridian which are compatible with glaucoma . the inclusion criteria for the subjects were as follows : 1 ) the subject had best - corrected visual acuity 0.2 . 2 ) the subject had spherical equivalent < + 6 or > 6 d. in this study , we included patients who had undergone a previous cataract surgery at least 2 years prior to the enrollment in the study . eyes with myopia over 6 d or hyperopia over + 6 d before cataract surgery were excluded . we defined the start of the follow - up period when the patients were diagnosed with glaucoma . for the patients who had underwent a cataract surgery during the follow - up period , we excluded the iop data obtained before cataract surgery , and used the iop data obtained after the cataract surgery . we included patients who met the requirement of > 2-year follow - up period after the cataract surgery . for the patients who had underwent selective laser trabeculoplasty , we used the iop data in the same way . we also included the cases in whom the glaucoma medications were changed or added during the follow - up period . the exclusion criteria were 1 ) history of glaucoma operation , 2 ) history of ocular trauma , 3 ) retinal diseases , and 4 ) ocular inflammatory diseases . physicians who had been trained in cls measurement conducted the placement and removal of cls in each subject in a hospital setting . one eye of each subject was monitored . if both eyes had glaucoma , we measured the eye with the worse vf which also met the inclusion criteria . the 24-hour iop was monitored with a triggerfish cls ( sensimed , lausanne , switzerland ) . the cls consists of a highly oxygen - permeable soft contact lens , the key elements of which are two sensing - resistive strain gauges that are capable of recording circumferential changes in the area of the corneoscleral junction . this device is based on a novel approach to iop monitoring in which changes in corneal curvature and circumference are assumed to correspond to changes in the iop . the unit of measurement used for the monitoring of iop fluctuation with the cls is not mmhg but mveq , which is unique to the cls . the median iop values were obtained every 5 minutes , providing 288 points over the 24-hour period . the range of iop fluctuations , which was defined as the difference between the maximum value ( mveq ) and the minimum value measured during the 24-hour course , was calculated from the iop fluctuation data . patients who were taking glaucoma medication continued to take the medication during the measurement period . we defined the number of glaucoma drops as the number of drops prescribed when the subject s iop was measured by cls . the long - term iop fluctuation was defined by four parameters : 1 ) the mean of the eligible iop ( mmhg ) determined during the follow - up ; 2 ) the iop difference ( mmhg ) , which we defined as the difference between the maximum iop and the minimum iop ; 3 ) the standard deviation of iop ( sd - iop , mmhg ) which was calculated from all eligible iop values ; and 4 ) the peak iop ( mmhg ) , which was defined as the maximum iop . all statistical analyses were performed using the jmp 10 software program ( sas institute inc . , we used the spearman rank correlation coefficient for determining the correlation of the long - term iop fluctuation index and short - term iop fluctuation . this was a retrospective study . fifty adult glaucoma patients ( 25 men , 25 women ) all 50 patients underwent a comprehensive ophthalmic examination , including test of refraction and visual acuity , goldmann gonioscopy , gat , a fundus examination , and automated perimetry ( humphrey field analyzer ; carl zeiss meditec ag , jena , germany ) . the study and the research protocol were approved by the institutional review board of the university of toyama ( approval no 23 - 100 ) , and the procedures used conformed to the tenets of the declaration of helsinki . after the nature and possible consequences of the study were explained to the subjects , written informed consent was obtained from each subject . the diagnosis of glaucoma was made if both of the following criteria were satisfied : 1 ) the presence of glaucomatous optic disk neuropathy ( a cup / disk ratio of 0.7 or the presence of notching ) accompanied by corresponding vf defects , and 2 ) a threshold sita 30 - 2 examination showing a glaucoma hemifield test result outside normal limits , and a cluster of three contiguous points on the pattern deviation plot depressed at the p<0.05 level ( occurring in age - matched normal subjects ) not crossing the horizontal meridian which are compatible with glaucoma . the inclusion criteria for the subjects were as follows : 1 ) the subject had best - corrected visual acuity 0.2 . 2 ) the subject had spherical equivalent < + 6 or > 6 d. in this study , we included patients who had undergone a previous cataract surgery at least 2 years prior to the enrollment in the study . eyes with myopia over 6 d or hyperopia over + 6 d before cataract surgery were excluded . we defined the start of the follow - up period when the patients were diagnosed with glaucoma . for the patients who had underwent a cataract surgery during the follow - up period , we excluded the iop data obtained before cataract surgery , and used the iop data obtained after the cataract surgery . we included patients who met the requirement of > 2-year follow - up period after the cataract surgery . for the patients who had underwent selective laser trabeculoplasty , we used the iop data in the same way . we also included the cases in whom the glaucoma medications were changed or added during the follow - up period . the exclusion criteria were 1 ) history of glaucoma operation , 2 ) history of ocular trauma , 3 ) retinal diseases , and 4 ) ocular inflammatory diseases . physicians who had been trained in cls measurement conducted the placement and removal of cls in each subject in a hospital setting . one eye of each subject was monitored . if both eyes had glaucoma , we measured the eye with the worse vf which also met the inclusion criteria . the 24-hour iop was monitored with a triggerfish cls ( sensimed , lausanne , switzerland ) . the cls consists of a highly oxygen - permeable soft contact lens , the key elements of which are two sensing - resistive strain gauges that are capable of recording circumferential changes in the area of the corneoscleral junction . this device is based on a novel approach to iop monitoring in which changes in corneal curvature and circumference are assumed to correspond to changes in the iop . the unit of measurement used for the monitoring of iop fluctuation with the cls is not mmhg but mveq , which is unique to the cls . the median iop values were obtained every 5 minutes , providing 288 points over the 24-hour period . the range of iop fluctuations , which was defined as the difference between the maximum value ( mveq ) and the minimum value measured during the 24-hour course , was calculated from the iop fluctuation data . patients who were taking glaucoma medication continued to take the medication during the measurement period . we defined the number of glaucoma drops as the number of drops prescribed when the subject s iop was measured by cls . the long - term iop fluctuation was defined by four parameters : 1 ) the mean of the eligible iop ( mmhg ) determined during the follow - up ; 2 ) the iop difference ( mmhg ) , which we defined as the difference between the maximum iop and the minimum iop ; 3 ) the standard deviation of iop ( sd - iop , mmhg ) which was calculated from all eligible iop values ; and 4 ) the peak iop ( mmhg ) , which was defined as the maximum iop . all statistical analyses were performed using the jmp 10 software program ( sas institute inc . , cary , nc , usa ) . we used the spearman rank correlation coefficient for determining the correlation of the long - term iop fluctuation index and short - term iop fluctuation . we classified the subtypes of glaucoma as primary open - angle glaucoma ( 30 eyes including 15 normal - tension glaucoma eyes ) , primary angle - closure glaucoma ( one eye ) , and pseudo - exfoliation glaucoma ( 19 eyes ) . we defined normal - tension glaucoma as untreated iop that had never been > 21 mmhg among the primary open - angle glaucoma eyes . we were able to successfully measure the 24-hour iop with the cls in all of the subjects . some subjects had minor complications : conjunctivitis , slight hyperemia , or peripheral corneal edema . these healed within a few days without any additional eye drops . as shown in figure 1 and table 2 , the short - term iop fluctuation was significantly correlated with the mean iop ( p=0.001 ) , the iop difference ( p=0.0207 ) , the sd - iop ( p=0.0061 ) , and the peak iop ( p=0.0017 ) . the short - term and the long - term iop fluctuations were moderately but significantly correlated . we classified the subtypes of glaucoma as primary open - angle glaucoma ( 30 eyes including 15 normal - tension glaucoma eyes ) , primary angle - closure glaucoma ( one eye ) , and pseudo - exfoliation glaucoma ( 19 eyes ) . we defined normal - tension glaucoma as untreated iop that had never been > 21 mmhg among the primary open - angle glaucoma eyes . we were able to successfully measure the 24-hour iop with the cls in all of the subjects . some subjects had minor complications : conjunctivitis , slight hyperemia , or peripheral corneal edema . as shown in figure 1 and table 2 , the short - term iop fluctuation was significantly correlated with the mean iop ( p=0.001 ) , the iop difference ( p=0.0207 ) , the sd - iop ( p=0.0061 ) , and the peak iop ( p=0.0017 ) . the short - term and the long - term iop fluctuations were moderately but significantly correlated . the prior studies on iop fluctuation were a mix of short - term and long - term studies . it has been difficult to determine the significance of these studies due to lack of standardization regarding the time between assessments , the methods of measurement , and the definition of iop fluctuation itself . to the best of our knowledge , this study is the first to analyze the relationship between long - term and short - term iop fluctuations with a cls . the mean iop as well as peak iop , the iop difference , and sd - iop of long - term iop fluctuation was correlated with the short - term iop fluctuation measured with a cls . thus , the short - term and the long - term iop fluctuations were significantly correlated . the long - term iop fluctuation was shown as a significant risk factor by multiple logistic regression analysis.8,15 another study showed that peak iop was a better predictor of progression than mean iop or iop fluctuation.9 some studies reported that the short - term and the long - term iop fluctuations were correlated in stable glaucoma patients.1519 these results are in agreement with our present findings . tan et al reported that short - term iop fluctuation correlated with thickness of retinal nerve fiber layer.17 agnifili et al compared iop fluctuations between healthy subjects and glaucoma patients . they reported that glaucoma patients showed larger iop fluctuation than normal subjects.18 the larger iop fluctuation in both long and short term could predict the rate of deterioration of vf . the only available treatment for glaucoma at present is lowering the iop , but it does not adequately suppress the progression of glaucoma.8,9 since a new treatment modality to reduce the iop fluctuation is required , it may be necessary to use a cls to measure iop fluctuations . measuring the iop fluctuation with a cls could thus become a standard method of measuring short - term iop fluctuation . this method has several merits : iop fluctuation can be measured every 5 minutes rather than every few hours . no restriction is imposed on the subject s posture during the measurement . even though the obtained values are objective ( mveq ) , short - term iop fluctuations measured with a cls might help to predict long - term iop fluctuations . it was reported that glaucoma medication could make the circadian iop fluctuation smaller.20 holl et al reported that a cls could not measure the effect of topical medication or body position.21 adherence of a cls to the cornea might alter the effects of topical medications . second , short - term iop fluctuation was assessed with a cls on different days , although some studies reported a good reproducibility of the results of short - term iop fluctuation with a cls.2225 third , the value in mveq measured with a cls can not be converted to mmhg . mottet et al reported that the iop measurement using a cls is an accurate and reproducible method to characterize the iop rhythm but does not allow estimating the iop value in mmhg corresponding to the relative variation of the electrical signal measured.14 to address these problems , further studies are necessary . in conclusion , long - term and short - term iop fluctuations were significantly correlated . measuring iop fluctuation with a cls could be useful to predict long - term iop fluctuation .

purposewe investigated correlations between short - term and long - term intraocular pressure ( iop ) fluctuations.methodswe examined 50 eyes of glaucoma patients who were followed for > 2 years . we measured short - term iop fluctuation using a triggerfish contact lens sensor ( cls ) . the short - term iop fluctuation ( mveq ) was defined as the difference between the maximum value and the minimum value measured during the 24-hour course with cls . the long - term iop fluctuation was defined by four parameters : 1 ) the mean iop ( mmhg ) determined during follow - up ; 2 ) the iop difference , which was defined as the difference between the maximum iop and the minimum iop ; 3 ) the standard deviation of iop ; and 4 ) the peak iop , which was defined as the maximum iop . correlations between these parameters and the short - term iop fluctuation were examined.resultsthe mean follow - up period was 5.4 years . the average iop was 15.04.0 mmhg . the range of short - term iop fluctuation identified with cls was significantly correlated with all the four long - term iop fluctuation parameters.conclusionshort-term iop fluctuations were found to be associated with long - term iop fluctuations . examination of 24-hour iop fluctuations with the cls might be useful for predicting the long - term iop fluctuation .

we reviewed outbreak reports to identify outbreaks associated with an imported food from the inception of the surveillance system in 1973 through 2014 , the most recent year for which data were available . we obtained additional data for some outbreaks ( e.g. , country of origin ) from the us food and drug administration ( fda ) and the us department of agriculture food safety and inspection service . we categorized implicated foods by using the schema developed by the interagency food safety analytics collaboration ( 3 ) . we conducted a descriptive analysis of the number of outbreaks over time , by food category , and by region of origin . during 19962014 , a total of 195 outbreak investigations implicated an imported food , resulting in 10,685 illnesses , 1,017 hospitalizations , and 19 deaths . outbreaks associated with imported foods represented an increasing proportion of all foodborne disease outbreaks where a food was implicated and reported ( 1% during 19962000 vs. 5% during 20092014 ) . the number of outbreaks associated with an imported food increased from an average of 3 per year during 19962000 to an average of 18 per year during 20092014 ( figure ) . number of outbreaks caused by imported foods and total number of outbreaks with a food reported , united states , 19962014 . reporting practices changed over time ; 19731997 , imported foods anecdotally noted in report comments ; 19982008 , contaminated food imported into u.s . included as a location where food was prepared ; 20092014 , reporting jurisdictions could indicate whether each food is imported ( yes / no ) and the country of origin . the most common agents reported in outbreaks associated with imported foods were scombroid toxin and salmonella ; most illnesses were associated with salmonella and cyclospora ( table ) . aquatic animals were responsible for 55% of outbreaks and 11% of outbreak - associated illnesses . outbreaks attributed to produce had a median of 40 illnesses compared with a median of 3 in outbreaks attributed to aquatic animals . most of the salmonella outbreaks ( 77% ) were associated with produce , including fruits ( n = 14 ) , seeded vegetables ( n = 10 ) , sprouts ( n = 6 ) , nuts and seeds ( n = 5 ) , spices ( n = 4 ) , and herbs ( n = 1 ) . other agents implicated were tetrodotoxin ( 3 outbreaks ) and campylobacter , chaconine , paragonimus , other virus , sulfite , and trichinella ( 1 outbreak each ) . foods implicated were a chicken dish , crab cake , creampuff , beer , and a wheat snack ( 1 outbreak each ) . latin america and the caribbean was the most common region implicated , followed by asia ( technical appendix ) . thirty - one countries were implicated ; mexico was most frequently implicated ( 42 outbreaks ) . other countries associated with > 10 outbreaks were indonesia ( n = 17 ) and canada ( n = 11 ) . fish and shellfish originated from all regions except europe but were most commonly imported from asia ( 65% of outbreaks associated with fish or shellfish ) . produce originated from all regions but was most commonly imported from latin america and the caribbean ( 64% of outbreaks associated with produce ) . all but 1 outbreak associated with dairy products involved products imported from latin america and the caribbean . outbreaks in this analysis were reported from 31 states , most commonly california ( n = 30 ) , florida ( n = 25 ) , and new york ( n = 16 ) . the number of reported outbreaks associated with imported foods , although small , has increased as an absolute number and in proportion to the total number of outbreaks in which the implicated food was identified and reported . although many types of imported foods were associated with outbreaks , fish and produce were most common . many outbreaks , particularly outbreaks involving produce , were associated with foods imported from countries in latin america and the caribbean . because of their proximity , these countries are major sources of perishable items such as fresh fruits and vegetables ; mexico is the source of about one quarter of the total value of fruit and nut imports and 45%50% of vegetable imports , followed by chile and costa rica . similarly , our finding that many outbreaks were associated with fish from asia is consistent with data on the sources of fish imports ( 6 ) . one quarter of the outbreaks were multistate , reflecting the wide distribution of many imported foods . systems like pulsenet have helped to improve detection and investigation of multistate outbreaks , resulting in an increased number of multistate outbreaks ( 7,8 ) . the increasing number of outbreaks involving globally distributed foods underscores the need to strengthen regional and global networks for outbreak detection and information sharing . the importance of having standard protocols for molecular characterization of isolates and systems for rapid traceability of implicated foods to their source was illustrated during the investigation of a listeriosis outbreak linked to italian cheese imported into the united states in 2012 ( 9 ) . newer tools like whole genome sequencing can also help to generate hypothetical transmission networks and in some instances facilitate traceback of foods to their origin ( 10 ) . moreover , new tools that aid visualization of supplier networks facilitate the investigation of outbreaks involving the increasingly complex global economy ( 11 ) . only a small proportion of fda - regulated foods are inspected upon entry into the united states . new rules under the food safety modernization act of 2011 , including the preventive controls rule for human food , produce safety rule , foreign supplier verification program , and accreditation of third party auditors , will help to strengthen the safety of imported foods by granting fda enhanced authorities to require that imported foods meet the same safety standards as foods produced domestically ( 12 ) . although data collection has improved in recent years , these findings might underestimate the number of outbreaks associated with imported foods because the origin of only a small proportion of foods causing outbreaks is reported . similarly , because of how data are collected and reported , the relative safety of imported and domestically produced foods can not be compared . because of changes in surveillance and changing import patterns , changes over time should be interpreted cautiously . our findings reflect current patterns in food imports and provide information to help guide future outbreak investigations . prevention focused on the most common imported foods causing outbreaks , produce and seafood , could help prevent outbreaks . efforts to improve the safety of the food supply can include strengthening reporting by gathering better data on the origin of implicated food items , including whether imported and from what country . region and country of origin of imported foods implicated in outbreaks , by food category , united states , 19962014 .

the proportion of us food that is imported is increasing ; most seafood and half of fruits are imported . we identified a small but increasing number of foodborne disease outbreaks associated with imported foods , most commonly fish and produce . new outbreak investigation tools and federal regulatory authority are key to maintaining food safety .

bulking agent injection is now a standard treatment for patients with grade 1 - 3 primary vesicoureteral reflux ( vur ) . patients with higher grades and secondary types of vur show an unacceptably high failure rate when treated with injections of a bulking agent . collagen has been used for many years as a bulking agent in ureterovesical orifice and bladder neck to treat vur and incontinence . collagen , however , can get calcified in the tissue , a process that can cause a series of problems for patients . we present a case of a patient in whom calcified injected collagen showed imaging features mimicking ureteral stone . a 15-year - old girl presented with moderate degree of right flank pain for 2 weeks . pain was localized on the right side , altered with change in position , and did not relate to any particular organ . the patient had been treated successfully for bilateral grade 2 vur when she was 3 years old . a kidney ureter bladder ( kub ) radiography was requested and the report indicated presence of bilateral ureteral stone [ figure 1 ] . ultrasonography showed bilateral ureterovesical junction ( uvj ) stone and mild stasis in both kidneys , which was compatible with the findings of the non - enhanced spiral abdomino - pelvic computed tomography ( ct ) scan [ figure 2 ] . however , diethylene triamine pentaacaetic acid ( dtpa ) scan with furosemide injection and radionuclide cystography ( rnc ) scan ruled out obstruction and relapse of vur . calcification of bilateral intravesical collagen injected to treat her vur 12 years earlier was responsible for the positive findings on kub , ultrasonography , and spiral ct . 15-year - old female with bilateral flank pain , which was suspected to be due to stones in bilateral ureterovesical junction , later diagnosed as due to calcification of collagen used 12 years earlier to treat vesicoureteral reflux . 15-year - old female with bilateral flank pain , which was suspected to be due to stones in bilateral ureterovesical junction , later diagnosed as due to calcification of collagen used 12 years earlier to treat vesicoureteral reflux . spiral ct scan of pelvis without intravenous and oral contrast shows bilateral uvj calcifications ( arrows ) . endoscopic therapy for vur has gained popularity because of elimination of invasive surgical procedures in children . in addition , it has an acceptable success rate and a low level of complications . although a transition has occurred in the material of choice from teflon to collagen and now to hyaluronic acid copolymers . however , we do find patients who were treated with collagen now in the second decade of their life . the injected material , being a foreign body , can induce inflammatory response that leads to tissue calcification . this calcification has been reported to be symptomatic with some patients reporting episodes of stone passing , hematuria , and even obstruction . this is the second case report of ureterovesical injection calcification presenting as a ureteral stone during differential diagnosis and the first report of its imaging . unlike the first case report where the patient presented with renal colic and passing of stone , our patient was completely asymptomatic , except for positional non - related flank pain . there is no difference between a ureteral stone and calcification of injected collagen , when viewed by different imaging modalities like kub [ figure 1 ] , ct scanning [ figure 2 ] , and also ultrasound examination . this similarity is because of the place where the bulking agent is injected . in doubtful cases , as in our case , uvj obstruction can be ruled out by dtpa nuclear scan in which glomerular filtration of injected nuclear radioisotope reveals any obstruction in the upper urinary tract . the diagnosis of uvj stone in the presence of uvj calcification , possibility of ureteral stone passing ( in a patient with renal colic ) after uvj injection therapy , and the probability of recurrent vur after ureteral intervention ( in which calcified ureteral orifice will tolerate dilatation to pass the ureteroscope ) remain to be studied . vur can be assessed by rnc in which a radioisotope passing to the bladder via the urethral catheter reveals presence of vur . the standard treatment for grade 1 and 3 primary vur is injection of a bulking agent into the uvj . clinicians should be aware that calcification of bulking agent like collagen can occur and they need to differentiate this from ureterovesical stones on imaging findings .

primary vesicoureteral reflux can be treated by injection of a bulking agent into the wall of the ureterovesical junction . over time , the bulking agent can get calcified . radiological images of the area show findings that mimic those seen in ureterovesical junction calculi . in this report , we present the imaging findings of this phenomenon and discuss its challenging aspects .

the mucopolysaccharidoses ( mps ) are a family of genetic disorders characterized by changes in an individual s ability to metabolize mucopolysaccharides , which are complex sugar molecules found in the connective tissue , mucosal fluids , synovial fluid , and other cells throughout the body . there are six types of mps , with some types having several subtypes . according to brown ( 1999 ) , epidemiological data are problematic to report , but range from 1 in 100 000 live births to 1 in 600 000 live births . this dysfunction in metabolism may result in a variety of distinct physical features , such as short stature , contracture of joints , claw - like hands , abnormal spine curvature , and hydrocephalus ( neufeld and muenzer 1995 ) . treatment options vary , but hematopoietic cell transplantation seems to offer the best long - term quality of life improvement and decreased mortality and morbidity options for children with some of the more severe forms of mps ( grewal et al 2002 ) . one subtype of mps is mps i , which includes hurler , scheie , and hurler - scheie syndromes . each of these disorders within mps i is caused by a deficiency of alpha - l - iduronidase . within mps i , hurler syndrome is generally associated with more severe features and poorer prognosis than either scheie or hurler - scheie syndromes ( brown 1999 ) . in most cases of hurler syndrome , motor skills are limited by age 18 months , and developmental regression occurs , including progressive loss of neurocognitive functioning , resulting in mental retardation . with hurler - scheie syndrome , these declines usually appear between 3 and 8 years of age , with milder neurocognitive losses and a low risk for early mortality . these neurocognitive losses typically include progressive dementia resulting from hydrocephalus and increasing deposits of nonmetabolized mucopolysaccharides ( shapiro et al 1995 ) . language and memory deficits after a period of initial developmental slowing have been associated with the more severe forms of mps i ( guffon et al 1998 ) . while neurocognitive deficits have been associated with mps , to date there are few neurocognitive reports of children with this disorder and no longitudinal studies , resulting in scant knowledge regarding appropriate assessment and intervention ( brown 1999 ) . many studies report cross - sectional assessments of these children at different stages . in this case report , we have the opportunity to follow a young lady with hurler - scheie syndrome over the course of 10 years , documenting the quantity and quality of neurocognitive effects associated with this disease . this represents a rare chance to longitudinally track the effects of hurler - scheie in a young person and allows recommendations to be made to health care professionals regarding potential neurocognitive and psychological care for children with this disorder . researchers are currently engaging in novel therapies designed to reduce and perhaps reverse the effects of hurler - scheie . however , while these therapies may have a beneficial effect on orthopedic , skeletal , or ophthalamic problems , their effect on neurocognitive functioning is not clearly understood . thus , we are in a position to begin documenting both the physiological and neurocognitive effects of these new treatments . the focus of this case report is to describe long - term neurocognitive changes in a young female with hurler - scheie syndrome that was treated using enzyme replacement therapy . to our knowledge , this is the first longitudinal case report of a patient receiving enzyme replacement therapy for hurler - scheie syndrome . the following information was collected with the approval of the institutional review board at the university of mississippi medical center . we report the case of a now 15-year - old female diagnosed with mps - i , specifically hurler - scheie syndrome , alpha ( not her real name ) . alpha s mother gave consent for her child to serve as a subject in this investigation , and alpha gave assent . her mother reported alpha grew well at first , but then seemed to slow down . her mother also noted alpha could not raise her arms over her head , even as an infant . at age 3.5 years , alpha complained of wrist pain , and was seen by her primary care physician . alpha began taking gymnastics class as part of her 4-year - old preschool ; her instructor noted that alpha could not touch her toes or raise her arms . alpha was then referred to a local orthopedic clinic and x - rays were obtained . according to alpha s mother , the clinic noted something wrong on the x - ray films , and referred alpha to a pediatric orthopedic surgeon for further evaluation . alpha could not actively abduct her shoulders beyond 90 degrees and passive abduction was limited to 130 degrees . she was noted to have good finger function and grip , and a full range of motion in her neck , hips , knees , ankles , and elbows , with no appreciable organomegaly . however , after reviewing the x - rays and noting atypical features such as broad ribs with some notching at the medial metaphyseal ends , and smaller than usual femoral heads bilaterally , the orthopedic surgeon suspected alpha had scheie syndrome , one of the mps i syndromes . alpha was referred to a geneticist to conduct urine and blood tests and was diagnosed with hurler - scheie syndrome . by age 8 , alpha remained a very happy , bright young lady who was active in theatre camp and swimming ( she used her own , special technique according to her mother ) . she could actively abduct her shoulders 80 degrees and had about 50% range of motion in her neck . she was wearing glasses , was an avid reader , and was doing very well in school . by age 10 in 1999 , alpha was 50 inches ( 124 cm ) tall and weighed 71 pounds ( 32 kg ) . she had restricted range of motion in her hips and knees , and was beginning to complain of neck pain . in 2000 , at the age of 12 , alpha was admitted for the placement of a vp shunt secondary to increasing hydrocephalus . she also began enzyme replacement therapy in 2000 on a pharmaceutical study at the age of 12 , as well as outpatient physical therapy to encourage range of motion in her extremities . tanner staging was noted as appropriate for her age ; in general , hurler - scheie and scheie patients have normal puberty and reproductive abilities . her neurocognitive functioning was assessed three times , in june 1994 when she was 5 years 11 months old , in june 2002 when she was 13 years 11 months old , and in july 2003 , when she was 15 years old . the initial assessment at age 511 was not conducted by the authors , but took place at another location . however , this assessment was done by a licensed clinical psychologist who used age - appropriate , valid measures . copies of the report from that assessment were reviewed from the patient s medical chart . given her young age ( 511 ) at her initial assessment , the patient was administered the stanford - binet intelligence scale , fourth edition , and the wide range achievement test , third edition ( see table 1 ) . these measures assess for intelligence ( iq ) and achievement , respectively . at the time , the patient had just completed kindergarten , had no behavior problems , and was considered by her teachers to be ready for the first grade . her mother likewise thought the patient was doing well academically , but was concerned that the patient could perhaps have been showing signs of mild forgetfulness . overall , the scores on these measures range from the average to the highly superior range , as she scored at or above the 90th percentile in most areas . at that time , it was clear that alpha was functioning in the average to superior range intellectually and academically , and had no significant behavioral or emotional problems . alpha s parents were aware of the impressive nature of these findings , but were also aware of the usual course of this disease , including gradual neurocognitive decline . they began both systematic and non - specific interventions designed to challenge alpha intellectually and stimulate her already significant cognitive abilities . alpha was enrolled in a school that stressed academic excellence , and her mother reported that alpha did well through the seventh grade . alpha was home - schooled for the second half of the seventh grade , as she was travelling frequently to receive treatment for her condition . when she began eighth grade back at her same school , her grades began to fall from consistent as up through the seventh grade to bs and cs in the eighth grade . this prompted alpha s parents to seek another neurocognitive evaluation . at the time of the second assessment , the patient was 13 years and 11 months , and this second battery consisted of parts of the wechsler intelligence scale for children third edition ( wisc - iii ) , woodcock - johnson tests of cognitive ability third edition ( wj - iii ) , the wide range assessment of memory and learning ( wraml ) , the california verbal learning test children s version ( cvlt - c ) , the stroop color and word test , the rey - osterrieth complex figure test ( rocf ) , the child behavior checklist ( cbcl - parent report ) , the youth self report ( ysr ) , the children s depression inventory ( cdi ) , and the revised children s anxiety and depression scale ( rcads ) . this battery is designed to be a comprehensive measure of intelligence , neurocognitive functioning , and behavioral and emotional adjustment . it should be noted that though different batteries of measures were used between neurocognitive assessments , the results from both assessments are comparable . specifically , overall intelligence quotient ( iq ) scores between the stanford - binet and the woodcock - johnson show good correlation ( sattler 1992 ) . results from this second assessment indicate a significant decline in overall abilities from alpha s initial level of functioning . her abilities were now within the average range of functioning ; however , she again demonstrated no significant behavioral or emotional problems . alpha s parents began implementing suggested interventions , including after school tutoring and structuring alpha s study time so that she studied for short blocks of time with frequent breaks . at the time of this report , alpha s mother described alpha as doing better in school ( generally bs and cs , a few as ) , and continuing to be emotionally and socially well - adjusted . she completed the same battery as the one she completed at age 1311 ( see table 3 ) . while declines in neurocognitive functioning are generally expected in children with mps , this case report of a young lady with hurler - scheie demonstrates that declines may proceed at different rates and are likely influenced by premorbid neurocognitive functioning and environmental behavioral vectors . in other words , alpha s current level of average neurocognitive functioning is probably the result of impressive capabilities prior to the onset of hurler s syndrome as well as the degree of intervention and cognitive expectations that characterize her family system . the idea of cognitive reserve as a protective factor in individuals who have some sort of neurological insult has been assumed in children with traumatic brain injury and brain tumors . thus , it could be that since alpha had such a high premorbid level of functioning , she had more to spare during the course of her illness and treatment ( taylor 2004 ) . it should be noted that many children with hurler - scheie show some degree of cognitive sparing , in comparison with other types of mps . it should be noted that alpha has been diagnosed with a milder form of mps , hurler - scheie , and that this subtype of mps has been shown to involve relative neurocognitive sparing ( brown 1999 ) . however , this report is the first of its kind to our knowledge to document longitudinal changes in a youngster . the issue of alpha s current level of neurocognitive functioning can not be fully addressed in this report . her initial decline is undoubtedly related to her diagnosis of hurler - scheie syndrome . however , the amount of decline and conversely the degree of neurocognitive sparing attributable to her premorbid functioning and behavioral interventions by her parents is indeterminable . the differences in her test performance may be attributable to regression to the mean , a common result of multiple assessments conducted on a single individual . consequently , we can not estimate the amount of variance accounted for by alpha s premorbid intellectual capabilities , hurler - scheie syndrome , and her parents level of cognitive and intellectual interventions . it should also be noted that the measured decline in iq might be related to progressive motor dysfunction , as this might affect some of the subtests on the neurocognitive measures . there are no reports of the possible neurocognitive - sparing or improving effects of enzyme replacement therapy , which alpha received . weaknesses of this case report include the use of different measures of intelligence and neurocognitive functioning . however , all measures used to assess this patient are well - established , well - validated measures , and hence should be interpreted as adequately reflecting her level of neurocognitive functioning at the time they were administered . this case represents a unique opportunity to study a genetic disorder with neurocognitive effects , in the light of apparently strong biological and environmental protective factors . children with hurler - scheie syndrome should be followed regularly with neurocognitive assessments in order both to document expected intellectual declines and to target areas for cognitive and behavioral interventions that might slow the decline associated with this disease . the strong behavioral and environmental support that alpha received from her parents can not be discounted . alpha s parents proactively began working with her in light of her diagnosis , and then followed through with a neurocognitive rehabilitation plan suggested by the first author ( tde ) . this neurocognitive rehabilitation plan was tailored to address the specific levels of functioning that alpha evidenced from her assessments . we strongly recommend neurocognitive assessment and intervention for these patients , alongside newer medical interventions . the lack of continued neurocognitive decline from age 1311 to age 150 may be attributable to ( 1 ) the milder form of mps with which alpha was diagnosed , ( 2 ) the newer treatments initiated by the team of physicians ( jm ) , ( 3 ) the fact that alpha probably began life with a higher - than - average neurocognitive capacity , or ( 4 ) the supportive and appropriately challenging environment of social support that alpha enjoyed . however , neurocognitive assessment adds a low burden to an overall treatment plan , and can help with behavioral interventions designed to improve long - term neurocognitive functioning .

an adolescent with hurler - scheie syndrome is reported . this now 15 year - old - young woman was initially diagnosed at age 4 . she was assessed for neurocognitive functioning at ages 5 , 13 , and 15 years . results show a significant decline in intellectual functioning from the superior range to the average range from age 5 to age 13 , and then no change from age 13 to age 15 . the relationship between hurler - scheie syndrome , premorbid intellectual functioning , and cognitive behavioral interventions are discussed in light of the longitudinal neurocognitive effects of this disease .

smoking cessation is crucial for a better prognosis in patients with coronary heart disease ( chd)13 and copd.2,4,5 acute heart attacks are associated with high smoking cessation rates . in patients with a first myocardial infarction ( mi ) in the 1970s,3 among whom 78% were smoking , the quitting rate was 55% . in a systematic review from 2003 , the mean rate of smoking cessation within a year after a new diagnosis of chd was found to be 45%.1 a lower quitting rate was observed in patients with copd , in particular in subjects not participating in smoking cessation programs . among smoking copd patients in a swedish study , 10% of those receiving usual care had stopped smoking after 3 years , compared to 38% of those who took part in smoking cessation groups.6 among hospitalized patients with respiratory and cardiac diseases who took part in an intensive smoking cessation program in singapore , 60% of the cardiac patients and 40% of the respiratory patients were still abstinent from smoking after 2 months.7 the mortality due to chd has decreased considerably in the western world in the last few decades.8 evidence shows that decreased smoking prevalence in the general population , particularly among patients with heart disease , is an important reason for this decrease.811 the mortality due to copd has decreased less.12 a stronger tendency of persistent smoking among copd patients may partly explain this difference . copd patients reluctance to quit smoking may lead to a feeling of hopelessness among the doctors who treat them.13 our aim was to determine how the quitting rate in adults is influenced by getting a diagnosis of obstructive lung disease in the population - based troms study . we also wanted to investigate whether influence from this life event differs from being diagnosed with chd . a large proportion of the adult population in troms has participated in health surveys ( the troms study ) since 1974 . the troms study so far consists of six surveys , and this study deals with subjects who participated in both troms 5 ( 20012002 ) and troms 6 ( 20072008 ) . the attendance rates ( of those invited ) in troms 5 and troms 6 were 79% and 66% , respectively . details of the participants have previously been described.14 we included participants who answered the questions on smoking in both surveys . the regional committee for medical and health research ethics and the norwegian data inspectorate approved the troms 5 and troms 6 surveys with a license for further analysis on non - identifiable data , like this study . the invitation included a questionnaire with questions on smoking habits , education , and diseases . we classified subjects reporting daily smoking in troms 5 , but not in troms 6 , as quitters . we divided length of education into three categories , 12 years , 1316 years , and 17 years . we classified diseases reported in troms 6 and not in troms 5 as new diagnoses . we categorized mi and angina pectoris as chd and asthma and copd as obstructive lung disease . the frequencies of current smoking at baseline ( in troms 5 ) and smoking cessation among smokers were calculated by subject characteristics , and the statistical differences between subgroups were analyzed by chi - square statistics . significant variables in univariable analysis ( p<0.1 ) , as well as age and sex , were entered in the multivariable analysis . for the statistical analyses , we used the ibm spss statistic , version 21 ( ibm corporation , armonk , ny , usa ) . a large proportion of the adult population in troms has participated in health surveys ( the troms study ) since 1974 . the troms study so far consists of six surveys , and this study deals with subjects who participated in both troms 5 ( 20012002 ) and troms 6 ( 20072008 ) . the attendance rates ( of those invited ) in troms 5 and troms 6 were 79% and 66% , respectively . details of the participants have previously been described.14 we included participants who answered the questions on smoking in both surveys . the regional committee for medical and health research ethics and the norwegian data inspectorate approved the troms 5 and troms 6 surveys with a license for further analysis on non - identifiable data , like this study . in both surveys , the invitation included a questionnaire with questions on smoking habits , education , and diseases . we classified subjects reporting daily smoking in troms 5 , but not in troms 6 , as quitters . we divided length of education into three categories , 12 years , 1316 years , and 17 years . we classified diseases reported in troms 6 and not in troms 5 as new diagnoses . we categorized mi and angina pectoris as chd and asthma and copd as obstructive lung disease . the frequencies of current smoking at baseline ( in troms 5 ) and smoking cessation among smokers were calculated by subject characteristics , and the statistical differences between subgroups were analyzed by chi - square statistics . significant variables in univariable analysis ( p<0.1 ) , as well as age and sex , were entered in the multivariable analysis . for the statistical analyses , we used the ibm spss statistic , version 21 ( ibm corporation , armonk , ny , usa ) . a total of 4,631 subjects participated in both the fifth and the sixth surveys of the troms study , and 4,497 subjects comprising 1,852 men and 2,645 women answered the questions on smoking in both surveys . the mean time difference between attending the two surveys was 6.2 years ( standard deviation 0.5 years ) . the mean age at troms 5 was 61.1 years ( range 3081 years ) ; 1,150 ( 25.6% ) reported daily smoking and 1,753 ( 39.0% ) reported former smoking . the highest prevalence of smoking ( 43.8% ) was found in subjects with self - reported copd , whereas the frequency among those with self - reported chd was 19.0% . the characteristics of the population in troms 5 and the respective frequencies of smoking are listed in table 1 . in the sixth survey , 765 subjects ( 17.0% ) reported current smoking , and 428 of the 1,150 who had been smoking in troms 5 had quit ( 37.2% ) . a significantly higher quitting rate was found in men compared to women , and the quitting rate increased with the length of education . self - reported diseases registered in troms 5 had no significant impact on the quitting rate ( table 2 ) . the number of days between attending the two surveys had no impact on the quitting rate ( odds ratio = 1 , p=0.8 ) . table 3 shows the frequencies of new self - reported diseases reported in troms 6 , but not in troms 5 , and the association between a new diagnosis and smoking cessation . new diagnoses of asthma / copd ( n=79 ) and chd ( n=73 ) were both associated with increased frequency of quitting , 50.6% ( p=0.01 ) and 52.1% ( p=0.008 ) , respectively . both men and women belonging to any of these diagnostic groups showed increased quitting rates , 54.9% vs 39.8% ( p=0.02 ) in men and 47.1% vs 32.7% ( p=0.009 ) in women . the impact of getting these new diagnoses was particularly strong among the subjects with education length no longer than 12 years ( p=0.001 ; figure 1 ) . subjects with education length up to 12 years were also more frequently diagnosed with these diseases compared to those with higher education , 16.0% and 9.5% ( p=0.01 ) , respectively . among subjects with higher education , a new diagnosis of both chd and obstructive lung disease was significantly associated with smoking cessation when analyzed by logistic regression and also after adjusting for sex and education level in multivariable logistic regression analyses ( table 4 ) . half of the participants with a new diagnosis of asthma / copd or angina / mi quit smoking during the 6 years of follow - up . however , the association between these new diagnoses and smoking cessation was found only in participants with education length up to 12 years . the much higher smoking rate among those with asthma and , in particular , those with copd at baseline than in those with chd indicates a shift in attitude toward smoking cessation among subjects with copd . the high number of participants and the high attendance rates among those invited are strengths of the study . the prevalences of daily smoking among all the attendees in troms 5 and troms 6 were 31% and 22% , respectively,11 which are close to the national prevalences of 29% and 21% , as registered by statistics norway ( https://www.ssb.no)15 in 2002 and 2008 , respectively . in our study sample , the prevalence was lower at both points of time , 25.6% and 17.0% , respectively , and a healthy survivor effect may have contributed to the low frequency of daily smoking in our subsample . the study is based on questionnaires and not on objective measurements of smoking , such as cotinine and thiocyanate . in a previous norwegian study , self - reported smoking was strongly related to serum thiocyanate if the question was asked in a neutral setting.16 although the questions on smoking were included in a self - administered questionnaire , underreporting of daily smoking among those with a new diagnosis can not be ruled out . shift in diagnosis based on the same illness from troms 5 to troms 6 , for instance , between asthma and copd , may have taken place in some subjects . this is no longer a problem when asthma and copd are combined into one category ( asthma and/or copd ) and subjects with a new diagnosis of any of these diseases are compared with those with neither of the diagnoses at both troms 5 and troms 6 . although we have found associations between a new diagnosis and smoking cessation , we do not know for sure whether the extra cessations in the subgroups with a new diagnosis were really preceded by a new diagnosis . subjects in these subgroups could have stopped smoking before the diagnosis was given . in these cases , it is likely that symptoms from a pulmonary or heart disease had raised their awareness of the risk of continued smoking . no previous study has , to our knowledge , described smoking cessation in relation to a new diagnosis of heart or lung disease . most previous studies evaluated smoking cessation programs , and control groups in such studies can be compared with our participants . however , the quitting rate of 41.7% among subjects with a copd diagnosis reported in troms 5 was more similar to the rate of 38% among the copd patients , who had taken part in the smoking cessation program in a swedish study , than the rate of 10% among the copd patients on usual care.6 in a study from primary care , where smokers received smoking cessation advice and were followed up annually with spirometry , higher rates of abstinence were found among those with copd than among those with normal lung function.17 this result is in line with our findings . we found that the quitting rate increased with increasing length of education , and this association is well known from previous studies.18 qualitative studies have shown that patients with a copd diagnosis may have several reasons for not quitting despite the knowledge of harming themselves,19 and they do not always believe that quitting would give them a better life.20 copd patients often show little interest in receiving help from medication and describe unassisted quitting as the best method to stop smoking , based on willpower , strong motivation , and internal strength.21 in our study , a new diagnosis of copd or chd might have given many participants the motivation they needed to quit without assistance . in another qualitative study , the interviewees who had stopped smoking emphasized that persons close to them had a strong influence on their decision to quit.22 this gives a reason to believe that the decreasing acceptance of smoking in the society also influences copd patients . half of the participants with a new diagnosis of asthma / copd or angina / mi quit smoking during the 6 years of follow - up . however , the association between these new diagnoses and smoking cessation was found only in participants with education length up to 12 years . the much higher smoking rate among those with asthma and , in particular , those with copd at baseline than in those with chd indicates a shift in attitude toward smoking cessation among subjects with copd . the high number of participants and the high attendance rates among those invited are strengths of the study . the prevalences of daily smoking among all the attendees in troms 5 and troms 6 were 31% and 22% , respectively,11 which are close to the national prevalences of 29% and 21% , as registered by statistics norway ( https://www.ssb.no)15 in 2002 and 2008 , respectively . in our study sample , the prevalence was lower at both points of time , 25.6% and 17.0% , respectively , and a healthy survivor effect may have contributed to the low frequency of daily smoking in our subsample . the study is based on questionnaires and not on objective measurements of smoking , such as cotinine and thiocyanate . in a previous norwegian study , self - reported smoking was strongly related to serum thiocyanate if the question was asked in a neutral setting.16 although the questions on smoking were included in a self - administered questionnaire , underreporting of daily smoking among those with a new diagnosis can not be ruled out . all diagnoses were self - reported , and their correctness could not be confirmed . shift in diagnosis based on the same illness from troms 5 to troms 6 , for instance , between asthma and copd , may have taken place in some subjects . this is no longer a problem when asthma and copd are combined into one category ( asthma and/or copd ) and subjects with a new diagnosis of any of these diseases are compared with those with neither of the diagnoses at both troms 5 and troms 6 . although we have found associations between a new diagnosis and smoking cessation , we do not know for sure whether the extra cessations in the subgroups with a new diagnosis were really preceded by a new diagnosis . subjects in these subgroups could have stopped smoking before the diagnosis was given . in these cases , it is likely that symptoms from a pulmonary or heart disease had raised their awareness of the risk of continued smoking . no previous study has , to our knowledge , described smoking cessation in relation to a new diagnosis of heart or lung disease . most previous studies evaluated smoking cessation programs , and control groups in such studies can be compared with our participants . however , the quitting rate of 41.7% among subjects with a copd diagnosis reported in troms 5 was more similar to the rate of 38% among the copd patients , who had taken part in the smoking cessation program in a swedish study , than the rate of 10% among the copd patients on usual care.6 in a study from primary care , where smokers received smoking cessation advice and were followed up annually with spirometry , higher rates of abstinence were found among those with copd than among those with normal lung function.17 this result is in line with our findings . we found that the quitting rate increased with increasing length of education , and this association is well known from previous studies.18 qualitative studies have shown that patients with a copd diagnosis may have several reasons for not quitting despite the knowledge of harming themselves,19 and they do not always believe that quitting would give them a better life.20 copd patients often show little interest in receiving help from medication and describe unassisted quitting as the best method to stop smoking , based on willpower , strong motivation , and internal strength.21 in our study , a new diagnosis of copd or chd might have given many participants the motivation they needed to quit without assistance . in another qualitative study , the interviewees who had stopped smoking emphasized that persons close to them had a strong influence on their decision to quit.22 this gives a reason to believe that the decreasing acceptance of smoking in the society also influences copd patients . it has been suggested that a hard core of smokers will constitute an increasing proportion of copd patients who still smoke.23 the high quitting rates among subjects with both an established and a new diagnosis of copd in our study contradicts this pessimism . the study supports pursuit of early diagnosis of copd and gives reasons for a more optimistic attitude among health workers when they discuss smoking cessation with their copd patients .

backgroundpatients with copd have had a lower tendency to quit smoking compared to patients with coronary heart disease ( chd ) . we wanted to investigate if this is still true in a norwegian population.methodsour data came from the fifth and sixth troms surveys , which took place in 20012002 and 20072008 . the predictors of smoking cessation were evaluated in a cohort of 4,497 participants who had stated their smoking status in both surveys.resultsof the 4,497 subjects in the cohort , 1,150 ( 25.6% ) reported daily smoking in troms 5 . in troms 6 , 428 had quit ( 37.2% ) . a new diagnosis of obstructive lung disease ( asthma or copd ) and chd were both associated with increased quitting rates , 50.6% ( p=0.01 ) and 52.1% ( p=0.02 ) , respectively . in multivariable logistic regression analysis with smoking cessation as outcome , the odds ratios ( ors ) of a new diagnosis of obstructive lung disease and of chd were 1.7 ( 1.12.7 ) and 1.7 ( 1.02.9 ) , respectively . male sex had an or of 1.4 ( 1.11.8 ) compared to women in the multivariable model , whereas the ors of an educational length of 1316 years and 17 years compared to shorter education were 1.6 ( 1.12.2 ) and 2.5 ( 1.54.1 ) , respectively.conclusionthe general trend of smoking cessation in the population was confirmed . increased rates of smoking cessation were associated with a new diagnosis of heart or lung disease , and obstructive lung disease was just as strongly linked to smoking cessation as was chd . this should encourage the pursuit of early diagnosis of copd .

cranial nerves were composed of two different histological portions , glial and non - glial portion . the transitional zone refers to the junction between glial portion and the non - glial peripheral portion . the most outer layer of brain cortex is coated by pia glial membrane called glial limiting membrane or glial limitans10 ) . skinner12 ) reported the extent of glial out growth into the various cranial nerves and glial out spread of facial nerve was extended as far as 2.6 mm in length from the origin of brain stem . in general , root exit zone or root entry zone ( rez ) of cranial nerves means the area nerves come from the brainstem . recently , some authors proposed to replace the term of rez by root exit point ( rexp ) , root detach point ( rdp ) , and root exit zone ( rexz ) in detail13 ) . the purposes of this study were to measure the length of the central myelin , especially from root detach point to transitional zone and determine histological structures of central myelin of facial nerve . twenty brain stems were obtained from formalin embedded cadavers . the 12 men and 8 women of cadavers ranged in age from 32 to 78 years ( mean , 58 years ) . during the brain stems were served , care should be taken to preserve the cisternal segment of facial nerve . we obtained 40 facial nerves but 17 facial nerves were put out of shape during preparation for histological study . twenty three facial nerves could be examined properly . to make a facial nerve - brainstem tissue block , we cut the midline of brain stem into halves and slice them along the facial nerve . first , one slice was stained with hematoxylin and eosin ( h&e ) for screening the shape of facial nerve and brain stem . and then , other slices were stained with periodic acid - schiff ( pas ) for peripheral myelin and glial fibrillary acid protein ( gfap ) for central myelin and glial membrane . after staining , photomicrographs of each section were taken and measure the distance of medial rez , from detach point of facial nerve at the brain stem to the most distal part of central myelin , and thickness of glial membrane of central myelin of rez . the thickness of glial membrane was obtained at two different points , the thickest point of proximal and distal area of central myelin . usually to correct for the shrinkage error during fixation , occulomotor nerve of same specimen was used as a standard . but we did not calculate the percentage of facial nerve shrinkage because of being used pre - fixed specimens with formalin . with the h&e staining , the shape of facial nerve was easily determined ( fig . special staining methods , such as pas and gfap , could be clearly distinguished the facial nerve into peripheral and central myelin . peripheral myelin was stained dark blue with pas staining and central myelin was stained dark brown with gfap ( fig . 2 ) . glial outgrowth from the brain stem , central myelin , ends in dome or cone formation . the outermost membrane surrounding the brain stem , glial limiting membrane , continues to the central myelin could be visible . the mean thickness of glial membrane of central myelin was 66.5 um ( 40 - 110 um ) at proximal and 7.4 um ( range of 5 - 10 um ) at distal ( table 1 ) . the length of medial rez was mean 2.6 mm ( range of 1.6 - 3.5 mm ) ( table 2 ) . there is no clear description of the relationship between the rez and the transitional zone . in the strict sense , rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . the term of rez was defined by jannetta as a junctional area between central and peripheral myelin6 ) . many authors used the terms of rez and transitional zone interchangeably1,3,11 ) . in a recent study , tomii et al.13 ) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . they have proposed the use of terms root exit point ( rexp ) , rdp ( medial detach point ) and transitional zone instead of rez . and they stressed the rdp appears to be a good landmark for the microvascular decompression surgery . in 1915 , henschen5 ) has been descried that cranial nerve was composed of two different segments , such as glial and non - glial portion . skinner12 ) also reported several characteristics of various cranial nerves as follows . from the brain there is an outgrowth of glia cells , astrocyte and oligodendroglia , along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve , central myelin , or central glial myelin2,10,13 ) . to measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . skinner12 ) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . adams1 ) described that the transitional zone is only 1 to 3 mm in length . jannetta6 ) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth , seventh , and ninth cranial nerves . in 2003 , he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm , respectively . we measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve . glial portion of the cranial nerve , central myelin , is composed of fibrillary astrocyte and oligodendroglia cells12 ) . and it has a structure similar to that of white matter of the cns , consisting of parallel traveling nerve fibers , and lacks funicular structure . nerve fibers are embedded in supporting tissue , but endo- , peri , and epineurium are absent . de ridder et al.2 ) stated the layer of pia mater surrounds the central myelin . glial limiting membrane , pia glial membrane , or glial limitans , is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10 ) . the main function of glial limiting membrane is to act as a physical barrier of the cns . we observed that the glial limiting membrane extending from the outermost layer of brain stem , strong stained with gfap , surrounds the central myelin . we called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . we also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . the glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . the cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . the mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9 ) . jannetta et al.7 ) reported that myelin defect exist in the rez in patient of hemifacial spasm . there is no clear description of the relationship between the rez and the transitional zone . in the strict sense , rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . the term of rez was defined by jannetta as a junctional area between central and peripheral myelin6 ) . many authors used the terms of rez and transitional zone interchangeably1,3,11 ) . in a recent study , tomii et al.13 ) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . they have proposed the use of terms root exit point ( rexp ) , rdp ( medial detach point ) and transitional zone instead of rez . and they stressed the rdp appears to be a good landmark for the microvascular decompression surgery . in 1915 , henschen5 ) has been descried that cranial nerve was composed of two different segments , such as glial and non - glial portion . skinner12 ) also reported several characteristics of various cranial nerves as follows . from the brain there is an outgrowth of glia cells , astrocyte and oligodendroglia , along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve , central myelin , or central glial myelin2,10,13 ) . to measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . skinner12 ) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . adams1 ) described that the transitional zone is only 1 to 3 mm in length . jannetta6 ) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth , seventh , and ninth cranial nerves . in 2003 , he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm , respectively . we measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve . glial portion of the cranial nerve , central myelin , is composed of fibrillary astrocyte and oligodendroglia cells12 ) . and it has a structure similar to that of white matter of the cns , consisting of parallel traveling nerve fibers , and lacks funicular structure . nerve fibers are embedded in supporting tissue , but endo- , peri , and epineurium are absent . de ridder et al.2 ) stated the layer of pia mater surrounds the central myelin . glial limiting membrane , pia glial membrane , or glial limitans , is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10 ) . the main function of glial limiting membrane is to act as a physical barrier of the cns . we observed that the glial limiting membrane extending from the outermost layer of brain stem , strong stained with gfap , surrounds the central myelin . we called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . we also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . the glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . the cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . the mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9 ) . jannetta et al.7 ) reported that myelin defect exist in the rez in patient of hemifacial spasm . our study shows the length of medial rez is mean 2.6 mm ( range of 1.6 - 3.5 mm ) and the rez is covered by glial sheath continued from glial limiting membrane of brain stem .

objectivethe aim of this study was to evaluate the microanatomy and histological features of the central myelin in the root exit zone of facial nerve.methodsforty facial nerves with brain stem were obtained from 20 formalin fixed cadavers . among them 17 facial nerves were ruined during preparation and 23 root entry zone ( rez ) of facial nerves could be examined . the length of medial rez , from detach point of facial nerve at the brain stem to transitional area , and the thickness of glial membrane of central myelin was measured . we cut brain stem along the facial nerve and made a tissue block of facial nerve rez . each tissue block was embedded with paraffin and serially sectioned . slices were stained with hematoxylin and eosin ( h&e ) , periodic acid - schiff , and glial fibrillary acid protein . microscopy was used to measure the extent of central myelin and thickness of outer glial membrane of central myelin . thickness of glial membrane was examined at two different points , the thickest area of proximal and distal rez.resultsspecial stain with pas and gfap could be differentiated the central and peripheral myelin of facial nerve . the length of medial rez was mean 2.6 mm ( 1.6 - 3.5 mm ) . the glial limiting membrane of brain stem is continued to the end of central myelin . we called it glial sheath of rez . the thickness of glial sheath was mean 66.5 m ( 40 - 110 m ) at proximal rez and 7.4 m ( 5 - 10 m ) at distal rez.conclusionmedial rez of facial nerve is mean 2.6 mm in length and covered by glial sheath continued from glial limiting membrane of brain stem . glial sheath of central myelin tends to become thin toward transitional zone .

engaging in high levels of physical activity is a key strategy for successful maintenance of weight loss . a study of nearly 4,000 participants in the national weight control registry ( nwcr ) , the largest longitudinal study of successful weight loss maintainers , indicated participants expend 2,621 2,252 kcal per week through physical activity , which is equivalent to approximately 60 minutes of moderate - intensity physical activity per day . additionally , long - term followup of participants in behavioral weight loss programs has shown that those who are most successful at maintaining their weight loss report activity levels similar to those of nwcr participants [ 3 , 4 ] . many individuals , particularly those who are obese , are insufficiently active , and increasing physical activity can be a challenge [ 5 , 6 ] . thus , strategies that assist individuals in adopting and sustaining high levels of physical activity to help facilitate a healthy body weight are needed . prior studies have identified multiple strategies for increasing physical activity adoption and maintenance , such as providing home or clinic - based exercise programs , increasing access to active behaviors ( e.g. , adding exercise equipment in the home ) , and reducing access to sedentary behaviors ( e.g. , limiting time to watch television ) , using pedometers to track activity and progress toward physical activity goals , and accumulating exercise throughout the day in multiple short bouts ( 10 min ) [ 11 , 12 ] . performing a variety of different types of activities may be another strategy to increase physical activity levels . the national health and nutrition examination survey ( nhanes ) showed that individuals who reported engaging in a variety of different activities ( i.e. , walking + other leisure - time activities ) were more likely to meet national physical activity recommendations compared to those who reported no variety ( i.e. , only walking ) . likewise , in an 18-month behavioral weight loss intervention , overweight participants who reported physical activity variety ( i.e. , 2 different activities ) at 6 months had higher self - reported activity - related energy expenditure and a lower body mass index ( bmi ) at 18 months than those who did not report physical activity variety ( i.e. , only 1 activity ) . thus , while the above findings suggest that physical activity variety may contribute to higher physical activity levels within the context of behavioral weight loss treatment , it is unclear whether physical activity variety is associated with higher physical activity in individuals who have achieved long - term success in controlling their body weight . in the current study , we examined the relationship between physical activity variety , defined as the number of different types of self - reported moderate - to - vigorous activities performed in one week , and minutes spent in objectively measured moderate - to - vigorous physical activity ( mvpa ) in two groups of individuals who have successfully maintained their body weight long - term : ( 1 ) weight loss maintainers with previous history of overweight / obesity and ( 2 ) normal - weight individuals without a history of overweight . normal - weight participants were included as a comparison group given that weight loss maintainers represent a unique group of individuals who report strict adherence to multiple behavioral strategies in order to maintain their body weight . due to their unique characteristics and history , it is possible that the association between physical activity variety and mvpa could be different for weight loss maintainers and normal - weight individuals . however , based on previous research showing a relationship between physical activity variety and higher physical activity levels across diverse groups and settings [ 14 , 15 ] , we predicted that engagement in a greater variety of moderate - to - vigorous activities would be associated with higher mvpa daily minutes in both the weight loss maintainer and normal - weight groups . participants were enrollees in the cross - sectional lite study that compared weight control behaviors of weight loss maintainers and normal - weight controls . a convenience sample of men and women was recruited through advertisements placed in national and local publications intended for a general audience . persons interested in participating were asked to either call a toll - free number or to visit a website . participants were recruited from across the united states , although most were from new england , california , and the washington , dc area . weight loss maintainers had a history of overweight or obesity ( bmi 25 ) but were currently normal weight ( bmi = 18.524.9 ) , having maintained a 10% loss of their lifetime maximum body weight for at least 5 years . normal - weight participants had a current bmi between 18.5 and 24.9 and no history of overweight or obesity . participants in both groups were weight stable ( 10 lb ) for at least 2 years prior to enrollment . of 813 individuals who responded to advertisements and a brief online screening tool , the study protocol was approved by the miriam hospital institutional review board , providence , ri , usa . participants reported information about age , gender , marital status , ethnicity / race , type of employment , and education . weight and weight history were assessed via self - report methods that have been previously validated . questions on the paffenbarger physical activity questionnaire regarding average number of city blocks walked per day , and weekly frequency and duration of sports and recreational activities performed were used to determine variety or number of different activities performed during the past 7 days . only activities that were performed at a moderate or vigorous intensity and for 10 minutes in duration were included given that engagement in these activities is considered necessary for improving health and achieving a healthy body weight [ 1 , 12 ] . the intensity of sports and recreational activities was determined using the paffenbarger coding scheme . for walking to be counted as an activity , participants had to report walking the equivalent of at least 12 blocks per day ( i.e. , 1 mile at a moderate intensity ) . treadmill walking reported as a sports and recreational activity was not distinguished as a separate activity from walking 12 blocks / day . climbing stair flights , walking < 12 blocks / day , and sports and recreational activities that were performed for < 10 minutes were not considered to contribute to variety . monrovia , ca , usa ) was used to objectively measure daily minutes spent in mvpa . this device converts accelerations or movements from vertical , horizontal , and anterior - posterior planes into counts , with greater magnitude or intensity of acceleration over a given time period generating a higher number of counts . participants were sent the device in a postage - paid envelope with instructions on how to activate the device and wear it on their waistband during all waking hours for 7 consecutive days , except while bathing or swimming . each device was programmed with the participant 's personal data ( sex , age , height , and weight ) and set to capture movements continuously in 1-minute intervals . the rt3 has shown to be a strong predictor of oxygen consumption during sedentary and treadmill activities [ 20 , 21 ] and a more precise measure of physical activity at the group level compared to its tri - trac predecessor . consistent with previously documented methods for analyzing rt3 data , a minimum of 4 days on which the device was worn for 10 hours each day was required for data to be considered valid [ 23 , 24 ] . rt3 nonwear times , defined as periods of 30 consecutive minutes of zero counts ( permitting intervals of up to 2 consecutive minutes registering 1100 counts / min ) , were deleted from analysis . the remaining time was partitioned according to intensity level . based on previous rt3 validation research and a recent study that used the rt3 to compare mvpa patterns in weight loss maintainers , normal - weight , and obese groups , we computed time spent in mvpa using a threshold of 984 counts / min . descriptive statistics are presented in tables as means sd for continuous measures and percentages for categorical responses . chi independent t - tests were conducted to assess differences between the groups on demographic characteristics , weight , accelerometer daily wear time , mvpa minutes / day , and reported variety / number of different types of moderate - to - vigorous activities performed . linear regression was used to examine the associations of physical activity variety and group status ( weight loss maintainers versus normal weight ) with objectively measured mvpa minutes / day , adjusting for age , gender , years of education , marital status , bmi , and accelerometer daily wear time . logistic regression was used to assess whether physical activity variety and group status were associated with achieving the 250 mvpa minutes / week recommendation for optimal long - term weight maintenance . for this analysis , mvpa minutes / week was calculated by multiplying average daily mvpa minutes by 7 ( days ) . of 413 participants who were sent an accelerometer and the ppaq , 394 ( 95% ) met valid accelerometer wear requirements and provided complete data on the ppaq . the characteristics of these 226 weight loss maintainers and 169 normal - weight participants are presented in table 1 . both groups were similar in age ( 48.8 12.9 years ) , gender ( 84% female ) , marital status ( 66% married ) , race / ethnicity ( 94% caucasian ) , employment status ( 82% employed ) , and job type ( 95% in professional or clerical positions ) . a greater proportion of normal - weight participants was college educated , compared to the weight loss maintainers . both groups were normal weight , although the weight loss maintainers had a slightly higher bmi . on average , weight loss maintainers had lost nearly 29 kg and maintained 10% weight loss for 13.7 9.6 years . there were no differences in accelerometer wear time between the groups , with the weight loss maintainers and normal - weight participants wearing the accelerometer for an average of 14.8 2.0 hours / day on 7.5 0.9 days . as reported previously , weight loss maintainers spent an average of 6 minutes more per day in mvpa , compared to normal - weight participants ( 58 versus 52 min / d ) . the variety / number of different activities performed by the weight loss maintainers and normal - weight participants was similar ( 1.8 1.2 versus 1.7 1.2 , p = 0.52 ) . additionally , when participants who reported no moderate - to - vigorous activities were excluded , the variety / number of activities performed by wlm ( n = 180 ) and nw ( n = 141 ) remained similar ( 2.2 1.0 versus 2.0 1.0 , p = 0.10 ) . we next examined the independent and joint associations of physical activity variety and group status with mvpa minutes per day . given that the physical activity variety group status interaction was not significant ( p = 0.73 ) , the results of the main effects linear regression model for mvpa minutes per day are shown ( table 2 ) . greater physical activity variety ( p < 0.001 ) and weight loss maintainer status ( p < 0.05 ) were independently related to greater daily time spent in mvpa , after adjustment for demographic characteristics ( age , gender , educational level , marital status , current bmi ) and daily accelerometer wear time . figure 1 presents estimated mvpa minutes per day in the weight loss maintainer and normal - weight groups by reported number of different moderate - to - vigorous activities performed . across both groups combined , the number of moderate - to - vigorous activities ranged from 0 to 4 , with 73 participants ( 18.5% ) reporting 0 activities , 105 reporting 1 activity ( 26.6% ) , 112 ( 28.4% ) reporting 2 activities , 74 reporting 3 activities ( 18.7% ) , and 30 ( 15.4% ) reporting 4 or more activities . each 1 unit increase in the number of different moderate - to - vigorous activities performed was associated with an additional 9.4 1.3 daily minutes spent in mvpa , representing an additional 56.2 8.3 kcal expended per day based on rt3-derived estimates . similarly , logistic regression analyses showed that greater physical activity variety ( or = 1.78 [ 1.372.30 ] , p < 0.001 ) , weight loss maintainer status ( or = 0.23 [ 0.080.69 ] , p = 0.002 ) , and lower bmi ( or = 0.81 [ 0.690.95 ] , p = 0.01 ) were independently associated with meeting the 250 mvpa minutes / week guideline for optimal weight maintenance . the physical activityvariety group status interaction was not significant ( p = 0.25 ) . given the continuing obesity epidemic and growing evidence that indicates greater amounts of physical activity are needed for successful long - term weight control , it is important to identify strategies that can assist individuals in adopting and maintaining high levels of physical activity . this study examined whether performing a greater variety of different types of moderate - to - vigorous physical activities was related to greater time spent in mvpa among weight loss maintainers and normal - weight individuals without a history of overweight / obesity . we found that , independent of group , greater variety was associated with higher daily mvpa minutes and meeting the 250 mvpa minutes per week recommendation for optimal long - term weight maintenance . these findings are consistent with previous studies of the general population and in overweight / obese individuals undergoing behavioral weight loss treatment . however , the present study is the first to show a relationship between greater physical activity variety and higher objectively measured mvpa duration and energy expenditure in two groups of individuals who have had long - term success in maintaining a normal body weight . we found that for each additional different type of moderate - to - vigorous activity performed , participants on average spent an additional 9 minutes in mvpa and expended 56 more kcal per day . thus , for example , participants who reported engaging in 3 different activities during the previous week spent on average an additional 18 minutes in mvpa and expended 112 more calories per day compared to participants who reported engaging in only 1 activity . while our findings do not imply causation , they do warrant additional longitudinal research to examine whether incorporating variety into a physical activity routine may be an efficacious strategy to achieve higher mvpa levels for enhanced weight control . the relationship between greater physical activity variety and engagement in higher mvpa levels may be potentially explained by a number of physiological and psychological factors . for example , alternating different physical activities that involve different muscle groups and energy systems ( aerobic , anaerobic ) might promote greater exercise consistency by affording more time for recovery and decreasing risk of overuse injuries [ 13 , 26 ] . participating in a variety of activities may also facilitate greater exercise adherence via increased enjoyment and decreased boredom [ 13 , 26 ] . greater access to a variety of activities may increase the likelihood that individuals will find an exercise activity or a combination of exercise activities that they like and will perform regularly . additionally , research based on the behavioral economics model suggests that motivation to exercise is enhanced when individuals can choose from a variety of physical activities versus only one physical activity [ 28 , 29 ] . consequently , adding a variety component to a physical activity prescription may aid individuals in achieving and maintaining high levels of physical activity . future research is needed to investigate potential mechanisms that underlie the relationship between variety and higher mvpa levels . whereas increased variety of healthy physical activities is associated with greater time spent in mvpa , it is also possible that decreased variety of unhealthy sedentary activities might contribute to lesser time spent being sedentary and higher overall physical activity levels . given that sedentary behaviors , independent of physical activity , have shown to be detrimentally associated with bmi and other cardiometabolic risk factors [ 30 , 31 ] , future studies that examine the association between variety and sedentary behaviors are needed . the cross - sectional nature of our study does not allow us to determine whether greater variety contributes to higher mvpa levels or alternatively whether individuals with higher mvpa levels naturally incorporate more variety into their physical activity routine . as variety in physical activity is rarely measured , the importance of factors such as the time frame for assessing variety ( i.e. , a week , month , year ) and frequency of occurrence of different activities within the time frame ( i.e. , once a week , twice a month ) are not known . while the use of an objective measure of physical activity is a strength of this investigation , it is important to note that hip - worn accelerometers like the rt3 used in this study may be limited in their ability to accurately estimate the intensity of activities not performed on flat surfaces . thus , it is possible that mvpa was underestimated in individuals who more frequently engaged in activities that involved an incline or greater upper body movement . additionally , the accelerometer count threshold we used to identify mvpa was determined in a leaner , younger male sample and thus may have affected validity in our older , largely female sample . given the homogeneity of our sample with respect to gender ( female ) and race ( white non - hispanic ) , our results may not be generalizable to men or other ethnic populations . similarly , participants in this study were all normal weight and had high physical activity levels on average . thus , future investigations should examine the potential importance of variety for increasing physical activity in overweight and inactive populations . finally , it is important to note that just as altering the variety of different types of activities performed could potentially increase mvpa duration and energy expenditure , so could altering the frequency , intensity , and duration of a single activity , independent of changes in physical activity variety . in summary , this study examined the relationship between physical activity variety and objectively measured mvpa levels in weight loss maintainers and normal - weight individuals who both had long - term success in maintaining their body weight . in both groups , physical activity variety was related to greater engagement in mvpa and likelihood of accumulating 250 mvpa minutes / week , consistent with physical activity guidelines for optimal long - term weight maintenance . future studies are needed to test whether incorporating variety or different types of activities ( e.g. , walking and cycling ) can facilitate engagement in higher levels of mvpa within interventions aimed at promoting and maintaining physical activity .

given the importance of physical activity ( pa ) for weight control , identifying strategies to achieve higher pa levels is imperative . we hypothesized that performing a greater variety of self - reported moderate - to - vigorous activities ( mvpas ) would relate to higher objectively measured mvpa minutes in two groups who were successfully maintaining their body weight : weight loss maintainers ( wlm / n = 226 ) and normal - weight individuals ( nw / n = 169 ) . the paffenbarger questionnaire and rt3 accelerometer were used to determine variety / number of different mvpas performed and mvpa minutes , respectively . the variety / number of different activities performed by wlm and nw was similar ( 1.8 1.2 versus 1.7 1.2 , p = 0.52 ) . regression analyses showed that greater variety ( p < 0.01 ) and wlm status ( p < 0.05 ) were each positively related to greater mvpa minutes / day and meeting the 250 mvpa minutes / week guideline for long - term weight maintenance . the association between greater variety and higher mvpa was similar in nw and wlm . future studies should test whether variety can facilitate engagement in higher mvpa levels for more effective weight control .

the protective effects of vagus nerve during ischemia / reperfusion ( i / r ) have been suggested ( 1 ) . vagus nerve stimulation decreases the inflammation and the injuries resulted from i / r through affecting neutrophils while vagotomy increases these processes ( 1 ) . moreover , it has been observed that stimulation of vagus nerve can cause protective effects on gastric injury through releasing prostaglandins , nitric oxide ( no ) and calcitonin gene related peptide ( cgrp ) ( 2 ) . it is probable that vagus nerve exerts its gastric protective effects through releasing the aforementioned mediators . also , it has been reported that cholinergic activity of the efferent vagus nerve can participate in immunity modulation ( 3 ) . another study has shown that nicotine in monocytes not only decreases the production of pre - inflammatory cytokines , but also changes the response to il-10 , an anti - inflammatory cytokine ( 4 , 5 ) . cho et al have shown that alternative stimulation of cervical vagus nerve increases intra - gastric pressure and also causes bleeding ulcers in mucosal glands of stomach , and these effects can be prevented by atropine administration or sub - diaphragmatic vagotomy ( 6 ) . it has been reported that gastric vagotomy is effective in treating gastric and duodenal ulcers ( 7 ) . melatonin as a neurohormone is basically produced in the pineal gland and also in epiphysis , gastrointestinal ( gi ) mucosa and other organs . melatonin has several physiological activities such as controlling circadian rhythm , sleep induction , regulation of seasonal reproduction , and improvement of immunity . the most important effect of melatonin is an anti - oxidant effect that protects living organisms against oxidative stress ( 11 ) . in several studies , the ability of melatonin in decreasing molecular injury has been shown during i / r . melatonin prevents myocardial infarction , necrotic cell death and renal abnormality after i / r ( 8 , 12 ) , reduces brain edema in ischemia ( 12 ) , and prevents acute gastric ulcers resulted from stress . these effects can be done through direct reactive oxygen species / reactive nitrogen species ( ros / rns ) detoxification , increase in anti - oxidative enzymes , and the prevention of electron leakage in mitochondrial inner membrane ( 11 , 13 ) . melatonin has a role in expression and function of nicotinic acetylcholine receptors and increases the efficacy of beta bungarotoxin - sensitive acetylcholine receptors ( 14 ) . it has been recognized that intra - cerebral injection of melatonin is effective in prevention of acid and pepsin secretion through cholinergic activity ( 15 ) . it has been identified that 5ht3 and 5ht2 receptors are involved in stimulatory effects of melatonin in releasing pancreatic enzymes ( 11 ) . protective effects of melatonin are not only due to antioxidant activity , but it also activates capsaicin - sensitive afferent fibers ( 1 , 16 ) . melatonin may have a potential impact on the treatment of peptic ulcer via significant increase in ghrelin expression ( 17 ) . melatonin increases the release of pancreatic amylase and proteins through vagus nerve ( 18 ) . the effects of melatonin on pancreatic enzymes are reversed by vagotomy and administration of capsaicin ( 18 , 19 ) . intra - lumen administration of melatonin increases plasma cholecystokinin ( cck ) and antioxidants ( 11 , 19 , 20 ) . it has been shown that sensory nerve fibers are involved in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer ( 16 ) . it has been reported that vagus nerve increases the secretion of bicarbonate from duodenum mucosa through an increase in melatonin secretion ( 21 ) . vagal pathways are the most important regulatory pathways in the gastrointestinal tract which is one of the most important sources of melatonin production . in previous studies , both protective and harmful effects of the stimulation of vagus nerve in digestive system have been reported . the role of sensory neurons in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer has been reported . there is no study about the combined effects of melatonin administration and intervention of vagus nerve during gastric i / r injury . probably , vagus nerve and melatonin have interactions in their protective effects during gastric i / r events so , the present study was performed to examine this interaction during gastric i / r . this study was performed in 42 male wistar rats weighing 180 - 220 g. animals were kept at room temperature 20 - 22 c and 12 hr-12 hr light - dark cycle . animals had free access to water and food and were randomly divided into 6 groups of 7 rats . rats were fasted for 12 hr prior to the experiment but had access to water . experimental groups were : 1 ) base+i / r+vehicle ; 2 ) base+i / r+melatonin ; 3 ) vagotomy+i / r+vehicle ; 4 ) vagotomy+i / r+melatonin ; 5)vagus nerve stimulation+i / r+vehicle ; 6)vagus nerve stimulation+i / r+ melatonin . base , means condition that neither vagus nerve was stimulated nor vagotomy was performed . animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) . after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec . stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve . then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk . occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) . melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) . melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) . one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) . mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation . optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) . glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein . superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) . catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) . the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks . the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece . comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test . animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) . after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec . stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve . then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk . occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) . melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) . melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) . one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) . mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation . optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) . glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein . superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) . catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) . the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks . the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece . comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test . mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) . / r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) . / r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) . a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mda level in the study groups is presented in figure 2 . as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) . moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) . / r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i / r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) . mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) . * p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity in the study groups are shown in figure 3 . catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) . a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) . / r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle figure 4 shows glutathione peroxidase activity in the studied groups . a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) . / r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) . level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 . as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity / r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group . this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) . superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) . / r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) . / r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) . a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) . moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) . / r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i / r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) . mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) . * p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) . a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) . / r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) . / r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) . level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 . as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity / r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group . this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) . superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle the present study was performed to investigate the protective effect of interaction between melatonin and vagus nerve during gastric i / r . the results of this study showed that melatonin administration decreased neutrophils infiltration and mda level and increased sod , cat and gpx activities in gastric tissue . also , vagus stimulation increased neutrophils infiltration and mda level in gastric tissue while melatonin administration along with vagus stimulation decreased the effect of vagus stimulation in gastric tissue . finally , vagotomy prevented some of the protective effects of melatonin during gastric i / r . according to the results of this study , melatonin can reduce gastritis during i / r , probably through decreasing the activity of oxidant enzymes and increasing the activity of anti - oxidant enzymes such as sod , cat and gpx . it has also been demonstrated that melatonin decreases mad level ( 27 - 29 ) . other probable mechanisms underlying the protective effect of melatonin include an increase in gastric microcirculation ( 1 ) , a reduction in acid and pepsin secretions ( 15 ) , a reduction in production of inflammatory cytokines ( 16 ) and an increase in the expression of anti - oxidant gene ( 17 , 30 - 32 ) . moreover , in our study , a stimulation of vagus nerve increased gastritis which was associated with an increase in mda level while vagotomy after i / r was ineffective . it is probable that the stimulation of vagus nerve following gastric i / r aggravated the condition and increased the activity of oxidant enzymes . since vagotomy in basal condition did not have any effect , it seems that the control of gastritis and the activity of oxidant and anti - oxidant enzymes are not under the control of vagus nerve in basal conditions . it has been demonstrated that the stimulation of vagus nerve increases output of gastric juice and acid ( 33 ) , and decreases the activity of anti - oxidant enzymes like sod ( 33 ) . on the other hand , vagotomy showed healing effects on duodenal ulcers ( 7 ) similar to the effect of atropine ( 34 ) . in a study , diaphragmatic vagotomy did not change the activity of catalase in the intestine ( 35 ) which was similar to the result of the present study . the results of some studies are not consistent with our data . for example , it has been identified that vagal afferent fibers decrease gastrointestinal tract inflammation through nicotinic receptors leading to a decrease in inflammatory cytokines ( 16 ) . cholinergic agonists such as neostigmine decrease superoxide anion and inflammation in i / r ( 36 ) . elevation of acetylcholine transferase activity and decrease in acetylcholine esterase inhibitors increase gpx ( 35 , 37 ) . treatment with acetylcholine esterase inhibitors increases catalase half - life ( 38 ) . in the present study , melatonin administration affected the outcomes in i / r+vagus stimulation group . melatonin decreased gastritis and mda level , and increased sod and gpx activity , but not in vagotomy group . these findings show that protective effects of melatonin are probably mediated through vagus nerve . also , interaction between melatonin and vagus stimulation is suggested since these show a synergic effect in relation with anti - oxidant factors . regarding this probable interaction , it is suggested that melatonin exerts its gastric protective effect through stimulation of gi tract neurons leading to cgrp release ( 1 ) . intra - cerebral injection of melatonin inhibits acid and pepsin secretions through vagus cholinergic activity ( 15 ) . melatonin increases the release of amylase and pancreas proteins through vagus nerve ( 16 ) . effect of melatonin administration accompanied with vagotomy has not suggested in the release of amylase(18 ) . studies have shown that intra - lumen administration of melatonin or its precursor induces the release of pancreatic enzymes , while this effect is not shown in isolated pancreas . moreover , these effects of melatonin are reversed by vagotomy and capsaicin administration ( 11 , 18 , 19 ) . it has been reported that melatonin effect is mediated indirectly through the release of cck followed by vagovagal reflex ( 11 , 19 , 39 ) . it has been shown that intra - lumen administration of melatonin increases plasma cck level ( 11 , 19 ) and cck acts through stimulation of vagal afferent fibers in gi tract ( 40 , 41 ) . it has been reported that vagotomy in combination with melatonin administration inhibits the release of pancreas proteins ( 7 , 11 , 16 ) . it has been reported that melatonin increases the expression and activity of ach receptors , and synergistic effects are induced during vagus stimulation ( 42 ) . according to a study , injection of phenylephrine causes melatonin release from mucosa enterochromaffin cells in the presence of normal vagus nerve and sympathetic paths ( 39 ) . melatonin increases the release of bicarbonate from gastric mucosa ( 39 ) and decreases the release of inflammatory cytokines of gi through increasing vagus activity ( 16 ) . it is suggested that melatonin is neuroprotective in gastric i / r probably by decreasing gastritis and mda and increasing the activities of cat , sod and gpx . these effects of melatonin are probably mediated by vagus nerve . moreover , in gastric i / r , melatonin can reverse harmful effects of vagus stimulation . it is suggested that further studies are required to find the mechanisms involved in this interaction .

objectives : vagal pathways in gastrointestinal tract are the most important pathways that regulate ischemia / reperfusion ( i / r ) . gastrointestinal tract is one of the important sources of melatonin production . the aim of this study was to investigate probable protective effect of the interaction between vagus nerve and melatonin after i / r.materials and methods : this study was performed in male rats that were divided into six groups . cervical vagus nerve was cut bilaterally after induction of i / r and the right one was stimulated by stimulator . melatonin or vehicle was injected intraperitoneally . the stomach was removed for histopathological and biochemical investigations.results:a significant decrease in infiltration of gastric neutrophils and malondialdehyde ( mda ) level after i / r was induced by melatonin and was disappeared after vagotomy . the stimulation of vagus nerve significantly enhanced these effects of melatonin . however , a stimulation of vagus nerve alone increased neutrophils infiltration and mda level . melatonin significantly increased the activities of catalase , glutathione peroxidase ( gpx ) , superoxide dismutases ( sod ) . unlike stimulation of vagus nerve , vagotomy decreased these effects of melatonin.conclusion:according to these results , it is probable that protective effects of melatonin after i / r may be mediated by vagus nerve . therefore , there is an interaction between melatonin and vagus nerve in their protective effects .

a stroke is defined by the who as rapidly developing clinical signs of a focal ( or global ) disturbance of cerebral function , with symptoms lasting 24 h or longer or leading to death , with no apparent cause other than of vascular origin . taking into consideration the cause , the mechanism and the character of morphological lesions , there are two types of stroke : ischemic and haemorrhagic [ 2 , 3 ] . eighty percent of strokes are of ischemic origin [ 2 , 4 ] , and their most common cause is thrombosis of the atherosclerotic internal carotid artery ( ica ) and/or cerebral arteries ( ca)20 % . a stroke , including an ischemic one , is of particular interest to the forensic pathologist aiming on establishing the cause of death and its mechanism , rather than for giving an opinion in cases of suspected medical malpractice . neck and/or head trauma may very rarely constitute a cause of thrombosis of the ica and ca without any pre - existing pathology [ 58 ] . it most commonly results from direct , penetrating or non - penetrating ( blunt ) neck trauma in the region of large cervical vessels ; less frequently it is attributed to indirect head trauma . in such cases the autopsy result and available clinical information has to allow the forensic pathologist to prove the existence of neck and/or head trauma and establish its causal connection with ica thrombosis and exclude other , known , non - traumatic causes of the latter . we present two cases of ica thrombosis with concomitant middle cerebral artery ( mca ) thrombosis secondary to the thrombus present in the ica lumen , in which the postmortem examination , the medical records regarding hospitalization and records of investigation indicated trauma as the cause of thrombosis . a 57-year old male carpenter sustained an injury in an occupational accident in the carpenter s warehouse . he was struck very hard in the face , in the region of his left cheek by an irregular - shaped wood fragment which looked like a big splinter ( measuring about 26 cm in length , and with the greatest cross - sectional dimensions of 3.5 1.5 cm ) that broke off from the board being processed on the carpenter s machine ( fig . 1 ) . first aid was provided in the emergency department in the local hospital , where the presence of a large , wooden fragment that thrust deep into the left cheek and penetrated through the parapharyngeal space into the neck was diagnosed . on admission he was then transferred to the otolaryngological ward , where a physical examination , including an evaluation of his neurological status revealed : maintained consciousness , narrow and symmetric pupils , circulatory and respiratory sufficiency and bp of 180/80 mmhg . the ct examination revealed the presence of a foreign body penetrating the left maxillary sinus with a comminuted fracture of the anterior and inferior wall below the external surface of the base of the skull , and a left occipital condyle fracture with concomitant breaking and impression into the posterior cranial fossa and soft tissues of the neck . no cerebral lesions , including traumatic , or features of raised intracranial pressure were observed ( fig . 2 ) . the ct angiography allowed the diagnosis of an occlusion of the ica by a thrombus localised at the level of the bifurcation of the common carotid artery ( cca ) and at the level of c1 , adjacent to the foreign body . the laboratory findings were as follows : d - dimer 20921.25 g / l ( n < 500 ) ; pt and aptt within normal limits . once the diagnostic procedures were finished , a surgical intervention was performed on the day of admission . the preparation of the internal jugular vein revealed the medial translocation and compression of the carotid vessels . the foreign body was removed . post - interventional ct revealed the thrombotic occlusion of the left ica and mca and a hypodense area supplied by the mca and the concomitant left hemisphere oedema of the brain . the patient died on the 6th post - operative day exhibiting symptoms of intracranial hypertension and a brain oedema.fig . 2case 1 : localisation of the foreign body ( ct ) case 1 : wooden splinter removed during the surgical procedure case 1 : localisation of the foreign body ( ct ) the findings of the medico - legal autopsy were as follows : a thrombus occluding the left ica and the proximal segment of the left mca , a large infarction area in the temporal lobe and adjacent parts of the frontal and occipital lobes of the left hemisphere of the brain , a large brain oedema with features of subfalcine herniation and transtentorian herniation , a comminuted fracture of the left maxilla and an occipital bone fracture in the region of the occipital condyle with the fragment impression . the histopathological examination revealed the occlusion of the left ica by the thrombus ( h + e ( fig . 3 ) , masson , gomori , verhoeff and ptah ) , focal intimal haemorrhages and features of a mechanical injury the rupture of the artery wall in the form of an irregular fissure in the tunica media ( fig . 4 ) . the samples of the macroscopically changed left cerebral hemisphere revealed ischemic necrosis with polymorphonuclear leukocytes reaction , oedema and hyperaemia , while secondary , focal , non - reactive perivascular haemorrhages were found in the brainstem . other organs showed no significant histopathological lesions , apart from morphological indices of circulatory disturbances.fig . 3case 1 : the internal carotid artery wall with the adjacent thrombus ( h + e)fig . 4case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h + e and b verhoeff ) case 1 : the internal carotid artery wall with the adjacent thrombus ( h + e ) case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h + e and b verhoeff ) a 32-year old female patient was brought to the hospital after being battered what resulted in police intervention . on admission the patient was conscious , complaining about a left upper extremity contracture that started 2 weeks prior to admission . she was generally healthy , with no chronic diseases , taking no medication , apart from being a smoker . for the previous month she had been repeatedly battered by her partner ( about twice a week ) with a fist and open hand to the face , neck and back , as well as pulled by the hair . the last battery that took place 1 day prior to admission resulted in a short - term loss of consciousness . on admission the following body injuries were described in her medical record : resorbing bruises on the face , including a wound of the lower lip mucosa , and the upper extremities , . on admission the patient was sleepy with left - sided hemiparesis with symptoms concerning mainly the upper extremity , blood pressure was 110/80 mmhg , a fundus examination revealed no pathological lesions . d - dimer 1 200 ng / ml ( n < 500 ) , aptt slightly shortened 25.69 s ( n : 2836 ) , aptt ratio and pt within normal limits . ct revealed the presence of a hypodense area in the right hemisphere region supplied by the mca , whereas mri scan confirmed the presence of an ischemic brain damage . thrombotic occlusion of the ica and mca was visible in mri ( fig . 5 ) and angioct ( fig . , the patient died on the 12th day after admission presenting symptoms of raised intracranial pressure with a concomitant brainstem injury secondary to a brain oedema.fig . 5case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri)fig . 6case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri ) case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) the findings of the medico - legal autopsy were as follows : a thrombus occluding the right ica and the mca , thrombosis of the internal jugular veins , as well as the superior sagittal sinus and transverse sinus , an extensive infarction area in the right hemisphere region supplied by the mca ; a brain oedema with features of right - sided subfalcine herniation and transtentorian herniation , carotid and basal ca with no atherosclerotic lesions . a histopathological examination revealed an occlusion of the right ica by the thrombus ( h + e , masson , gomori , verhoeff and ptah)a muscular type artery with normal wall structure , the lumen filled with thrombus fragments , mechanical injury of the arterial wall rupture of the intima and tunica media ( fig . 7 ) . internal jugular veins normal wall structure , occluded by thrombi . there were dominating circulatory disturbances in the brain in the form of very significant capillary - venous hyperaemia resulting from an impaired cerebral venous return secondary to jugular vein and dural sinuses thrombosis.fig . 7case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h + e and b verhoeff ) case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h + e and b verhoeff ) observed in both cases , focal ischemic brain injury was secondary to the thrombosis of the ica and mca . cerebral vascular lesions may be causally associated with the sustained head and/or neck trauma with concomitant ica thrombosis and ischemic brain damage [ 5 , 713 ] . they can , however , accompany head and/or neck trauma , yet be independent of trauma and have other morbid origins [ 2 , 4 ] . in both cases we considered the most common , known , possible non - traumatic causes for ica thrombosis , including atherosclerosis , which is the most common cause for ica and ca thrombosis . the endothelium and the connective tissue cap injury expose the blood coagulation activating factors , which induce thrombus formation . the endothelium injury itself with exposure of the basal membrane may predispose to thrombus formation as well . the presence of stable atherosclerotic plaque may be complicated by arterial thrombosis , and this refers to cerebral vessels as well . however , vessel stenosis has to be significant , i.e. the blood flow transforms from being laminar into post - stenotic turbulent [ 1417 ] both the in vivo neuroradiologic diagnostic imaging modalities , including ct angiography , and the postmortem examination with a histopathological assessment did not reveal the atherosclerosis of ica or mca in any of the discussed cases . other factors favouring carotid arteries thrombosis include : hypertension , diabetes , hypercholesterolaemia , obesity and smoking [ 2 , 4 , 18 ] . such factors accelerate the course of atherosclerosis , of which a common complication is thrombosis . the analysis of both patients medical records , including the laboratory findings obtained during hospitalisation allowed diabetes and lipid metabolism disorders to be ruled out . the woman did not manifest features of hypertension , whereas unstable aterial pressure values , i.e. labile hypertension , was observed in the male patient . no lesions in the small - sized cerebral vessels that could have been responsible for the ischemic stroke were found during the postmortem examination , including the neuropathologic examination , within the ischemic , as well as in the non - ischemic areas . no arteritis , amyloid angiopathy , vascular malformations nor lesions observed in the course of hypertensive encephalopathy were found . the presence of a thrombus inside the ica and/or ca , and secondary focal ischemic brain damage may result from embolism [ 2 , 4 , 18 , 19 ] . the most common source of such embolic material are thrombi localised in the left side of the heart ( auricle of the left atrium in patients with atrial fibrillation ) , parietal thrombi in the left ventricle whose formation is associated with myocardial infarction or more frequently aneurysm of the heart in the area of an extensive post - infarction scar or left ventricle dilatation , e.g. in the course of dilated cardiomyopathy or endocarditis , especially of bacterial origin the embolism in the ica and ca may then develop in patients with atrial septal defect and deep vein thrombosis . the postmortem examination did not , however , reveal any of these conditions in the discussed patients . a significantly less common cause of stroke are haematological disorders , which are responsible for about 1 % of all strokes and occur slightly more frequently in young subjects . the laboratory findings performed during hospitalisation and autopsies allowed haematological disorders to be ruled out as factors predisposing to thrombosis . the elevation of d - dimer level was the sole abnormal parameter observed on the coagulation profile and was associated with present ica and mca thrombosis in both cases . oral contraceptives ( oc ) should be considered as a possible cause for thrombosis in the female patient . oc are described in neurology as one of the ethological factors of ica and ca thrombosis . in the presented case the anamnesis collected on the patient s admission to the hospital indicated that she did not take any medication , most probably including oc . however , should she have taken oc , they did not significantly influence the development of ica thrombosis . arterial thrombosis in such cases is very rare in women below 35 years of age [ 21 , 22 ] . the annual incidence of ischemic stroke in women in this age group is 13 cases/100 000 women . the following risk factors are considered to increase the risk of developing thrombosis in subjects using oc : hypertension , diabetes , hyperlipidemia and smoking [ 2025 ] . oc predispose more strongly to the development of venous , rather then arterial thrombosis , which results from the haemodynamic properties of blood flow in these two types of vessels . other risk factors for venous thrombosis are slowed blood flow and thrombophilia , whereas vessel wall injury ( endothelium ) , including the atherosclerotic plaque are characteristic risk factors for arterial thrombosis . despite doubts concerning her taking oc , the patient s age ( 32 years old ) and lack of other , above - mentioned risk factors and also atherosclerosis allow oc to be ruled out as a possible cause of ica and mca thrombosis . the above - mentioned exclusions and the circumstances preceding the disease established in the course of the investigation indicate the traumatic background of the ica thrombosis in both cases . the ct and ct angiography performed in the first patient revealed the presence of a foreign body adjacent to the left ica on the level of c1 , whereas medial translocation and compression of carotid vessels caused by the foreign body were found during the surgical procedure . the histopathological examination revealed features of mechanical arterial injury , namely the rupture of the tunica media with focal haemorrhages into the intima and tunica media . the above - mentioned conditions indicate direct trauma as the cause of thrombosis . apart from the arterial occlusion by the thrombus found in the histopathological sections obtained from the second patient , the mechanical injury to the arterial wall in the form of the intima and tunica media rupture were also noted . such findings and the information obtained during investigation indicate indirect trauma as the cause of arterial thrombosis . post - traumatic ica thrombosis may result from a penetrating ( e.g. knife , bullet or other foreign body splinter , as in case 1 ) or non - penetrating ( blunt ) trauma . there are two types of the latter , namely direct ones concerning the neck in the region of the carotid artery , and indirect concerning distant regions , e.g. head . penetrating neck trauma caused by the above - mentioned tools , leads to arterial wall injury in the form of contusion or vessel wall dissection with the formation of intramural haematoma , outer arterial layer damage ( adventitia ) and the formation of pseudoaneurysm or segmental vessel spasm . suppurative inflammation may also develop in the periarterial space , should the patient survive long enough after the trauma . the above - mentioned primary vessel wall injuries , associated with an injury limited to at least endothelium and intima , predispose to the development of thrombosis with subsequent vessel occlusion and ischemic stroke . the ica segment localised a few centimetres over the cca bifurcation is the vessel segment most commonly injured in the course of non - penetrating ( blunt ) ica traumas , both direct and indirect . they result in the rupture of the intima and/or tunica media , with the possible formation of intramural haematoma , i.e. a condition called dissecting aneurysm . the thrombotic occlusion of the vessel lumen occurs a few hours or even days or weeks after the trauma [ 5 , 27 , 28 ] . six to ten percent of patients manifest such symptoms within the first hour after trauma , another 50 % after 10 h and in 5773 % of cases the asymptomatic period lasts more than 24 h. only 1735 % of such patients develop ischemic symptoms after such a period . the above - mentioned facts indicate that in some cases of traumatic ica thrombosis there is a variably long asymptomatic period between the trauma and the manifestation of neurological symptoms of ischemic brain damage . some authors compare it to intervallum lucidum occurring in patients with epi- and/or subdural haematoma . it results from thrombus propagation within the damaged vessel and may hamper the process of establishing the causal connection between the trauma and the ica thrombosis , especially , as according to some authors , the process itself may last up to several years [ 8 , 9 , 27 ] , which in our opinion is hardly probable . according to unterharnscheidt traumatic ica thrombosis may result from : ( 1 ) direct neck trauma ( e.g. boxers ) ; ( 2 ) direct head trauma ( including insignificant ) with force transfer to the neck region ; ( 3 ) oral cavity trauma ; ( 4 ) fractures of the neuro- and splanchnocranium bones ; ( 5 ) whiplash injuries ; ( 6 ) strangling or other manual procedures in the carotid artery region ( e.g. massage ) and ( 7 ) compression by seatbelt in the course of road traffic accidents . non - penetrating ica injuries result from blunt head and/or neck trauma . a head trauma with a violent and powerful head rotation and hyperextension of the cervical spine results in some topographic alterations , including the ica course . it may lead to ica compression by the transverse processes of c3 or c1c2 vertebral bodies and predisposes to thrombosis due to the secondary rupture of intima and tunica media by an intact adventitia . ica thrombosis may result from neck trauma , as well . by certain head positions , in which the artery is covered only with skin and fascia , it would be pressed against the c1 and c2 or transverse processes of c3c5 with secondary damage to intima , and even tunica media . in both of the discussed cases the thrombosis could have been caused by a segmental artery spasm on the injury site , which is impossible to establish during a postmortem examination . in 1974 crissey and bernstein identified four basic mechanisms of ica injuries , and thus four types of damage . type i damage results from a direct blow to the neck and most commonly concerns older patients , frequently having advanced ica atherosclerosis . a similar mechanism occurs by a sudden powerful hyperflexion of the cervical spine with a compression of the ica between the mandible and spine . type ii ica damage is caused by the hyperextension of the cervical spine with a head and neck rotation in the opposite direction . such a mechanism results in injury to the ica segment that is overstretched over the lateral masses of the first two cervical vertebrae . the discussed mechanisms of ica injuries , apart from a direct blow to its region , are most commonly observed in motorcyclists participating in road traffic accidents . type iii ica damage is most commonly observed in children and accompanies the intraoral trauma caused by a foreign body e.g. by falling on a hand - held or more frequently mouth - held object , e.g. pencil . the establishment of the causal connection between neck and/or head trauma and ica thrombosis and its cerebral consequences requires the exclusion of non - traumatic factors in the first line disease - associated , being the most common cause of thrombosis . this is very important as such trauma is most frequently a result of a criminal offence and brings the perpetrator to criminal and civil liability . the establishment of a causal connection may be problematic or even impossible in victims who apart from traumatic injuries suffer from diseases or factors predisposing to thrombosis .

the following manuscript presents two cases of ischemic stroke secondary to traumatic internal carotid artery thrombosis with concomitant middle cerebral artery thrombosis occurring very rarely in the medico - legal practice . penetrating neck trauma due to an occupational accident and multiple head and neck trauma secondary to battery were described . the autopsy and histopathological examination as well as the analysis of available medical records , including radiological examinations , and records of investigation indicated the sustained trauma to be the cause of the thrombosis .

pathological tissue fibrosis is the abnormal accumulation of collagen - rich extracellular matrix ( ecm ) after a chronic or misregulated response to injury that progressively disrupts tissue architecture , leading to tissue stiffness , impaired organ function , and eventually organ failure . fibrosis is featured in diverse conditions , accounts for as much as 45% of all deaths worldwide , and appears to be increasing in prevalence . fibrosis complications arise in very different disease settings , from autoimmunity and environmentally induced inflammation to cancer , spanning multiple organs . to date , the treatment options are extremely limited for attenuating or reversing this process . thus , there is an urgent need to delineate underlying pathological mechanisms that may lead to new therapeutic approaches . both the initiation and persistence of pathological fibrosis involves the activation and differentiation of progenitors to myofibroblasts , the key effectors in fibrosis . myofibroblasts play an important role in executing physiologic tissue repair , leading to matrix deposition , wound contraction , and healing on one hand , and pathological fibrogenesis leading to chronic fibrosing conditions on the other . accordingly , prominent molecular pathways in acute tissue repair often recapitulate their fibrogenic function in chronic fibrotic conditions , essentially conditions in which wound healing has gone awry ; these include platelet derived growth factor receptor beta ( pdgfr ) and transforming growth factor beta ( tgf- ) . however , there are still many gaps in our understanding of the mechanisms underlying fibrogenesis . due to their morphology and function , these cells are incredibly difficult to study in vitro . recently , elegant fate mapping studies pointed to a role for pericytes as a significant progenitor pool for myofibroblasts after injury in a variety of organ systems . liver pericytes , also known as hepatic stellate cells ( hscs ) , have been shown to be the major progenitor pool for myofibroblasts after carbon tetrachloride ( ccl4)-induced liver injury and fibrosis,5 , 6 and gene expression profiling of these cells isolated at various time points after ccl4 administration identified molecular alterations that might be functionally relevant to fibrogenesis . likewise , myofibroblasts and their precursors have been transcriptionally profiled during kidney fibrosis in mice using translational ribosome affinity purification technology . isolation of this cell type induces a lot of alteration in what these cells express ; therefore , it is important to study the function of the genes in their native disease environment without disrupting cell - cell and cell - matrix interactions . we aimed to identify novel modifiers of tissue fibrosis expressed in myofibroblasts or their progenitors through rna silencing in vivo . although much more challenging than conducting a cell - based screen in vitro , this in vivo approach was pursued to enable the interrogation of gene function in the context of the complex tissue environment and thereby yield physiologically relevant fibrosis modifiers . in order to achieve this objective , we employed recently generated transcriptomes from myofibroblasts and their precursors in models of liver and kidney injury and fibrosis.6 , 7 to achieve effective gene silencing in vivo , we used small interfering rna ( sirna ) delivery with lipid nanoparticles ( sirna - lnps ) , an emerging technology for gene silencing in vivo , particularly in the liver , and hence deployed this technology in a model of liver fibrogenesis . the sirna - lnp technology has been previously used to show that silencing of the collagen type i alpha 1 gene ( col1a1 ) reduced its mrna levels and collagen accumulation in liver fibrosis models.9 , 10 however , the use of sirna - lnp technology in a targeted assay for identifying novel fibrosis modifiers has not yet been reported . herein , we report our use of a platform composed of novel sirna targets , sirna - lnp delivery technology , and ccl4-induced liver fibrosis , resulting in the identification of novel modifiers of fibrogenesis in vivo . we aimed to silence genes in key fibrogenic cell lineages , myofibroblasts , and pericytes to identify novel mediators of tissue fibrosis . candidate genes were selected by the intersection of gene expression datasets for this cell lineage in two different organ systems . we identified genes that were commonly at least 2-fold upregulated in activated hscs isolated from livers of mice treated with ccl4 for 2 months and myofibroblasts and pericytes , their precursors , in the mouse kidney 2 and 5 days after unilateral ureteral obstruction ( uuo ) . we excluded transcripts that were not associated with a gene product , had no human homolog , or had well - known functions in fibrosis ( figure 1 ) . ultimately , we unbiasedly tested 24 genes by sirna - mediated gene knockdown ( kd ) in vivo ( table s1 ) . ccl4-induced liver injury and fibrosis in mice is a well - characterized model consisting of repeated ccl4 administration that injures hepatocytes , followed by a fibrogenic reaction . hscs are activated to expand , differentiate to myofibroblasts , and produce increased levels of ecm and pro - inflammatory mediators , thereby also promoting macrophage accumulation ; all of these reactions constitute a coordinated response to tissue injury and the progressive accumulation of collagen . we found that animals dosed orally with 1 ml / kg of ccl4 in mineral oil on day 0 and day 7 and euthanized on day 10 exhibited significant liver fibrosis based on increased picrosirius red ( psr ) and collagen immunopositivity in liver tissue as compared to vehicle - treated mice . percent area of tissue immunoreactive with asma , the hallmark of myofibroblasts , and iba-1 , a macrophage - specific marker , were also increased in ccl4-treated animals , suggesting an increase in myofibroblasts and macrophage numbers in the tissue ( figures s1a s1c ) . corresponding to increased collagen deposition in tissue , col1a1 mrna was markedly upregulated ( figure s2a ) . the assessment of col1a1 mrna levels was used as an expedient approach to functionally assess a relatively large number of sirna targets and flag fibrogenic genes of interest . given the sirna - lnp delivery system targets multiple liver cell types , including hscs , hepatocytes , kupffer cells , but not endothelial cells,11 , 12 , 13 , 14 we validated the ability to kd genes expressed in hscs in mice using sirna - lnps ( figures s1d s1h ) . mice were injected with a single dose of sirna - lnp ( 1 mg / kg ) against reelin ( reln - lnps ) , a gene prominently expressed in hscs . animals receiving reln - lnps , but not luc - lnps ( negative control ) , showed significant reduction in reln mrna in both oil- and ccl4-treated animals ( figure s1e ) . we also demonstrate the ability to kd the hsc - specific gene col1a1 in liver . col1a1-lnp reduced col1a1 mrna by 50% , with no effect on reln mrna . because the tgf- pathway is a recognized fibrogenic mediator , we tested whether kd of transforming growth factor beta receptor 1 ( tgfbr1 ) decreased the transcription and accumulation of collagen indeed , administration of tgfbr1-lnps and col1a1-lnps , but not luc - lnps , reduced the transcription of col1a1 mrna ( figure s1f ) as well as collagen accumulation ( figures s1 g and s1h ) . to test the role of genes identified in our transcriptomic analysis , we modified the sirna administration protocol to allow pre - existing protein to be turned over and therefore achieve pre - clearance of proteins encoded by the genes of interest and ensure continued kd throughout the experiment ( figure 2a ) . we first used luc - lnps to validate this protocol for detecting differences between oil- and ccl4-treated cohorts , with respect to the levels of col1a1 mrna , our primary parameter for defining target genes . we similarly evaluated differences in the mrna levels for a panel of other genes ( figure s2a ) . the strictly standardized mean difference ( ssmd ) values for col1a1 , collagen type iii alpha 1 ( col3a1 ) , tissue inhibitor of metalloproteinases 1 ( timp1 ) , and platelet derived growth factor receptor beta ( pdgfrb ) , but not tgfbr1 , integrin subunit alpha m ( itgam ) , or alpha - actin-2 ( acta2 ) , suggested that these genes were suitable to use as readout genes ( figure s2b ) . having established the suitability of the ccl4 treatment with the sirna - lnp kd platform , we proceeded to test our candidate genes . we tested the effect of kd of 24 individual genes in vivo ( figure 2a ) . we identified seven genes that significantly reduced the amount of col1a1 mrna ( table 1 ) . five of these seven genes , namely , early growth response 2 ( egr2 ) , fk506-binding protein ( fkbp10 ) , atpase na / k transporting subunit alpha 2 ( atp1a2 ) , follistatin like 1 ( fstl1 ) , and hyaluronan synthase 2 ( has2 ) , were silenced by 75%100% in whole liver tissue and significantly reduced col1a1 mrna levels . silencing of these genes did not elicit any overt toxicity , as measured by body weight loss ( figure s3 ) . because the other 19 genes did not meet these criteria ( tables 1 and s1 ) , we focused on further analyzing the five modifiers of fibrosis . the kd of the transcription factor ( tf ) egr2 had the broadest effect by reducing the levels of col1a1 and col3a1 mrna as well as the percent area immunopositive for asma , iba-1 , and collagen proteins , although the reduction in collagen by half did not achieve significance ( figure 2 ; table 1 ) . egr2 silencing also reduced pdgfrfb mrna levels ( figure 3a ) , suggesting a mechanism of decreased fibrosis and macrophage accumulation due to reduced expansion of activated hscs . given that egr2 has structural similarities to its family members , egr1 and egr3 , we confirmed the specificity of the egr2 sirna by transfecting human 293 t cells with plasmids encoding mouse egr1 , egr2 , or egr3 under the transcriptional control of the cmv promoter . in the tested construct , the expression of mouse egr1 , egr2 , and egr3 is not under the control of the endogenous promoter . thus , reduction in gene expression is due to the binding of the sirna egr2 to the mrna . using the same egr2 sirna that was used for the in vivo experiments , we found that this sirna reduced egr2 mrna levels , but not egr1 or egr3 mrna , unambiguously demonstrating that egr1 and egr3 were not targeted by our egr2 sirna ( figures s4a s4c ) . interestingly , even though the sirna against egr2 was specific , egr2-lnp treatment in vivo also reduced egr1 and egr3 mrna levels ( figures s4d s4f ) , suggesting transcriptional co - regulation of egr family members . similar to egr2 , kd of fkbp10 or atp1a2 reduced the collagen and iba-1 positive areas as did that of egr2 ( figure 2 ; table 1 ) ; however , kd of fkbp10 or atp1a2 did not alter the percent immunopositivity for asma . fkbp10 kd also decreased pdgfrb mrna levels , but this effect was somewhat weaker than that of egr2 ( 28% versus 45% reduction ; figures 3a and 3b ) , possibly accounting for its lack of effect on asma immunopositivity . these results suggest that egr2 , fkbp10 , and atp1a2 regulate fibrogenesis through different mechanisms . kd of either of the two other fibrosis modifiers , fstl1 and has2 , decreased col1a1 mrna levels as well as macrophage and collagen accumulation in the tissue , although has2 kd did not achieve significance for the latter ( figure 2 ) . notably , kd of these genes showed increased amounts of asma immunopositivity , which was highly significant for has2 kd , suggesting increased numbers of myofibroblasts . corresponding with the increased myofibroblasts , we detected higher levels of the timp1 mrna in the livers of both fstl1 and has2 kd animals ( figures 3d and 3e ) . despite the increased myofibroblast and timp1 mrna levels , , we also conducted in parallel in vitro studies and showed that three independent fstl1 sirnas reduced col1a1 mrna levels . thus , the direct reduction of collagen transcription may be sufficient to reduce overall collagen accumulation , despite the elevated number of myofibroblasts and reduced collagen degradation ( figure s5 ) . egr2 emerged as the strongest modifier of fibrosis in the ccl4 liver fibrosis / sirna kd platform . we aimed to directly demonstrate that egr2 kd in an hsc would reduce col1a1 and pdgfrb mrna levels . we tested the effect of egr2 kd in the human hsc line lx-2 using three independent sirnas ( figure 4 ) . this experiment required a different sirna to be used because no species cross - reactive sirna was designed ( see materials and methods ) . all sirnas against erg2 reduced its mrna levels as well as col1a1 and pdgfrb mrna levels . these results are consistent with the effect of egr2 kd in our in vivo ccl4 assay platform . as expected , kd of col1a1 reduced its own expression , but not that of egr2 . interestingly , col1a1 silencing also reduced pdgfrb mrna levels , suggesting a regulatory feedback loop . taken together , these data support that egr2 is fibrogenic in both rodents and humans . myofibroblasts are key effectors of fibrosis and therefore the elucidation of pathways that promote this cell lineage would be a significant advance in the field . here , we report the use of a targeted in vivo sirna kd platform to identify novel mediators of fibrogenesis . we leveraged novel biology - targeting genes upregulated during liver and kidney fibrosis in myofibroblasts and their progenitors and sirna - lnp technology to silence these genes in ccl4-induced liver injury and fibrosis in mice . we identified five genes , namely egr2 , atp1a2 , fkbp10 , fstl1 , and has2 , which modified fibrogenesis in this system because their kd reduced col1a1 mrna levels and collagen accumulation in the liver . prior to our study , egr2 , atp1a2 , and fkbp10 had not been functionally validated in fibrosis in vivo , and our study is the first to validate fstl1 and has2 in liver fibrosis . we further directly demonstrated that egr2 kd in human hscs reduced the expression of fibrotic genes . we conclude that in vivo sirna kd using sirna - lnp is a powerful tool to identify disease - relevant modifiers in vivo . studying myofibroblasts is extremely difficult because they are in direct contact with multiple cell types , including other myofibroblasts , myeloid cells , mesothelial cells , endothelial cells , epithelial cells , and circulating fibrocytes in situ . myofibroblasts respond to chemo - mechanical stimuli , and therefore disruption of their native environment during cell isolations alters their activation state . to circumvent these shortcomings this approach allowed us to study functional consequences of silencing of specific genes of interest in vivo in their native environment . it is of note that our sirna - lnp delivery approach allows us to silence gene expression in hscs ; however , this lnp formulation also targets other cells in the liver , including kupffer cells and hepatocytes , but not endothelial cells.11 , 12 , 13 , 14 therefore , we can not rule out the contribution of gene silencing in kupffer cells and hepatocytes on the anti - fibrotic outcome . our strategy to identify novel players in tissue fibrosis took advantage of recent transcriptomic datasets for myofibroblasts and their precursors isolated from fibrotic liver or kidney in mouse models.6 , 7 using sirna - lnp delivery technology , we tested the effect of gene silencing in a physiologically relevant context in vivo . we employed a ccl4 administration model that featured significant collagen accumulation and established several robust rna and histological measures of fibrogenic activity to identify fibrosis modifiers . using the criteria of significant reduction of col1a1 mrna levels of greatest interest are those that also reduced collagen accumulation in the tissue , namely , egr2 , atp1a2 , fkbp10 , fstl1 , and has2 . notably , these five genes were the most strongly silenced in vivo ( by 75% or greater ) that significantly reduced col1a1 mrna levels . it is possible that we might have achieved a greater degree of in vivo gene silencing for other target genes with a higher dose or a different dosing paradigm . thus , although we can not draw conclusions about the functional role of genes whose silencing was incomplete , our results clearly identify five genes as novel fibrogenic mediators in liver fibrosis in vivo . these genes fell into different groups based on the effects of their silencing on a mini mrna array and histological measures . we propose grouping them as follows : egr2 , a gene with potential function in hsc expansion ; atp1a2 and fkbp10 , genes that regulate mechanosensing ; and fstl1 and has2 , genes involved in matrix - cell signaling . egr2 , a tf of the egr family , emerged as the strongest effector in the sirna screen . egr tfs are important for the induction of cellular programs , including cell proliferation , differentiation , and death , in response to stimuli such as cytokines , growth factors , and toxic substances.17 , 18 , 19 egr2 has not been previously reported to play a role in tissue fibrosis in vivo . rather , egr2 is prominently known for its role in early myelination of the peripheral nervous system in humans and mice.21 , 22 , 23 egr2 kd silenced its expression by 94% , reduced col1a1 and col3a1 mrna levels , and diminished accumulation of myofibroblasts and macrophages , each to 15% of control . thus , our demonstration of reduced fibrogenic activity by egr2 silencing reveals a novel egr2 function . the downregulation of pdgfrb mrna levels by 45% suggests that egr2 may promote hsc expansion . this mechanism would explain the reduced asma - expressing hsc and thereby reduced signals for macrophage accumulation , all of which may have contributed to the reduction in fibrosis . importantly , we also directly demonstrate that egr2 kd in a human hsc line reduces col1a1 and pdgfrb transcripts , supporting the regulation of a fibrogenic program in hscs by this tf . the comparatively weaker effect of col1a1 silencing in vitro ( 50% ) versus in vivo sirna - lnp- mediated kd ( 80% ) of egr2 is due to the use of different delivery methods as well as the use of different sirna sequences to achieve gene silencing in human cells versus in vivo in mice . interestingly , col1a1 kd also reduced pdgfrb mrna levels . because ecm accumulation and expression of pdgfr are linked , it is feasible that decreased collagen production would decrease pdgfr expression in a feedback loop . the relevance of our finding to fibrogenesis in humans is also supported by a prior in vitro study , in which overexpression of egr2 in human skin fibroblasts upregulated collagen gene expression and myofibroblast differentiation , and these profibrotic tgf--induced responses were attenuated by egr2-depletion . we unambiguously confirmed that our sirna is specific for egr2 ( figures s4a s4c ) and also found that egr2 kd in human hscs reduces col1a1 and egr2 , but not egr1 or egr3 mrna ( data not shown ) , confirming the anti - fibrotic effect . however , in vivo egr2 kd also decreased the mrna levels of egr1 and egr3 , supporting the co - regulation of these transcription factors . it has been shown previously in vitro that overexpression of egr1 induced egr2 and egr3 expression and likewise egr3 overexpression induced egr1 and egr2 expression , whereas overexpression of egr2 did not have such an effect.25 , 26 , 27 here , we report that egr2 kd also reduces egr1 and egr3 mrna levels in vivo , a novel aspect of egr tf co - regulation . although egr2 has not been previously reported to play a role in tissue fibrosis in vivo , egr1 and egr3 have been implicated in the fibrotic response in vitro and in vivo previously.27 , 28 , 29 therefore , the extent of the anti - fibrotic effect may be due the co - regulation of egr tf . atp1a2 and fkbp10 were identified as fibrogenic modulators , with mechanisms of action distinct from that of egr2 . atp1a2 is the large catalytic subunit of an enzyme that catalyzes the hydrolysis of atp , coupled with the exchange of na and k ions across the plasma membrane . atp1a2 has a known function in the contractility of skeletal and cardiac muscle . on the basis of our results , it is intriguing to speculate that atp1a2 silencing may have reduced the contractility of myofibroblasts , effectively mimicking their behavior in a more compliant matrix environment and hence resulting in a less fibrogenic program.31 , 32 supporting this theory , we found that atp1a2 kd reduced fibrogenesis in vivo by significantly limiting the transcription of the col1a1 and timp1 genes , macrophage accumulation , and collagen deposition . however , in contrast to egr2 , atp1a2 silencing had no effect on asma expression . this theme of altered mechanosensing may also explain the alleviated fibrosis upon silencing of fkbp10 . fkbp10 is a molecular chaperone in the endoplasmatic reticulum that directly interacts with collagen i , contributing to the maturation and molecular stability of type i procollagen , promoting normal secretion , stable inter - molecular crosslinking , and collagen deposition in the ecm . fkbp10 has a well - established role in bone mass in humans and mice , but its role in tissue fibrosis is largely unexplored . fkbp10 mouse embryos are post - natally lethal and display reduced collagen crosslinking in calvarial bones . in this study , we report our novel finding that fkbp10 silencing reduces collagen deposition to 64% ; however , we found no reduction in asma expression . we speculate that fkbp10 silencing alters the quantity or crosslinking of the ecm and thereby alters mechanosensing . consistent with our in vivo results , a prior in vitro study showed that inhibition of fkbp10 in primary human fibroblasts from pulmonary fibrosis patients attenuates expression of pro - fibrotic genes and decreases collagen secretion . thus , our findings expand the scope of atp1a2 and fkbp10 functions by demonstrating their novel roles in fibrogenesis in vivo . fstl1 and has2 silencing also affected multiple fibrogenic readouts in our assay platform , but resulted in a pattern distinct from that of egr2 , atp1a2 , and fkbp10 . both fstl1 and has2 silencing reduced col1a1 mrna , macrophage accumulation , and collagen deposition but counterintuitively increased myofibroblast density as well as the level of timp1 mrna , a profibrotic pathway . fstl1 is a tgf--inducible , secreted , matricellular glycoprotein belonging to the secreted protein acidic rich in cysteines ( sparc ) family , with pleiotropic functions.37 , 38 , 39 during the course of our current study , it was reported that haplodepletion of fstl1 or a neutralizing anti - fstl1 antibody reduced bleomycin - induced pulmonary fibrosis . we found that fstl1 gene silencing reduced collagen transcription and accumulation in the context of liver fibrosis but surprisingly increased myofibroblast density , possibly reflecting a compensatory feedback loop . we hypothesize that the overall reduction in collagen accumulation is a direct result of decreased collagen production by fstl1 silencing , so that the increased number of myofibroblasts and reduced activity of collagen degradation would still result in decreased overall collagen accumulation . indeed , kd of fstl1 in the human hsc line lx-2 resulted in decreased levels of col1a1 mrna . like fstl1 , silencing of has2 resulted in unexpected effects , also likely through a matrix - modifying mechanism . has2 is one of three isoenzymes responsible for cellular hyaluronan ( ha ) synthesis , a matrix component normally highly abundant in joint synovium . ha is well known for its role in wound healing.40 , 41 has2 has been previously reported to be pathological in the bleomycin - induced lung fibrosis model , in which mesenchymal cell - specific deletion of has2 abrogated myofibroblast accumulation and inhibited the development of lung fibrosis . we show that has2 silencing also reduces liver fibrosis . has2 silencing also unexpectedly augmented myofibroblast density . taken together , our studies have identified fstl1 and has2 as fibrosis modifiers in our in vivo liver fibrosis platform and reveal surprising effects of silencing these genes , supporting their complex role in the regulation of fibrogenesis . to the best of our knowledge , our study is the first example of a targeted sirna assay for the discovery of novel fibrosis modifiers in vivo . the results implicate five novel modulators of the fibrogenic process in the liver and demonstrate the feasibility of sirna - lnp for interrogating gene function in the liver . because these genes are commonly upregulated in pericytes and myofibroblasts in both liver and kidney fibrosis models , these genes warrant follow - up to further explore their function and mechanism of action in liver and other organs and to inform their role in both physiological and pathophysiological fibrosis . all animal procedures were conducted in accordance with cambridge laws and the institutional animal care and use committee approved protocol no . 657 . 8- to 10-week - old male balb / c mice ( taconic bioscience ) were given ccl4 by oral gavage ( sigma - aldrich ; 1 ml / kg dissolved in mineral oil ) on days 0 and 7 . sirna - lnps were either given on day 6 at a concentration of 1 mg / kg or on days 7 , 3 , 2 , and 6 at 0.5 mg / kg . these experiments were controlled with cohorts of mice that received sirna targeting luciferase ( luc - lnps ) with oil or ccl4 , respectively . animals were sacrificed on day 10 by co2 asphyxiation , followed by cardiac perfusion with 10 ml of pbs , and liver lobes were harvested for qpcr and histopathological analysis . target fibrosis modifier genes were selected by intersecting transcriptomic data generated from isolated hscs of mice treated with ccl4 for 2 months , and genes specifically expressed in pericytes / myofibroblasts in the mouse kidney 2 and 5 days after uuo.6 , 7 169 genes were common to both datasets and significantly upregulated at least 2-fold . among these , we excluded transcripts that were not associated with a gene product , genes with no known human homolog , and well - known modifiers in fibrosis ( validated in vivo by independent investigators ) , resulting in 24 genes for further evaluation . a set of ten sirnas with canonical sirna structures was defined for each target gene . all sirnas were directed against the coding sequence of their respective target mrna and perfectly matched all known mrna transcript variants of the target gene available in the ncbi reference sequence database ( refseq db , release 65 , may 2014 ) . the sirnas used in this study were designed to be a perfect match only to their target mrna and to have 2 mismatches within positions 218 of the 19-mer sequence to any other genes . the design as described above is sufficient to ensure specificity.44 , 45 specifically , at least four mismatches between the sirna sequences of each of the genes of interest ( atp1a2 , egr2 , fkbp10 , fstl1 , and has2 ) and the readout genes col1a1 , col3a1 , tgfbr1 , pdgfrb , and timp1 . further , there were three mismatches between egr2 sirna and egr1 sequence and more than four mismatches between egr2 sirna and egr3 sequence . the sirna antisense strands lacked a seed region ( nucleotides 27 ) identical to a seed region ( nucleotides 27 ) of known mouse mirnas ( mirbase , release 20 , june 2013 ) . additional similarity analysis showed that there were at least four mismatches between the sirna sequences of each of the genes of interest ( atp1a2 , egr2 , fkbp10 , fstl1 , and has2 ) and the readout genes col1a1 , col3a1 , tgfbr1 , pdgfrb , and timp1 . further , there were three mismatches between egr2 sirna and egr1 sequence and more than four mismatches between egr2 sirna and egr3 sequence . from sirnas fulfilling those criteria , the sirnas for each final screening were selected for predicted activity based on analysis with proprietary algorithms ( axolabs , gmbh ) . in vivo grade sirnas each of the ten aforementioned sirna molecules / target gene was screened for its potency to kd gene expression in a cell - based reporter assay . for each target gene , the regions of their coding sequences targeted by the sirnas were synthesized and inserted to the 3 utr of the firefly luciferase gene in pcmvluc(v ) validation vector ( origene ) , generating an mrna transcript that encodes both the luciferase ( luc ) and the target gene when expressed in cells . 293 t cells ( atcc ) were co - transfected with the validation vector and the sirnas of interest using lipofectamine 2000 ( thermo fisher scientific ) at 0.1- , 1- , and 10-nm concentrations to assess kd potency . sirna binding to the target gene leads to degradation of the mrna that encode both luc and the target gene , resulting in reduced luciferase expression . luciferase expression was measured 24 hr post - transfection in a functional assay using the promega luciferase assay system ( promega ) , and luciferase signal was detected using a perkinelmer envision plate reader ( perkinelmer ) . for each sirna tested , the efficiency of kd was calculated as percent of luciferase signal compared to cells that were transfected with the validation vector for the target gene alone . the most potent sirna for each target gene ( > 90% luciferase reduction at 1 nm sirna in vitro ) was then formulated as lnps for in vivo application . the sequences of the sirnas that resulted in efficient kd ( 75% ) of their target gene in vivo are provided in table s2 . sirna - lnps were synthesized by mixing together an sirna - containing aqueous phase with a lipid - containing ethanol phase . the aqueous phase contained sirna in 10 mm citrate buffer ( ph 3 ) . the ethanol phase contained ionizable lipid c12 - 200 ( wuxi apptec ) , 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc , avanti polar lipids ) , cholesterol ( sigma ) , and 1,2-dimyristoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy(polyethylene glycol)-2000 ] ( c14 peg 2000 , avanti ) at a 50:10:38.5:1.5 molar ratio and 5:1 c12 - 200:sirna weight ratio . the aqueous and ethanol phases were mixed together at a 3:1 volume ratio in a microfluidic chip device using syringe pumps as previously described to a final sirna concentration of 1 mg / ml . the resultant sirna - lnps were dialyzed overnight in a 20,000 molecular weight cut - off ( mwco ) cassette against 1x pbs at 4c . on average , sirna - lnps had a mean diameter of approximately 100150 nm , with a polydispersity index between 0.1 and 0.2 as measured by dynamic light scattering ( zetasizer , malvern instruments ) . the sirna encapsulation efficiency ( approximately 60% ) was determined using a modified quant - it ribogreen assay ( invitrogen ) as previously described . this sirna delivery system can target multiple liver cell types , including hepatocytes , monocytes , tissue macrophages , dendritic cells ( dcs ) , and myofibroblasts / pericytes . rna was extracted from liver lobes with trizol ( thermo fisher scientific ) , followed by on - column dnase digestion using rneasy mini kits and rnase free dnase sets ( both from qiagen ) . cdna was generated using the high - capacity cdna reverse transcription kit , and qpcr analysis was performed using pre - designed taqman primer probes ( table s3 ) on the quantstudio 12k flex real - time pcr system ( all from thermo fisher scientific ) to determine the expression levels of sirna target genes and a panel of readout genes . the relative expression of each gene was normalized to mouse or human glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) using the 2 method , and the mean expression of each gene in the control group was set to 100% . 5-m sections were stained with psr using an automated leica system according to standard protocols . anti - alpha - smooth muscle actin ( sma ) ihc was performed with fluorescein isothiocyanate ( fitc)-labeled mouse monoclonal antibody 1a4 , followed by an anti - fitc antibody and periodic acid - schiff ( pas ) counterstain ( abcam ) . anti - iba-1 and anti - col1a1 ihc was performed using rabbit polyclonal antibodies ( cat . # 019 - 19741 , wako , and genetex , cat . the iba-1 positive area was limited to infiltrating macrophages by differentiating isolated single cells ( largely kupffer cells lining sinusoids ) from larger round cells in clusters ( periportal , largely infiltrating macrophages , sometimes filled with mineral ) . de novo interstitial asma was differentiated from endogenous asma in muscular arteries by performing a pas counterstain and then programming the algorithm to distinguish vascular morphology for exclusion from quantification . the human hsc line lx-2 ( emd millipore ) was transfected with sirnas against egr2 , col1a1 , or luc with lipofectamine rnaimax reagent following the manufacturer s protocol . we used the following predesigned sirnas ( listed as sirna i d ) : egr2 : s4540 , s4541 , and s4542 ; col1a1 : s3276 ; luc : am4629 ; and fstl1 : s22032 , s22033 , and s22034 ( thermo fisher scientific ) . rna was isolated on day 2 after transfection using rneasy kits with on - column dnase digestion ( qiagen ) . 293 t cells ( atcc ) were co - transfected with pcmv - egr1 , pcmv - egr2 , or pcmv - egr3 ( origene ) and either silencer select negative control no . 1 sirna ( thermo fisher scientific ) or sirna against egr2 ( axolabs ) . rna was isolated 24 hr after co - transfection using rneasy plus kits ( qiagen ) . statistical significance was assessed by two - tailed , unpaired student s t test for comparison of two groups or by one - way anova , followed by the dunnett s multiple comparisons test for more than two groups . we used an ssmd method to analyze the suitability of the mrna readout genes in our sirna - lnp / ccl4 liver fibrosis platform ( figure s2 ) . the ssmd method was used to calculate the median of differences divided by the standard deviation of the differences between the group treated with oil and the control sirna targeting luciferase ( oil / luc - lnp ) and versus ccl4/luc - lnp . it represents the average fold change penalized by the variability of the fold change across animals . ssmd is a suitable method because it takes the variability between animals into account . for our study , a threshold of < 1.5 was taken as the cut - off value for suitable readout genes .

fibrotic diseases contribute to 45% of deaths in the industrialized world , and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed . we aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through rna silencing in vivo . we leveraged novel biology , targeting genes upregulated during liver and kidney fibrosis in this cell lineage , and employed small interfering rna ( sirna)-formulated lipid nanoparticles technology to silence these genes in carbon - tetrachloride - induced liver fibrosis in mice . we identified five genes , egr2 , atp1a2 , fkbp10 , fstl1 , and has2 , which modified fibrogenesis based on their silencing , resulting in reduced col1a1 mrna levels and collagen accumulation in the liver . these genes fell into different groups based on the effects of their silencing on a transcriptional mini - array and histological outcomes . silencing of egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line . prior to our study , egr2 , atp1a2 , and fkbp10 had not been functionally validated in fibrosis in vivo . thus , our results provide a major advance over the existing knowledge of fibrogenic pathways . our study is the first example of a targeted sirna assay to identify novel fibrosis modifiers in vivo .

heterogenous vancomycin intermediate staphylococcus aureus ( hvisa ) strains have been reported as indicators for reduced vancomycin susceptibility in s. aureus , and various studies associated its presence with vancomycin treatment failure . it has been shown that methicillin resistant s. aureus ( mrsa ) has the propensity to evolve into hvisa phenotype during in vitro exposure to subinhibitory concentrations of vancomycin . during the last decade , hvisas had been isolated in many countries including those in south east asia ; nevertheless , in our knowledge , its emergence has not been reported in malaysia . as a pilot study , we investigated the prevalence of hvisa among mrsa strains isolated at hospital kuala lumpur ( hkl ) in a 3-month period and determined factors associated with its infections . hospital kuala lumpur is the largest hospital in malaysia with the highest mrsa burden in the country . in this hospital , vancomycin is used as the standard first line treatment for mrsa infection ; however , recently , its efficacy has been a subject of discussion due to several anecdotal vancomycin treatment failure cases in hospital kuala lumpur . we also wondered if some of the mrsas isolated in the hospital were actually hvisas with reduced susceptibilities to vancomycin that could not be detected by routine microbiological tests used in our hospital diagnostic laboratory . to investigate this , from 25 february to 25 may 2009 , we collected a total of 320 index mrsa isolates ( first mrsa isolated from the corresponding patients ) and established them as strains for vancomycin resistance testing . as it is cost , time and labor consuming to perform vancomycin population analysis on all 320 strains to test for heterogenous vancomycin resistance , strains were first screened for the phenotype using glycopeptide resistance detection ( grd ) etest antibiotic strips ( ab biodisk , sweden ) . after grd screening , a total of 8 strains were defined as presumptive hvisa , no visa strain was detected . following that , to confirm the results of the grd screening interestingly , area under the curve ( auc ) analyses of the strains ' population analysis profiles confirmed that 7 out of the 8 tested strains were hvisa ( table 1 ) , giving a prevalence rate of 2.19% . table 1heterogenous vancomycin intermediate staphylococcus aureus strains and their corresponding patients in this study.specimen noagegenderprimary diagnosisspecimendiabetes mellitusrenal failuremalignancyadmission to icudays of hospital staydays of iv vcmon beta - lactamarea under curve ratio68214maleright hip osteomyelitisnasal swabnononoyes44 days0yes0.9058253femaleleft diabetic foot ulcerpus swabyesyesnoyes52 days14yes0.9318258maleacute ventriculitiscsfyesnonoyes98 days14yes1.0125220malegluteal sarcoma with hapsputumnonoyesyes24 days10yes0.9097871malepemphigus folliaceouspus swabyesnonono34 days7yes1.012154femaleacute encephalitis with haptracheal aspiratenononoyes32 days14yes0.9646029femalemeningo - encephalitis with hapsputumnononoyes38 days10yes0.98icu , intensive care unit ; iv , intravenous ; vcm , vancomycin ; csf , cerebrospinal fluid ; hap , hospital acquired pneumonia . icu , intensive care unit ; iv , intravenous ; vcm , vancomycin ; csf , cerebrospinal fluid ; hap , hospital acquired pneumonia . all hvisa strains isolated in this study were hospital acquired as they were isolated from their corresponding patients after 48 hours of hospital admission . to determine factors associated with the 7 hvisa infections , demographic data of all corresponding patients of each index mrsa isolate were retrieved from medical records . medical history of each patient such as diabetes mellitus , renal failure , malignancy , together with prescription history of vancomycin and beta - lactam antibiotics ( as these were the only classes of antibiotics prescribed to the corresponding patients of the study isolates during this investigation ) , length of hospitalization and intensive care unit ( icu ) admission were recorded . continuous variables were then assessed by independent samples t - test , while categorical variables were analyzed using pearson 's chisquare . calculations were performed using statistical package for social science ( spss ) 12.0 ( spss inc . , chicago , usa ) where a p - value of < 0.05 was considered as significant . after performing multivariate linear regression , we found that icu admission ( p<0.004 ) , hospitalization of more than 14 days ( p<0.014 ) and vancomycin administration of more than 7 days ( p<0.016 ) were independent factors associated with hvisa infections in our group of patients . our findings were in line with those of charles et al . in 2004 , where hvisa / visa infections were found to be associated with longer antibiotic treatment periods and longer hospitalization . in a separate report it seems that patients who are severely ill , hospitalized for long durations with icu admissions might have a higher chance of developing hvisa infections . as many patients in hkl fulfill some or all of the above criteria , taking it together , we suspect that the prevalence of hvisa in hkl might be high ; however , these strains are not being actively detected by the hospital diagnostics laboratory . as hvisa and mrsa with reduced vancomycin susceptibility has been reported to cause treatment failure , given the hvisa prevalence rate detected in this study , it is not surprising that vancomycin treatment failure cases among mrsa infected patients are increasing in hkl . in our study , we employed the grd test as a screening tool for hvisa before confirming the resistance with population analysis , and found that the grd etest was fairly specific with only one false positive result . in a review , howden and colleagues have reported the test 's sensitivity as 9394% with a 8295% specificity for hvisa detection . therefore , the grd might be considered a good screening tool for hvisa in hospitals where most hospitalized patients are severely ill with long hospitalization durations . once identified as hvisa infected , optimal treatment could be prescribed to the corresponding patient to prevent vancomycin treatment failure , thereby increasing the chance of a good clinical outcome for the patient . as the strains used in this study were collected in a short span of 3 months , and that vancomycin treatment failure is on the rise in hkl , we suspect that the actual prevalence of hvisa in this hospital might be even higher . we found the grd test useful for hvisa screening , nevertheless pap - auc analysis still remains the gold standard for hvisa confirmation . a more comprehensive , case control study involving major hospitals in the country would be important to better understand the significance and distribution of hvisa in malaysian hospitals .

in a 3-month study done in hospital kuala lumpur ( hkl ) , 7 out of 320 methicillin resistant staphylococcus aureus isolates were confirmed as heterogeneous vancomycin intermediate s. aureus ( hvisa ) using the glycopeptide resistance detection e - test and population analysis , giving a prevalence rate of 2.19% . this is the first report of hvisa in malaysia .